OA11213A - Cox-2 selective carprofen for treating pain and inflammation in dogs - Google Patents

Abstract

Treating or preventing inflammatory processes and diseases in dogs associated with the activity of inducible cyclo-oxygenase-2 (COX-2), while at the same time reducing or eliminating undesirable side effects associated with simultaneous inhibition of the activity of constitutive cyclo-oxygenase-1 (COX-1) by selectively inhibiting COX-2 activity with reference to COX-1 activity, wherein the selectivity ratio or COX-2 : COX-1 activity inhibition is at least 3 : 1 based on ex vivo inhibition levels measured in whole blood; the inhibitor is a member selected from the group of anti-inflammatory compounds consisting essentially of salicylic acid derivatives, p-aminophenol derivatives, indole and indene acetic acids, heteroaryl acetic acids, arylpropionic acids, anthranilic acids, enolic acids, and alkanones; the inhibitor in particular is comprised of (+)(S)-enantiomer of 6-chloro- alpha -methyl-9H-carbazole-2-acetic acid.

Description

-1-

f\ A fi fi·. Λ -JI > i_ I vJ

> COX-2 SELECTIVE CARPROFEN FOR

TREATING PAIN AND INFLAMMATION IN DOGS

5 FIELD OF THE INVENTION

The présent invention concerns the treatment of pain and inflammation in dogs with anti-inflammatory agents which are non-steroidal anti-inflammatory drugs (NSAIDs), and inparticular such agents having a reduced incidence of adverse gastro-intestinal side effects,since such side effects are a prévalent and potentially severe problem in dogs. 10

BACKGROUND OF THE INVENTION

As is well known to artisans of ordinary skill in this field, e.g., veterinarians, thecanine species, i.e., dogs, especially older dogs, are very susceptible to chronicinflammatory processes such as degenerative joint disease. Because of the very large 15 number of dogs which are kept as pets or for utilitarian purposes such as guard dogs andseeing-eye dogs, there has been an ongoing effort to find pharmaceutical agents which willimpede or altogether stop the progress of such inflammatory disease processes in dogs, orat least ameliorate the symptoms of the inflammation such as pain and edema. One class ofsuch pharmaceutical agents which has been investigated extensively for anti-inflammatory 20 and analgésie use in humans, and more recently in dogs, is that of the non-steroidal anti-inflammatory drugs (NSAIDs). This type of anti-inflammatory drug has been explored widelyand new, improved agents of this type for use in humans hâve been discovered anddeveloped over a period of décades.

However, the use of NSAIDs in dogs has been more limited, e.g., only two such 25 NSAIDs hâve been approved by the Food and Drug Administration, Committee onVeterinary Medicine (FDA/CVM), for use in dogs in the United States, i.e., ARQUEL®,meelofenamie acid, and RIMADYL®, carprofen. Consequently, there is less expérience andknowledge in veterinary medicine about safety and efficacy issues surrounding the use ofNSAIDs in dogs. In veterinary medicine, for example, the most common indication for 30 NSAIDs is the treatment of degenerative joint disease (DJD), which in dogs often resuttsfrom a variety of developmental diseases, e.g., hip dysplasia and osteochondrosis, as wellas from traumatic injuries to joints. In addition to the treatment of chronic pain andinflammation, NSAIDs are also useful in dogs for treating post-surgical acute pain, as well asfor treating ciinical signs associated with osteoarthritis. 35 This demand for canine NSAID therapy, combined with the absence of any approved NSAIDs for this purpose, has resulted in substantial off-label use in dogs of NSAIDs approved for humans, sometimes with disastrous conséquences. The veterinary -2- 011213 litêrature is replete with reports of gastrointestinal hemorrhage, perforation and peritonitis indogs associated with the use of NSAIDs approved for human use such as indomethacin,naproxen, aspirin, ibuprofen, and phenylbutazone. Although such gastrointestinal adversereactions afflict human patients as well, dogs often receive inappropriately high dosesbecause of the lack of information about proper dosing, and because of the inherently highdegree of canine susceptibility to such gastrointestinal adverse reactions. There is,accordingly, a pressing need for safe yet effective NSAIDs in the treatment of pain andinflammation in dogs.

While the search for safe and effective NSAID agents in canine therapy must dealwith the potential for serious adverse gastrointestinal reactions, other adverse reactionsinclude kidney and liver toxicity. However, the most serious of these are the gastrointestinaleffects such as single or multiple ulcérations, including perforation and hemorrhage of theesophagus, stomach, duodénum or small and large intestine. These adverse reactions areusually debilitating, but can often be severe, and occasionally can even be life-threatening.Indeed, the therapeutic index for the use of NSAIDs in dogs can be so low as tocontraindicate such treatment.

The expression “therapeutic index” is sometimes generally defined as the ratio ofthe LD50 to the ED50 of a drug, and is intended to be a statement of how sélective the drug isin producing its desired effects. As used herein, however, the expression “therapeutic index"is more consistent with the définition utilized in the animal health field, which is the ratio ofthe maximum tolerated dose in the animal to the minimum effective dose in the animal. Inthe présent invention the term “animal” refers, of course, to dogs. ' The maximum tolerateddose in a particular canine subject would typically be determined by a number of differentassays and techniques. For example, gastrointestinal hemorrhage may be determined byassay methods commonly used to detect occult blood in stool specimens, while endoscopycan be used to detect the occurrence of ulcération or perforation. Where the animal iseuthànized as part of the study, autopsy can provide valuable information as well.

It has been the expectation in the art heretofore that any NSAID candidate,especially one for canine therapy, would hâve a low therapeutic index. The hope has alwaysbeen that the therapeutic index was not so low so as to render the candidate unacceptablefor use in dogs. Thus, an important aspect of the présent invention was the surprisingdiscovery that the anti-inflammatory compositions described herein hâve an extremely hightherapeutic index when used for treating pain and inflammation in dogs, and further that saidanti-inflammatory compositions hâve unique possession of this unexpected property,virtually to the exclusion of ail other NSAIDs. -3- Ûi 1213 A significant body of knowledge has accumulated in recent years regarding themechanisms of action whereby NSAIDs achieve their anti-inflammatory therapeutic results,as well as whereby they produce serious gastrointestinal adverse reactions at the sametime. While most of this body of knowledge has been gathered with respect to NSAIDmechanisms of action in humans, it is applicable to a large extent to the same mechanismsof action in dogs, although there is apparently some species specificity, as is further below-detailed. With regard to the therapeutic efficacy of NSAIDs, it has long been known that themechanism of action whereby NSAIDs reduce inflammation is their ability to disrupt thearachidonic acid cascade, which leads to the endogenous production of prostaglandine,thromboxanes and leukotrienes. These lipid compounds are referred to collectively as“eicosanoids" because they are commonly derived from C20 polyunsaturated fatty acids, theeicosenoic acids, the most abundant of which is arachidonic acid. Arachidonic acid, which isc/s-Δ5, c/s-Δ8, c/s-Δ11, c/s-Δ14 eicosatetraenoic acid, is the dominant precursor for manyprostaglandins and leukotrienes which are mediators of inflammation.

In the first stage of the arachidonic acid cascade, arachidonic acid is released as aresuit of tissue-specific stimuli by hormones or proteases, or by membrane perturbance, andinvolves the action of a spécifie phospholipase A2. A free arachidonate results which in thesecond stage of the cascade is acted on by the bifunctional enzyme prostaglandinendoperoxide synthase, also referred to as prostaglandin H synthase (hereafter PGHsynthase), the first activity of which is as a cyclo-oxygenase, while the second activityinvolves a two-electron réduction. Most NSAIDs act as inhibitors of the cyclo-oxygenaseactivity of PGH synthase, and thereby block the production of various prostaglandins, whichare locally acting hormones which carry out their fonctions by binding to spécifie cellularreceptors. The prostaglandins are very potent but are also quickly catabolized. Some ofthese prostaglandins are mediators of the inflammatory process; however, some of theseprostaglandins also hâve a gastrointestinal protective fonction. Blocking production of thesebénéficiai prostaglandins is one of the chief factors contributing to the adversegastrointestinal reactions which are experienced with the use of NSAID therapy.Accordingly, there has been an ongoing search for pharmaceutieal agents which, whileacting as cyclo-oxygenase inhibitors, also by some additional mode of action or another, hâve substantially reduced gastrointestinal adverse reactions and resulting side effects.

It has more recently been discovered that in humans and virtually ail other mammalian species which hâve been studied, that cyclo-oxygenase (COX) comprises two isozymes, a constitutive enzyme (COX-1) and an inducible enzyme (COX-2), which hâve different activities in various Systems. The identification of the COX-2 isozyme led to conjecture early on that it might be responsible for the production of prostaglandins -4- 011213 éxclusively or primarily at inflammatory sites. Since this has now been shown to be thecase, the sélective inhibition of the COX-2 isozyme will reduce inflammation without the sideeffects of gastrointestinal toxicity. COX-1 and COX-2 hâve a 60% homology, similarvalues, and the same arachidonic acid binding sites, but COX-2 accepts a wider range ofsubstrates than does COX-1.

Another metaboiic pathway leads from the above-mentioned arachidonate to theproduction of leukotrienes through the action of a lipoxygenase. Some of these leukotrienesare also mediators of inflammation; accordingly, much effort has been expended in thesearch for pharmaceutical agents which are dual inhibitors of both cyclo-oxygenase andlipoxygenase. A particular NSAID which has been used to treat inflammatory diseases in dogs, andthe only one of two up to the présent time which has been approved for use in the UnitedStates for that purpose by the Food and Drug Administration, Committee on VeterinaryMedicine (hereafter FDA/CVM), is the above-mentioned carprofen. Carprofen, racemic 6-chloro-a-methylcarbazole-2-acetic acid, belongs to the aryl propionic acid class of NSAIDs.Other members of this class are, e.g., benoxaprofen, cicioprofen, fenoprofen, flurbiprofen,furaprofen, indoprofen, ketoprofen, pirprofen, and suprofen. While these compounds areclosely related in structure, they may stiil possess different anti-inflammatory and otherbiological properties. Carprofen, for example, has been shown to be a relatively weakinhibitor of cyclo-oxygenase, but has also been shown, in humans and various animalmodels, to decrease significantly the pain and swelling, and other symptoms of inflammation.These évaluations of carprofen are described in the technical literature, some of which iscited and discussed further below. Carprofen has also been shown to be inactive withrespect to lipoxygenase in the rat, and does not, presumably, block production ofleukotrienes. While the mode of action of carprofen stiil appears to be unknown, it has beendemonstrated to hâve some activity against phopholipase A2.

DESCRIPTION OF THE PRIOR ART

The current State of knowledge in the art, as shown by the disclosures of the below-discussed référencés, has been largeiy confused when trying to explain the mechanisms ofaction in dogs whereby carprofen is able to possess good anti-inflammatory activity while atthe same time exhibiting diminished adverse gastrointestinal and other reactions. The priorart has characterized carprofen as having weak to no cyclo-oxygenase inhibitor activity, andhas concluded that it must, therefore, be operating by some altogether different mechanismof action. -5- 01 121 3

As already mentioned further above, recently the existence of the constitutive COX-1 and inducibie COX-2 isozymes has been reported, including their diverse rôles inprotecting the gastrointestinal mucosa and in mediating inflammation, respectively. This hasled, naturally, to the investigation of compounds in a search for any which might, when usedin dogs as well as other animais and humans, inhibit oniy the inducibie COX-2 isozyme, i.e.,be sélective COX-2 inhibitors. These investigations hâve included, in particular, évaluationof the inhibitory activity of the enantiomers of various NSAlDs, including especiallyketoprofen, ketorolac, and fiurbiprofen. These particular NSAlDs were examined fordifférences in inhibitory potency in terms of one enantiomer vs. the other, as well asinhibitory potency in the case of each enantiomer treated separately, against the COX-1enzyme as compared to inhbitory potency against the COX-2 enzyme. The results of theseinvestigations showed that for ail three NSAlDs, the enantiomers were equally potent againstboth the COX-1 and COX-2 enzymes. Thus, neither the R- nor the S-enantiomer of any ofthese NSAlDs showed any selectivity toward COX-1 or COX-2. While indeed there weredifférences in potency between the enantiomers, with the S-enantiomer being the morepotent in each case, with regard to COX-1 vs. COX-2 inhibition, each enantiomer showedequal inhibitory potency, i.e., neither enantiomer was able to discriminate between the twoisozymes.

Accordingly, as a resuit of these investigations in the prior art, the current State ofthe art is that neither carprofen, nor any of the other classical NSAlDs having a carboxylicacid moiety, hâve been found to be sélective COX-2 inhibitors in dogs or any other species.These conclusions hâve been reinforced by the disclosure in the prior art of the conformationof the sequenced structures of the COX-1 isozyme, as well as of the COX-2 isozyme,complexed with various inhibitors, at the level of their basic functional molecularconfigurations. As a resuit of these reported studies, the art now teaches that carboxylic-acid-group-containing inhibitors such as carprofen are inherently incapable of being sélectiveCOX-2 inhibitors. Thus far, only sulfonyl-moiety-containing compounds and nabumetone, anaphthalenyl-2-butanone compound, hâve been reported to be sélective inhibitors of theCOX-2 isozyme.

Also recently in the art, with the above-mentioned discovery of the existence of theconstitutive COX-1 and inducibie COX-2 isozymes, studies hâve been conducted usingvarious species in order to ascertain the existence of any stereoselective inhibition spécifieto one or more of said species. These investigations of the individual activity of the R- andS-enantiomers of certain NSAlDs on COX-1 vs. COX-2 inhibition in various species hâvereported that there is a consistent potency différence between R- and S-enantiomers of ailchiral NSAlDs investigated. However, these studies hâve also reported that there is no -6- 011213 species spécifie selectivity by either the R- or the S-enantiomer of any of the chiral NSAIDsinvestigated, with respect to COX-1 vs. COX-2. For example, it has been shown that whilethe S-enantiomer is, e.g., three-times as potent as the R-enantiomer in inhibiting both theCOX-1 and the COX-2 isozymes in a given species, that with respect to either of these sameisozymes, the S-enantiomer shows equal potency in inhibiting both the COX-1 and the COX-2 isozymes, i.e., the S-enantiomer shows no sélective inhibition of COX-2 in that species.

In view of the above-described state of the art, especially where dogs hâve been thespecies investigated, it was wholly unexpected that, in accordance with the présentinvention, carprofen has been found to be a surprisingly potent inhibitor of the COX-2isozyme in the dog; and further that it is a sélective COX-2 inhibitor in the dog. Moreover,the selectivity of carprofen against COX-2 is as much as two fold greater than that of virtuallyail other NSAIDs, including carboxylic-acid-moiety-containing NSAIDs, and purportedlyCOX-2 sélective sulfonyl-moiety-containing NSAIDs. The sélection of carprofen as theprééminent sélective inhibitor of the COX-2 isozyme in dogs, from among ail other NSAIDs,not only runs counter to the current teachings in the art, but is also a wholly unexpecteddiscovery in terms of the surprising results achieved. The fact that ail of the représentativeother NSAIDs evaluated herein hâve been approved for administration to humans in theUnited States, including even those that are presently available in commerce, lends furthercredence to the soundness of these conclusions. Carprofen stands out from ail of the otherNSAIDs evaluated herein, including those which hâve been approved for human use orévaluation in clinical trials.

In view of the above-described state of the art, especially where dogs were thespecies investigated, it was further wholly unexpected that, in accordance with the présentinvention, the S-enantiomer of carprofen has been found to be a highly sélective inhibitor ofthe COX-2 isozyme vs. the COX-1 isozyme in the dog, and that this is the case to asignificantly greater extent than ail other NSAIDs or their S-enantiomers, includingcarboxylic-acid-moiety-containing NSAIDs, and purportedly COX-2 sélective sulfonyl-moiety-containing NSAIDs. The sélection of the S-enantiomer of carprofen as the prééminent andsélective inhibitor of the COX-2 isozyme in dogs, which at the same time exhibits little or noadverse gastrointestinal or other reactions in dogs, from among ail other NSAIDs, not onlyruns counter to the current teachings in the art, but is also a wholly unexpected discovery interms of the surprising results achieved. The fact that ail of the représentative other NSAIDsevaluated herein hâve been approved for administration to humans in the United States,including even those that are presently available in commerce lends further credence to thesoundness of these conclusions. -7- 011213

In view of the above-described State of the art, especially where dogs were thespecies investigated, it was still further wholly unexpected that, in accordance with theprésent invention, the S-enantiomer of carprofen has been found to hâve, by reason of itsbeing a highly setective inhibitor of the COX-2 isozyme, a surprisingly improved level of anti-inflammatory, analgésie and anti-pyretic activity compared to that of ail other NSAIDs,including those having a carboxylic-acid-moiety, or their S-enantiomers, as well as asurprisingly reduced level of adverse gastrointestinal and other reactions compared to that ofail other NSAIDs, including those having a carboxylic-acid-moiety, or their S-enantiomers.

As already mentioned, carprofen belongs to the aryl propionic acid class of NSAIDs, and is as well a member of the subclass of such compounds which are substituted carbazole acetic acids. These compounds and their use as anti-inflammatory, analgésie and anti- rheumatic agents are described in U.S. 3,896,145. The anti-inflammatory activity of carprofen in dogs was investigated by McKellar et al. and reported in “Pharmacokinetics, tolérance and sérum thromboxane inhibition of carprofen in the dog", Journal of Small

Animal Practice, 31, 443-448, 1990. The biological activities of the individual (-)(R) and (+)(S) enantiomers of carprofen, as well as of racemic carprofen in dogs were further investigated by McKellar ef al. and reported in “Stereospecific pharmacodynamies and pharmacokinetics of carprofen in the dog", J. Vet. Pharmacol. Therap. 17, 447-454, 1994.

Reporting on the results of this investigation, the authors concluded that: “[t]he mode of action of CPF [carprofen] remains unknown. ... CPF administered asa racemic mixture or as either the (+)(S) or {-)(R) enantiomer did not significantly inhibitthe génération of TxB2 from blood or PGE2 and 12-HETE in inflammatory exudate,suggesting that it does not act as a conventional NSAID. A principal mode of action of most NSAIDs is known to be inhibition of the enzymecyclooxygenase in the génération of inflammatory prostaglandins from arachidonic acid. ... The large number of mediators now known to be involved in inflammation providemany possible targets for anti-inflammatory drugs, and it is probable that CPF has itsprincipal action activity on one or a number of mediators not yet identified."

Thus, it was concluded by these workers in the art that carprofen has a mode ofaction which is “not primarily attributable” to cyclo-oxygenase inhibition; and indeed, that the“poor activity of carprofen against cyclo-oxygenase, lipoxygenase and phospholipasesuggest that its major mode of action may be by mechanisms other than eicosanoidinhibition." Such findings and conclusions hâve instructed other workers in the art away fromthe surprising discovery of the présent invention, that carprofen dosed dogs show near totalinhibition of PGE2 synthesis, as is demonstrated further below in a working example.

