Cyclooxygenases are enzymes that take part in a complex biosynthetic cascade that results in the conversion of polyunsaturated fatty acids to prostaglandins and thromboxane(s). Their main role is to catalyze the transformation of arachidonic acid into the intermediate prostaglandin H2, which is the precursor of a variety of prostanoids with diverse and potent biological actions. Cyclooxygenases have two main isoforms that are called COX-1 and COX-2 (as well as a COX-3). COX-1 is responsible for the synthesis of prostaglandin and thromboxane in many types of cells, including the gastro-intestinal tract and blood platelets. COX-2 plays a major role in prostaglandin biosynthesis in inflammatory cells and in the central nervous system. Prostaglandin synthesis in these sites is a key factor in the development of inflammation and hyperalgesia. COX-2 inhibitors have analgesic and anti-inflammatory activity by blocking the transformation of arachidonic acid into prostaglandin H2 selectively.King, F. D., Ed. (2002). Medicinal chemistry Principles and practice. Cambridge, The royal Society of Chemistry.

The rise for development of selective COX-2 inhibitors DuP-697

The impetus for development of selective COX-2 inhibitors was the adverse gastrointestinal side-effects of NSAIDs. Soon after the discovery of the mechanism of action of NSAIDs, strong indications emerged for alternative forms of COX, but little supporting evidence was found. COX enzyme proved to be difficult to purify and was not sequenced until 1988. In 1991 the existence of the COX-2 enzyme was confirmed by being cloned by Dr. Dan Simmons at Brigham Young University. Before the confirmation of COX-2 existence, the Dupont company had developed a compound, DuP-697, that was potent in many anti-inflammatory assays but did not have the ulcerogenic effects of NSAIDs. Once the COX-2 enzyme was identified, Dup-697 became the building-block for synthesis of COX-2 inhibitors. Celecoxib and rofecoxib, the first COX-2 inhibitors to reach market, were based on DuP-697. It took less than eight years to develop and market the first COX-2 inhibitor, with Celebrex (celecoxib) launched in December 1998 and Vioxx (rofecoxib) launched in May 1999.FDA, Center for Drug Evaluation and Research (2008). "FDA approved drug products – Celebrex." Retrieved 18.10., 2008, from FDA, Center for Drug Evaluation and Research (2008). "FDA approved drug products – Vioxx." Retrieved 18.10., 2008, from Celecoxib and other COX-2 selective inhibitors, valdecoxib, parecoxib, and mavacoxib, were discovered by a team at the Searle division of Monsanto led by John Talley.

Development of COX-2 inhibitors Early studies showed that, when inflammation is induced, the affected organ unexpectedly develops an enormous capacity to generate prostaglandins. It was demonstrated that the increase is due to de novo synthesis of fresh enzyme. In 1991, during the investigation of the expression of early-response genes in fibroblasts transformed with Rous sarcoma virus, a novel mRNA transcript that was similar, but not identical, to the seminal COX enzyme was identified. It was suggested that an isoenzyme of COX had been discovered. Another group discovered a novel cDNA species encoding a protein with similar structure to COX-1 while studying phorbol-ester-induced genes in Swiss 3T3 cells. The same laboratory showed that this gene truly expressed a novel COX enzyme. The two enzymes were renamed COX-1, referring to the original enzyme and COX-2. Building on those results, scientists started focusing on selective COX-2 inhibitors. Enormous effort was spent on the development of NSAIDs between the 1960s and 1980 so there were numerous pharmacophores to test when COX-2 was discovered. Early efforts focused on modification on two lead compounds, DuP-697 and NS-398. These compounds differ greatly from NSAIDs that are arylalkonic acid analogs. Encouraged by the "concept testing" experiments with selective inhibitors, and armed with several solid leads and clear idea of the nature of the binding site, development of this field was rapid. In vitro recombinant enzyme assays provided powerful means for assessing COX selectivity and potency and led to the discovery and clinical development of the first rationally designed COX-2 selective inhibitor, celecoxib. Efforts have been made to convert NSAIDs into selective COX-2 inhibitors such as indometacin by lengthening of the alkylcarboxylic acid side-chain, but none have been marketed.

Structure Activity Relationship (SAR) DuP-697 was a building-block for synthesis of COX-2 inhibitors and served as the basic chemical model for the coxibs that are the only selective COX-2 inhibitors on the market today. DuP-697 is a diaryl heterocycle with cis-stilbene moiety. Structure activity relationship (SAR) studies for diaryl heterocyclic compounds have indicated that a cis-stilbene moiety and changes in the para-position of one of the aryl rings play an important role in COX-2 selectivity. Celecoxib and parecoxib have a sulfonamide substituent (SO2NH2) in para-position on one of the aryl rings while etoricoxib and rofecoxib have a methylsulfone (SO2CH3). The oxidation state on the sulfur is important for selectivity; sulfones and sulfonamides are selective for COX-2 but sulfoxides and sulfides are not. The ring system that is fused in this stilbene system has been extensively manipulated to include every imaginable heterocyclic and carbocyclic skeleton of varying ring sizes. It is known that a SO2NHCOCH3 moiety as in parecoxib, which is a prodrug for valdecoxib, is 105 – 106 more reactive acetylating agent of enzyme serine hydroxyl groups than simple amides. Due to the fact that varying kinetic mechanisms affect potency for COX-1 versus COX-2, relying Potency and selectivity in human whole blood is used by many groups and has been accepted as a standard assessment of COX-2 potency and selectivity.

