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  • 1. ISSN 1040-4597 A Quarterly Clinical Review Volume VIII, Number 4–2001 Cyclooxygenases Cyclooxygenases Randy C. Mifflin, Ph.D. and Don W. Powell, M.D. Department of Internal Medicine University of Texas Medical Branch Galveston, Texas Key Concepts • Cyclooxygenase (COX) enzymes catalyze the rate limiting steps in prostaglandin synthesis. Prostaglandins play major roles in diverse physiological processes such as maintenance of GI mucosal integrity and pathological processes such as inflammation and neoplasia. • Two COX isoforms, COX-1 and COX-2 exist in higher organisms. They are highly similar in struc- ture and enzymatic activity. The main differences between the two lie in their genetic regulation and biological roles. COX-1 expression is constitutive in most cell types and is thought to carry out “housekeeping” roles in the various tissues. In contrast, COX-2 expression is induced in Figure 1. Prostaglandin Biosynthesis. response to inflammatory and proliferative stimuli. The Cyclooxygenase • COX enzymatic activity is the Reaction CONTENTS target of widely used non- steroidal anti-inflammatory Cyclooxygenase (COX) Cyclooxygenases ...................49 drugs NSAIDs). COX-2-selective Randy C. Mifflin, Ph.D. and enzymes, also referrred to as NSAIDs are currently being Don W. Powell, M.D. prostaglandin H synthases or developed in the hopes of limit- prostaglandin endoperoxide Cyclooxygenases and GI ing inflammation without adverse Mucosal Protection ................56 synthases, catalyze the rate limit- GI and renal effects. Mark Feldman, M.D. ing steps in prostaglandin (PG) • The cellular source of inducible and thromboxane (TX) synthesis Cyclooxygenase-2 and COX-2 activity in acute and Colon Carcinogenesis............60 (Fig. 1). Enzymatic COX chronic GI inflammation and Raymond N. DuBois, M.D. and neoplasia is currently poorly substrates are 20 carbon polyun- Moss Mann, M.D. understood. saturated fatty acids, most often 49
  • 2. for the Cox 2 isoform. In the last Introduction profile will require post-market- part of the article, the authors ing studies of the millions of also review less well-known patients with inflammatory areas of cyclooxygenase biology diseases who use the drugs. such as the regulation of Cox In the last article, Drs. R.N. gene expression in gastrointesti- DuBois and M. Mann review the nal tissues. The latter is impor- concept that inhibition of tant because an inhibition of cyclooxygenase enzymes has a cyclooxygenase in the stomach therapeutic role in cancer risk and intestine leads to gastroin- reduction in the gastrointestinal testinal damage; side effects tract, especially for human which limit the clinical effective- colorectal carcinoma. The ness of cyclooxygenase authors review the role of Cox inhibitors. inhibition in the prevention of The second article by Dr. sporadic colorectal cancer and Mark Feldman reviews the role their therapeutic effect in reduc- Don W. Powell, M.D. of cyclooxygenase in maintain- ing the size and number of Issue Editor ing the barrier function of the colonic adenomas in a genetic gastrointestinal tract. Dr. Feld- form of colon cancer, familial The development of clinically man reviews the idea of the adenomatous polyposis (FAP). effective inhibitors specific for “cytoprotective effect” of These authors also review the cyclooxygenase 2 (Cox 2) is a prostaglandins in the gastroin- potential mechanisms for the dramatic example of the power testinal mucosa. He lists the chemopreventive effect of nons- of modern biological research at evidence for the concept that teroidal anti-inflammatory drugs the turn of the 20th Century. In cyclooxygenase 1 is the “house- (NSAIDs) in the prevention of less than ten years after this keeping” form of the prostaglan- colorectal cancer. Studies in second isoform of cyclooxyge- din-forming enzymes that main- animals suggest that the chemo- nase was cloned, our understand- tains structure and basic function preventive effects of NSAIDs ing of the formation of prosta- in tissues, whereas Cox 2 is the may occur through specific inhi- glandins, their roles in normal isoform that produces prosta- bition of Cox 2 enzymes. If this biology and disease, and poten- glandins during inflammatory is the case, then Cox 2 specific tial ways to inhibit their forma- states. The prostaglandins are the inhibitors might have a positive tion have been revolutionized as cause of pain, erythema, warmth chemopreventive effect without a result of studies of the molecu- and the edema of inflammation. the gastrointestinal side effects lar biology and structure of these These proposed differences in of Cox 1 inhibition. While it is enzymes. This is a prime exam- the two Cox isoforms have led to too early to be sure if this varia- ple of the application of molecu- the concept of “good” cyclooxy- tion of the Cox 2 therapeutic lar biology and structural biol- genase (Cox 1) and “bad” cyclo- hypothesis is correct, certainly it ogy to drug development. oxygenase (Cox 2). He reviews is an exciting idea that will result The first article in this issue of the Cox 2 therapeutic hypothe- in much basic and clinical inves- the Regulatory Peptide Letter by sis, which says that highly selec- tigation in the coming few years. R.C. Mifflin and D.W. Powell tive Cox 2 inhibitors should be The cloning of a second summarizes the biology of the efficacious in inflammatory isoform of the cyclooxygenase two cyclooxygenase enzymes. disease without causing the enzyme, as well as molecular The authors briefly describe the severe side effects, particularly and structural studies of both biochemistry of prostaglandin GI toxicity, which limits the proteins, have led to a new class (PG) formation, the known func- usefulness of this class of drugs. of therapeutic agents in less than tions of the many members of Dr. Feldman points out that a decade. While the ultimate the PG family, and the various although early reports of the promise and efficacy of these distinct receptors through which clinical trials of the specific Cox drugs remains to be determined the PGs have their biologic 2 inhibitors now approved in the through their extensive use in effects. They review the molecu- United States, Celecoxib® and humans with disease, the lar and structural organization of Rofecoxib®, tend to support the creation of specific Cox 2 the Cox isoforms and how the inhibitors represents an impor- concept of a Cox 2 therapeutic molecules that make up the tant paradigm for the develop- hypothesis, it is still early in the enzyme determine its structure. ment of pharmacologic agents use of these drugs. Their ulti- This structure has allowed indus- s in the 21st Century. mate efficacy and side effect try to create specific inhibitors 50
  • 3. arachidonic acid (AA), released isotype switching to IgG1 and IgE from phospholipid of cellular in B cells. Important roles for PGs Editorial Board membranes by a phospholipase in CNS function and development A2. The first step involves addi- have also been identified. For tion of O2 atoms to C-11 and C-15 example, increased PGE2 synthe- Chung Owyang, M.D. to yield PGG2. Since this results sis by hypothalamic endothelial Editor-in-Chief in cyclization of the fatty acid this cells is involved in the febrile Chief, Division of is referrred to as the cyclooxy- response and PGs generated at Gastroenterology genase activity. The 15-hydro- sensory nerve terminals cause Professor of Internal Medicine peroxide group of PGG2 is then hyperalgesia. Increased PG University of Michigan Medical Center converted to an alcohol forming synthesis has also been correlated Ann Arbor, MI PGH2 by the peroxidase activity with seizure activity. of the enzyme. PGH2 is subse- PGs have been implicated in a quently converted to other PGs wide variety of disease processes. Haile T. Debas, M.D. (PGD2, PGE2, PGF2 , PGI2) or The huge annual market for non- Dean thromboxanes (TXA2 ) by specific steroidal antiinflammatory drugs School of Medicine cellular synthases (Fig. 1). (NSAIDs) which inhibit COX University of California activity is a testament to