Another study of carprofen in dogs was reported by Holtsinger ef al. in “TheTherapeutic Efficacy of Carprofen (Rimadyl-V™) in 209 Clinical Cases of CanineDegenerative Joint Disease", V.C.O.T. 1992; 5:140-4. The authors theorized from in vitro -8- 011213 studies that carprofen might exert ils anti-inflammatory action, at least in part, by inhibitingneutrophil migration, and that this might explain how carprofen could be equipotent withindomethacin as an anti-inflammatory agent, and yet hâve an ulcerogenic potential whichwas 16 times less. However, the authors voiced many réservations regarding theirconclusions and thereby failed to point in the direction of the discovery on which the présentinvention is based. A study of the use of carprofen to treat osteoarthritis in dogs was reported byVasseur et al. in “Randomized, controlied trial of the efficacy of carprofen, a nonsteroidalanti-inflammatory drug, in the treatment of osteoarthritis in dogs", J Am Vet Med Assoc,206(6): 807-811, 1995. These investigators were also unable to explain the activity ofcarprofen. They concluded, on the one hand, that prostaglandins are protective of thegastrointestinal mucosa, and that "carprofen, like other NSAID, inhibits prostaglandinsynthetase, blocking prostaglandin biosynthesis.” However, this conclusion was inçonsistentwith the results of their study, which determined that “carprofen has minimal or no harmfuleffects on the gastrointestinal mucosa in dogs." A study of the use of carprofen to treat acute postoperative pain in dogs wasreported by Lascelles étal, in “Postoperative analgésie and sédative effects of carprofen andpethidine in dogs”, Veterinary Record, 134: 187-191, 1994. These investigators wêresimilarly confused as to the mode of action of carprofen, noting on the one hand that“Carprofen . . . at therapeutic doses, seems to be a poor inhibitor of prostaglandinsynthetase (or cyclo-oxygenase), the enzyme responsible for the synthesis of infiammatorymediators produced by tissue damage", while acknowledging on the other hand that“[n]evertheless, studies hâve shown it to be a good analgésie for both acute and chronic. pain.” . A much earlier study which compared the biological activities of indomethacin tothose of the stereoisomers and racemate of carprofen in humans was done by Gaut ef al.and reported in “Stereoisomeric Relationships Among Anti-inflammatory Activity, Inhibition ofPlatelet Aggregation, and Inhibition of Prostaglandin Synthetase”, PROSTAGLANDINS, Vol.10, N0. 1, July 1975. The study concluded that the carprofen racemate, unlikeindomethacin, would hâve no effect on platelet aggregation and thus would produce noprolongation of bieeding time at doses possessing anti-inflammatory activity. The data fromthe study also suggested that the carprofen racemate and (S) isomer hâve greater specificitytoward anti-arthritic activity and are less ulcerogenic than indomethacin. A portion of the advances in prostaglandin research which were presented in 1994at the 9th international Conférence on Prostaglandins and Related Compounds focused onCOX-2 selectivity. A meeting report presented by Battistini ef al. entitled “COX-1 and COX- -9- 011213 2: Toward the Development of More Sélective NSAIDs" and published in DN&P, 7(8),October 1994, contained comparative data obtained from a large variety of cell types,including human, mouse or rat types, using different stimuli, as reported in the technicalliterature reviewed and cited by Battistini et al. Data demonstrating IC50 values againstCOX-1 and COX-2 for numerous anti-infiammatory compounds, including carprofen, waspresented, and the ratio of COX-2/COX-1 was used to détermine COX-2 selectivity.Inverting the ratios reported by Battistini et al. so as to be consistent with those used hereinto facilitate comparison, the most COX-2 sélective compounds had ratios of 1428.57 to50,000.00. The ICS0 (μΜ) values of carprofen for both the COX-1 and COX-2 isozymes wereshown as being exactly the same (10.96) to give a ratio of 1.00, ciearly demonstrating thatcarprofen has no COX-2 selectivity. The values cited by Battistini ef al. were originallyreported by Akarasereenont ef al. in “Relative Potency of Nonsteroid Anti-InflammatoryDrugs As Inhibitors of Cyclo-oxygenase-1 or Cyclo-oxygenase-2", Br. J. Pharmacol.,Proceedings Supp. No. 183P, 5-7 January 1994; and by the same group in Mitchell et al.,“Selectivity of nonsteroidai antiinflammatory drugs as inhibitors of constitutive and induciblecyclooxygenase”, Proc Nat Acad Sci USA, 90; 11693-7, 1993. The IC50 (pg/ml) values (n =9) of carprofen inhibition of COX-1 (derived from bovine aortic endothélial cells) and COX-2(derived from lipopolysaccharide stimulated J774.2 macrophages) were 3 ± 0.41 and 3 +1.72, respectively, to give a ratio of 1.00. This is in complété contrast to the data for severalcompounds which were shown to hâve from 1000 to 4000 times more selectivity for COX -2.The report observed that none of the sélective COX-2 inhibitors described are carboxylicacids, like the vast majority of existing NSAIDs, including carprofen. Indeed, ail of the COX-2 sélective inhibitors hâve a sulfonyl group in the molécule, which in the case of meloxicamis incorporated into its 1,2-benzothiazine-1,1-dioxide ring structure. If this corrélation holds,the report spéculâtes, the Arg 150 residue of the COX protein would not be an essentialbinding site for sélective inhibitors of COX-2. Arg is essential for COX activity because itbirids the terminal carboxyl group of arachidonic acid, and is thus most likely the binding sitefor the carboxylic acid functional group of most existing NSAIDs, Accordingly, an inhibitorwhich was sélective for COX-2 would be expected to bind to a feature which was unique tothe COX-2 isozyme protein structure, and not to a feature which was common to both the COX-1 and COX-2 isozyme protein structures.

An even more spécifie interprétation of the molecular interactions of the classical NSAIDs and COX-2 sélective inhibitors with the protein structure of the PGH synthaseenzyme, more commonly known as the cyclo-oxygenase enzyme, was reported byKurumbail ef al. in “Structural Basis for Sélective Inhibition of Cyclo-oxygenase-2 by Anti-Inflammatory Agents”, NATURE 384, 644-648, December 1996. This interprétation is based -10- 011213 on the reported structures of unliganded murine COX-2 and complexes with flurbiprofen,indomethacin, and SC-558, a sélective COX-2 inhibitor having a phenylsulfcnamide groupbut no carboxylic acid group, determined at 3.0 to 2.5Â resolution.

The subject report indicates that the human and murine COX-2 enzymes areexpected to be very similar because of the 87% identity and strict sequence conservation inthe cyclo-oxygenase active site. Flurbiprofen, a siow-binding compétitive inhibitor of bothCOX-1 and COX-2, binds in the long hydrophobie channel and excludes substrate from thecyclo-oxygenase active site. SC-558 is a diaryl heterocyclic inhibitor with a central pyrazolering and a sulfonamide group attached to one of the aryl rings. In COX-2 the channel thatleads from membrane to the cyclo-oxygenase active site forks at the SC-558 binding site,with one branch forming a cavity that accepts the bromophenyl ring of SC-558, while theother branch forms a pocket which is virtually inaccessible in the COX-1 structure, but whichaccommodâtes the entire phenylsulfonamide moiety in COX-2. This pocket is moreaccessible in COX-2 because valine is substituted for isoleucine, which has a larger sideChain, at position 523. Access of the phenylsulfonamide group to the new pocket in COX-2is facilitated by another isoleucine to valine change at position 434, which forms a moleculargâte extending across the new hydrophilic pocket. Finally, at position 513 histidine in COX-1 is replaced by arginine in COX-2, and superposition of the two enzymes suggests that theimidazole ring of histidine in the COX-1 enzyme would not extend sufficiently for directinteractions with the sulfonamide group of SC-558, as is the case with arginine in the COX-2enzyme. The subject report notes that in each of the above-described three instances, theinhibition profile of COX-2 is altered dramatically by mutation of a single amino acid.

The subject report goes on to conclude that it appears probable that the primarydéterminant of COX-2 selectivity in the diaryl heterocyclic class of inhibitors to which SC-558belongs is the phenylsulfonamide moiety. However, the absence of a carboxylate group isalso significant. The arginine at position 120 with its guanidinium group is one of the fewcharged residues in the hydrophobie cyclo-oxygenase channel, and it stabilizes thecarboxylate of classical NSAIDs such as flurbiprofen by way of charge-charge interaction.The absence of such a carboxylate group in SC-558 is probably also a significantcomponent of its COX-2 selectivity. This conclusion is supported by the results of attemptsto improve its potency against COX-2 by incorporating an acidic group on the pyrazole of thediaryl heterocyclic structure, which has consistently led to poor selectivity.

The subject report embodies the first example of a membrane protein beingsuccessfully studied as a target in structure-based drug design, and approximates thecurrent State of the art conceming the structure/activity relationships of NSAIDs to the cyclo-oxygenase isozymes and their resuiting anti-infiammatory activity vs. their adverse -11- 011213 gastrointestinal reactions. It is within the context of this State of the art that the présentinvention will be seen to be a wholly unexpected development which provides, surprisingly,an optimum efficacy/safety profile for the treatment of inflammation in dogs.

The possibility of enantioselective inhibition of the COX-2 isozyme was investigatedand reported by Carabaza et al. in “Stereoselective Inhibition of Inducible Cyclooxygenaseby Chiral Nonsteroidal Antiinflammatory Drugs", J Clin Pharmacol 1996; 36:505-512. Thestereoselective inhibition of COX-2 by ketoprofen, flurbiprofen, and ketorolac was studied inthree different in vitro Systems, and the results were compared with the inhibition of COX-1in three parallel in vitro models. It was found that both isozymes were inhibited by the S-enantiomers of ail three NSAIDs on an equal potency basis; but that the R-enantiomers of ailthree NSAIDs inhibited both isozymes with significantly and correspondingly less potency.Put another way, ail three R-enantiomers exhibited equal potency in inhibiting both COX-1and COX-2, but ail of their potency levels were much lower than those of the correspondingS-enantiomers in ail cases. The “significant degree of enantioselectivity" referred to in thisreported study refers only to R- vs. S-, and does not refer to the COX-2 selectivity uniquelyexhibited by the S-enantiomer of carprofen, in accordance with the présent invention.

SUMMARY OF THE INVENTION

There is provided in accordance with the présent invention a method of treating orpreventing pain and inflammatory processes and diseases associated with the activity ofinducible cyclo-oxygenase-2 (COX-2) in a member of the species Canis familiaris in need ofsuch treatment, while at the same time reducing or eliminating undesirable side effectsàssociated with simultaneous inhibition of the activity of constitutive cyclo-oxygenase-1(COX-1) by selectively inhibiting COX-2 activity with référencé to COX-1 activity, wherein theselectivity ratio of COX-2 : COX-1 activity inhibition is at least 3 : 1 based on ex vivoinhibition levels in whole blood measured at a dose giving s 80% COX-2 inhibition, andpreférably > 90% COX-2 inhibition, comprising administering to said member of the speciesCanis familiaris an amount therapeutically effective for treating pain and inflammation inaccordance with the above-recited limitations, of an anti-inflammatory sélective COX-2ïnhibitory compound comprising a member selected independently from the group of anti-inflammatory compounds consisting essentially of salicylic acid dérivatives; p-aminophenoldérivatives; indole and indene acetic acids; heteroaryl acetic acids; arylpropionic acids;anthranilic acids; enolic acids; and alkanones.

It is also within the scope of the présent invention to carry out the above-described method of treating or preventing pain and inflammatory diseases and processes by administering more than one member selected from the above-recited group of anti- -12- 011213 inîlammatory compounds. The desirability of any one such combination of anti-infiammatoryactive agents will probably be based on obtaining a favorable balance of thepharmacokinetic properties of the individual agents involved. For example, rapidity of onsetof action may be balanced with extended therapeutic half-life, or a tendency toward theformation of large cellular réservoirs in certain tissues may be balanced with higher rates ofplasma protein binding. Ail such combinations are contemplated to be within the scope ofthe présent invention.

The présent invention provides any anti-inflammatory sélective COX-2 inhibitorsatisfying the above-described limitations. Among such sélective inhibitory compounds is apreferred subgenus of carprofen compounds useful in treating or preventing pain andinflammatory processes and diseases in a member of the species Canis familiaris of theformula:

wherein: R2 is·

O

II

-C-+C-A

I

_yJ n where A is hydroxy, (C^ - C4)alkoxy, amino, hydroxy-amino, mono-(C, -C2)alkylamino, di-iC,-C2)alkylamino; X and Y are independently H or (C, - C2)alkyl; and n is 1 or 2; RÊ is halogen, (C5 - C3)alkyl, trifluoromethyl, or nitro; R9 is H; (C, - C2)alkyl; phenyl or phenyl-{C1 - C2)alkyl, where phenyl is optionallymono-substituted by fiuoro or chloro; -C(=O)-R, where R is (C, - C2)alkyl or phenyl,optionally mono-substituted by fiuoro or chloro; or -C(=O)-O-R1, where R1 is (C! - C2)alkyl, where X and Y are different, the and (+)(S) enantiomers thereof; and ailpharmaceutically acceptable sait forms, prodrugs and métabolites thereof which aretherapeutically active for treating or preventing pain and inflammation. Where the inhibitor ofFormula (I) exists as (-)(R) and (+)(S) enantiomers, in accordance with the présent inventionthere is provided the (+)(S) enantiomer alone, or where both enantiomers are présenttogether, there is provided a racemic or a non-racemic mixture thereof.

The présent invention further provides in a preferred aspect of the above-describedmethod of treating or preventing pain and inflammatory processes and diseases in a -13- 011213 member of the species Canis familiaris in need of such treatment, administration to saidmember of an amount therapeutically effective for treating pain and inflammation, of an anti-inflammatory sélective COX-2 inhibitory compound for which COX-2 in vitro ICS0 potency insaid member is at least 30 fold greater, preferably at least 40 fold greater, more preferably atleast 50 fold greater, more preferably still at least 60 fold greater, even more preferably stillat least 80 fold greater, and most preferably at least 100 fold greater than its in vitro IO50potency against COX-1 in said member, wherein said inhibitor is a member selected fromthe group of anti-inflammatory compounds consisting essentially of salicylic acid dérivatives;p-aminophenol dérivatives; indole and indene acetic acids; heteroaryl acetic acids;arylpropionic acids; anthranilic acids; enolic acids; and alkanones.

The présent invention still further provides in another preferred aspect of the above-described method of treating or preventing pain and inflammatory processes and diseases ina member of the species Canis familiaris in need of such treatment, administration to saidmember of an amount therapeutically effective for treating pain and inflammation, of an anti-inflammatory sélective COX-2 inhibitory compound which selectively inhibits substantiallyonly inducible COX-2, with substantially no inhibition of corresponding constitutive COX-1.

There is further provided the above-described methods wherein said therapeuticallyeffective amount of an anti-inflammatory compound of Formula (l) as defmed, and especiallyof said (+)(S)-enantiomer of 6-chloro-a-methyl-9H-carbazole-2-acetic acid, is administeredsystemically to said member of Canis familiaris, wherein said systemic administrationcomprises; (1) injection or infusion into suitable body tissues or cavities of a pharmaceuticalcomposition containing said inhibitor in suitable liquid form for delivering said inhibitor bysystemic administration which is intraarterial, intra- or transdermal, subcutaneous,intramuscular, intraspinal, intrathecal, or intravenous, where said inhibitor is: (a) contained insolution as a soluté; (b) contained in the discontinuous phase of an émulsion, or thediscontinuous phase of an inverse émulsion which inverts upon injection or infusion, saidémulsions containing suitable emulsifying agents; or (c) contained in a suspension as asuspended solid in colloïdal or microparticulate form, said suspension containing suitablesuspending agents; (2) injection or infusion into suitable body tissues or cavities of apharmaceutical composition containing said inhibitor in suitable liquid form to serve as adepot for delivering said inhibitor by systemic administration, wherein said compositionprovides storage of said inhibitor and thereafter delayed-, sustained-, and/or controlled- release of said inhibitor for systemic distribution; (3) instillation, inhalation or insufflation intosuitable body tissues or cavities of a pharmaceutical composition containing said inhibitor insuitable solid form for delivering said inhibitor, where said inhibitor is; (a) contained in a solidimplant composition which is installed in suitable body tissues or cavities, said composition -14- 011213 providing detayed-, sustained-, and/or controlled-release of said inhibitor; (b) contained in aparticulate composition which is inhaled into the lungs; or (c) contained in a particuiatecomposition which is blown into suitable body tissues or cavities, where said compositionoptionally provides delayed-, sustained-, and/or controlled-release of said inhibitor, or (4)ingestion of a pharmaceutical composition containing said inhibitor in suitable solid or liquidform for pérorai delivery of said inhibitor, where said inhibitor is: (a) contained in a soliddosage form; or (b) contained in a liquid dosage form. Suppositories may be regarded as aspécial type of implant, since they comprise bases which are solid at room température butmelt at body température, slowly releasing the active ingrédient with which they areimpregnated into the surrounding tissue of the body, where the active ingrédient becomesabsorbed and transported to effect systemic administration. Dosage forms which permittransdermal and transmucosal administration to achieve systemic delivery are alsocontemplated, especially including transdermal patch technology.

There is further provided the above-described method of treating or preventing painand inflammation comprising ingestion or administration of a solid pérorai dosage formselected from the group consisting of delayed-release oral tablet, capsule, caplet, lozenge,troche, and multiparticulates, enteric-coated tablets and capsules which prevent release andabsorption in the stomach to facilitate delivery distal to the stomach of the dog, sustained-release oral tablets, capsules and microparticulates which provide systemic delivery of theactive ingrédient in a controlled manner over at least a 10-hour period, a fast-dissolvingtablet, encapsulated solutions, an oral paste, a granular form incorporated in or to beincorporated in the food of the dog being treated, and a chewable form in which said activeingrédient is consumed along with the palatable chew, or may altematively be delivered byleaching from the body of the chew which is not consumed, during mastication by the dogbeing treated. Also included for use with the above-described dosage forms aremicroencapsulated formulations of the active ingrédient, which may then be incorporatedinto a tablet, capsule, or other final formulation. Still further, there is provided said methodcomprising ingestion of a liquid pérorai dosage form selected from the group consisting of asolution, suspension, émulsion, inverse émulsion, élixir, extract, tincture, and concentrate,optionally to be added to the drinking water of the dog being treated. Any of these liquiddosage forms, when formulated in accordance with methods well known in the art, can eitherbe administered directly to the dog being treated, or may be added to the drinking water ofthe dog being treated. The concentrate liquid form, on the other hand, is formulated to beadded first to a given amount of water, from which an aliquot amount may be withdrawn foradministration directly to the dog or addition to the drinking water of the dog. -15- 011213

There is still further provided the above-described methods wherein saidtherapeutically effective amount of an anti-inflammatory compound of Formula (I) as defined,is administered locally to a site of inflammation in said member of Canis famiiiaris. There isstill further provided said method of local administration wherein said local administrationcomprises: (1) injection or infusion into a local site of inflammation of a pharmaceuticalcomposition containing said inhibitor in suitable liquid form for delivering said inhibitor bylocal administration which is intraarterial, intraarticular, intrachondrial, intracostal, intracystic,intra- or transdermal, intrafasicular, intraligamentous, intramedulary, intramuscular,intraneural, intraosteal, intrapelvic, intrapericardial, intraspinal, intrastemal, intrasynovial,intratarsal, or intrathecal; inciuding components which provide delayed-reiease, controlled-release, and/or sustained-release of said inhibitor into said local site of inflammation; wheresaid inhibitor is contained: (a) in solution as a soluté; (b) in the discontinuous phase of anémulsion, or the discontinuous phase of an inverse émulsion which inverts upon injection orinfusion, said émulsions containing suitable emulsifying agents; or (c) in a suspension as asuspended solid in colloïdal or microparticulate form, said suspension containing suitablesuspending agents; (2) injection or infusion of a pharmaceutical composition containing saidinhibitor in suitable liquid form to serve as a depot for delivering said inhibitor to said localsite of inflammation; wherein said composition provides storage of said inhibitor andthereafter delayed-, sustained-, and/or controlled-release of said inhibitor into said local siteof inflammation; or (3) instillation, inhalation or insufflation of a pharmaceutical compositioncontaining said inhibitor in suitable solid form for delivering said inhibitor to said local site ofinflammation, where said inhibitor is contained: (a) in a solid implant composition which isinstalled in said local site of inflammation, said composition optionally providing delayed-,sustained-, and/or controlled-release of said inhibitor to said local site of inflammation; (b) ina particulate composition which is inhaled into a local site of inflammation comprising thelungs; or (c) in a particulate composition which is blown into a local site of inflammation,where said composition optionally provides delayed-, sustained-, and/or controlled-release ofsaid inhibitor to said local site of inflammation. Other spécifie dosage forms for localadministration are also within the scope of the présent invention. For example, compositionsto be applied to the skin, preferably with enhancement of absorption by mechanical working of the composition into the skin as by rubbing, may be used to deiiver the inhibitor activeingrédient into a local area, such as an inflammed joint, in need of such treatment. Suchcompositions may be in the form of gels, lotions, balms, ointments, and other formulationsdesigned for topical application.