The relationship between amino acid profile of COX-2 enzyme and inhibition mechanism COX-2 receptor site and its amino acid profile along with celecoxib in the binding site

One of the keys to developing COX-2 selective drugs is the larger active site of COX-2, which makes it possible to make molecules too large to fit into the COX-1 active site but still able to fit the COX-2. The larger active site of COX-2 is partly due to a polar hydrophilic side-pocket that forms because of substitution of Ile523, His513, and Ile434 in COX-1 by Val523, Arg513, and Val434 in COX-2. Val523 is less bulky than Ile523, which increases the volume of the active site. Substitution of Ile434 for Val434 allows the side-chain of Phe518 to move back and make some extra space. This side-pocket allows for interactions with Arg513, which is a replacement for His513 of COX-1. Arg513 is thought to be a key residue for diaryl heterocycle inhibitors such as the coxibs. The side-chain of Leu384, at the top of the receptor channel, is oriented into the active site of COX-1, but, in COX-2, it is oriented away from the active site and makes more space in the apex of the binding site. The bulky sulfonamide group in COX-2 inhibitors such as celecoxib and rofecoxib prevent the molecule from entering the COX-1 channel. For optimal activity and selectivity of the coxibs, a 4-methylsulfonylphenyl attached to an unsaturated (usually) five-membered ring with a vicinal lipophilic group is required (rofecoxib). The SO2CH3 can be replaced by SO2NH2, wherein the lipophilic pocket is occupied by an optionally substituted phenyl ring or a bulky alkoxy substituent (celecoxib). Within the hydrophilic side-pocket of COX-2, the oxygen of the sulfonamide (or sulfone) group interacts with Hist90, Arg513, and Gln192 and forms hydrogen bonds. The substituted phenyl group at the top of the channel interacts with the side-chains of amino acid residues through hydrophobic and electrostatic interactions. Tyr385 makes for some sterical restrictions of this side of the binding site so a small substituent of the phenyl group makes for better binding. Degrees of freedom are also important for the binding. The central ring of the coxibs decides the orientation of the aromatic rings and, therefore, the binding to COX enzyme even though it often has no electrostatic interactions with any of the amino acid residues. The high lipophilicity of the active site does require low polarity of the central scaffold of the coxibs.

Mechanism of binding Studies on the binding mechanism of selective COX-2 inhibitors show that they have two reversible steps with both COX-1 and COX-2, but the selectivity for COX-2 is due to another step that is slow and irreversible and is seen only in the inhibition of COX-2, not COX-1. The irreversible step has been attributed to the presence of the sulfonamide (or sulfone) that fits into the side-pocket of COX-2. This has been studied using SC-58125 (an analogue of celecoxib) and mutated COX-2, wherein the valine 523 residue was replaced by isoleucine 523. The irreversible inhibition did not happen, but reversible inhibition was noticed. A model has been made to explain this three-step mechanism behind the inhibitory effects of selective COX-2 inhibitors. The first step accounts for the contact of the inhibitor with the gate of the hydrophobic channel (called the lobby region). The second step could account for the movement of the inhibitor from the lobby region to the active site of the COX enzyme. The last step probably represents repositioning of the inhibitor at the active site, which leads to strong interactions of the phenylsulfonamide or phenylsulfone group of the inhibitor and the amino acids of the side pocket. It is directly inhibition to postaglanding

Pharmacokinetics of coxibs The coxibs are widely distributed throughout the body. All of the coxibs achieve sufficient brain concentrations to have a central analgesic effect, and all reduce prostaglandin formation in inflamed joints. All are well absorbed, but peak concentration may differ between the coxibs. The coxibs are highly protein-bound, and the published estimate of half-lives is variable between the coxibs.Burke, A., E. Smyth, et al. (2005). Ch.26: Analgesic-antipyretic agents; pharmacotherapy of gout. The Pharmacological Basis of THERAPEUTICS. L. L. Brunton, J. S. Lazo and K. L. Parker, McGraw-Hill companies: 679-680 and 702-705.

Celecoxib Celecoxib was the first specific inhibitor of COX-2 approved to treat patients with rheumatism and osteoarthritis. A study showed that the absorption rate, when given orally, is moderate, and peak plasma concentration occurs after about 2–4 hours. However, the extent of absorption is not well known. Celecoxib has the affinity to bind extensively to plasma proteins, especially to plasma albumin. It has an apparent volume of distribution (VD) of 455 +/- 166 L in humans and the area under the plasma concentration-time curve (AUC) increases proportionally to increased oral doses, between 100 and 800 mg. Celecoxib is metabolized primarily by CYP2C9 isoenzyme to carboxylic acid and also by non-CYP-dependent glucuronidation to glucuronide metabolites. The metabolites are excreted in urine and feces, with a small proportion of unchanged drug (2%) in the urine. Its elimination half-life is about 11 hours (6–12 hours) in healthy individuals, but racial differences in drug disposition and pharmacokinetic changes in the elderly have been reported. People with chronic kidney disease appear to have 43% lower plasma concentration compared to healthy individuals, with a 47% increase in apparent clearance, and it can be expected that patients with mild to moderate hepatic impairment have increased steady-state AUC.

Celecoxib Peak [drug] 2–4 hours Protein binding 97%MetabolitesCarboxylic acid and glucuronide conjugatesHalf-life [t1/2]6–12 hours

Parecoxib and valdecoxib