the role San Francisco, CA Functions of of PGs in acute inflammation and chronic inflammatory diseases Prostaglandins Robert T. Jensen, M.D. such as asthma, rheumatoid/osteo Chief, Digestive Diseases PGs play critical roles in arthritis, and inflammatory bowel Branch normal physiological processes. disease (IBD). Epidemiological National Institutes of Platelet-derived TXA2 is an and animal studies indicate that Diabetes, Digestive and important mediator of platelet inhibition of PG synthesis is Kidney Diseases aggregation and thus hemostasis. efficacious in the prevention of National Institutes of Health During periods of stress, PGs of Bethesda, MD coronary artery thrombosis, the E and I series are important Alzheimer’s disease, and gastro- regulators of renal blood flow. intestinal and breast cancer. The Gabriel M. Makhlouf, M.D., Ph.D. PGs likewise are important in properties of PGs that contribute Professor of Medicine modulating many aspects of to disease progression include Director of Gastroenterology reproductive biology including their thrombotic activity, ability to Research ovulation, fertilization, fetal modulate cellular apoptosis and Division of Gastroenterology development, and parturition. The other cell cycle parameters, Medical College of Virginia opposing actions of different PG Richmond, VA angiogenic activity, and other classes help to maintain bronchial functions yet to be identified. tone. The processes of bone In the accompanying article, Don W. Powell, M.D. formation and resorption are also Dr. Dubois will cover the role of Edward Randall and Edward subject to regulation by PGs. COX enzymes and PGs in the Randall Jr. Professor and Chair Macrophage differentiation is development of colorectal cancer Department of Internal Medicine likewise modulated by PGs. As and discusses their role in neo- Professor of Physiology and discussed in Dr. Feldman’s article plasia in more detail. Biophysics to follow, PGs are vital to the The University of Texas maintenance of mucosal integrity Medical Branch PG Receptors in the GI tract and also play a role Galveston, TX The effects of PGs upon cells in the regulation of motility and are realized when each binds its secretion. PGs also affect immune James C. Thompson, M.D. specific membrane-bound recep- function in a number of ways. Ashbel Smith Professor tor (Table 1). These constitute a Through its ability to differen- of Surgery homologous family of G protein- tially inhibit cytokine synthesis Department of Surgery coupled receptors containing by TH1 cells, PGE2 can shift the The University of Texas seven transmembrane domains. balance of an immune response in Medical Branch Differential responses to specific favor of TH2 cells. PGE2 also Galveston, TX PGs are determined by the type of synergizes with IL-4 to activate 51
  • 4. Table 1. Membrane-Based Eicosanoid Receptors. (Adapted from E.J. Goetzl, S. An, and W.L. Smith FASEB J. 9: 1051–1058, 1995.) Receptor Tissue Transductional Eicosanoid Name Distribution* Signal Effects Ca++ PGE2 EP1 Smooth Muscle, Fibroblasts SM Contraction Mobilizatiion Proliferation PGE2 EP2 Smooth Muscle, Epithelial Cells, cAMP Increase SM Relaxation, Stimulate Intestinal Mast Cells, Neurons, Fibroblasts Secretion, Sensation, Inhibit Mast Cell Degranulation PGE2 EP3 Smooth Muscle, Adipocytes, cAMP Decrease SM Contraction, Inhibit Lipolysis, Ca++ Mobilization Neurons, Epithelial Cells. Neurotransmitter Release, Stimulate Kidney Renal H20 Reabsorption PGE2 EP4 Fibroblasts, Myofibroblasts, cAMP Increase SM relaxation, Induction of Smooth Muscle Stellate Morphology in Myofibroblasts PGD DP Platelets, Smooth Muscle, cAMP Increase SM Relaxation, Inhibition of Neurons Neurotransmitter Release, Platelet Aggregation PGI IP Platelets, Smooth Muscle, cAMP Increase SM Relaxation, Inhibition of Platelet Neurons Aggregation, Stimulate Intestinal Secretion Ca++ Mobilization PGF2 FP Kidney, Myofibroblasts, SM Contraction, Myofibroblast Astrocytes, Smooth Muscle Contraction Ca++ Mobilization TXA2 TP Platelets, Smooth Muscle SM Contraction, Platelet Aggregation, Glomerular Filtration, Intestinal Secretion * not comprehensive G protein (G s, G i, G q, G 12) associated receptors (PPARs). heme-containing proteins with a coupled to each receptor. At least These receptors are members of molecular weight of roughly 71 four distinct PGE2 receptors exist the nuclear hormone receptor KDa. They share 63% identity at which couple to different signal- superfamily of ligand activated the amino acid level. COX-2 ing pathways. As a general rule, transcription factors which target contains an 18 amino acid inser- G s-coupled receptors result in to the nucleus upon ligand bind- tion in its carboxyl terminal increased levels of intracellular ing. Thus certain PGs are able to region while COX-1 contains an 8 cyclic adenosine monophosphate directly modulate transcription of amino acid insertion at the amino (cAMP); G i-coupled receptors specific genes via interaction with terminus of the mature protein. result in inhibition of cAMP PPARs. Both proteins are glycosylated; generation; and G q-coupled three conserved N-linked glyco- COX-1 and COX-2 receptors result in intracellular sylation sites exist in both Ca2+ mobilization. Signaling via enzymes and COX-2 contains an Two distinct COX enzymes G 12-coupled receptors is not additional site within the 18 exist. COX-1 was first purified completely undersood. Further amino acid insertion. and characterized in the 1970s diversity in the response to each The mature proteins contain and the gene was isolated in 1988. PG is achieved through alternate three distinct domains. The first is The discovery and cloning of the splicing of PG receptor mRNAs a conformation which is highly second COX isoenzyme, COX-2, generating different carboxyl similar to that of epidermal in 1991 initiated a revolution in termini. growth factor (EGF) and is termed our understanding of PGs and Recent evidence also indicates the EGF-like domain. The func- their functions in normal physiol- that certain PGs, such as PGJ2 and tion of this domain in COX ogy and disease. its derivatives, are also potent enzymes is poorly understood but The two enzymes are highly ligands for a class of receptors is thought to facilitate recruitment similar in structure and enzymatic termed peroxisome proliferator- and interaction with other cellular activity. Both are homodimeric 52
  • 5. proteins. The second domain contains a series of amphipathic helices which comprise the membrane attachment site. COX enzymes are unlike other integral membrane proteins in that they are not anchored via transmembrane domains. Instead, they associate with the endoplasmic reticulum (ER) membrane via hydrophobic interactions and are thus mono- topic membrane proteins. It is interesting that while both enzymes are associated with the luminal face of the ER, COX-2 is also enriched in the perinuclear region. The association of COX-2 with the nucleus raises questions about a direct role of COX-2- derived PGs on gene expression Figure 2. A: Diagrammatic representation of the orientation of COX-1 and COX-2 in via association with PPARs. The the ER membrane. Shown are a COX-1 and a COX-2 homodimer demonstrating third domain is a large globular the association with the lumenal ER surface via the amphipathic helices. In the region which contains the cyclo- central portion of each monomer is shown a cutout section demonstrating NSAID oxygenase and peroxidase active binding to the active site. In the case of COX, the active site is narrower allowing access only to NSAIDs with smaller side chains. COX-2 specific NSAIDs contain sites. The COX active site lies in larger side chains not accommodated by the COX-1 pocket. B: Diagrammatic a narrow hydrophobic channel representation of the molecular structure of flurbiprofen, a nonselective NSAID, and framed by the membrane attach- celecoxib, a COX-2 specific NSAID. ment helices which allows arachi- differences in this channel that Mechanisms of donic acid cleaved by PLA2 direct also play a role in determining COX Inhibition access from the ER membrane substrate and inhibitor