There is still further provided the above-described methods wherein thetherapeutically effective amount of anti-inflammatory inhibitor is administered to said member -16- 01 1 21 3 oî the species Canis famiiiaris in an amount, expressed as mg per kg of body weight of saidmember per day, ranging from about 0.01 mg/kg to about 20.0 mg/kg/day, preferably fromabout 0.1 mg/kg to about 12.0 mg/kg/day, more preferably from about 0.5 mg/kg to about10.0 mg/kg/day, and most preferably from about 0.5 mg/kg to about 8.0 mg/kg/day.Administration of 6-chloro-a-methyl-9/7-carbazole-2-acetic acid is typically provided bydosing at a rate of about 4.0 mg/kg/day.

There is additionally provided in accordance with the présent invention apharmaceutical composition for treating or preventing pain and inflammatory processes anddiseases in a member of the species Canis famiiiaris in need of such treatment, comprisinga pharmaceutically acceptable carrier together with an amount therapeutically effective fortreating pain and inflammation, of an anti-inflammatory sélective inhibitor of cyclo-oxygenase-2 (COX-2) wherein the selectivity ratio of COX-2 to COX-1 is at least 3 :1 basedon ex vivo inhibition levels in whole blood measured at a dose giving > 80% COX-2inhibition, and preferably > 90% COX-2 inhibition. In a preferred aspect of saidpharmaceutical composition for treating or preventing pain and inflammatory processes anddiseases, said therapeutically effective anti-inflammatory sélective COX-2 inhibitor has an invitro 1CSO potency of at least 30 fold greater, preferably at least 40 fold greater, morepreferably at least 50 fold greater, more preferably still at least 60 fold greater, even morepreferably at least 80 fold greater. and most preferably at least 100 fold greater than cyclo-oxygenase-1 (COX-1) in vitro IC^ potency; wherein said inhibitor is a member selected fromthe group of anti-inflammatory compounds consisting essentially of salicylic acid dérivatives;p-aminophenol dérivatives; indole. and indene acetic acids; heteroaryl acetic acids;•arylpropionic acids; anthranilic acids; enolic acids; and alkanones.

There is further provided the above-described pharmaceutical composition whereinsaid inhibitor is a member selected from the group consisting of arylpropionic acids; andfurther still, said inhibitor is a compound of above-defined Formula (l). There is furtherprovided the above-described pharmaceutical composition wherein the cyclo-oxygenase-2(COX-2) in vitro ICso potency of the inhibitory compound of Formula (I) is at least 100 foldgreater than the cyclo-oxygenase-1 (COX-1) in vitro ICsq potency thereof; and wherein oneof X and Y is H and the other is methyl; and wherein when both resulting enantiomers areprésent, (+)(S) enantiomer is présent in amount of at least 75%. In particular, there isprovided the above-described pharmaceutical composition wherein for Formula (I), for R2 ,n = 1, one of X and Y is H and the other is methyl, and A is hydroxy, (C, - C2) alkoxy, oramino; R6 is chloro or trifluoromethyl; and R9 is H, methyl, acetyl, benzoyl, or acetyloxy; andwherein when both resulting enantiomers are présent together, (+)(S) enantiomer is -17- 011213 present in amount of at least 85%, preferably at least 90%, more preferably at least 95%,and most preferably at least 99%.

There is still further provided the above-described pharmaceutical compositions inwhich said inhibitor comprises 6-chloro-a-methyI-9F/-carbazole-2-acetic acid; and whereinwhen both resulting enantiomers are présent together, (+)(S) enantiomer is présent in anamount of at least 85%, preferably at least 90%, more preferably at least 95%, and mostpreferably at least 99%. In particular, there is provided the above- and below-describedpharmaceutical composition in which said inhibitor is comprised entirely of (+)(S) enantiomerof 6-chloro-a-methyl-9/7-carbazole-2-acetic acid.

There is also provided the above-described pharmaceutical compositions whereinthe therapeutically effective amount of anti-inflammatory inhibitor présent is sufficient, in thecontext of the dosage regimen and administration parameters employed, to provide amember being treated with an amount of said inhibitor, expressed as mg per kg of bodyweight of said member per day, ranging from about 0.01 mg/kg to about 20.0 mg/kg/day,preferably from about 0.1 mg/kg to about 12.0 mg/kg/day, more preferably from about 0.5mg/kg to about 10.0 mg/kg/day, and most preferably from about 0.5 mg/kg to about 8.0mg/kg/day. Administration of 6-chloro-a-methyl-9/7-carbazole-2-acetic acid is typicallyprovided by dosing at a rate of about 4.0 mg/kg/day.

In a still further preferred aspect of the pharmaceutical compositions of the présentinvention there is provided an anti-inflammatory sélective COX-2 inhibitor which selectivelyinhibits substantially only inducible COX-2, with substantially no inhibition of correspondingconstitutive COX-1. In particular a preferred embodiment thereof comprises apharmaceutically acceptable carrier together with a therapeutically effective amount fortreating pain and inflammation, of an anti-inflammatory sélective inhibitor of COX-2comprising a compound of Formula (I) wherein for R2, n = 1, one of X and Y is H, and theother is methyl, and A is hydroxy, (C, -C2) alkoxy, or amino; R6 is chloro or trifluoromethyl;and'R9 is H, methyl, acetyl, benzoyl, or methoxycarbonyl; and (+)(S) enantiomer is présentin amount of at least 99%. Especially, said inhibitor is comprised entirely of (+)(S)-enantiomer of 6-chloro-a-methyl-9H-carbazole-2-acetic acid. Preferably in saidpharmaceutical composition, said therapeutically effective amount of said inhibitor is

I sufficient, in the context of the dosage regimen and administration parameters employed, toprovide a member being treated with an amount of said inhibitor, expressed as mg per kg ofbody weight of said member per day, ranging from about 0.5 mg/kg/day to about 8.0mg/kg/day.

There is still further provided the above-described pharmaceutical composition in adosage form which can provide the required therapeutically effective amount for treating pain 01 1213 -18- and inflammation, of anti-inflammatory sélective COX-2 inhibitor in a convenient regimen.However, a number of the above- and below-described pharmaceutical compositions areintended to be long-acting, Le., providing inhibitor activity for longer than just hours or asingle day, and instead providing such activity for several days up to a week or more. Theimplants and depots in particular are examples of such long-acting pharmaceuticalcompositions, and some of these are intended to provide inhibitor activity for up to a monthor more. The required therapeutically effective amount of inhibitor necessary to treat orprevent pain and inflammation, expressed as the mg per kg of body weight of said memberof species Canis familiaris per day, ranges from about 0.01 mg/kg to about 20.0 mg/kg/day,preferably from about 0.1 mg/kg to about 12.0 mg/kg/day, more preferably from about 0.5mg/kg to about 10.0 mg/kg/day, and most preferably from about 0.5 mg/kg to about 8.0mg/kg/day. Administration of 6-chloro-a-methyl-9W-carbazole-2-acetic acid is typicallyprovided by dbsing at a rate of about 4.0 mg/kg/day.

In particular, there is further provided the above-described pharmaceuticalcompositions wherein said therapeutically effective amount for treating pain andinflammation, of an anti-inflammatory inhibitor, is provided in a dosage form suitable forsystemic administration to said member of Canis familiaris, wherein said pharmaceuticalcomposition contains said inhibitor in suitable liquid form for delivering said inhibitor by: (1)injection or infusion which is intraarterial, intra- or transdermal, subcutaneous, intramuscular,intraspinal, intrathecai, or intravenous, wherein said inhibitor: (a) is contained in solution as asoluté; (b) is contained in the discontinuous phase of an émulsion, or the discontinuousphase of an inverse émulsion which inverts upon injection or infusion, said émulsionscontaining suitable emulsifying agents; or (c) is contained in a suspension as a suspended.solid in colloïdal or microparticulate form, said suspension containing suitable suspending, agents; (2) injection or infusion into suitable body tissues or cavities as a depot, wherein saidcomposition provides storage of said inhibitor and thereafter delayed-, sustained-, and/orcontrolled-release of said inhibitor for systemic distribution; (3) instillation, inhalation orinsufflation into suitable body tissues or cavities of said pharmaceutical composition insuitable solid form, where said inhibitor (a) is contained in a solid implant compositionproviding delayed-, sustained-, and/or controlled-release of said inhibitor; (b) is contained ina particulate composition to be inhaled into the lungs; or (c) is contained in a particulatecomposition to be blown into suitable body tissues or cavities, where said composition optionally provides delayed-, sustained-, and/or controlled-release of said inhibitor, or (4)ingestion of said pharmaceutical composition in suitable solid or liquid form for péroraidelivery of said inhibitor, where said inhibitor: (a) is contained in a solid dosage form; or (b) iscontained in a liquid dosage form. -19- 011213

In an especially preferred aspect of the above-described pharmaceuticalcompositions which provide delayed-, sustained-, and/or controlled-release of said anti-inflammatory seiective COX-2 inhibitor, there is included ail such dosage forms whichproduce > 80% inhibition of COX-2 isozyme activity and resuit in a plasma concentration ofsaid inhibitor of at least 10 pg/mL for at least 4 hrs; preferably for at least 8 hrs; morepreferably for at least 12 hrs; more preferably still for at least 16 hrs; even more preferablystill for at least 20 hrs; and most preferably for at least 24 hrs. Preferably, there is includedthe above-described dosage forms which produce > 80% inhibition of COX-2 isozymeactivity and resuit in a plasma concentration of said inhibitor of at least 15 pg/mL for at least4 hrs, preferably for at least 8 hrs, more preferably for at least 12 hrs, still more preferably forat least 20 hrs, and most preferably for at least 24 hrs. More preferably, there is included theabove-described dosage forms which produce > 90% inhibition of COX-2 isozyme activityand resuit in a plasma concentration of said inhibitor of at least 20 pg/mL for at least 4 hrs,preferably for at least 8 hrs, more preferably for at least 12 hrs, still more preferably for atleast 20 hrs, and most preferably for at least 24 hrs.

Particular dosage forms of the above-described pharmaceutical compositionsinclude suppositories as a spécial type of implant, comprising bases which are solid at roomtempérature but melt at body température, slowly releasing the active ingrédient with whichthey are impregnated into the sumounding tissue of the body, where the active ingrédientbecomes absorbed and transported to effect systemic administration; solid pérorai dosageforms seiected from the group consisting of delayed-release oral tablet, capsule, caplet,lozenge, troche, and multiparticulates, enteric-coated tablets and capsules which preventrelease and absorption in the stomach to facilitate delivery distal to the stomach of the dog,sustained-release oral tablets, capsules and microparticulates which provide systemicdelivery of the active ingrédient in a controlled manner up to a 24-hour period, a fast-dissolving tablet, encapsulated solutions, an oral paste, a granular form incorporated in or tobe incorporated in the food of the dog being treated, and a chewable form in which saidinhibitor active ingrédient is consumed along with the palatable chew, or may alternatively bedelivered by leaching from the body of the chew which is not consumed, during masticationby the dog being treated; liquid pérorai dosage forms seiected from the group consisting ofsolutions, suspensions, émulsions, inverse émulsions, élixirs, extracts, tinctures, andconcentrâtes, optionally to be added to the drinking water of the dog being treated. Any ofthese liquid dosage forms, when formulated in accordance with methods well known in the art, can either be administered directly to the dog being treated, or may be added to the drinking water of the dog being treated. The concentrate liquid form, on the other hand, is formulated to be added first to a given amount of water, from which an aliquot amount may -20-

0112H be withdrawn for administration directly to the dog or addition to the drinking water of thedog.

There is further provided the above-described pharmaceutical compositions whereinsaid therapeutically effective amount for treating pain and inflammation, of said anti-inflammatory inhibitor, is provided in a dosage form suitable for local administration to a siteof inflammation in said member of Canis familiaris, wherein said pharmaceutical compositioncontains said inhibitor in suitable liquid form for delivering said inhibitor by: (1) injection orinfusion into a local site of inflammation, which is intraarterial, intraarticular, intrachondrial,intracostal, intracystic, intra- or transdermal, intrafasicular, intraligamentous, intrameduiary,intramuscular, intranasal, intraneural, intraocular, i.e., opthalmic administration, intraosteal,intrapelvic, intrapericardial, intraspinal, intrasternal, intrasynovial, intratarsal, or intrathecal;including components which provide delayed-release, controlled-release, and/or sustained-release of said inhibitor into said local site of inflammation; where said inhibitor is contained;(a) in solution as a soluté; (b) in the discontinuous phase of an émulsion, or thediscontinuous phase of an inverse émulsion which inverts upon injection or infusion, saidémulsions containing suitable emulsifÿing agents; or (c) in a suspension as a suspendedsolid in colloïdal or microparticulate form, said suspension containing suitable suspendingagents; or (2) injection or infusion as a depot for delivering said inhibitor to said local site ofinflammation; wherein said composition provides storage of said inhibitor and thereafterdelayed-, sustained-, and/or controlled-release of said inhibitor into said local site ofinflammation, and wherein said composition also inciudes components which ensure thatsaid inhibitor has predominantly local activity, with little systemic carryover activity; orwherein said pharmaceutical composition contains said inhibitor in suitable solid form fordelivering said inhibitor by; (3) instillation, inhalation or insufflation to said local site ofinflammation, where said inhibitor is contained: (a) in a solid implant composition which isinstalled in said local site of inflammation, said composition optionally providing delayed-,sustained-, and/or controlled-release of said inhibitor to said local site of inflammation; (b) ina particulate composition which is inhaled into a local site of inflammation comprising thelungs; or (c) in a particulate composition which is blown into a local site of inflammation,where said composition inciudes components which will ensure that said inhibitor haspredominantly local activity, with insignificant systemic carryover activity, and optionally provides delayed-, sustained-, and/or controlted-release of said inhibitor to said local site of inflammation.

There is provided in accordance with the présent invention combinations of one or more other therapeutically active agents together with the active ingrédients for treating pain and inflammation which make up the above-described pharmaceutical compositions of the -21- 011213 présent invention. Where a joint has become seriously infiammed and infected at the sametime by microorganisms, e.g., bacteria, fungi, protozoa, virus and the iike, the activeingrédient of the présent invention will desirably be administered in combination with one ormore antibiotic, antifungal, antiprotozoal, antiviral or similar therapeutic agents. Further, theactive ingrédient of the présent invention may be administered not only in combination withother NSAIDs, but in combination as well with inhibitors of other mediators of inflammation,comprising one or more members selected from the group consisting essentially of theclasses of such inhibitors and examples thereof which include, H, -receptor antagonists;kinin-Bi - and B2 -receptor antagonists; prostaglandin inhibitors such as PGD-, PGF- PGI2 -,and PGE-receptor antagonists; thromboxane A2 (TXA2-) inhibitors; 5- and 12-lipoxygenaseinhibitors; leukotriene LTC4 -, LTD4/LTE4 -, and LTB4 -inhibitors; PAF-receptor antagonists;gold in the form of an aurothio group together with various hydrophilic groups;immunosuppressive agents, e.g., cyclosporine, azathioprine, and methotrexate; anti-inflammatory glucocorticoids; penicillamine; hydroxychloroquine; anti-gout agents, e.g.,colchicine, xanthine oxidase inhibitors, e.g., allopurinol, and uricosuric agents, e.g.,probenecid, sulfinpyrazone, and benzbromarone. It is further provided that the anti-inflammatory agents of the présent invention are administered in combination withtherapeutic agents intended for the treatment of disease conditions, syndromes andsymptoms found in older dogs, comprising one or more members selected from the groupconsisting essentially of the therapeutic agents and conditions being treated which includecognitive therapeutics to counteract memory loss and impairment; anti-hypertensives andother cardiovascular drugs intended to offset the conséquences of atherosclerosis, includinghypertension, myocardial ischemia including angina, congestive heart failure, andmyocardial infarction, selected from diuretics, vasodilators such as hydralazine, β-adrenergicreceptor antagonists such as propranolol, angiotensin-II converting enzyme inhibitors (ACE-inhibitors) such as enalapril used to treat gériatrie dogs with mitral insufficiency, and enalapriialone and in combination with neutrat endopeptidase inhibitors, angiotensin II receptorantagonists such as losartan, renin inhibitors, calcium channel blockers such as nifedipine,sympatholytic agents such as methyldopa, a2-adrenergic agonists such as clonidine, a-adrenergic receptor antagonists such as prazosin, and HMG-CoA-reductase inhibitors (anti-hypercholesterolemics) such as lovastatin or atorvastatin; antineoplastic agents, especiallyantimitotic drugs including the vinca alkaloids such as Vinblastine and vincristine; growth hormone secretagogues; strong analgésies; local and systemic anesthetics; and H2 - receptor antagonists and other gastroprotective agents. It is stiil further provided that the above combinations of therapeutic agents are used to treat acute conditions in dogs, -22- 011213 irîcluding bacterial infections occurring simultaneously with degenerative joint disease; and totreat chronic conditions in dogs, wherein the regimen used for this purpose comprisesadministration of the anti-inflammatory agents of the présent invention in combination withother médications used on a regularly scheduled basis for treating chronic conditionsincluding osteoarthritis; formulation of the anti-infiammatory agents of the présent inventionwith one or more other therapeutic agents which are to form the intended combination, into aconvenient dosage form containing ail of the drugs forming the combination, includingwherein said different drugs hâve varying half-lives, by creating controlled-release forms ofsaid drugs with different release times which achieves relatively uniform dosing; a medicatedfeed dosage form in which said drugs used in the combination are présent together inadmixture in said feed composition. There is further provided in accordance with the présentinvention co-administration in which the combination of drugs is achieved by thesimultaneous administration of said drugs to be given in combination; including co-administration by means of different dosage forms and routes of administration; the use ofcombinations in accordance with different but regular and continuous dosing scheduleswhereby desired plasma levels of said drugs involved are maintained in the dog beingtreated, even though the individual drugs making up said combination are not beingadministered to said dog simultaneously.

It is also contemplated that in accordance with the présent invention there will alsobe provided a package suitable for use in commerce for the therapeutic treatment orprévention of pain and inflammation processes and diseases in a member of the speciesCanis familiaris in need of such treatment, comprising a suitable container which may be inthe form of an outer package and an inner container removably housed therein; enclosed insaid container a suitable dosage form of an active ingrédient comprising a sélective inhibitor•of COX-2 wherein the selectivity ratio of COX-2 to COX-1 is at least 3 : 1 based on ex vivoinhibition levels in whole blood measured at a dose giving > 80% COX-2 inhibition,preferably > 90% COX-2 inhibition, as described elsewhere herein; and associated with saidcontainer printed instructional and informational material, which may be attached to saidcontainer, enclosed in said container, or displayed as an intégral part of said container, saidinstructional and informational material stating in words which convey to a reader thereofthat said active ingrédient, when administered to a dog to be treated, effectively inhibitsCOX-2 induced at an existing or expected site of pain and inflammation in said dog, whereinthe selectivity ratio of COX-2 to COX-1 is at least 3 : 1 based on ex vivo inhibition levels inwhole blood measured at a dose giving > 80% COX-2 inhibition, thereby treating or preventing said pain and inflammation which would otherwise resuit therefrom. In apreferred aspect of said package suitable for use in commerce, said instructional and -23- 011213 informational material wilt State in words which convey to a reader thereof that said activeingrédient when administered to a dog to be treated will provide effectively complétéinhibition of induced COX-2 in said dog, thereby treating or preventing pain and inflammationtherein; but also that when said ingrédient is thus administered to said dog it will causesubstantially no inhibition of constitutive COX-1 in said dog, whereby undesirablegastrointestinal and other adverse effects resulting from substantial inhibition of said COX-1wilt be avoided in effectively most dogs.

It is also contemplated that in accordance with the présent invention there will furtherbe provided a package of the type described immediateiy above, comprising a suitablecontainer as described; enclosed in said container an oral dosage form of a compound ofFormula (I), which may be in the form of a chewable or ingestible oral tablet, a unit dosepacket referred to as a sachet, a suspension made from a unit dose packet, a powder fororal suspension, or an oral suspension perse, which does not exhibit an adverse food effect;and associated with said container printed instructional and informational material asdescribed, which is free of any express or implied limitation with respect to whether said oraldosage form can be taken with or without food.

DETAILED DESCRIPTION OF THE INVENTION

The object of the présent invention is to find a solution to a serious probiem whichhas confronted the veterinary field for décades with regard to the need for an effective butsafe anti-inflammatory treatment for dogs. The seriousness and intractability of this probiemarises from the fact that virtually ail anti-inflammatory agents, especially the NSAIDs, whichhâve been tested for use in dogs, hâve had altogether unacceptable and sometimesdangerous adverse reactions in dogs which hâve greatly curtailed their use. By far the mostwide-spread and threatening of these adverse reactions is disturbance and irritation of thegastrointestinal mucosa leading to ulcération, hemorrhage and eventually perforation andperitonitis. These undesirable adverse reactions are mediated by the inhibition of variousprostaglandine by the NSAID inhibitors, resulting in a restricted blood supply to the protectivegastrointestinal muscosa, which in tum is seriously diminished both in total mass and inprotective functioning.

Gastric damage by NSAIDs is brought about by at least two distinct mechanisms.Local irritation by orally administered NSAIDs permits back diffusion of acid into the gastricmucosa, inducing tissue damage. Parentéral administration, on the other hand, can alsocause damage and bleeding, which has been correlated with inhibition of the biosynthesis ofgastric prostaglandins that serve a cytoprotective fonction in the gastric mucosa. Theseprostaglandins, especially PGI2 and PGE2, inhibit acid sécrétion by the stomach, increase -24- 011213 mucosal blood flow, and promote the sécrétion of cytoprotective mucus in the intestine.Other undesirable side effects of the NSAIDs include disturbances in platelet function, theprolongation of gestation or spontaneous labor, changes in rénal function, andhypersensitivity reactions. 5 Ail of the above-described undesirable side effects of NSAIDs probably dépend upon blockade of the synthesis of endogenous prostaglandins. Accordingly, there issignificant interest in the discovery of NSAIDs which do not resuit in the systemic productionof such undesirable side effects. Dogs are not only especially vulnérable to inflammatoryprocesses and diseases, such as degenerative joint disease, but they are also particularly 10 ·. susceptible to complications from the adverse gastrointestinal reactions which ensue.

As used herein, the term “dog(s)" dénotés any member of the species Canis familiaris , of which there are a large number of different breeds. While laboratorydéterminations of biological activity may hâve been carried out using a particular breed, it iscontemptated that the inhibitory compounds of the présent invention will be found to be 15 useful for treating pain and inflammation in any of these numerous breeds.

In its broadest aspects, the gist of the présent invention is the surprising discovery that a small genus of anti-inflammatory agents, of which carprofen, 6-chloro-a-methyl-9H-carbazole-2-acetic acid, is the best example, possesses a high degree of canine cyclo-oxygenase-2 (COX-2) selectivity, and that this selectivity is unique among the large class of 20 carboxyl- and carboxy(C-, -C4)alkyl aryl and/or heteroaryl anti-inflammatory agents to whichthe carprofen genus of compounds also belongs. This unique and unexpected selectivity indogs has far-reaching implications for the safe and effective treatment of dogs suffering fromany one of a number of inflammatory processes and diseases. lt is now reasonabiy well accepted in the art of treating inflammatory processes and 25 diseases, especially in dogs, that the cascade of endogenous reactions which producesvarious prostaglandin compounds in the body, beginning with arachidonic acid, is carriedforward by an essential enzyme called cyclo-oxygenase. It has been established that thisenzyme exists in two isozyme forms which are separate and act independently, aconstitutive cyclo-oxygenase-1 (COX-1) isozyme and a cyclo-oxygenase-2 (COX-2) 30 isozyme. The COX-1 isozyme in dogs is responsible for producing prostaglandins whichperform important functions in the stomach, intestine, kidney and blood platelets, some ofwhich are protective in nature, especially with respect to the gastrointestinal mucosa. TheCOX-2 isozyme in dogs is responsible for producing prostaglandins such as PGE2 whichmédiate acute and chronic inflammation within neutrophils, macrophages, endothélial cells 35 and fibroblasts, in which the COX-2 gene is expressed. COX-2 can be induced byendotoxin, lipopolysaccharide (LPS), various cytokines, e.g., IL-1 and TNF, growth factors, -25- 011213 ë.g., EGF and PDGF, and many other agents. For example, the COX-2 isozyme can bedetected by immunoblotting in mononuclear cells of the pleural exudate after carrageenan-induced pleurisy in a rat model.

Ultimately, the objective in the art has been to discover compounds which are ableto inhibit the activity of the COX-2 isozyme in dogs on effectively a 100% basis, while at thesame time inhibiting the activity of the COX-1 isozyme on effectively a 0% basis. As apracticai matter, this has amounted to a search for compounds which selectively inhibit theCOX-2 isozyme, /.e., compounds which provide optimal anti-infiammatory therapy throughrelatively high ievels of COX-2 isozyme inhibition at a dose which also produces minimalundesirabie side effects through relatively low Ievels of COX-1 isozyme inhibition. Until thediscovery of the présent invention described herein, the only compounds which hâve beenshown to exhibit COX-2 selectivity are those having a sulfonyl or sulfonamido group, ratherthan a carboxyl group, as is characteristic of the vast majority of ciassical NSAIDs. Theseobservations hâve led to spéculation that the Arg 150 residue of the COX-1 isozyme is notan essential binding site for sélective inhibitors of COX-2, whereas the Arg 150 residue isessential for COX-1 activity because it binds the terminal carboxyl group of arachidonic acidand analogously, the carboxylic acid fonction of the above-referred to ciassical NSAIDs. Itwould be expected that COX-2 sélective inhibitors would bind to some unique feature of thecanine COX-2 isozyme, rather than to a feature that was common to the canine COX-1 andCOX-2 isozymes.

Accordingly, the présent invention provides a method of selectively inhibitinginducible cyclo-oxygenase-2 (COX-2) with respect to inhibition of corresponding constitutivecyclo-oxygenase-1 (COX-1), and thereby treating or preventing pain and inflammatoryprocesses and diseases associated therewith in a member of the species Canis familiaris inneed of such treatment, wherein the selectivity ratio of COX-2 to COX-1 is at least 3 : 1based on in vivo and ex vivo inhibition Ievels in whole blood measured at the dose or rangeof doses giving > 90% COX-2 inhibition, comprising administering to said member anamount therapeutically effective for treating pain and inflammation in accordance with theabove-recited limitations, of an anti-infiammatory compound.

The présent invention forther provides the above-described method of treating orpreventing pain and inflammatory processes and diseases in a member of the species Canisfamiliaris wherein said anti-infiammatory sélective COX-2 inhibitor comprises a compound ofthe formula: -26- 0 Ί1213

wherein: R2 is

O

II

C-A 10 15 20 25 n where A is hydroxy, (C, - C4)alkoxy, amino, hydroxy-amino, mono-fC, -C2)alkylamino, di-(C.|-C2)alkylamino; X and Y are independently H or (C-, - C2)alkyI; and n is 1 or 2; R® is halogen, (C, - C3)aikyl, trifluoromethyl, or nitro; R9 is H; (C! - C2)alkyl; phenyl or phenyl-(C, - C2)alkyl, where phenyl is optionailymono-substituted by fluoro or chloro; -C(=O)-R, where R is (C, - C2)alkyl or phenyl,optionaily mono-substituted by fluoro or chloro; or -C(=O)-O-R1, where R1 is (C5 - C2)alkyl; where X and Y are different, the {-)(R) and (+)(S) enantiomers thereof; and ailpharmaceutically acceptable sait forms, prodrugs and métabolites thereof which aretherapeutically active for treating or preventing pain and inflammation. Where the inhibitor ofFormula (I) existe as (-)(R) and (+)(S) enantiomers, in accordance with the présent inventionthere is provided the (+)(S) enantiomer alone, or where both enantiomers are présenttogether, there is provided a racemic or a non-racemic mixture thereof.

It is quite surprising that the preferred carprofen genus of compounds, characterizedby an α-methyl-acetic acid functionality, has many times greater canine COX-2 selectivitythan any of the carboxyl-containing classical NSAIDs, as well as many times greater canineCOX-2 selectivity than some of the sulfonyl and sulfonamide-containing NSAIDs accepted inthe art as being highly COX-2 sélective compounds. This aspect of the présent invention isembodied in a method of treating or preventing inflammatory processes and diseases in adog comprising administering to them an anti-inflammatory therapeutically effective amountof an inhibitor of canine cyclo-oxygenase-2 (COX-2) for which in vitro ICso potency in saiddog is at least 50 fold greater than canine cyclo-oxygenase-1 (COX-1) in vitro ICso potencyin said dog; wherein said inhibitor is a member selected from the group of anti-inflammatorycompounds consisting essentially of salicylic acid dérivatives; p-aminophenol dérivatives;indole and indene acetic acids; heteroaryl acetic acids; arylpropionic acids; anthranilic acids;enolic acids; and alkanones; and in particular is a member selected from the group 30 -27- 011213 consisting of carboxyl- and carboxyfC, -C4)alkyl aryl and/or heteroaryl anti-inflammatoryagents. In particular, said inhibitor is a member selected from the group consisting of thearyl propionic acid class of non-steroidal anti-inflammatory drugs.

Carprofen and the genus of carprofen dérivatives preferably utilized in the methodsand compositions of the présent invention may be prepared in accordance with methods ofsynthesis well known to the organic chemist of ordinary skill. For example, compounds ofFormula (I) where R6is halogen, (C, - C3)alkyl, trifluoromethyl, or nitro; and where R9is H ormethyl; may be prepared by reacting (1) a phenylhydrazine in which the phenyl portion hasthe desired R6 substitution and the α-nitrogen of the hydrazine has the desired R9substitution; with (2) a cyclohexanone having the desired R2 substitution. The resulting1,2,3,4-tetrahydrocarbazole is then aromatized to produce the desired carbazole of Formula (1) . The aromatization may be carried out using (1) an aromatizing agent, e.g., p-chloranil, o-chloranil, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), sulfur, palladium on carbon, or leadoxide; in the presence of (2) a solvent such as xylene, benzene, toluene, quinoline,dimethylsulfoxide (DMSO), and dimethylformamide (DMF); (3) at a température in the rangefrom room température to reflux of the reaction mixture, preferably the latter.

Compounds of Formula (I) which are acids, i.e., where A is hydroxy, and salts ofsuch acids with bases, can be converted to amides of Formula (I) where A is amino,hydroxyamino, mono-(C1 -C2)alkyiamino, and di-(C5 -C2)alkylamino; by (1) forming thecorresponding acid chloride by treatment with phosphores pentachloride (PCI5); followed by (2) reaction with the appropriate amine reactant to form the desired amide, carried out in thepresence of an équivalent of pyridine or triethylamine to serve as the base for the protontransfer step and thereby remove the Η+ΟΓ which is formed. The same acid chlorides whichare formed in step (1) can be reacted with the appropriate alkanol to provide the esters ofFormula (I) where A Is (C, -C4)alkôxy. This reaction is also desirably carried out in thepresence of a base such as pyridine which can neutralize the H+CF being formed so that itdoes not interfère with any acid sensitive alkanol reactant.

The above-described synthetic approaches to préparation of the preferred carprofengenus of compounds utilized in the methods and compositions of the présent invention aredescribed in detail in U.S. Patent No. 3,896,145, which is incorporated herein by référencé inits entirety.

When "X" and Ύ" are different in the définition of the “R2" substituent, then a chiral (asymmetric) carbon atom exists. A racemic mixture of (R)- and (S)-enantiomers results when there is a 50:50 mixture of the two enantiomers. In accordance with the présent invention it has been discovered that the (S)-enantiomer of the preferred carprofen genus of compounds of Formula (I) having a chiral carbon is the enantiomer which possesses the -28- 011213 sûrprising degree of unexpected canine COX-2 selectivity, a unique biological activity notpossessed by virtuaily any of the classical NSAlDs having a carboxylic acid moiety, andespecially not possessed by their S-enantiomers. Therefore, the (S)-enantiomer of thepreferred carprofen genus of compounds of Formula (I) having a chiral carbon wouldpossess a surprisingly reduced level of adverse gastrointestinal and other reactions in dogscompared to that of virtuaily ali other NSAlDs having a carboxylic acid moiety, and especiallycompared to their S-enantiomers. Thus, it would also be wholly unexpected that the S-enantiomer of carprofen would hâve, by reason of its being a very highly sélective inhibitor ofthe canine COX-2 isozyme, a surprisingly improved level of anti-inflammatory, analgésie andanti-pyretic activity in dogs compared to that of virtuaily ail other NSAlDs characterized by acarboxylic acid moiety.

One especially preferred embodiment of the présent invention is to use only the (S)-enantiomer of carprofen, 6-chloro-a-methyl-9H-carbazoie-2-acetic acid, as the activeingrédient or treating agent in the methods and compositions of the présent invention.However, other embodiments are contemplated to be within the scope of this preferredgenus of the présent invention as well. For example, non-racemic mixtures of the {R}- and(S)-enantiomers can be used, and in that event the (S)-enantiomer is présent in amount of atleast 85%, preferably at least 90%, more preferably at least 95%, and most preferably atleast 99%. Since the (R)- and (S)-enantiomers are identical in molecular weight, density,etc., it is unnecessary to State any basis for the above-recited percentages. In other words,they could be percentages by weight, volume, Chemical equivalency, etc. The reason forincluding the above-indicated amounts of the (R)-enantiomer may be as simple as thepracticalities of not being required to remove absolutely every last trace of the (R)-enantiomer from the racemic mixture. There can also be reasons for doing so which relateto bénéficiai overall biological properties.

It will also be appreciated by those in the art that the ranges of dosage amountsrecited elsewhere herein for the preferred genus of carprofen compounds are beingdescribed with respect to a 50:50 racemic mixture of enantiomers, where a chiral compoundis involved. This has been done largely as a matter of convenience. Where the activeingrédient being used as a therapeutic agent comprises a mixture of enantiomers differentfrom a 50:50 mixture, or where the therapeutic agent comprises substantially 100% of the(+)(S) or (-)(R) enantiomer alone, the person of ordinary skill in this art will be able tocalculate the actual amount of dosage required in a very straightforward manner, simply bymultiplying the dosage amounts recited by a factor which reflects the ratio of the amount ofenantiomer being used to the amount présent for the recited dosage based on a 50:50mixture of the enantiomers. Accordingly, where the recited dosage is 4mg/kg/day for the -29- 011213 50:50 racemic mixture, the corresponding dosage amount when substantially 100% of (+)(S)enantiomer is used one-half of the recited amount, i.e., 2mg/kg/day.

Since the pharmaceutical compositions of the présent invention containing amember of the preferred genus of carprofen compounds contemplate the use of racemicmixtures containing 50% of (S)-enantiomer, as weli as non-racemic mixtures of about 99%or less of the (S)-enantiomer along with less than 50% of the (R)-enantiomer, résolution ofracemates of the carprofen genus of compounds of Formula (I) having a chiral carbon intothe optically active isomers must be carried out. This can be readily accomplished usingknown procedures and techniques in the art. For example, some racemic mixtures can beprecipitated as eutectics after which they can be separated. However, it is usually preferredto use Chemical procedures for resolution, in accordance with which diastereomers areformed from the racemic mixture with an optically active resolving agent. For example, anoptically active base, e.g., D-a-methylbenzy lamine, which can be reacted with the carboxylgroup. The diastereomers thus formed are then separated by sélective crystallization andconverted to the comesponding optical isomer.

Included within the scope of the présent invention are ail of the anti-infiammatorytherapeutically active and phamnaceutically acceptable sait forms, prodrugs and métabolitesof the preferred carprofen genus of compounds used in the présent invention. Thisespecially includes acid addition salts thereof, where “A" is defined as anything other than“hydroxy", formed by treating the compounds of Formula (I) with pharmaceuticallyacceptable organic and inorganic acids, e.g., hydrohalides such as hydrochloride,hydrobromide, hydroiodide; other minerai acids and their corresponding salts such assulfate, nitrate, phosphate, etc.; and alkyl- and mono-arylsulfonates such asethanesulfonate, toluenesulfonate, and benzenesulfonate; and other organic acids and theircorresponding salts such as acetate, tartrate, maleate, succinate, citrate, benzoate,salicylate, ascorbate, etc.

Where “A" is defined as “hydroxy” in the preferred carprofen genus of compoundsused in the présent invention, salts thereof may be formed by treatment withpharmaceutically acceptable bases. Examples of such bases are alkali métal hydroxidesincluding potassium hydroxide, sodium hydroxide, and lithium hydroxide; alkaline earth métalhydroxides such as barium hydroxide and calcium hydroxide; alkali métal alkoxides, e.g.,potassium ethanolate and sodium propanolate; and various organic bases such aspiperidine, diethanoiamine, and W-methylglutamine. Also included are the aluminum salts ofthe compounds of Formula (I).

In addition to the use of the various above-described sait forms of the compounds ofFormula (I), there is included within the scope of the présent invention the use as active -30- 011213 ingrédients of ali analgésie and anti-inflammatory therapeutically active and phamnaceuticallyacceptable prodrugs and métabolites of the above-recited compounds. In particular, thisincludes those dérivatives where R9 is defined as (C, - C2)alkyl, especially methyl; phenyl orphenyl-fC, -C2)alkyl, especially benzyl, where phenyl is optionally mono-substituted by fluoroor chloro, especially 4-fluoro-phenyl; -C(=O)-R, where R is (C! -C2)alkyl or phenyl, especiallyacetyl and benzoyl, where phenyl is optionally mono-substituted by fluoro or chloro; or-C(=O)-O-R1 , where R1 is (C4 -C2)alkyI, especially acetyloxy. These N-moieties are readilycieaved during metabolism of the compound of Formula (I), making these particulardérivatives désirable prodrugs.