specificity without having to transit a hydro- Based upon their inhibitory include histidine (COX-1) to argi- philic environment (Fig. 2). The mechanisms COX inhibitors nine (COX-2) at position 513, amino acids involved in substrate can be grouped into four classes and serine (COX-1) to alanine binding and catalysis are by and (Table 2). All but the first class (COX-2) at position 516. large identical between the two are reversible inhibitors in that Another interesting difference enzymes. Two important differ- once the drug is removed, COX between COX-1 and COX-2 is ences are found at residues 434 activity is restored, albeit at that each enzyme apparently and 523 (COX-1 numbering) different rates depending upon the utilizes a distinct source of cellu- where isoleucine occupies each compound. The first class, which lar arachidonate as substrate position in COX-1 and valine is includes aspirin and recently resulting in a functional compart- present in each position in COX-2. developed COX-2-specific mentalization of COX-1 versus These amino acids are part of the aspirin-like molecules irreversibly COX-2 activity. For example, substrate binding channel and one inactivate COX activity by acety- aggregation of IgE receptors on consequence of these substitutions lating an active site serine. mast cells results in a biphasic is that COX-2 has a wider channel. Aspirin is considered COX-1 release of PGD2. The first phase This channel difference is the selective since doses 10 to 100 is mediated by COX-1 utilizing basis behind the broader substrate fold higher than those required for arachidonate released by a form specificity of COX-2 and, as COX-1 are necessary to acetylate of phospholipase A2 called secre- discussed below, the basis behind the COX-2 active site. Although tory PLA2 while the second phase the design of drugs specifically aspirin-acetylated COX-1 retains is mediated by COX-2 utilizing targeted to inhibit COX-2. In no enzymatic activity, due to its arachidonate released by a differ- fact, changing isoleucine 523 in larger substrate binding channel, ent phospholipase A2 termed COX-1 to valine renders it sensi- acetylated-COX-2 retains its cytosolic PLA2. tive to some COX-2-selective peroxidase activity, and is effec- inhibitors. Other amino acid 53
  • 6. sible for generation of PGs which Table 2. Four Modes of COX Inhibition by NSAIDs. mediate homeostatic or “house- keeping” functions such as main- Mode of tenance of vascular tone and Inhibition Selectivity Examples Comments mucosal integrity in the GI tract. Covalent COX-1 Aspirin Acetylation of active The human COX-1 promoter Modification COX-2 (APHS) APHS* site serine region resembles that of other Compete with AA† for Reversible, COX-1 and 2 Ibuprofen housekeeping genes in that it Competitive Mefenamate active site. lacks a TATA box and is generally Inhibition not subject to transcriptional Slow, Time- COX-1 and 2 Indomethacin Salt bridge formation induction. However, COX-1 dependent Flurbiprofen with Arg. 120 expression is subject to develop- Inhibition mental and inducible regulation under certain circumstances. For Time-dependent COX-2 Celecoxib Larger side groups to COX-2 Inhibition Rofecoxib occupy extra side example, stem cell factor (SCF) SC58125 pocket in COX-2 treatment of immature murine bone marrow derived mast cells • o-(acetoxyphenyl)hept-2-ynyl sulfide † arachidonic acid results in a 6–8 fold induction of COX-1 mRNA and protein levels. tively converted into a lipoxyge- inhibitor. These agents exhibit a Differentiation inducing stimuli nase enzyme capable of generat- slow, time-dependent inhibition (eg. transforming growth factor- , ing 15-R-hydroxyeicosatetraenoic of both COX isoforms. The phorbol esters) have also been acid (HETE). Recently, the aspirin delayed kinetics of inhibition by shown to result in transient 1.5 to derivative, o-(acetoxyphenyl)- this class probably reflects the 3-fold induction of COX-1 expres- hept-2-ynyl sulfide (APHS) has time necessary for formation of a sion in monocytes and macro- been developed which exhibits salt bridge between the carboxy- phages. Estrogen-induced expres- increased selectivity and potency late of the drug and arginine 120 sion of COX-1 is responsible for toward COX-2. APHS, like (COX-1 numbering). the increase in PGI2 synthesis in aspirin, irreversibly inactivates The fourth class of COX perinatal pulmonary vascular beds the cyclooxygenase activity of inhibitors selectively inhibit COX- and is partly responsible for the COX-2. APHS-modified COX-2 2. These include recently-devel- pulmonary vasodilation seen retains the ability to generate oped drugs such as celecoxib and during this period. 15-R-HETE. The development of SC58125 which incoporate In contrast COX-2 expression APHS as a COX-2-selective agent sulphonamide or sulphone groups is undetectable in most normal was based upon structural data of in place of carboxylic acid. They tissues. Important exceptions to the COX-2 substrate channel and also contain larger side groups this rule are the brain and renal the observation that effective which penetrate the larger binding cortex where constitutive COX-2 COX-2-selective inhibitors have pocket of COX-2, but their size expression occurs. COX-2 expres- sulfur-containing side chains in prevents them from entering the sion in many cell types is highly place of a carboxylic acidic group. smaller pocket of COX-1. These induced in response to proinflam- APHS represents a parent com- compounds are effective time- matory stimuli such as IL-1, pound which is certain to be dependent inhibitors of COX-2; TNF , and bacterial lipopolysac- followed by more effective, irre- the time dependence is thought to charide (LPS). Nucleotide versible, COX-2 specific NSAIDs. reflect the time required for opti- sequence analysis of the human The second class of inhibitors mal insertion of the inhibitor into COX-2 gene promoter reveals the consists of reversible, competi- the deeper pocket of COX-2 presence of potential binding sites tive inhibitors of both enzymes. (Table 2). for a variety of transcription These compounds compete with factors activated by inflammatory Regulation of COX arachidonic acid for binding to and proliferative stimuli. These the cyclooxygenase active site. include NF-1, AP-2, STATs, Gene Expression Ibuprofen and mefenamate are NFkB, NFIL6/cEBP, CREB/ATF, COX-1 expression is constant examples of this class of inhibitor. and E-box-binding proteins. (constitutive) in most tissues and Indomethacin and flurbiprofen Signaling pathways which play a cell types. COX-1 is, therefore, exemplify the third class of COX role in COX-2 induction include considered as the isoform respon- 54
  • 7. generation of cAMP, activation of elevations in COX-2 expression prevention of certain GI cancers protein kinase C isoforms, gener- occur in response to acute or (see accompanying article by ation of inositol trisphosphates, chronic mucosal inflammation Dr. Dubois). However, recent generation of ceramide, activation and ulceration. studies indicate that COX-2- of mitogen activated protein The cell types in which COX-2 derived PGs play a beneficial role kinases (MAPKs) such as c-Jun expression increases in response in the healing of gastric and N-terminal kinase (JNK), P38 to mucosal injury and inflamma- intestinal ulcers, and thus COX-2 kinase, and extracellular signal tion have not been clearly identi- inhibition in patients with already regulated kinases (ERKs), as well fied and defined. Epithelial cell existing GI lesions could be as Janus-associated kinases COX-2 expression has been detrimental. Dr. Feldman in the (JAKs). COX-2 gene expression demonstrated following invasion accompanying article discusses is also subject to negative regula- by bacteria, in patients suffering the COX-2 therapeutic hypothesis tion. The anti-inflammatory from IBD, and at the latter stages in more detail. cytokines IL-4, IL-10, and IL-13, of carcinogenesis. However, stud- In summary, great strides have and corticosteroids inhibit COX-2 ies from several laboratories show recently been made on the struc- expression. COX-2 expression is that the