The présent invention has been described in the paragraphs immediately aboveparticularly with regard to the preferred genus of carprofen compounds of Formula (I).However, the présent invention contemplâtes a wider scope with regard to providing anti-inflammatory sélective COX-2 inhibitors. As already indicated, such sélective COX-2inhibitors comprise those wherein the selectivity ratio of COX-2 to COX-1 is at least 3 : 1based on in vivo and ex vivo inhibition levels in whole blood measured at the dose or rangeof doses giving > 80% COX-2 inhibition. In accordance with pharmacokinetic (PK) datadescribed further below, it has been demonstrated that a relationship exists between thepharmacological or toxic response in dogs to the anti-inflammatory sélective COX-2inhibitors of the présent invention and the accessible concentration of said inhibitors, e.g., inthe blood or plasma of said dogs. For purposes of the instant exposition of the présentinvention, said pharmacological response in dogs is regarded herein as the anti-inflammatory response resulting from inhibition of the COX-2 isozyme; while said toxicresponse in dogs is regarded herein as the undesirable side effect, e.g., gastrointestinal,response resulting from inhibition of the COX-1 isozyme. Thus, the concentration ofinhibitory drug in the systemic circulation of said dogs will be related to the concentration ofinhibitory drug at its sites of action.

As is well known, the most important pharmacokinetic parameters are bioavailability,the fraction of said drug which is absorbed into the systemic circulation; volume ofdistribution, relating to the amount of space in the body which is available to contain saiddrug; and clearance, relating to the body’s ability to eliminate a particular drug. Inaccordance with the présent invention, the selectivity ratio for COX-2 to COX-1, which mustbe at least 3 ; 1, is determined on the basis of ex vivo measurement of the percentage (%)inhibition of each of said isozymes in canine whole blood. The procedures for making saidex vivo measurements may be briefly summarized as consisting of first administering thesélective inhibitor test compound at a predetermined total dose level, e.g., 2 mg/lb topreselected dogs. The dose is administered in accordance with a predetermined dosing -3 Ι- ΟΙ 1 21 3 rêgimen, e.g., once a day (s./.d.), twice a day (b.i.d.), etc., after which whole blood samplesare collected from each test animal, Assays for COX-1 and COX-2 activity are based onstimulation of said blood with either a calcium ionophore for thromboxane B2 (COX-1 activity)or lipopolysaccharide (LPS) for prostaglandin E2 (COX-2 activity), respectively. Theseprocedures are described in more detail in the further below exemplary descriptions. COX-2 selectivity is an essential feature of the anti-inflammatory inhibitors useful inthe présent invention, and is required to be in a ratio of at least 3 :1 compared to inhibition ofCOX-1 activity. It is insufficient for a given compound simpiy to possess a 3 : 1 sélectiveratio, since a given compound might possess the required sélective ratio at some point overits total dosage range, but still fail to provide adéquate inhibition of COX-2 activity overall.Accordingly, it is additionally required that for an anti-inflammatory inhibitor to be within thescope of the présent invention it must also provide > 80% COX-2 activity inhibition, andpreferably > 90% COX-2 activity inhibition. This is deemed to be the minimum level ofinhibition adéquate to provide satisfactory pharmacological activity in terms of treating andpreventing pain and inflammation.

It is further insufficient for a given compound simpiy to possess a 3:1 sélective ratioat a given dose and to provide > 80% COX-2 activity inhibition, preferably > 90% COX-2activity inhibition at some other dose. But rather, both criteria must be met at some level ofexposure and at the same time, post-dose. Accordingly, it is also required that the 3 : 1sélective ratio occur at the dosage concentration or range of dosage concentrations whichalso provides > 80% COX-2 activity inhibition.

In order to demonstrate the manner in which the above-described limitations wouldbe applied to a potential sélective COX-2 inhibitor, the following example is to be considered.Said candidate is evaluated in accordance with the above-described procedures formeasuring COX-1 and COX-2 activity inhibition in whole blood samples at a total dose of 2mg/lb administered s./'.d., with activity levels being determined at 2, 4 and 12 hours afteradministration. When data points for COX-1, COX-2 and untreated samples are plotted on agraph of % inhibition vs. time, it is found that the COX-1 curve drops fairly steeply from a %inhibition at 2 hrs of 10 -15% to no observed inhibition at 12 hrs. The COX-2 curve, on theother hand, is found to drop only slightly during this same period of time, declining from a %inhibition at 2 hrs of about 95% to a % inhibition at 12 hrs of about 90%.

Accordingly, it should be noted that at the dose of 2 mg/lb administered s.i.d. the potential inhibitor has satisfied (1) the at least 3:1 selectivity ratio, and (2) the > 80% COX-2 inhibition limitations required for it to be an anti-inflammatory sélective COX-2 inhibitor falling within the scope of the présent invention. Because the data from both of these graphs has been compared over the same dose (2 mg/lb) and the same time period of 12 hrs, the third -32- limitation has also been satisfied, namely that the first two limitations be satisfied at the samedose or range of doses.

Similar déterminations can be made and graphs plotted of the resulting data for anumber of different dosage amounts of said candidate compound. However, the combinedresults are demonstrated by means of a graph which plots % inhibition of COX-1 and COX-2vs. dose or plasma concentration, rather than time. The curve representing the COX-2 dataappears first and rises steeply, starting at the least dosage concentration which produces anidentifiable % inhibition, and as the dosage approaches higher concentrations, reachinginhibition levels à 80% and becoming asymptotic thereafter. The curve representing theCOX-1 data appears later and rises less steeply than the curve of COX-2 data, becomingasymptotic at a significantly lower level of % inhibition as well. A candidate compound fallswithin the scope of the présent invention if a line parallel to the y-axis and intercepting bothcurves at some dosage concentration results in data points which satisfÿ both the 3 :1 ratioand > 80% inhibition limitations.

The scope of the présent invention with respect to those anti-inflammatory sélectiveCOX-2 inhibitors which are included therein can be viewed or expressed in different,although essentially équivalent terms to those above-used. For example, the présentinvention may be considered to provide particularly a method of treating or preventing painand infiammatory processes and diseases in a member of the species Canis familiaris inneed of such treatment, which comprises administering to said member a therapeuticallyeffective amount for treating pain and inflammation, of an anti-inflammatory inhibitor of cyclo-oxygenase-2 (COX-2) for which in vitro ICS0 potency in said member is at least 30 foldgreater, preferably at least 40 fold greater, more preferably at least 50 fold greater, morepreferably still at least 60 fold greater, even more preferably at least 80 fold greater, andmost preferably at least 100 fold greater than cyclo-oxygenase-1 (COX-1) in vitro ICr,potency in said member, wherein said inhibitor is a member selected from the group of anti-inflammatory compounds consisting essentially of salicyiic acid dérivatives; p-aminophenoldérivatives; indole and indene acetic acids; heteroaryl acetic acids; arylpropionic acids;anthranilic acids; enolic acids; and alkanones.

In accordance with the above-described methods of the présent invention, there ismore particularly provided the above-described method of treating or preventing pain andinfiammatory processes and diseases wherein said cyclo-oxygenase-2 (COX-2) in vitro IC^potency of the inhibitory compound of Formula (I) is at least 100 fold greater than said cyclo-oxygenase-1 (COX-1) in vitro IC50 potency thereof; wherein one of X and Y is H and theother is methyl; and wherein when both resulting enantiomers are présent together, (+)(S)enantiomer is présent in amount of at least 75%. In particular, there is provided the above- -33- 011213 tiescribed method wherein for Formula (I), for R2, n = 1, one of X and Y is H and the otheris methyl, and A is hydroxy, (C, - C2)alkoxy, or amino; R6 is halo, especially chloro, ortrifluoromethyl; and R9 is H, methyl, acetyl, benzoyl, or methoxycarbonyl; and wherein whenboth resulting enantiomers are présent togefher, (+)(S) enantiomer is présent in amount of atleast 85%, preferably at least 90%, more preferably at least 95%, and most preferably atleast 99%.

There is still further provided the above-described methods in which said inhibitorcomprises 6-chloro-a-methyl-9H-carbazole-2-acetic acid; and wherein when both resultingenantiomers are présent together, (+)(S) enantiomer is présent in amount of at least 85%,preferably at least 90%, more preferably at least 95%, and most preferably at least 99%. Inparticular, there is provided the above-described method in which said inhibitor is comprisedentirely of (+)(S)-enantiomer of 6-chloro-a-methyl-9H-carbazole-2-acetic acid.

The présent invention may also be described as providing a method of selectivelyinhibiting substantially only inducible cyclo-oxygenase-2 (COX-2), with substantially noinhibition of corresponding constitutive cyclo-oxygenase-1 (COX-1), and thereby treating orpreventing pain and infiammatory processes and diseases associated therewith in a memberof the species Canis familiaris in need of such treatment, comprising administering to saidmember a therapeutically effective amount for treating pain and inflammation, of an anti-inflammatory compound of Formula (I) above where R2 , X , Y , n , A , R6, and R9 are asdefined; including the (-)(R) and (+)(S) enantiomers; and ail anti-inflammatory therapeuticallyactive and pharmaceutically acceptable sait forms, prodrugs and métabolites of the above-recited compounds. Here again, where the inhibitor of Formula (I) exists as (-)(R) and (+)(S)enantiomers, in accordance with the présent invention there is provided the (+)(S)enantiomer alone, or where both enantiomers are présent together, there is provided aracemic or a non-racemic mixture thereof.

In a manner essentially parallel to the other above-descriptions thereof, the présentinvention may be further described as including the above-recited method of selectivelyinhibiting substantially only inducible cyclo-oxygenase-2 (COX-2), wherein said inhibitorcomprises 6-chloro-a-methyl-9H-carbazole-2-acetic acid; and wherein when both resultingenantiomers are présent, (+)(S) enantiomer is présent in amount of at least 85%, preferablyat least 90%, more preferably at least 95%, and most preferably at least 99%. In particular,there is provided the above-described method in which said inhibitor is comprised entirely of(+)(S)-enantiomer of 6-chloro-a-methyl-9H-carbazole-2-acetic acid.

When the compounds of Formula (I), or their enantiomers or salts, are to be used as active ingrédients in the methods and compositions of the présent invention, they can be incorporated into standard pharmaceutical dosage forms. For example, they are useful -34- 011213 when administered in systemic or local, oral or parentéral applications and for this purposeare combined with the usual pharmaceutical excipients, diluents and adjuvants, e.g., organicand inorganic inert carrier materials such as water, gelatin, lactose, starch, magnésiumstéarate, talc, vegetable oils, gums, polyalkyleneglycols, etc. These pharmaceuticalpréparations can be employed in a solid form, e.g., as tablets, troches, suppositories,capsules, and especially in combination with or for admixture with a palatable food itemsuitable for dogs; or they can be administered in liquid form, e.g., as solutions, suspensions,standard and inverse émulsions, and élixirs. Pharmaceutical excipients and adjuvants whichcan be added include preservatives, antioxidants, antimicrobial agents and other stabilizers;wetting, emulsifying, and suspending agents, and anticaking compounds; fragrance andcoloring additives; compositions for improving compressibility, or to create a delayed-,sustained-, or controlled-release of the active ingrédient; and various salts to change theosmotic pressure of the pharmaceutical préparation or to act as buffers. Particular dosageforms which hâve been used with success include a 5% mixed-micelle solution of carprofenfor intravenous injection, a 3% palatable paste, and oral tablets in 25 mg, 75 mg, and 100mg dosages.

In the methods and compositions of the présent invention, especially those whereinthe inhibitor comprises 6-chloro-a-methyl-9H-carbazole-2-acetic acid and both resultingenantiomers are présent together, it is a preferred embodiment to use a non-racemicmixture. Particularly, in such preferred non-racemic mixtures, it is désirable to hâve the(+)(S) enantiomer présent in amount of at least 85%, preferably at least 90%, morepreferably at least 95%, and most preferably at least 99%. Thus, in such non-racemicmixtures the (+)(S) enantiomer will be the prédominant component, not only because it issignificantly more potentthan the (-)(R) enantiomer in inhibiting cyclo-oxygenase-2 (COX-2),but also because it is highly sélective with respect to inhibiting cyclo-oxygenase-2 (COX-2)as compared to cyclo-oxygenase-1 (COX-1). The correspondingly smaller amounts of the (-)(R)'enantiomer, i.e., less than 15%, less than 10% and less than 5%, respectively, areoptionally included where a balance of cyclo-oxygenase or other enzyme inhibitoryproperties is deemed désirable. Where the amount of (-)(R) enantiomer présent is less than5% and less than 1%, the reason for the inclusion will usually reflect the practïcalities of themethod used to résolve the enantiomers. Where this method is time consuming ordemanding of resources, it will often be désirable, from a practical standpoint, to simply allowthis smaller proportion of the (-){R) enantiomer to be carried over into the final, non-racemicmixture final producL

The anti-inflammatory inhibitors of Formula (I) of the présent invention may be administered systemically to a dog to be treated as a pharmaceutical composition in suitable -35- 011213 liquid form by injection or infusion. There are a number of sites and organ Systems in thebody of the dog which will allow the properly formutated pharmaceutical composition, onceinjected or infused, to permeate the entire body and ail of the organ System of the dog beingtreated. An injection is a single dose of the pharmaceutical composition forced, usually by asyringe, into the tissue involved. The most common types of injections are intramuscular,intravenous, and subcutaneous. By contrast, an infusion is the graduai introduction of thepharmaceutical composition into the tissue involved. The most common type of infusion isintravenous. Other types of injection or infusion comprise intraarterial, intra- or transdermal(including subcutaneous), or intraspinal especially intrathecal. In these liquid pharmaceuticalcompositions, the anti-inflammatory inhibitor may be contained in solution as the soluté.This is the most common and most preferred type of such composition, but requires aninhibitor in a sait form that has reasonably good aqueous solubility. Water (or saline) is byfar the most preferred solvent for such compositions. Occasionally supersaturated solutionsmay be utilized, but these présent stability problems that make them impractical for use onan everyday basis.

If it is not possible to obtain a form of some compound of Formula (I) that has therequisite degree of aqueous solubility, as may sometimes occur, it is within the skill of theartisan to préparé an émulsion, which is a dispersion of small globules of one liquid, thediscontinuous or internai phase, throughout a second liquid, the continuous or extemalphase, with which it is immiscible. The two liquids are maintained in an emulsified State bythe use of emulsifiers which are pharmaceutically acceptable. Thus, if the anti-inflammatoryinhibitor is a water-insoluble oil, it can be administered in an émulsion of which it is thediscontinuous phase. Also where the inhibitor is water-insoluble but can be dissolved in asolvent which is immiscible with water, an émulsion can be used. While the inhibitor wouldmost commoniy be used as the discontinuous or internai phase of what is referred to as anoil-in-water émulsion, it could also be used as the discontinuous or internai phase of aninverse émulsion, which is commoniy refemed to as a water-in-oil émulsion. Here the anti-inflammatory inhibitor is soluble in water and could be administered as a simple aqueoussolution. However, inverse émulsions invert upon injection or infusion into an aqueousmedium such as the blood, and offer the advantage of providing a more rapid and efficientdispersion of the inhibitor into that aqueous medium than can be obtained using an aqueoussolution. Inverse émulsions are prepared by using suitable, pharmaceutically acceptableemulsifying agents well known in the art. Where the anti-inflammatory inhibitor has limitedwater solubility, it may also be administered as a suspended solid in colloïdal ormicroparticulate form in a suspension prepared using suitable, pharmaceutically acceptable -36- 011213 suspending agents. The suspended solids containing the inhibitor may also be formulatedas delayed-, sustained-, and/or controlled-release compositions.

While systemic administration will most frequently be carried out by injection orinfusion of a liquid, there are many situations in which it will be advantageous or evennecessary to deliver the anti-inflammatory inhibitor as a solid. Systemic administration ofsolids is carried out by instillation, inhalation or insufflation of a pharmaceutical compositionin suitable solid form containing the inhibitor. Instillation of the inhibitor may entail installing asolid implant composition into suitable body tissues or cavities. The implant may comprise amatrix of bio-compatible and bio-erodible materials in which particles of a solid inhibitor aredispersed, or in which, possibly, globules or isolated cells of a liquid inhibitor are entrapped.Desirably, the matrix will be broken down and completely absorbed by the body. Thecomposition of the matrix is also preferably selected to provide controlled-, sustained-,and/or delayed release of the inhibitor over extended periods of time, even as much asseveral months. A substantial number of the dosage forms described herein may be formulated so asto provide controlled-, sustained-, and/or delayed release of the active ingrédient from saiddosage form. In an especially preferred aspect of the pharmaceutical compositions of theprésent invention which provide delayed-, sustained-, and/or controlled-release of the anti-inflammatory sélective COX-2 inhibitor active ingrédient, there is included ail such orallyadministered dosage forms which produce > 80% inhibition of COX-2 isozyme activity andresuit in a plasma concentration of said inhibitor of at least 10 pg/mL for at least 4 hrs;preferably for at least 8 hrs; more preferably for at least 12 hrs; more preferably still for atleast 16 hrs; even more preferably still for at least 20 hrs; and most preferably for about 24hrs. Preferably, there is included the above-described dosage forms which produce > 80%inhibition of COX-2 isozyme activity and resuit in a plasma concentration of said inhibitor ofat least 15 pg/mL for at least 4 hrs, preferably for at least 8 hrs, more preferably for at least12'hrs, still more preferably for at least 20 hrs, and most preferably for about 24 hrs. Morepreferably, there is included the above-described dosage forms which produce > 90%inhibition of COX-2 isozyme activity and resuit in a plasma concentration of said inhibitor ofat least 20 pg/mL for at least 4 hrs, preferably for at least 8 hrs, more preferably for at least12 hrs, still more preferably for at least 20 hrs, and most preferably for about 24 hrs.

Accordingly, a useful controlled release dosage form of carprofen in accordance with

the présent invention is one which maintains a carprofen plasma level greater than 2 pg/mL for most of the day after a single oral dose at 2 mg/lb. Preferred oral controlled release dosage forms of carprofen in accordance with the présent invention are ones which maintain a plasma carprofen concentration greater than 10 pg/mL for a period of time greater than -37- 011213 that for which an immédiate release dosage form of carprofen maintains a comparableplasma level, when said immédiate release dosage form and controlled release dosage formare administered at the same dose, e.g. 2, 1.8, 1.6, or 1.4 mg/lb. For instance, preferred2mg/lb oral controlled release dosage forms of this invention maintain a plasma carprofenconcentration greater than 10 pg/mL for greater than 10.5 hrs.

Immédiate release carprofen dosage forms containing doses of 1.8, 1.6, and 1.4mg/lb maintain a plasma carprofen concentration above 10 pg/mL for 9,5 hrs, 8.5 hrs, and7.5 hrs, respectively. Preferred 1.8 mg/lb oral controlled release carprofen dosage formsmaintain a plasma carprofen concentration above 10 pg/mL for greater than 9.5 hrs.Likewise, the threshold durations for 1.6 mg/lb and 1.4 mg/lb doses are 8.5 hrs and 7.5 hrs,respectively. The performance characteristics for preferred oral controlled release carprofendosage forms at doses higher than 2 mg/lb or less than 1.4 mg/lb can be similarly calculated,assuming linear pharmacokinetics. More preferred oral controlled release carprofen dosageforms are those which maintain a plasma carprofen concentration greater than 10 pg/mL fora period of time greater than or equal to that observed when an immédiate release carprofendosage form is dosed at any higher dose. These controlled release dosage forms thusmaintain at least 80% COX-2 inhibition in canine blood for a period of time longer than thatachieved by an immédiate release dosage form at a higher dose.

Most preferred oral controlled release carprofen dosage forms are those which areable to maintain plasma carprofen levels above approximately 10 pg/mL for a period of timegreater than or equal to the time observed for an immédiate release 2 mg/lb carprofendosage form (10.5 hrs), when said oral controlled release carprofen dosage forms areadministered at a dose less than 2 mg/lb. The performance of a 2 mg/lb oral immédiaterelease dosage form is taken as the fondamental standard for purposes of this comparisonsince 2 mg/lb/day is the currently recommended and accepted efficacious oral dose inaccordance with the présent invention as described herein.

The term “implant” always dénotés a solid pharmaceutical composition containingthe anti-infiammatory inhibitor, while the term “depot” usually implies a liquid pharmaceuticalcomposition containing the anti-inflammatory inhibitor, which is deposited in any suitablebody tissues or cavities to form a réservoir or pool which slowly migrâtes to surroundingtissues and organs and eventually becomes systemically distributed. However, thesedistinctions are not always rigidly adhered to in the art, and consequently, it is contemplatedthat there is included within the scope of the présent invention liquid implants and soliddepots, and even mixed solid and liquid forms for each. Suppositories may be regarded asa type of implant, since they comprise bases which are solid at room température but melt atbody température, slowly releasing the active ingrédient with which they are impregnated -38- 011213

Into the surrounding tissue of the body, where the active ingrédient becomes absorbed andtransported to effect systemic administration.

Systemic administration can also be accomplished by inhalation or insufflation of apowder, i.e., particulate composition containing the inhibitor. For example, the inhibitor inpowder form may be inhaled into the lungs using conventional devices for aerosolizingparticulate formulations. The inhibitor as a particulate formulation may also be administeredby insufflation, i.e., blown or otherwise dispersed into suitable body tissues or cavities bysimple dusting or using conventional devices for aerosolizing particulate formulations. Theseparticulate compositions may also be formulated to provide delayed-, sustained-, and/orcontrolled-release of the anti-inflammatory inhibitor in accordance with well understoodprinciples and known materials.

Other means of systemic administration which may utilize the inhibitors of theprésent invention in either liquid or solid form include transdermal, intranasal, and opthalmicroutes. In particular, transdermal patches prepared in accordance with well known drugdelivery technology may be prepared and applied to the skin of a dog to be treated,whereafter the active agent by reason of its formulated soiubility characteristics migrâtesacross the epidermis and into the dermal layers of the dog’s skin where it is taken up as partof the general circulation of the dog, ultimately providing systemic distribution of the activeingrédient over a desired, extended period of time. Also included are implants which areplaced beneath the epidermal layer of the skin, i.e. between the epidermis and the dermis ofthe skin of the dog being treated. Such an implant will be formulated in accordance with wellknown principles and materials commonly used in this delivery technology, and may beprepared in such a way as to provide controlled-, sustained-, and/or delayed-release of theactive ingrédient into the systemic circulation of the dog. Such subepidermal (subcuticular)implants provide the same fecility of installation and delivery efficiency as transdermalpatches, but without the limitation of being subject to dégradation, damage or accidentairemoval as a conséquence of being exposed on the top layer of the dog’s skin.

Pharmaceutical compositions of spécial types suitable for oral administration to dogsmay also be devised. Pharmaceutical compositions suitable for pérorai administration, i.e.,ingestion by mouth or administration through the mouth, may be solid or liquid. Preferredpérorai dosage forms for systemic administration are solids, e.g., palatable oral compositionssuch as fast dissolving palatable wafers, tablets, capsules, caplets, lozenges, troches, etc.,and liquids, e.g., solutions, suspensions, émulsions, élixirs, tinctures, etc. Pharmaceuticalcompositions of spécial types suitable for oral administration to dogs may be used, andinclude, but are not limited to such items as an oral paste to be delivered to the back of thetongue of the dog being treated, a granular form to be delivered through incorporation in the -39- 01 1213 ‘dog’s food, and a chewable form wherein the active ingrédient is consumed along with thepalatabie chew, or a chewable form which may deliver the active ingrédient by leaching fromthe body of the chew which is not consumed, during mastication by the dog being treated.As is known in the art, the formulation of such palatabie compositions takes into accountcanine behavior regarding the extent of mastication of the dosage form which will take place,and the résultant level of dosing.

As with the other routes of administration and corresponding dosage formsdescribed herein, dosage forms intended for oral administration are also suitably formulatedto provide controlled-, sustained-, and/or delayed release of the active ingrédient. Typically,these would include delayed-reiease oral tablets, capsules and multiparticulates, as well asenteric-coated tablets and capsules which prevent release and absorption of the activeingrédient in the stomach of the dog and facilitate enteric delivery distal to the stomach, /'.e.,in the intestines of the dog. Other typical oral dosage forms would include sustained-releaseoral tablets, capsules, and multiparticulates which provide systemic delivery of the activeingrédient in a controlled manner over a prolonged period of time, e.g., a 24-hour period.Where rapid delivery of the active ingrédient is required or désirable, a controlled-releaseoral dosage form may be prepared in the form of a fast-dissolving tablet, which would alsopreferably include highly soluble sait forms of the active ingrédient.

The description herein of the dosage forms which are contemplated to be within thescope of the présent invention has, largely as a matter of convenience, dassified such formsinto those for local and systemic administration, as well as into solid and liquid forms.However, these distinctions are fairly arbitrary and should not be taken as in any way limitingthe scope of the présent invention with respect to routes of administration and dosage forms.For example, the description herein has already made it évident that some routes ofadministration, while ostensibly local, may also hâve systemic action or conséquences. Theline drawn herein between liquid and solid dosage forms may also be obscured in actualpractice. For example, a suitable oral dosage form for use in the présent invention includesencapsulated solutions, a mixed solid and liquid formulation. Microemulsion formulations,also within the scope of the présent invention, may also be characterized as a mixed solidand liquid dosage form.

The anti-inflammatory inhibitor can be administered locally to a site of inflammationin a dog to be treated. Local vs. systemic administration entails a more focused vs. a moregeneralized manner of delivering the anti-inflammatory-inhibitor-containing pharmaceuticalcomposition to the dog suffering from pain and inflammation. However, the use of depotsand implants as well as delayed-, sustained-, and controlled-release formulations has tendedto blur these distinctions. Accordingly, the above-described liquid and solid pharmaceutical -40- 011213 compositions containing the anti-inflammatory inhibitor can, for the most part, be used forlocal administration as well, but with an emphasis on choosing components for saidcompositions which will tend to promote absorption of the inhibitor into the local tissues atthe site of administration, but which will also tend to prevent infiltration and migration of theinhibitor into more outlying and distant tissues, resulting in systemic carryover.

Local administration is focused on suitable tissues and body cavities into which theanti-inflammatory inhibitor may be injected, infused, implanted, deposited, inserted, instilled,or insufflated. Such administration may include, but is not limited to, that which isintraarterial, intraarticular, intrachondrial, intracostal, intracystic, intra- or transdermal,intrafasicular, intraligamentous, intramedulary, intramuscular, intranasal, intraneural,intraocular, i.e. opthalmic administration, intraosteal, intrapelvic, intrapericardial, intraspinal,intrasternal, intrasynovial, intratarsal, intrathecal, or intravenous.

Pharmaceutical compositions in liquid form containing the inhibitor offer theadvantage of permitting injections of the liquid into or in close proximity to the site ofinflammation. This is particularly the case where inflamed joints and degenerative jointdisease are involved. By injection of the inhibitor directly into the joint, it is possible toachieve a high concentration of inhibitor in a short period of time, thus not only substantiallyenhancing access of the inhibitor to the site of inflammation, and thus the therapeutic activityof the inhibitor, but also at the same time minimizing the occurrence of untoward adversereactions that might otherwise occur, The resuit is a high local concentration of the inhibitorwith a correspondingly low systemic carryover concentration.

Injections may also be made of pharmaceutical compositions containing theinhibitor, where the pharmaceutical composition is in delayed-release, controlled-release, orsustained-release form. These formulations of recognized composition may be a solids,semi-solids, gels or other liquid/solid combinations in which an erodible matrix or sériés ofcoatings is used to provide a continuous release of the inhibitor at a predetermined rate or atvariable rates if desired. The terms “extended-release" and “long-acting” as well as othersare used to describe these formulations. Ail of these employ various combinations ofbioerodible polymers, e.g., various cellutosic polymers, and naturel matériels, e.g., cornstarch and magnésium stéarate, to obtain slow and/or uniform dispensing of the inhibitorcontained within the matrix. These pharmaceutical compositions may be injected into thesite if suitably liquid or suspendable, or may be delivered by other means if more solid innature.

The therapeutically effective amount for treating pain and inflammation of inhibitory compounds of Formula (I) is administered to a dog being treated in an amount expressed as milligrams per kilogram of body weight of said dog, per day: “mg/kg/day". The expression -41- 011213 "per day” as used herein should not be interpreted as necessarily requiring that anyparticular dosage form be administered on a daily basis to the dog being treated. Theexpression “per day" is merely an indication of the smallest convenient but arbitrary segmentof time which is being used as part of the overall unit for measuring the dose of anti-inflammatory inhibitor being administered. The dose, i.e., the therapeutically effectiveamount for treating pain and inflammation of the inhibitor will usually range from about 0.01mg/kg/day to about 20.0 mg/kg/day, preferably from about 0.1 mg/kg/day to about 12.0mg/kg/day, more preferably from about 0.5 mg/kg/day to about 10.0 mg/kg/day, and mostpreferably from about 0.5 mg/kg/day to about 8.0 mg/kg/day. For instance, a 50 Ib. dogweighs 23 kg (1 kg = 2.2 Ib.), and thus would be treated most preferably with fforn about 10mg to about 180 mg of therapeutic agent per day. The fractional amounts are not significantand the dosages would appropriately be rounded to a number which corresponds to unitdosage amounts which are conveniently available. Where the dosage form is, e.g., aninjectable Iiquid, the preferred dosage amounts may be achieved more precisely. On theother hand, where the dosage form is, e.g., an oral tablet, it will be necessary to make moreof an approximation of the preferred dosage. Thus, the 10 mg dose could be approximatedby halving a 25 mg tablet, and the 180 mg dose could be approximated by using a 100 mgtablet together with a 75 mg tablet or three 25 mg tablets, since these are typical dosageamounts for oral tablets. As will be apparent to those skilled in this art, where the dosageform most frequently employed is the oral tablet and a large number of dogs are treated on adaily basis, added convenience will be obtained through the use of a dispenser containing ailof the available dosage amounts of said tablets, e.g., 25 mg, 75 mg, and 100 mg tablets. Inthis way virtually any preferred dosage amount may be approximated using a combination of said tablets and/or halves thereof.

It is necessary for the skilled artisan, such as a veterinarian, not only to déterminethe preferred route of administration and the corresponding dosage form and amount, butsaid artisan must also détermine the dosing regimen, i.e., the frequency of dosing. Ingeneral terms it is most likely that the choice will be between once-a-day (s./.d.) dosing andtwice-a-day (b.i.d.) dosing, and that the former will provide more rapid and profound therapy,while the latter will provide less profound but more sustained therapy. Kowever, thisgeneralization does not take into account such important variables as the spécifie type ofpain or inflammation involved, the spécifie therapeutic agent involved and itspharmacokinetics, and the spécifie patient (dog) involved. For an approved product in themarketplace, much of this information is already provided by the results of clinical studiescarried out to obtain such approval. In other cases, such information may be obtained in astraightforward manner in accordance with the teachings and guidelines contained in the -42- 011213

Instant spécification taken in light of the knowledge and skill of the artisan. The results whichare obtained can also be correlated with data from corresponding évaluations of anapproved product in the same assays.

The above-recited ranges of dosage amounts, which are also recited elsewhereherein, are for racemic mixtures of compounds of Formula (I) having a chiral carbon, or forsingle compounds of Formula (I) where no chiral carbon atom is présent. As will beappreciated by the person of ordinary skill in this art, i.e., a practicing veterinarian or aperson with an advanced degree and expérience in animal health issues, where other than aracemic mixture of compounds of Formula (I) is involved, the anti-inflammatorytherapeutically effective amount will vary. For example, if 85% of the mixture is (S)-enantiomer, that will ordinarily tend to reduce the necessary dosage. These considérationsare based on an assumed equal potency, and the fact that the (S)-enantiomer is significantlymore active than the (R)-enantiomer. However, the degree of différence between theactivities of the two enantiomers must also take into account other différences, especiallydifférences in pharmacokinetics between the two enantiomers, in determining the properdosage. For example, it has been found that there is a significant différence in clearancerates between the (+)(S) and (-)(/?) enantiomers. This, in turn, will hâve a calculable impacton the amount of active compound to be administered. Ordinarily, such déterminations willbe made on a case-by-case basis by the artisan, but these are well within the ordinary skillof the art, as is instituting the methods whereby data necessary for the supportingcalculations may be obtained.

Typical dosage forms and amounts would include (1) intravenous administration ofcarprofen at a dose rate of 4.0 mg/kg/day of bodyweight, injected into the right cephalic vein;(2) oral administration of carprofen at a dose rate of 4.0 mg/kg/day of bodyweight as an oralpaste syringed on the back of the tongue, given one hour before feeding; and (3) oraladministration of carprofen at a dose rate of 4.0 mg/kg/day of bodyweight as 25 mg, 75 mg,and 100 mg tablet préparations, placed on the back of the tongue of the dog being treated,given one hour before feeding.

The active ingrédients of the présent invention may also be combined with othertherapeutically active ingrédients which would be readily apparent to the skilled artisan in thisfield, and which will usually be determined by the circumstances under which the therapeuticagent of the présent invention is administered. For example, where a joint has becomeseriously inflammed and infected atthe same time by microorganisms, e.g., bacteria, fungi,protozoa, virus and the like, the active ingrédient of the présent invention will desirably beadministered in combination with one or more antibiotic, antifungal, antiprotozoal, antiviral orsimilar therapeutic agents. The active ingrédient of the présent invention may be -43- 011213 "administered not only in combination with other NSAIDs of the type described in furtherdetail herein, but in combination as well with inhibitors of other mediators of inflammation, inorder to obtain a multi-fold inhibitory action against inflammation. Additional classes of suchinhibitors and examples thereof include, e.g., H, -receptor antagonists; kinin-B-, - and B2 -receptor antagonists; prostaglandin inhibitors such as PGD-, PGF- PGI2 -, and PGE-receptorantagonists; thromboxane A2 (TXA2-) inhibitors; 5- and 12-lipoxygenase inhibitors;leukotriene LTC4 -, LTD4/LTE4 -, and LTB4 -inhibitors; PAF-receptor antagonists; gold in theform of an aurothio group together with various hydrophilic groups; immunosuppressiveagents, e.g., cyclosporine, azathioprine, and methotrexate; anti-inflammatoryglucocorticoids; penicillamine; hydroxychloroquine; anti-gout agents, e.g., coichicine,xanthine oxidase inhibitors, e.g., allopurinol, and uricosuric agents, e.g., probenecid,sulfinpyrazone, and benzbromarone.

Because inflammation is most prévalent among gériatrie dogs, it will be appreciatedby those skilled in the art that the anti-inflammatory agents of the présent invention may alsobe administered in combination with therapeutic agents intended for the treatment of diseaseconditions, syndromes and symptoms which are also found in abundance in older dogs.Such therapeutic agents and the conditions which they are used to treat include, e.g.,cognitive therapeutics to counteract memory loss and impairment. Another large class ofsuch therapeutic agents includes anti-hypertensives and other cardiovascular drugsintended to offset hypertension, myocardial ischemia including angina, congestive heartfailure, and myocardial infarction, e.g., diuretics, vasodilators such as hydralazine, β-adrenergic receptor antagonists such as propranolol, angiotensin-ll converting enzymeinhibitors (ACE-inhibitors) such as enalapril used to treat gériatrie dogs with mitralinsufficiency, and enalapril alone and in combination with neutral endopeptidase inhibitors,angiotensin 11 receptor antagonists such as losartan, renin inhibitors, calcium channelblockers such as nifedipine, sympatholytic agents such as methyldopa, a2-adrenergicagonist such as clonidine, α-adrenergic receptor antagonists such as prazosin, and HMG-CoA-reductase inhibitors (anti-hypercholesterolemics) such as lovastatin.

Still other classes of such therapeutic agents include antineoplastic agents,especially antimitotic drugs including the vinca alkaloids such as Vinblastine and vincristine,for treating various cancers; therapeutic agents for treating rénal failure; anti-obesity drugsfor treating excess weight problems in dogs; anti-parasitic drugs for treating both endo- andecto-parasites which commonly afflict dogs; and anti-pruritic drugs for treating various typesof pruritis in dogs.

Other types of drugs which can be used in combination with the anti-inflammatoryagents of the présent invention include growth hormone secretagogues; strong analgésies; -44- 011213 focal and systemic anesthetics; and H2 -receptor antagonists and other gastroprotectiveagents. It will be recognized by those of ordinary skill in this art that some of the abovecombinations of therapeutic agents will be used most frequently to treat various acuteconditions in dogs, e.g., bacterial infections occurring simultaneously with degenerative jointdisease. However, there would be an equal if not greater intererst on the part of such skilledpersons in treating chronic conditions in dogs.

In accordance with a regimen which would be used for this purpose, it iscontemplated that the anti-inflammatory agents of the présent invention would beadministered in combination with other médications used on a reguiarly scheduled basis fortreating chronic conditions such as osteoarthritis. It is also envisioned that adminstration incombinations could assume a number of different forms and still be within the scope of theprésent invention. For example, the anti-inflammatory agents of the présent invention mightsimply be formulated with one or more of the other therapeutic agents which are to form theintended combination, into a convenient dosage form, such as an oral tablet, containing ail ofthe drugs forming the combination. Varying half-lives for the different drugs could beaccommodated by the person skilled in preparing formulations by creating controlled-releaseforms of said drugs with different release times so that relatively uniform dosing wasachieved. A medicated feed used as the dosage form could also be prepared in accordancewith well known principles in the art of formulation, in which the drugs used in thecombination were simply présent together in admixture in the feed composition. The présentinvention also contemplâtes co-administration in which the combination of drugs is achievedby the simultaneous administration of the drugs to be given in combination. Such co-administration could even be by means of different dosage forms and routes ofadministration. The présent invention further contemplâtes the use of such combinations inaccordance with different but regular and continuous dosing schedules whereby desiredplasma levels of the drugs involved were maintained in the dog being treated, even thoughthe individual drugs making up the combination were not being administered to said dogsimultaneously. Ail such combinations would be well within the skill of the art to devise andadminister.

The methods and compositions of the présent invention are useful for treating orpreventing pain and/or inflammation in dogs. Pain, which is a more or less localizedsensation of discomfort, distress, or agony, resulting from the stimulation of specializednerve endings, may occur as or be viewed as separate and distinct from inflammation. Forexample, chronic pain and pain induced and/or associated with surgery, e.g., peri-operativeand postoperative pain, initially may hâve little association with inflammation. Opioidanalgésies are effective in the treatment of postoperative pain but do not affect inflammation. -45- 01 1213

Tlowever, NSAlDs can be superior to such opioid analgésies in the treatment of some formsof postoperative pain, and they are particularly effective in settings in which inflammation hascaused sensitization of pain receptors to normally painless mechanical or Chemical stimuli.While NSAIDs inhibit the biosynthesis and release of protaglandins, which are inflammatorymediators, there is data to suggest that the analgésie affect of NSAlDs may occur by way ofmechanisms other than inhibition of protaglandin synthesis.

Accordingly, the présent invention has been described in terms of being useful in thetreatment and prévention of pain and inflammation, since these most often occur together intissue injury and disease processes and conditions mediated by COX-2. However, there isno intention to inextricably link pain and inflammation with regard to the usefulness of theprésent invention in treating and preventing them; and they are thus to be regarded in thecontext of the instant spécification as being the object, either individually and separately, ortogether in combination, of the methods, compositions and other facets of the présentinvention.