vast majority of intestinal ture and genetic regulation of the also regulated at post-transcrip- PG production in inflammatory two COX isoforms. Significant tional levels by various mecha- conditions occurs in the lamina advances have also been made in nisms including mRNA splicing, propria and submucosa. In a rat defining the role played by each message stability, and translation. model of colitis, increased levels in normal biological processes The 3 untranslated region of the of COX-2 mRNA are seen and the and disease. However, many COX-2 mRNA contains multiple bulk of immunoreactive COX-2 is unanswered questions still remain copies of the pentanucleotide localized to cells of the lamina regarding the cellular sources of motif AUUUA which confers propria in regions occupied by inducible PG synthesis in GI message instability upon a subepithelial myofibroblasts, inflammation and cancer and the number of cytokine and proto- mast cells, neutrophils, and therapeutic value of recently oncogene mRNAs. Such motifs smooth muscle cells, and in the developed COX-2 selective represent potential targets by muscularis of the colon. Like- NSAIDs. which agents such as IL-1 stabi- wise, recent studies using rat lize, and corticosteroids destabi- models of NSAID-induced gastric lize the COX-2 message, thus ulceration localized COX-2 promoting elevated or decreased expression to the lamina propria levels of enzymatic activity, of regenerative regions. Interest- respectively. ingly, in a murine model of famil- ial adenomatous polyposis coli, COX Expression Oshima et. al. localized COX-2 transcription in early adenomas, in GI Tissues not in epithelial cells, but to a Numerous studies have docu- location directly subjacent to the mented expression of COX-1 epithelial cells in the area occu- throughout the length of the GI pied by intestinal subepithelial tract. COX-1 immunoreactivity myofibroblasts. has been demonstrated in crypt The notion that COX-1 repre- epithelial cells, endothelial cells sents the “good COX” and COX- of blood vessels, lamina propria 2 represents the “bad COX” is mast cells, macrophages, lympho- probably an oversimplification. cytes, fibroblasts, and smooth Prolonged COX-1 inhibition muscle cells to name a few. Using certainly can produce adverse sensitive detection methods, GI side effects (ulcers) while COX-2 mRNA can be found in recently developed COX-2- normal stomach and intestinal specific inhibitors (see below) tissue and occasional COX-2 result in fewer ulcers. Further- immunoreactive inflammatory more, COX-2 inhibition may cells are seen. However, dramatic prove to be beneficial for chemo- 55
  • 8. Suggested Reading Cyclooxygenases leukotrienes via an alternate 5-lipoxygenase (5-LOX) pathway. and GI Mucosal Goetzl, E.J., An, S., Smith, W.L. The enzyme prostaglandin H Specificity of expression and effects Protection synthase (COX) actually performs of eicosanoid mediators in normal two sequential reactions: a cyclo- physiology and human diseases. FASEB. J. 9, 1051–1058, 1995. oxygenase reaction, which Mark Feldman, M.D. An excellent review which covers the converts arachidonic acid to Professor and Vice Chairman biology of other eicosanoid media- Department of Internal Medicine PGG2, followed by a peroxidase tors, in addition to PGs, as well as University of Texas Southwestern reaction, which converts PGG2 to their receptors. Medical School PGH2. In the GI mucosa, PGH2 Kalgutkar, A.S., Crews, B.C., Rowlin- Dallas, Texas is then converted to various son, S.W., Garner, C., Seibert, K., Marnett, L.J. Aspirin-like molecules prostaglandins, including PGE2, Key Concepts that covalently inactivate cyclooxyge- PGF 2 , and PGI2 (prostacyclin) nase-2. Science 280, 1268–1270, • Cyclooxygenases, particularly and, to a lesser extent, to PGD2. 1998. cyclooxygenase-1 (COX-1), are Platelets, on the other hand, An original research report describ- important in protecting the ing the development of APHS, an irre- convert PGH2 to thromboxane A2. gastrointestinal mucosa by versible COX-2 selective inhibitor. Leukocytes convert arachidonic catalyzing synthesis of mucosa- protective prostaglandins. Kurumbail, R.G., Stevens, A.M., acid to PGs (such as PGE2 ) via Gierse, J.K., McDonald, J.J., Stege- • Inhibitors of COX-1, such as the COX pathway and to leuko- man, R.A., Pak, J.Y., Gildehaus, D., aspirin and non-steroidal anti- trienes via the 5-LOX pathway. Miyashiro, J.M., Penning, T.D., Seib- inflammatory drugs (NSAIDs), ert, K., Isakson, P.C., Stallings, W.C. The chemistry of COX is reduce endogenous PG synthe- Structural basis for selective inhibition sis and increase the incidence of described in more detail by of cyclooxygenase-2 by anti-inflam- gastrointestinal ulceration. Drs. Mifflin and Powell in the matory agents. Nature 384, 644–648, • Highly selective inhibitors of accompanying article. 1996. COX-2 are anti-inflammatory and An original research report describ- Acetylsalicylic acid (aspirin) analgesic, with reduced GI toxic- ing the crystal structure of COX-2 irreversibly blocks COX activity ity. However, the safety and cost- complexed with various NSAIDs. effectiveness of these agents is by acetylating a serine residue under ongoing evaluation. Reuter, B.K., Asfaha, S., Buret, A., near the active site of the enzyme. Sharkey, K.A., Wallace, J.L. Exacer- Non-salicylate nonsteroidal anti- bation of inflammation-associated Introduction inflammatory drugs (NSAIDs) colonic injury in rat through inhibition of cyclooxygenase-2. J. Clin. Invest. such as indomethacin, naproxen, Prostaglandin H synthase, 98, 2076–2085, 1996. and ibuprofen reversibly inhibit An original research report which more commonly referred to as COX activity by binding at sites localizes COX-2 expression to lamina cyclooxygenase, or COX, is the different than the aspirin site. propria cells in a rat model of colitis. In addition, this report demonstrates rate-limiting enzyme for cellular Acetylation of the key serine an adverse effect of COX-2 inhibition moiety of COX by aspirin synthesis of prostaglandins (PGs) on the course and severity of disease prevents arachidonic acid from and thromboxane A2 (TxA2). in this model. reaching the active (catalytic) site Arachidonic acid, the precursor Smith, W.L. & DeWitt, D.L. Prostaglan- of COX. Because the platelet is din endoperoxide H synthases-1 and of endogenous PGs and TxA2, is -2. Adv. Immun. 62, 167–215,1996. not nucleated, it cannot generate a polyunsaturated fatty acid An excellent review which includes a new enzyme after its COX has (C20:4) that is a component of discussion of the mechanisms of been irreversibly acetylated and COX catalysis. phospholipid in cell membranes inactivated by aspirin. Thus, Vane, J.R., Bakhle, Y.S., Botting, R.M. throughout the body. Under an thromboxane A2 production from Cyclooxygenases 1 and 2. Annu. Rev. appropriate stimulus, arachidonic arachidonic acid is curtailed for Pharmacol. Toxicol. 38, 97–120, acid is released from the cell 1998. the life of the platelet (7 to 10 An excellent review covering many membrane by the action of the days). Nucleated cells, such as GI aspects of COX structure, inhibition, epithelial cells, can produce COX enzyme phospholipase A2. regulation and role in physiology and s mRNA and new COX protein, disease. Arachidonic acid is then converted permitting gradual restoration of to either prostanoids (PGs, TxA2) COX-catalyzed PG synthesis via this prostaglandin H synthase once the aspirin has been excreted (COX) pathway and/or to or metabolized to salicylate. It is 56