The inflammatory process itself may hâve a number of precipitating causes,including infectious agents, ischemia, antigen-antibody interactions, and thermal or otherphysicai injury. The response to each of these causes is characteristically different, but theyail hâve a strong commonality. Clinical symptoms include erythema, edema, tendernessand pain. Three distinct phases can be recognized, but each of these is mediated bydifferent mechanisms. The first, acute transient phase involves local vasodilation andincreased capillary permeability; the second, delayed, subacute phase involves infiltration ofleukocytes and phagocytic cells; and the third, chronic proliférative phase involves tissuedegeneration and fibrosis. NSAlDs as a therapeutic class of anti-inflammatory agents,appear to act by inhibiting the enzymatic production and release of prostaglandins, whichparticipate in the pathogenesis of inflammation and fever. However, the NSAlDs do notinhibit the formation of eicosanoids such as the leukotrienes, which also contribute toinflammation, nor do they interfère with the formation of numerous other mediators ofinflammation.

It has been discovered, in accordance with the présent invention, that the carprofengenus of compounds of Formula (1), and especialiy carprofen itself, and more especially the(S)-enantiomer of carprofen, alone among the NSAlDs having a carboxylic acid moiety, hâvea surprising and unexpectedly high degree of selectivity for the COX-2 isozyme. While thisparticuiar isozyme is an important mediator of inflammation, there are many other importantmediators of inflammation that either hâve no interaction with NSAlDs, or no well understoodrelationship to the action of NSAlDs. Such mediators include several classes of leukocytes;cell adhesion molécules; soluble mediators such as C5a, PAF and leukotriene B4 ; cytokines -46- 0112 Ί 3 such as IL-1 and TNF; growth factors such as GM-CSF and TGF-β, ; histamine, bradykininand 5-HT. While the compounds of Formula (i) are shown herein to be unique inhibitors ofCOX-2, there is no intention thereby to be bound to any particular mechanism of action bywhich the compounds of Formula (I) might exert their anti-inflammatory activity.

Indeed, it has been pointed out further above that the anti-inflammatory mode ofaction of carprofen and the other compounds of Formula (I) is not well understood, and thatthere has been spéculation heretofore that the actual mode of action might involveneutrophils, also known as polymorphonuclear leukocytes. PAF stimulâtes such cells toaggregate, to reiease leukotrienes and lysosomal enzymes, and to generate superoxide, ailof which promote inflammation.

The uniqueness of carprofen and the compounds of Formula (!) among NSAlDs hasalready been referred to in general terms. While the compounds of Formula (!) are clearlyNSAlDs, they are not readily placed in any of the recognized Chemical classification ofNSAlDs:

Salicylic acids........... .. aspirin; p-Aminophenols......... . .acetaminophen; lndole/indene acetic acids.. .. Indomethacin, sulindac, etodolac; Heteroaryl acetic acids.... .. .tolmetin, diclofenac, ketorolac; Arylpropionic acids....... .. .ibuprofen, naproxen, flurbiprofen, ketoprofen; Anthranilic acids......... .. .mefenamic acid, meclofenamic acid; Enoiic acids............ .. oxicams, e.g., piroxicam, tenoxicam; Pyrazoiidinediones....... .. phenylbutazone; Alkanones.............. .. nabumetone.

Carprofen and the compounds of Formula (I), although they are propionic acids, donot belong to the subclass of arylpropionic acids because the carbazole group of thecarprofens is heteroaryl, not aryl. The carprofens do not belong to the subclass of heteroarylacetic acids, because the carprofens are propionic acids, not acetic acids. The carprofenscannot be placed in any of the other subclasses without doing some violence to the bases ofclassification. The only NSAID approved for treatment of humans which is recognized tohâve human COX-2 sélective activity is nabumetone in the above list, which is not an acid atail but a 2-butanone. Although the active species is the acid métabolite, this métabolite hasonly a small fraction of the COX-2 selectivity of carprofen in dogs.

Exemplary of the numerous classical NSAlDs within this broad class are thecompounds set out in the beiow table, which gives the common and IUPAC names of each -47- 011213 compound and its structure. Ali of the enumerated compounds appear in USP Dictionary ofUSAN and International Drug Names, 1995, C. A. Fleeger, ed., United States PharmacopeialConvention, inc., Rockville, MD. The USAN (United States Adopted Names) programproduces simple and useful non-proprietary names for drugs, and the name-selection 5 process is initiated when the drug enters the clinical investigation stage. The name andstructure of carprofen are given atthe beginning of the table in order to facilitate comparison. TABLE 1 COMMONNAME IUPAC NAME STRUCTURE Carprofen 6-chIoro-a-methyl-9/7-carbazole-2-acetic acid H . 0 Ci Alclofenac 3-chloro-4-(2-propenyloxy)-benzeneacetic acid ^°'OXVH Cl Aspirin 2-(acetyloxy)-benzoic acid 0 O Diflunisal 2’ ,4’-difluoro-4-hyd roxy-3-biphenylcarboxylic acid U. Diclofenac 2-[(2,6-dichlorophenyl)amino]-benzeneacetic acid 0 Cl -48- 011213

GOMMONNAME; IUPACNAME : STRUCTURE . Etodolac 1,8-diethyl-1,3,4,9-tetrahydro-pyrano[3,4-b]indole-1-acetic acid O Felbinac [1,1'-biphenyl]-4-acetic acid Flufenamic acid 2-{{3-(trifluoromethyl)phenyi]amino}-benzoic acid ° on Flunixin Meglumine(Banamine®)1 2-{[2-methyl-3-(trifluoromethyl)-phenyl]amino}-3-pyridinecarboxylic acid;compounded with 1-deoxy-1-(methylamino)-D-glucitol (1:1) \<cf· οζΡ OH Flurbiprofen 2-fluoro-a-methyl-[1,1 ’-biphenyl]-4-aceticacid Ibuprofen a-methyi-4-(2-methylpropyl)benzeneacetic acid àjoV 1 Registered Trademark; approved for use in dogs outside the United States. -49- 011213 COMMON NAME ; IUPAC NAME STRUCTURE / llonidap 6-chloro-5-fluoro-2,3-dihydro-(hydroxy-2-thienylmethy lene)-2-oxo-1 B-indole-1 -carboxamide °γΝΗ2 N Cl-/ Λ F 0 su A) indomethacin 1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1 B-indole-3-acetic acid jf —cr"' 'W Æo OH Indoprofen 4-(1,3-dihydro-1 -oxo-2H-isoindol-2-yl)*benzeneacetic acid Π 0 oz Isoxepac 6,11 -dihydro-11-oxo-dibenz[b,e]oxepin-2-acetic acid 0 OH 0 Χ—0 ) 0 Ketoprofen 3-benzoyl-a-methyl-benzeneacetic acid 0 /y' -X.OH Il Π (Ketofen®)1 jj 0 Meclofenamic acid (Arquel®)1 2-[(2,6-dichioro-3-methyiphenyl)amino]-benzoic acid δ Cl -50- 011213 : COMMON NAME IUPACNAME STRUCTURE Mefenamic acid 2-[(2,3-dimethylphenyl)amino]-benzoic acid OH O ΥΎ s J U Meloxicam (Metacam®) 4-hydroxy-2-methyl-N-(5-methyl-2- thiazolyl)-2H-1,2-benzothiazine-3- carboxamide OH 0 N' I jl ji I H 0 0 sz Nabumetone and 6-MNA 4-(6-methoxy-2-naphthalenyl)-2-butanone; prodrug; metabolized in vivo toactive 6-methoxy-2-naphthyl-acetic acid(6-MNA) depicted at right —O"' O Π' 0 Y0H —0" O Naproxen 6-methoxy-a-methyl-2-naphthaleneacetic acid —0' A A VOH 0 Niflumic acid 2-[3-(trifluoromethyl)anilino]nicotinic acid k 0 π OH CFS Nimesulide 4'-nitro-2’-phenoxymethanesulfonanilide HjCSOjNH (Suiidene®)1 ή r°y V J NO 2 -51- 011213

COMMON NAME; ' IUPACNAME STRUCTURE NS-398 N-[2-(cyclohexyloxy)-4-nitrophenyl]- methanesulfonamide 0 °γ^γΝ°2 H3CSO2N'/<X^ H Phenylbutazone 4-butyl-1,2-diphenyl-3,5pyrazolidinedione Piroxicam (Feldene®)1 4-hydroxyy-2-methyl-W-2-pyridinyl-2H-1,2-benzothiazine-3-carboxamide-1, 1-dioxide OH O l<i|l H //\\ O 0 Tenidap 5-chloro-2,3-dihydro-3-(hydroxy-2- thienylmethylene)-2-oxo-1H-indole-1- carboxamide °γΝΗ2 /AV0 S Tolfenamic acid A/-(3-chloro-o-tolyl)-anthranilic acid 0-, OH CW -52- 011213

COMMON NAME IUPACNAME STRUCTURE Vedaprofen (±)-4-cyclohexyl-a-methyl-1-naphthaleneacetic acid /-v H. /CH· O DESCRIPTION OF PREFERRED EMBODIMENTSThe carprofen genus of compounds, characterized by an α-methyl-acetic acid functionality, has many times greater COX-2 selectivity in dogs than any of the carboxyl- 5 containing or sulfonyl- or sulfonamide-containing NSAIDs set out in the above table. In orderto demonstrate this unexpected property, a comparison was made between the COX-2selectivity of carprofen, a compound of the présent invention, and the COX-2 selectivity ofcertain selected compounds from the above table. The resuits are illustrated in the below-described working examples. 10 As described above, the selectivity for COX-2 is conventionally determined as the ratio of COX-1 inhibition to COX-2 inhibition, or vice versa. In the présent description, theratio of COX-1 inhibition to COX-2 inhibition in dogs is utilized essentially for the sake ofsimplicity. Both inhibition values are IC50 values, which means that the more active a testcompound is, the smaller will be the IC50 value. This, in effect, inverts the ratio so that where 15 it is COX-1 : COX-2, and a test compound is very sélective for canine COX-2, the ratio willbe that of a larger number over a much smaller number. Thus, the most canine COX-2sélective test compounds will be those with the highest ratio numbers. EXAMPLE 1 20 Cornparative évaluation of canine cyclo-oxygenase-1 and -2 inhibition by carprofen andother NSAIDs

Protocol for Evaluation of Canine COX-1 Activity

Test drug compounds were solubilized and diiuted the day before the assay was to 25 be conducted with 0.1 mL of DMSO / 9.9 mL of Hank's balanced salts solution (HBSS), andstored ovemight at 4° C. On the day that the assay was carried out, citrated blood wasdrawn from a donor dog, centrifuged at 190 x g for 25 min at room température, and theresulting platelet-rich plasma was then transferred to a new tube for further procedures. Theplatelets were washed by centrifuging at 1500 x g for 10 min at room température. The 30 platelets were washed with platelet buffer comprising Hank’s buffer (Ca free) with 0.2% -53- 011213 *bovine sérum albumin (BSA) and 20 mM HEPES. The platelet samples were then adjustedto 1.5 x 107 / mL, after which 50 μΙ of calcium ionophore (A23187) together with a calciumchloride solution were added to 50 μΐ of test drug compound dilution in plates to produce finalconcentrations of 1.7 μΜ A23187 and 1.26 mM Ca. Then, 100' μΐ of canine washed plateletswere added and the samples were incubated at 37° C for 15 min, after which the reactionwas stopped by adding 20 μ| of 77 mM EDTA. The plates were then centrifuged at 2000 x gfor 10 min at 4° C, after which 50 μΙ of supernatant was assayed for thromboxane B2 (TXB2)by enzyme-immunoassay (EIA). The pg/mL of TXB2 was calculated from the standard lineincluded on each plate, from which it was possible to calculate the percent inhibition of COX- 1 and the ICS0 values for the test drug compounds.

Protocol for Evaluation of Canine COX-2 Activity A canine histocytoma (macrophage-like) cell line from the American Type Culture

Collection designated as DH82, was used in setting up the protocol for evaluating the COX-2inhibition activity of various test drug compounds. There was added to fiasks of these cells10 pg/mL of LPS, after which the flask cultures were incubated overnight. The same testdrug compound dilutions as described above for the COX-1 protocol were used for the COX- 2 assay and were prepared the day before the assay was carried out. The cells wereharvested from the culture fiasks by scraping, and were then washed with minimal Eagle’smedia (MEM) combined with 1% fêtai bovine sérum, centrifuged at 1500 rpm for 2 min, andadjusted to a concentration of 3.2 x 105 cells/mL. To 50 μΙ of test drug dilution there wasadded 50 μΙ of arachidonic acid in MEM to give a 10 μΜ final concentration, and there wasadded as well 100 μΙ of cell suspension to give a final concentration of 1.6 x 105 cells/mL.The test sample suspensions were incubated for 1 hr and then centrifuged at 1000 rpm for10 min at 4° C, after which 50 μΙ aliquots of each test drug sample were delivered to EIAplates. The EIA was performed for prostaglandin E2 (PGE2), and the pg/mL concentration ofPGE2 was calculated from the standard line included on each plate. From this data it waspossible to calculate the percent inhibition of COX-2 and the IC50 values for the test drugcompounds. Repeated investigations of COX-1 and COX-2 inhibition were conducted overthe course of several months. The results were averaged, and a single COX-1 : COX-2 ratiowas calculated. The data obtained, together with an indication of the number of testsconducted for each test sample, are set forth in the following table of values. -54- TABLE 2 011213 NSAID No. oftests COX-1 IC50 pM CÔX-2 IC50 pM COX-1/COX-2 Ratio Carprofen (rac.) 9 13.2 0.102 129 Carprofen (S-) 3 6.71 0.0371 181 Carprofen (R-) 4 >25.0 5.97 >4.19 Flufenamic acid 6 2.31 0.0475 48.6 Nimesulide 6 2.15 0.0565 38.0 Niflumic acid 6 1.03 0.0464 22.2 Meclofenamic acid 5 0.737 0.0478 15.4 Tolfenamic acid 4 0.206 0.0137 15.0 Naproxen 3 7.08 0.626 11.3 Mefenamic acid 4 0.403 0.0362 11.1 Felbinac 3 2.54 0.362 7.01 6-MNA 6 28.3 4.21 6.72 NS-398 7 0.587 0.137 4.28 Flurbiprofen 4 0.505 0.123 4.10 Diciofenac 3 0.246 0.0778 3.16 Meloxicam 5 0.891 0.307 2.90 Phenyibutazone 5 >10.0 3.79 >2.46 Ibuprofen 4 1.03 0.391 2.63 Tenidap 12 0.469 0.228 2.06 Alclofenac 3 13.2 7.41 1.78 llonidap 16 0.472 0.270 1.75 Flunixin 5 0.00768 0.0121 0.635 Etodolac 3 1.33 2.57 0.517 Piroxicam 6 0.223 0.585 0.381 Ketoprofen 5 0.0286 0.123 0.232 Indomethacin 6 0.0558 0.366 0.152 Aspirin 3 34.3 >100 <0.343 Vedaprofen -55- 011213 EXAMPLE 2

Canine whole blood ex vivo déterminations of COX-1 and COX-2 activity inhibition bycarprofen

The objective of this study was to evaiuate the inhibitory potency of carprofenagainst COX-1 and COX-2 activity using an ex vivo procedure on canine whole blood.Three dogs were dosed with 10 mg/kg of racemic 6-chloro-a-methyl-carbazole-2-acetic acid(carprofen) administered by mouth (PO) in capsule dosage form, three dogs were dosedwith 2 mg/kg of carprofen on the same basis, and three dogs were untreated. A zero-hourblood sample was collected from ail dogs in the study prior to dosing, followed by 1-, 3-, and6-hour post-dose blood sample collections. Test tubes were prepared containing 2pL ofeither (A) calcium ionophore A23187 giving a 50 μΜ final concentration, which stimulâtes theproduction of thromboxane B2 (TXB2) for COX-1 activity détermination; or of (B)lipopolysaccharide (LPS) to give a 10 pg/mL final concentration, which stimulâtes theproduction of prostaglandin E2 (PGE2) for COX-2 activity détermination. Test tubes used asControls contained vehicle and were unstimulated by the addition of any agent. A 500 pLsample of blood was added to each of the above-described test tubes, after which they wereincubated at 37°C for one hr in the case of the calcium ionophore-containing test tubes, andovemight in the case of the LPS-containing test tubes. After incubation, 10 pL of EDTA wasadded to give a final concentration of 0.3%, in order to prevent coagulation of the plasmawhich sometimes occurs after thawing frozen plasma samples. The incubated sampleswere centrifuged at 4°C and the resulting plasma sample of -200 pL was collected andstored at -20°C in polypropylene 95-well plates. In order to détermine endpoints for thisstudy, enzyme immunoassay (EIA) kits available from Cayman were used to measureproduction of TXB2 and PGE2 , utilizing the principle of compétitive binding of tracer toantibody and endpoint détermination by colorimetry. Plasma samples were diluted toapproximate the range of standard amounts which would be suppiied in a diagnostic orresearch tools kit, i.e., 1/500 for TXB2 and 1/750 for PGE2.

The data set out in Table 3 further below demonstrate the percent inhibition of COX-1 and COX-2 activity based on their zéro hour values. The data is expressed as treatmentgroup averages in pg/mL of TXB2 and PGE2 produced per sample. Plasma dilution was notfactored in said data values.

The data in Table 3 show that at the 2 mg/kg dose there was significant COX-2 inhibition at ail timepoints. At 3- and 6-hours post-dose, there is observed to hâve been a slight décliné in COX-2 inhibition compared to the data obtained for the 10 mg/kg dose. The data in Table 3 atso show that at the 2 mg/kg dose there was no significant inhibition of -56- 011213 COX-1 activity at any of the timepoints involved. This resuit was consistent with theobserved excellent toleration of carprofen by the dogs in the study. The data for the 10mg/kg dose show that there was complété inhibition of COX-2 activity at every timepoint,and very strong inhibition of COX-1 activity beginning at 1 hr and plateauing over the 3- to 6- 5 hour timepoints. Accordingly, the data in Table 3 clearly demonstrate that at a 2 mg/kgdosage concentration carprofen possesses good COX-2 selectivity. As the dose isincreased from 2 to 10 mg/kg, increasing inhibition of COX-1 activity becomes évident. TABLE 3 COX-1 ACTIVITY INHIBITION - GrouD Averaoes ?HourL Untreated 2 mg/kg 10 mg/kg TXB, Pa/mL/Well 0-hour 1-hour 3-hour 6-hour 102 134 77 65 72 75 66 46 51 31 6 7 Percent Inhibition 1-hour 3-hour 6-hour 0% 25% 36% 0 % 8% 36% 39% 88% 86% COX-2 ACTIVITY INHIBITION - Grouo Averaqes • Hour Untreated 2 mg/kg 10 mg/kg PGE, Pa/mL/Well O-hour 1-hour 3-hour 6-hour '""'"'"393 900 349 405 511 33 98 190 336 19 12 12 Percent Inhibition 1-hour 3-hour 6-hour 0% 11% ' 0% 94% 81% 63% 94% 96% 96% 10 COX inhibition is observed when the measured percent inhibition is greater than thatmeasured for untreated contrais. The percent inhibition in the above table is caiculated in astraightforward manner in accordance with the following équation: (PGE2 at t = 0) — (PGE2 at t = 1) 15 % Inhibition (1-hour) = - X 100 (PGE2 att = O) EXAMPLE 3

Canine whole blood ex vivo déterminations of COX-2 activity inhibition by carprofen 20

This study followed the procedures described in Example 2 above, but with some modifications of detail below described.