  • 9. remarkable that aspirin remains misoprostol in preventing human A2 production, which leads to in the bloodstream for around gastric ulcers caused by NSAIDs. platelet aggregation and vasocon- three hours after oral dosing, with The observation that NSAIDs also striction, aiding the hemostatic presumably transient suppression cause ulcers in the jejunum and process. Likewise, constitutive of mucosal PG synthesis, yet ileum implies that gastric acid is expression of COX-1 in the once-a-day, low dose aspirin less important than prostaglandin gastrointestinal (GI) tract mucosa therapy results in significant depletion in the pathogenesis of is responsible for production of gastric mucosal damage, as NSAID ulcers. An exception PGs such as PGE2 which, through described below. appears to be NSAID-induced a variety of mechanisms duodenal ulcers in humans which (enhanced bicarbonate and mucus Cyclooxygenase and GI are readily prevented with hista- secretion, increased mucosal blood flow, increased cell prolif- mine-2 receptor antagonists or Mucosal Cytoprotection eration, and perhaps others), misoprostol, indicating that gastric PGs such as PGE2, produced protect the mucosa against ulcera- acid may play a role in the patho- from arachidonic acid in the tion. NSAIDs that interfere with genesis of this type of ulcer. normal GI tract mucosa by the the action of COX-1 reduce Profound inhibition of gastric actions of cyclooxygenase, play a constitutive prostanoid synthesis acid secretion by proton pump critical role in protecting both the in these tissues and, as a conse- inhibitors in humans virtually mucosa of stomach and small quence, have the potential to eliminates duodenal ulcers caused intestine against injury. The interfere with the normal physio- by NSAIDs and also reduces, but evidence that endogenous GI logic processes that prostanoids does not eliminate, NSAID- prostaglandins are “cytoprotec- mediate. Thus, side effects of induced gastric ulcers. tive” is two-fold. NSAIDs that inhibit COX-1 A second observation which First, when GI mucosal synthe- include excessive bleeding indicates that endogenous GI PGs sis of PGs is blocked by NSAIDs through impaired platelet-medi- are cytoprotective is that lethal GI that inhibit COX, ulcers frequently ated hemostasis, and GI ulcer ulcers develop in experimental develop in the stomach and/or formation through impaired GI animals in whom PGs have been small intestine of both humans and mucosal “cytoprotection.” A depleted by specific PG antibod- experimental animals. Further- combination of these two toxici- ies. GI ulcers in rabbits or dogs more, when humans and experi- ties may result in life-threatening can be produced by active or mental animals given NSAIDs are bleeding ulcers. Unfortunately, passive immunization against aspirin and traditional NSAIDs co-treated with PGE analogs, there PGE2, PGF2 , PGD2, or 6-keto inhibit COX-1 at customary doses is remarkable protection against PGF1 . These animal experi- and thus may cause life-threaten- GI ulcers. In animals, low doses of ments indicate that deficiency of ing ulcer complications. PGE analogs that are cytoprotec- even a single endogenous COX-2, unlike COX-1, is tive against NSAID-induced ulcers prostaglandin promotes the present in undectable or very low do not decrease gastric acid secre- development of ulcers in the amounts in most tissues, except tion. In humans, the PGE analog stomach and intestine. kidney and brain. However, most studied, misoprostol, does COX-2 production can be reduce gastric acid secretion in COX-1 and COX-2 increased dramatically in cells by doses that prevent NSAID ulcers. inducers of this enzyme, particu- In 1991, it was discovered that However, the protective effect of larly bacterial lipopolysaccharides there are two different isoforms of misoprostol on gastric ulcer (endotoxin) and certain cytokines COX, COX-1 and COX-2. Differ- formation in NSAID users is most and growth factors. At inflamma- ences between COX-1 and COX-2 likely a consequence of replace- tory sites, cytokines such as tumor are described in the accompany- ment of PGE and not simply a necrosis factor alpha (TNF ) and ing article by Drs. Mifflin and consequence of acid secretion interleukin-1 (IL-1) can induce Powell. Briefly, COX-1 is present inhibition. This is because hista- synthesis of large quantities of in virtually all tissues, is mine-2 receptor antagonists such COX-2, which enhances local expressed at a fairly constant as ranitidine or famotidine, which synthesis of PGs by inflammatory level, and plays a physiological are equipotent or even slightly cells. Thus, NSAIDs that interfere role in several tissues (“house- more potent than misoprostol in with the activity of induced keeper”). For example, COX-1 in inhibiting gastric acid secretion in COX-2 have the potential to platelets catalyze thromboxane humans, are not as effective as 57
  • 10. technically a selective COX-2 inhibitor with no GI toxicity. The COX-2 Therapeutic Hypothesis The current strategy for clinical research in this field has been to (a) develop COX-2 specific or highly selective drugs, (b) test them for efficacy in disease states (e.g., osteoarthritis or rheumatoid arthritis) that have been benefited by conventional, non-selective, COX-inhibiting NSAIDs, and then (c) determine whether the incidence of GI toxicity with the COX-2 selective drug is less than Figure 1. Correlation of gastric IC50 with COX-1 IC50 in blood for 25 different NSAIDs the comparison (non-selective) and anti-inflammatory/analgesic compounds. ASA = acetysalicylic acid (aspirin); drug. The COX-2 hypothesis is 6-MNA = 6-methoxy napthalene acetic acid, the active metabolite of nabumetone (Relafen®). From Cryer and Feldman (see references). Published with permission that the COX-2 selective from The American Journal of Medicine, Excerpta Medica, Inc., New York, NY. inhibitors will maintain clinical efficacy without GI toxicity. reduce local PG production at example, the expression of COX-2 Unfortunately, there is no sites of inflammation, thus mRNA is induced at the edges of uniform way of defining or deter- ameliorating the inflammatory gastric ulcers in rodents, but not mining the COX-2 selectivity of response. If, however, the same in the adjacent non-ulcerated an NSAID. In the past few years, NSAID also blocks COX-1 (i.e., mucosa. This interesting observa- many investigators around the the NSAID is not specific for tion suggests that PGs generated world (including my own labora- COX-2 at the dose employed), by newly generated COX-2 tory) have utilized fairly standard side effects from COX-1 inhibi- protein may play an important whole blood assays to address this tion discussed earlier are possible. role in the ulcer healing process, issue. A venous blood sample is Even highly COX-2 selective and that interference with this obtained and then exposed to the NSAIDs such as diclofenac may pathway by a selective COX-2 drug in question as it is allowed to reduce COX-1 activity at clini- inhibitor may impair healing. clot. The ability of increasing cally prescribed doses and cause Thus, a peptic ulcer caused by the concentrations of the drug to COX-1 related toxicity. What is bacterium Helicobacter pylori or reduce serum thromboxane B2 desired is a COX-2 specific agent by a COX-1 inhibitor (such as (TxB2 ) generation during clotting that has little or no COX-1 effect low-dose aspirin) conceivably in a test tube may be used as a at clinically prescribed doses. could heal less rapidly if a specific COX-1 assay because virtually all Such an agent could prove to be COX-2 inhibitor is also being of the TxB2 found in serum anti-inflammatory, anti-prolifera- used. COX-2 induced by growth during clotting is derived from tive, analgesic, and/or antipyretic, factors may also play a role in constitutive COX-1 in platelets. yet free of GI toxicity. However, a tumor growth. Thus, COX-2 The drug concentration which COX-2 specific drug may have inhibitors could be useful as reduces serum TxB2 by 50% is a unique toxicity that is not anti-tumor agents. referred to as the COX-1 IC50. presently apparent. For example, Acetaminophen (Tylenol, Our laboratory has recently COX-2 may play a role in renal others) is a fairly potent inhibitor shown that there is a correlation development, in the renin-angio- of bacterial lipopolysaccharide- between the ability of a drug to tensin system, in ovulation, and in induced COX-2 activity in human inhibit COX-1 in whole blood and uterine function during pregnancy. white blood cells, with an IC50 its ability to reduce gastric similar to the IC50 for aspirin COX-2 induction by tissue mucosal PGE2 synthesis (Fig. 1). (between 10 and 15 µM). Unlike growth factors (e.g., epidermal This correlation is not surprising, aspirin, acetaminophen has no growth factor or basic fibro- since the majority of COX in the inhibitory effect on gastric COX blast growth factor) may be criti- normal gastric mucosa is the activity. Thus, acetaminophen is cal in normal wound healing. For 58
  • 11. COX-1 isoform. We have also Table 1. Examples of NSAIDs that have little or no COX-2 Selectivity or are found that the IC50 for gastric COX-2 Selective in Human Whole Blood Assays COX in human gastric mucosa for NSAIDs such as aspirin corre- Little or no COX-2 Selectivity COX-2 Selective sponds very closely with the IC50 Naproxen 6-MNA* Diclofenac Meloxicam in vivo. Thus, effects of new COX Indomethacin Aspirin Nimesulide Celecoxib inhibitors on the stomach can be Ibuprofen Ketoprofen NS-398 Rofecoxib predicted to some extent by an COX-1 assay of whole blood. * 6-methoxy naphthalene acetic acid, the active metabolite of nabumetone In addition to measuring the (Relafen®). whole blood COX-1 IC50 for a given NSAID, the ability of Table 2. Therapeutic Serum Concentrations After Usual Dosing and Human increasing concentrations of the Gastric Mucosal IC50 of Two COX-2 Selective NSAIDs Listed in Table 1. same drug to reduce PGE2 synthe- sis when a blood sample is NSAID Therapeutic Gastric Ratio* exposed to bacterial lipopolysac- Concentration (µM) IC50 (µM) charide (endotoxin) can also be Nimesulide 14.6 1.50 10 determined. Virtually all of the Diclofenac 6.1 0.23 27 PGE2 produced in blood under these experimental conditions is * Ratio of therapeutic concentration to gastric IC50. From reference by Cryer and derived from COX-2 that has been Feldman. induced by endotoxin in leuko- cytes, particularly blood mono- concentrations than they inhibit Journal of Rheumatology and cytes. Once the COX-2 IC50 is COX-1 activity in whole blood, Immunology in 1994. One such determined, the ratio of IC50’s for nimesulide and diclofenac study compared nimesulide to the drug in question is then calcu- concentrations at customary doses naproxen for 2 weeks in 200 lated (COX-2/COX-1). A ratio are still well above the IC50 for patients with tendonitis and bursi- close to one indicates little or no gastric COX. The second reason tis. Adverse GI events occurred in COX selectivity. Some examples to question the ultimate GI safety 16% with 200 mg/day nimesulide of nonselective NSAIDs are given of COX-2 selective inhibitors is and 22% of patients on 1100 in Table 1. An NSAID with a low that two of these agents, meloxi- mg/day of naproxen, an insignifi- COX-2/COX-1 ratio is COX-2 cam (whole blood IC50 ratio of cant difference. selective; some examples are 0.09) and nimesulide (whole Two new COX-2 selective listed in Table 1. Some of the new blood IC50 ratio of 0.02–0.06), agents have recently been COX-2 selective inhibitors have have already been evaluated in approved in the United States. COX-2/COX-1 ratios in human Celecoxib (Celebrex®) was several clinical trials, and the whole blood assays of 0.1 or less. anticipated GI safety has not been approved in 1998 for osteo- Examples include rofecoxib, cele- realized. Several studies of arthritis and rheumatoid arthritis. coxib, DuP-697, flosulide, Rofecoxib (Vioxx®) was meloxicam were published in a flurbinitroxybutylester, L-745,337, supplement to British Journal of approved in 1999 for osteo- meloxicam, nimesulide, NS-398, Rheumatology in 1996; interested arthritis and for the short-term and SC-58125. However, there are readers are referred to this supple- management of acute pain in two reasons to question the idea ment for more details. The largest adults, including menstrual pain. that COX-2 selective drugs will of these studies compared meloxi- Very little data is published as yet not be toxic to the GI tract. First, cam to naproxen (a non-selective on these compounds, but early some currently marketed NSAIDs COX inhibitor) in 370 patients reports indicate that both drugs that are at least 10-fold COX-2 with rheumatoid arthritis who (a) cause slightly, but not signifi- selective (COX-2/COX-1 IC50 were treated for 6 months. cantly, more upper GI ulcers ratio < 0.1) are still able to inhibit Adverse GI effects occurred in detected by endoscopy than COX-1 in the blood and in the 35.5% of patients on 750 mg/day placebo; (b) cause much fewer stomach at clinically prescribed naproxen and in 26.6% of patients ulcers detected by endoscopy than doses and concentrations on 7.5 mg/day of meloxicam, an equally-effective doses of tradi- (Table 2). Thus, even though insignificant difference. Several tional NSAIDs; (c) cause signifi- nimesulide and diclofenac reduce studies of nimesulide were cantly fewer clinically important COX-2 activity at much lower published in the European ulcer events, including GI bleeds, 59
  • 12. than traditional NSAID compara- Cyclooxygenase-2 number of groups have initiated tors. Serious ulcer events such as research efforts focused on eluci- and Colon bleeding have been reported with dating the molecular basis of the Carcinogenesis both celecoxib and rofecoxib, anti-neoplastic effects of aspirin although cause and effect have and other NSAIDs. Many of these not been established. Careful efforts suggest that inhibition of Raymond N. DuBois, M.D., Ph.D. post-marketing surveillance stud- the enzyme cyclooxygenase-2 and Moss Mann, M.D. ies will be needed to determine (COX-2) by NSAIDs plays some Departments of Medicine whether celecoxib and rofecoxib role in cancer risk reduction. and Cell Biology Vanderbilt University prove the COX-2 hypothesis. Hopefully, continued study of Medical Center the role of the cyclooxygenase Veteran Affairs Medical Center Suggested Reading enzymes in colorectal carcino- Nashville, TN genesis will determine whether Cryer, B. & Feldman, M. Cyclooxyge- COX-2 selective inhibitors can be nase-1 and cyclooxygenase-2 selec- Key Concepts used in future cancer prevention tivity of widely used NSAIDs and regimens. • Colorectal cancer is the second other anti-inflammatory or analgesic leading cause of cancer death in drugs: Studies in whole blood and the U.S. gastric mucosa of healthy humans. Evidence for the role of Am. J. Med. 104:413-421, 1998. • Regular NSAID use appears to COX in colon cancer This study compares whole blood reduce the risk of developing prevention COX-1 and COX-2 IC50’s, and gastric colorectal cancer. IC50’s for 25 different NSAIDs and • There are data suggesting that Risk reduction in human anti-inflammatory or analgesic NSAIDs reduce the risk of compounds, and develops evidence sporadic colorectal carcinoma developing colorectal cancer that gastric COX is mainly COX-1. by inhibiting cyclooxygenase-2 Of the several observational (COX-2). Lecomte, J. et al. Treatment of studies of the effects of exposure tendonitis and bursitis: a comparison • Selective COX-2 inhibitors show to NSAIDs (usually aspirin) and of nimesulide and naproxen sodium in promise as chemopreventive the subsequent development of a double-blind parallel trial. Eur. J. agents with fewer gastrointesti- Rheumatol. Inflamm. 14:29-32, 1994. colorectal cancer, all but one have nal side effects; however, their A 2-week trial showing no significant safety record is not yet proven. demonstrated a protective effect difference in adverse GI events of NSAIDs. The studies were between the former COX-2 selective performed in a variety of settings agent and the latter non-selective Introduction agent. in the US and Australia, utilizing both colorectal cancer occurrence Colorectal cancer is a major Patrignani, P., Panara, M.R., Sciulli, M.G., Santini, G., Renda, G., Patrono, and mortality as outcomes. In cause of illness and death in the C. Differential inhibition of human the studies, exposure to NSAIDs United States and other parts of prostaglandin endoperoxide synth- was measured by interview or the world. In 1998 there were over ease-1 and -2 by nonsteroidal anti- computerized pharmacy records. 130,000 new cases of colorectal inflammatory drugs. J. Physiol. Phar- macol. 4:623-631, 1997. In the Nurses Health Study, a cancer and about 55,000 deaths A report of whole blood assays for protective effect was seen only from the disease (Table 1). Ameri- COX-1 and COX-2 activity of a wide after 10–15 years of aspirin use. cans have a 1 in 20-lifetime risk of variety of NSAIDs, including meloxi- Similar studies have revealed a developing colorectal cancer, and cam, nimesulide, and NS-398. protective effect of NSAIDs in approximately one in ten has a Wojtulewski, J.A. et al. A six-month relation to adenomatous polyp family member who develops this double-blind trial to compare the effi- detection. Additionally, a small cacy and safety of meloxicam 7.5 mg disease. Epidemiologic research daily and naproxen 750 mg daily in number of observational studies indicates that there is a 40–50% patients with rheumatoid arthritis. Br have shown a significant risk reduction in mortality from colo- J. Rheum. 35(S 1):22-28, 1996. reduction with use of non-aspirin rectal cancer in persons who take A trial of 370 patients showing similar NSAIDs. adverse GI events with meloxicam, a aspirin or other non-steroidal anti- COX-2 selective agent, and naproxen, The effect of aspirin use on the inflammatory drugs (NSAIDs) on a non-selective agent. Only 2 ulcers development of colorectal cancer a regular basis. Clearly, an effect s occurred, both with naproxen. has been assessed in a randomized of this magnitude could have a clinical trial that had a principal significant impact on health care, goal of evaluating aspirin for the both in terms of lives saved and prevention of myocardial infarc- health care dollars recovered. A 60
  • 13. tion. A secondary analysis of this Table 1. U.S. Colorectal cancer statistics (estimated) by gender for 1998. study of 22,071 male physicians randomized to placebo or aspirin Men Women Total 325 mg every other day demon- Estimated # of new 64,600 67,000 131,600 strated no protective effect against crc cases the development of colorectal % of all new ca cases* 10% 111% 10% cancer. It is possible that certain characteristics of the study group Rank vs other 3rd (behind lung 3rd (behind lung 2nd overall ca types* and prostate) and breast) (such as diet, exercise regimen, age, and gender) or the relatively Estimated # deaths 27,900 28,600 56,500 from crc low dose of aspirin could have obscured a protective effect. % of all ca deaths 9% 11% 10% Unfortunately, the low Rank vs other causes 3rd (behind lung 3rd (behind lung 2nd overall frequency of colorectal cancer of ca death and prostate) and breast) makes a large scale randomized Abbreviations: crc= colorectal cancer; ca= cancer; vs = versus. clinical trial financially and temporally difficult. More defini- * Excluding non-melanoma skin cancers and non-bladder carcinoma-in-situ. tive recommendations concerning Data from Landis, S.H., et al., Cancer Statistics, 1998. CA Cancer J. Clin. 48:6–29. aspirin use likely will be based on the results of an ongoing random- lar activities, but differing in that regular use of the NSAID ized clinical trial of aspirin use expression characteristics and sulindac led to regression of rectal which utilizes adenomatous polyp inhibition profiles by NSAIDs. adenomas in four patients with incidence as an intermediate COX-1 mRNA and protein are FAP, and this phenomenon was endpoint. This multi-center study expressed constitutively in many confirmed in several other case tests the effect of aspirin at one of tissues. A second, inducible reports. This observation was then two doses versus placebo on the isoform of cyclooxygenase, confirmed in randomized, placebo development of adenomatous referred to as cyclooxygenase-2 controlled, double-blinded, polyps among patients who have (COX-2) was independently crossover studies of sulindac use undergone prior colonoscopy cloned by two groups. COX-2 in FAP patients. These studies, with polypectomy. Hopefully, expression is induced by a collectively, indicate that sulindac data from that study will help number of extracellular and intra- has a significant effect on polyp determine the degree of benefit cellular stimuli. The formation of regression in FAP patients. and optimal dose of aspirin in a COX-2 protein parallels the chemopreventive regimen. Potential mechanisms increase in prostaglandin produc- NSAID use and reduction of tion following stimulation with for chemoprevention of adenoma size and number in mitogens or tumor promoters in a intestinal tumors by FAP patients wide variety of cell types. aspirin and other NSAIDs Does dysregulation of COX-2 Familial adenomatous polypo- Inhibition of cyclooxygenases expression coincide with develop- sis (FAP) is an autosomal domi- ment of gastrointestinal malig- The anti-inflammatory proper- nant inherited disease with vari- nancy? We have previously ties of NSAIDs are most likely able phenotypic expression that is reported increased COX-2 expres- due to their inhibition of cyclo- associated with an increased risk sion in human colorectal adenocar- oxygenase enzymes. These of colorectal cancer at a young cinomas when compared to normal enzymes catalyze key steps in the age. FAP is responsible for only adjacent colonic mucosa; these conversion of arachidonic acid to 1% of colorectal carcinomas findings have been confirmed by endoperoxide (PGH2 ), which is a detected in the general popula- other investigators using different substrate for a variety of prosta- tion. The genetic mutation techniques and patient populations. glandin synthases which catalyze responsible for this disease Additionally, COX-2 mRNA and the formation of prostaglandins resides in the adenomatous poly- protein levels are increased in and other eicosanoids (see accom- posis coli (APC) gene. Somatic intestinal tumors that develop in panying article by Drs. Mifflin mutations in the APC gene have rodents following carcinogen treat- and Powell). Two isoforms of been reported also in up to 50% of ment and in adenomas taken from cyclooxygenase have been identi- spontaneous colorectal cancer. multiple intestinal neoplasia (Min) fied to date, each possessing simi- Wadell and Loughry first reported 61
  • 14. prove to be effective chemopre- ventive agents with fewer Prostaglandin & Phospholipases Thromboxane Synthases gastrointestinal side effects than non-selective NSAIDs. Membrane bound Prostaglandins Arachidonate PGG2 PGH2 Cyclooxygenase independent fatty acid & TxA2 mechanisms Epidemiologic data strongly COX-1 support the chemoprotective effects of NSAIDs on gastroin- COX-2 testinal malignancies, while the data supporting their benefit in other solid tumors are not as well COX-2 inhibitor developed. The precise mecha- acts here nism by which NSAIDs prevent and/or cause regression of colorec- Figure 1. Role of the cyclooxygenase enzymes in prostaglandin synthesis, and tal tumors is not known. Despite putative site of action of selective COX-2 inhibitors. different chemical structures, inhi- mice. When intestinal epithelial Another study has provided bition profiles, and drug half lives, cells are forced to express COX-2 compelling genetic evidence all NSAIDs in clinical use possess constitutively, they develop pheno- which directly links COX-2 cyclooxygenase inhibitory activ- typic changes which include expression to intestinal tumor ity. Some investigators have increased adhesion to extracellular promotion. This report shows that reported effects of NSAIDs which APC∆716 mice, which develop membrane (ECM) and a resistance likely are not due to their inhibi- to butyrate-induced apoptosis. hundreds of tumors per intestine, tion of cyclooxygenase activity. Both of these phenotypic changes bred with COX-2 null mice have For example, certain NSAIDs are consistent with an increased an 80–90% reduction in tumor induce apoptosis and alter expres- neoplastic potential. COX-2 multiplicity in the homozygous sion of cell cycle regulatory genes expression has been detected in COX-2 null offspring. These in some cell lines when adminis- 80–90% of colorectal adenocarci- results suggest that: 1) COX-2 tered at relatively high concentra- nomas, but in only 40-50% of may act as a tumor promoter in tions (200–1000 µM). By using premalignant adenomas. These the intestine; and, 2) increased cyclooxygenase-deficient cell data suggest that elevation of levels of COX-2 expression may lines or drug metabolites lacking COX-2 expression is secondary to result directly or indirectly from COX-inhibitory activity, these other initiating events, such as disruption of the APC gene. studies rule out the involvement mutations of the APC gene or Recently, a new class of of cyclooxygenase enzymes in possibly dysfunction of other NSAIDs has been developed. the growth inhibitory effect. genes. (Fig. 1) These drugs are highly selective Certainly, this class of drugs Our observation of elevated for inhibition of the COX-2 appears to affect biochemical COX-2 expression in three differ- enzyme, but do not strongly pathways unrelated to cyclooxy- ent models of colorectal carcino- inhibit COX-1. They were primar- genase enzymes, and these effects genesis have led us to consider ily developed as anti-inflamma- are likely to occur in a dose- the possibility that COX-2 expres- tory agents with the goal of less- dependent fashion (some effects sion may be related to colorectal ening the gastrointestinal side occurring only at toxic doses). tumorigenesis in a causal way. effects caused by inhibition of The specific mechanisms of these Work by two independent groups COX-1. These selective COX-2 COX-independent effects, and has shown a reduction in tumor inhibitors have proven effective in their therapeutic implications, are multiplicity in Min mice treated inhibiting tumor growth in animal not yet well understood. with sulindac or piroxicam, both studies, and these agents also have Risks of chronic NSAID potent cyclooxygenase inhibitors. been shown to possess anti-angio- therapy for cancer prevention Recent studies have demonstrated genic activity in vitro which may a significant reduction in prema- contribute to their anti-neoplastic The role of NSAIDs for lignant and malignant lesions in effects in vivo. Although the gastrointestinal cancer prevention carcinogen-treated rats that were safety profiles of these drugs are will be determined by the side given a selective COX-2 inhibitor. not clearly established, they may effects of chronic use of these 62
  • 15. drugs. There are concerns about chronic NSAID therapy. The Suggested Reading the safety of long term use of aforementioned side effects of Oshima, M., Dinchuk, J.E., Kargman, aspirin and other NSAIDs in aspirin therapy may preclude S. L., Oshima, H., Hancock, B., humans. Long term aspirin use prophylactic use of that drug in Kwong, E., Trzaskos, J.M., Evans, can result in serious gastrointesti- all but the highest risk popula- J.F., Taketo, M.M. 1996. Suppression nal and renal adverse effects, even tions. The deleterious effects of of intestinal polyposis in APC716 knockout mice by inhibition of at relatively low doses of drug. non-selective NSAIDs on the prostaglandin endoperoxide These side effects tend to gastrointestinal tract, including synthase-2 (COX-2). Cell increase in older patients. The gastric erosions, ulcerations, and 87:803–809. most significant side effects, in blood loss, are postulated to This study provides direct genetic evidence that COX-2 plays a major terms of morbidity and mortality, result from inhibition of COX-1. role in the development of colorectal are gastrointestinal, and include While COX-2 selective NSAIDs tumors. dyspepsia, peptic ulcer, and are available, these drugs do not Giardiello, F.M., Hamilton, S.R., Krush, gastrointestinal bleeding. It is yet have established safety A.J., Piantadosi, S., Hylind, L.M., estimated that regular users of profiles. The side effects of any Celano, P., Booker, S.V., Robinson, NSAIDs have roughly a three- chemoprotective agent must be C.R., Offerhaus, G.J. Treatment of fold greater relative risk of devel- low to insure compliance and to colonic and rectal adenomas with sulindac in familial adenomatous oping serious gastrointestinal achieve the desired result, since polyposis. N. Eng. J. Med. 328:1313– complications when compared to the absolute risk of colorectal 1316, 1993. non-users of NSAIDs. Further- cancer in the general population This study is among the first random- more, the consequences of is quite low. On the other hand, if ized, double-blind, placebo-controlled trials to show a reduction in intestinal NSAID-associated gastrointesti- high risk populations can be iden- polyp size and number in familial nal complications are often dire. tified readily, then the use of adenomatous polyposis (FAP) A British study of 235 patients these agents in those populations patients. with severe peptic ulceration may be more reasonable, because Tsujii, M., & DuBois, R.N. Alterations found that the mortality rate from of a much more favorable risk to in cellular adhesion and apoptosis in ulceration among regular users of benefit ratio. epithelial cells overexpressing prosta- glandin endoperoxide synthase-2. NSAIDs was greater than twice The risk-benefit ratio for Cell 83:493–501, 1995. that of non-users. chemoprevention of colorectal Rat intestinal epithelial (RIE)cells The cost of NSAID-associated cancer likely would improve if were transfected with a COX-2 complications also has been accessible techniques were avail- expression vector, leading to overex- pression of COX-2 protein. The considerable. A retrospective able to identify groups that are at resulting phenotypic changes cohort study of 75,350 elderly high risk for the subsequent included increased adhesion to extra- (>65) Tennessee Medicaid recipi- development of colorectal cancer. cellular matrix (ECM) and resistance ents by Smalley, et al., found Advances in the discovery and to apoptosis. that the adjusted mean annual testing of colorectal cancer genes Waddell, W.R., & Loughry, R.W. payment for medical care related hopefully will make the identifi- Sulindac for polyposis of the colon. J. Surg. Oncol. 24:83–87, 1983. to gastrointestinal disorders was cation of cohorts at high risk for This is one of the first observational $134 for non-users of NSAIDs developing cancer more likely. studies to report NSAID-induced and was $244 among regular Additionally, if agents such as the polyp regression in familial adenoma- users of NSAIDs. These findings selective COX-2 inhibitors prove tous polyposis (FAP). may well underestimate the to have fewer adverse effects than Smalley, W.E., & DuBois, R.N. actual, current costs of NSAID- non-selective NSAIDs, the risk to Colorectal cancer and nonsteroidal related gastroenteropathy, as benefit ratio might improve. anti-inflammatory drugs. Adv. Pharm. 39:1–20, 1997. omeprazole and misoprostol were Human clinical trials evaluating This review discusses the potential not on the Medicaid formulary at the anti-neoplastic effects of roles of COX-2 and NSAIDs in that time. selective COX-2 inhibitors will s colorectal tumorigensis. As new data become available, need to be completed before the we must constantly reassess the role of these agents in cancer risks versus the benefits of prevention can be determined. 63
  • 16. U.S. POSTAGE Prepared by Ann Arbor, MI Permit No. 87 BULK RATE MedPub Inc. PAID Ms. Lori Ennis, Managing Editor 110 Miller Ave., Suite 100 P.O. Box 130589 Ann Arbor, MI 48103-0589 Editorial Policy The Editorial Board has complete independence in selection of topics and authors. The opinions expressed are those of the authors and are not to be construed as the opinions or recommendations of the publisher or the funding organization. Readers are urged to check the current prescribing information pertaining to any marketed drug or device mentioned in this publication. The Regulatory Peptide Letter Prepared with the assistance of a grant from Novartis, The Regulatory Peptide Letter bridges the gap between basic research and clinical application. Published four times annually by MedPub, Inc., and circulated to selected participating physicians. All rights reserved. Copyright © 2001, MedPub, Inc. No portion of this publication may be reproduced without permission from the publisher. Comments and questions from readers are welcome. Please address letters to: Ms. Lori Ennis, Managing Editor 110 Miller Ave., Suite 100 P.O. Box 130589 Ann Arbor, MI 48103-0589