Three dogs were dosed with 2 mg/kg of racemic 6-chloro-a-methyl-carbazole-2- acetic acid (carprofen) administered by mouth (PO) in tablet dosage form at zéro hour; three -57- 011213 cfogs were dosed with 4 mg/kg of carprofen on the same basis at zéro hour; and three dogswere untreated. A zero-hour blood sample was collected from ail dogs in the study prior todosing, followed by 2- and 4-hour post-dose blood sample collections. Test tubes wereprepared containing 2pL of lipopolysaccharide (LPS) to give a 10 pg/mL final concentration,Untreated test tubes were used as Controls. A 500 pL sample of blood was added to each ofthe above-described test tubes, after which they were incubated at 37°C ovemight. Afterincubation 10 pL of EDTA was added to give a final concentration of 0.3%. The incubatedsamples were centrifuged at 4°C and the resulting plasma sample of -200 pL was collectedand stored at -20°C in propylene 96-well plates. Enzyme immunoassay (EIA) kits availablefrom Cayman were used to measure production of PGE2, with endpoint détermination bycolorimetry. Plasma samples were diluted to 1/750 for PGE2.

The plasma samples were also assayed for total plasma carprofen concentrationusing HPLC with a 5 micron, 100x4.6mm Chromtech chiral AGP column and a mobile phasecomposed of 10:90 v/v 2-propanol:0.1M phosphate buffer pH 6.0, and fluorescencedétection (285 nm excitation, 345 nm émission). Plasma samples (0.2 mL) were bufferedwith 0.05 M citric acid pH 5.1 following the addition of (S)-naproxen as the internai standard,then were extracted with 4:1 v/v diethyl etherdichloromethane. The ether layer wasseparated, then back-extracted with 0.005 M Na2CO3, after which the organic phase wasdiscarded by aspiration. The aqueous phase was buffered with 0.05 M citric acid pH 5.1,then was again extracted with diethyl etherdichloromethane. The ether was thentransferred to clean tubes, evaporated under a stream of nitrogen, and the residuereconstituted in HPLC mobile phase for analysis.

The data set out in Table 4 further below show production of PGE2 (pg/mL/well),percent inhibition of COX-2 activity based on each dog’s zéro hour values, as well as plasmaexposure for individual dogs.

The data in Table 4 show that at 2- and 4-hours post-dose, there is observed tohâve been a small différence between the doses, with the 4 mg/kg dose showing slightlygreater COX-2 inhibition. Ex vivo COX-2 inhibition is well correlated with plasmaconcentrations of carprofen. -58- 011213 TABLE 4 COX-2 ACTIVITY INHIBITION and CARPROFEN PLASMA CONCENTRATIONS Dog number Dose (mg/kg) 0-hour pge2 Pq/mL/Well : 2-hour 4-hour Percent Inhibition 2-hour 4-hour Plasma concentration ua/mL 2-hour 4-hour 35769 0 426 340 439 20% 0% — —» 05690 0 130 239 219 0% 0% — — 06044 0 860 660 632 23% 27% — — 35773 2 293 35 69 88% 77% 21.9 13.4 35742 2 541 113 223 79% 59% 8.6 5.7 36184 2 409 46 90 89% 78% 17.2 10.6 05717 4 471 25 20 95% 96% 37.6 29.1 05704 4 393 21 25 96% 94% 29.9 21.1 36185 4 622 14 14 98% 98% 29.0 27.0

The percent inhibition in the above table is calculated in a straightforward manner inaccordance with the following équation: 5 (PGE2 at t - 0) — (PGE2 at t = 2) % Inhibition (2-hour) == - X 100 (PGE2 at t = 0) EXAMPLE 4 10 Canine in vivo déterminations of COX-2 activity inhibition by carprofen in a carrageenan-induced inflammation model

This objective of this study was to monitor in vivo COX-2 activity in asubcutaneously implanted chamber during an induced inflammation. The COX-2 enzyme 15 can be detected by western analysis as early as 5 hours after carageenan treatments (T.Kirchner et al., J. Pharmacol. Exp. Ther. (1997) 282,1094-1101). In addition, COX-1 activitymay be simuitaneously determined by ex vivo methods as described in Example 2. Sixbeagle dogs had polyethylene “whiffle” golf balis approximately 4.2 cm in outer diametersurgically implanted under the skin just behind the shoulder blades. They were allowed to 20 recover from the surgery for 1 month before being assigned to experimental groups.

The experiment was run in two replicates. On the day of the experiment, three dogs were given 2mg/kg of carprofen PO 30 min. before the start of the inflammation and theother three dogs were used as contrais. The haïr around the bail was shaved and the areawas probed to locate a hole in the whiffle bail. This area was then marked and it was -59- 011213 sterilized with 2% iodine tincture. A needle was inserted into the hole and 1.5 cc of fluid wasremoved, hereinafter referred to as the exudate fluid "EF." After removing 1.5 cc of EF, 1.5ccof a 0.33% carrageenan solution in H2O was added to the bail to induce an inflammatoryevent. EF samples were taken at 0, 5, and 24 hours after carrageenan injection. Blood 5 samples were also taken so that an ex vivo COX-1 whole blood assay could be run, asdescribed in Example 2.

The EF sample needed to be purified prior to assay, due to the low amount of PGE2found in the in vivo sample. This was accomplished by the use of 4 mL PGE2 affinitycolumns (Cayman Chemical). The cotumn was first washed with 10 mL of 0.1 M phosphate 10 buffer then 10 mL water. The EF was then diluted 1:5 with 0.1 M phosphate buffer andadded to the column. The column was washed with 10 mL of the phosphate buffer, then 10mL of water. Finally, the PGE2was removed from the column with 2.5 mL 95% éthanol. Thesample was evaporated under a stream of nitrogen and then diluted for analysis of PGE2 bythe Cayman EIA PGE2 (COX-2) kit, as described in Examples 2 and 3. 15 Table 5 illustrâtes that carrageenan induced almost a 4-fold increase in PGE2 synthesis in the whiffle bail over background levels. This increase was seen at 5 hours andremained until at least 24 hours. The carprofen dosed animais at 2 mg/kg show near totalinhibition of PGE2 synthesis that was statistically significant at 5 hours and 24 hours, with p-values of .013 and .015, respectively. The ex vivo COX-1 data showed no inhibition of TXB2 20 at any of the timepoints tested. This indicates that carprofen showed in vivo COX-2inhibition at the same time it showed no effect on ex vivo COX-1 activity. TABLE 5 IN VIVO COX-2 ACTIVITY INHIBITION - Grouo Averaaes Dose ima/ka) PSÊ2 Percent Inhibition Pa/mL/Well 0-hour , 5-hour 24-hour 5-hoiir 24-hour 0 3.4 17.1 13.0 0% 0% 2 6.5 1.9 3.2 71% 51% 25 -60- 011213 EXAMPLE 5

Computation of carprofen controlled release dosage form oral drug delivery input rates anddoses which give Cox-2 inhibition of 80% in dogs for a length of time longer than animmédiate release carprofen dosage form A study was carried out to obtain data from which to détermine useful, preferred,more preferred, and most preferred oral carprofen controlled release dosage forms for use inthe présent invention.

The plasma samples collected from dogs in above-recited Example 3 were alsoassayed for total plasma carprofen concentration using HPLC with a 5 micron, 100x4.6mmChromtech chiral AGP column and a mobile phase composed of 10:90 v/v 2-propanoI:0.1Mphosphate buffer pH 6.0, and fluorescence détection (285 nm excitation, 345 nm émission).Plasma samples (0.2 mL) were buffered with 0.05 M citric acid pH 5.1 following the additionof (S)-naproxen as the internai standard, then were extracted with 4:1 v/v diethylether.dichloromethane. The ether layer was separated, then back-extracted with 0.005 MNa2CO3, after which the organic phase was discarded by aspiration. The aqueous phasewas buffered with 0.05 M citric acid pH 5.1, then was again extracted with diethylether;dichloromethane. The ether was then transferred to clean tubes, evaporated under astream of nitrogen, and the residue reconstituted in HPLC mobile phase for analysis.

The carprofen plasma levels were plotted against % COX-2 inhibition (from Example3). Inspection of this plot indicated that 80% COX-2 inhibition occurred at a plasmacarprofen concentration of approximately 10 pg/mL, and that 50% COX-2 inhibition occurredat a plasma carprofen concentration of approximately 2 pg/mL, and that 90% COX-2inhibition occurred at a plasma carprofen concentration of approximately 20 pg/mL.

Following oral dosing to dogs of an immédiate release carprofen formulation at adose of 2 mg/lb, plasma carprofen concentrations were above 10 pg/mL for approximately 10.5 hr. In accordance with the présent invention, 2 mg/lb/day is within the range ofpreferred doses of carprofen. A useful controlled release dosage form of carprofen in accordance with the présentinvention is one which maintains a carprofen plasma level greater than 2 pg/mL for most ofthe day after a single oral dose at 2 mg/lb.

Preferred oral controlled release dosage forms of carprofen in accordance with theprésent invention are ones which maintain a plasma carprofen concentration greater than 10pg/mL for a period of time greater than that for which an immédiate release dosage form ofcarprofen maintains a comparable plasma level, when said immédiate release dosage formand controlled release dosage form are administered at the same dose, e.g. 2, 1.8, 1.6, or -6 ι- ΟΙ 1 21 3 ΐ.4 mg/lb. For instance, preferred 2mg/lb oral controlled release dosage forms of thisinvention maintain a plasma carprofen concentration greater than 10 pg/mL for greater than 10.5 hrs.

Immédiate release carprofen dosage forms containing doses of 1.8, 1.6, and 1.4mg/lb maintain a plasma carprofen concentration above 10 pg/mL for 9.5 hrs, 8.5 hrs, and 7.5 hrs, respectively.

Preferred 1.8 mg/lb oral controlled release carprofen dosage forms maintain aplasma carprofen concentration above 10 pg/mL for greater than 9.5 hrs. Likewise, thethreshold durations for 1.6 mg/lb and 1.4 mg/lb doses are 8.5 hrs and 7.5 hrs, respectively.The performance characteristics for preferred oral controlled release carprofen dosageforms at doses higher than 2 mg/lb or less than 1.4 mg/lb can be similarly calculated,assuming linear pharmacokinetics.

More preferred oral controlled release carprofen dosage forms are those whichmaintain a plasma carprofen concentration greater than 10 pg/mL for a period of timegreater than or equal to that observed when an immédiate release carprofen dosage form isdosed at any higher dose. These controlled release dosage forms thus maintain at least80% COX-2 inhibition in canine blood for a period of time longer than that achieved by animmédiate release dosage form at a higher dose.

Most preferred oral controlled release carprofen dosage forms are those which areable to maintain plasma carprofen levels above approximately 10 pg/mL for a period of timegreater than or equal to the time observed for an immédiate release 2 mg/lb carprofendosage form (10.5 hrs), when said oral controlled release carprofen dosage forms areadministered at a dose less than 2 mg/lb. The performance of a 2 mg/lb oral immédiate• release dosage form is taken as the fondamental standard for purposes of this comparisonsince 2 mg/lb/day is the currently recommended and accepted efficacious oral dose inaccordance with the présent invention as described herein.

As described below, controlled release oral dosage form carprofen release rateswere calculated which resuit in canine carprofen plasma concentrations greater than 10pg/mL. For ease of computation, these calculated rates were “zéro order” rates; thus thecomputed rates were for controlled release devices which release carprofen at a constant(i.e. zéro order) rate, it will be appreciated by those skilled in the art that practical dosageforms release at “zéro order" for only a portion of their drug release time, whereafter theyrelease at “first order”, or release at “mixed order". In order to make the définition ofcarprofen deiivery rates (or release rates) even more clear to the skilled artisan in theanalysis below, usefol release rates hâve been defined according to the time at which 80% -62- 011213 ôf the carprofen has exited from the dosage form, regardless of the drug release mechanisminvolved.

Zero-order release rates resulting in simulated plasma carprofen concentrationsabove 10 pg/mL were determined by the method of Zhou and Notari. See Zhou, M. and N. 5 Re, "Methodology for using oral dose pharmacokinetic data to select drugs for prolongedrelease formulations and validation of the method using simulated data”, Biopharm DrugDisp, 1995. 16, 319-331. Average data resulting from the oral administration of 2 mg/lbcarprofen to canines in an immédiate release formulation were fitted to the équation C=C((e-s':' 10 - where Cj, S2, and S! are parameters, and t is time. During zero-order release the plasmacarprofen concentrations were simulated by the foilowing équation: C = (*0Ç ! Dn, )[(<<*·' - 1) / S, - (β*'1 -1) ! S2 ] where ko is the zero-order release rate and Dre( is the reference dose. After release iscompleted, the foilowing équation is applicable: 15 C = (i0C, -[(1-?'’)/S,K1·’] where T = DCR / k0, and DCR is the controlled release dose.

Preferred Oral Controlled Release Carprofen Dosage FormsTable 6 shown below demonstrates that oral controlled release carprofen dosage 20 forms (2 mg/lb dose) which release 80% of their incorporated carprofen in a time periodranging from 1.6 to 19.2 hrs hâve the capacity to give carprofen plasma levels greater than10 pg/mL for greater than 10.5 hrs. Accordingly, these are preferred dosage forms at 2mg/lb.

Table 7 shown below demonstrates that oral controlled release carprofen dosage 25 forms (1.8 mg/lb dose) which release 80% of their incorporated carprofen in a time periodranging from 1.6 to 19.2 hrs hâve the capacity to give carprofen plasma levels greater than10 pg/mL for greater than 9.5 hrs. Accordingly, these are preferred dosage forms at 1.8mg/lb.

Table 8 demonstrates that oral controlled release carprofen dosage forms (1.6 mg/lb 30 dose) which release 80% of their incorporated carprofen in a time period ranging from 1.6 to 19.2 hrs hâve the capacity to give carprofen plasma levels greater than 10 pg/mL for greaterthan 8.5 hrs. Accordingly, these are preferred dosage forms at 1.6 mg/lb. -63- 01 121 3

Table 9 demonstrates that oral controlled release carprofen dosage forms (1.4 mg/lbdose) which release 80% of their incorporated carprofen in a time period ranging from 1.6 to 19.2 hrs hâve the capacity to give carprofen plasma levels greater than 10 pg/mL for greaterthan 7.5 hrs. Accordinly, these are preferred dosage forms at 1.4 mg/lb.

More Preferred Oral Carprofen Controlled Release Dosage Forms

Controlled release carprofen dosage forms of this invention are particularly usefulbecause they are able to maintain carprofen plasma levels greater than 10 pg/mL for greaterthan 10.5 hrs, even when they are administered at doses lower than the preferredefficacious use dose of 2 mg/lb. Table 7 demonstrates that oral controlled release carprofendosage forms (1.8 mg/lb dose) which release 80% of their incorporated carprofen in a timeperiod ranging from 4.8 to 19.2 hrs hâve the capacity to give carprofen plasma levels greaterthan 10 pg/mL for greater than 10.5 hrs. These are more preferred dosage forms at a doselevel of 1.8 mg/lb.

Table 8 demonstrates that oral controlled release carprofen dosage forms (1.6 mg/lbdose) which release 80% of their incorporated carprofen in a time period ranging from 4.8 to 19.2 hrs hâve the capacity to give carprofen plasma levels greater than 10 pg/mL for greaterthan 9.5 hrs, i.e., for longer than the duration which a 1.8 mg/lb immédiate release dosageform is able to achieve. These are, consequently, more preferred dosage forms at a doselevel of 1.6 mg/lb.

Table 9 demonstrates that oral controlled release carprofen dosage forms (1.4 mg/lbdose) which release 80% of their incorporated carprofen in a time period of 4.8 to 19.2 hrshâve the capacity to give carprofen plasma levels greater than 10 pg/mL for greater than orequal to 8.5 hrs, i.e., for longer than the duration which a 1.6 mg/lb immédiate releasedosage form is able to achieve. These are, consequently, more preferred dosage forms at adose level of 1.4 mg/lb.

Most Preferred Oral Carprofen Controlled Release Dosage Forms

Most preferred oral controlled release carprofen dosage forms are those which areable to maintain plasma carprofen levels above approximately 10 pg/mL for a period of timegreater than or equal to the time observed for an immédiate release 2 mg/lb carprofendosage form (10.5 hrs), when said oral controlled release carprofen dosage forms areadministered at a dose of iess than 2 mg/lb. The performance of a 2 mg/lb oral immédiaterelease dosage form is taken as a fundamental standard for purposes of comparison since 2mg/lb/day is the currently recommended efficacious oral dose in accordance with the présent -64- 011213 invention. The data in Tables 6-9 are used to define the characteristics of the most preferredoral controlled release dosage forms of the présent invention.

At 1.6 mg/lb dose, most preferred oral controlled release carprofen dosage formsare those which release 80% of their incorporated carprofen over the range 6.4 to 19.2 hrs. 5 At 1.4 mg/lb dose, most preferred oral controlled release carprofen dosage forms are thosewhich release 80% of their incorporated carprofen over approximately 12.8 hrs (10-14 hrs). TABLE 6.

Number of hours for which carprofen plasma concentrations are above 10 pg/mL, after10 dosing with controlled release dosage forms which release carprofen at various rates (computed); dose = 2 mg/lb.

Zero-order Time for 80% release of Duration (hrs) for which plasma carprofen release rate carprofen from dosage form concentration isabovel0 pg/mL. (mg/lb/hr) (hrs) 0.084 19.2 17.50 0.100 16 16.5 0.125 12.8 15.5 0.165 9.6 13.5 0.25Ô 6.4 12.5 0.330 4.8 12 1.0 1.6 11 15 TABLE 7.

Number of hours for which carprofen plasma concentrations are above 10 pg/mL, afterdosing with controlled release dosage forms which release carprofen at various rates(computed); dose = 1.8 mg/lb.

Zero-orderrelease rate(mg/lb/hr) Time for 80% release ofcarprofen from dosage form /(hrs) Duration (hrs) for which plasma carprofenconcentration is above 10 pg/mL 0.075 19.2 14 . 0.090 16 14.5 0.113 12.8 14.5 0.150 9.6 13 0.225 6.4 11.5 ..... 0.3 4.8 11 0.9 1.6 10 20 -65- 011213 TABLE 8.

Number of hours for which carprofen plasma concentrations are above 10 pg/mL, afterdosing with controlled release dosage forms which release carprofen at various rates(computed); dose = 1.6 mg/lb. 5

Zero-orderrelease rate(mg/lb/hr) Time for 80% release ofcarprofen from dosage form L (hr) ; ' Duration (hr) for which plasma carprofenconcentration is above 10 pg/mL 0.067 19.2 11 0.080 16 13 0.100 12.8 12.5 0.135 9.6 1Ï.5 0.2 6.4 ïï 0.27 4.8 Ï0 0.8 1.6 9 TABLE 9.

Number of hours for which carprofen plasma concentrations are above 10 μg/mL, after10 dosing with controlled release dosage forms which release carprofen at various rates (computed). Dose =1.4 mg/lb.

Zero-orderrelease rate(mg/lb/hr) Time for 80% release ofcarprofen from dosage form(hr) Duration (hr) for which plasma carprofenconcentration is above 10 pg/mL 0.059 19.2 0 0.07 16 9 0.088 12.8 10.5 0.115 9.6 10 0.175 6.4 9.5 0.23 4.8 8.5 0.7 1.6 8 15 EXAMPLE 6

Carprofen implants for dogs.

Carprofen implants are useful for delivery of carprofen over extended time periods,e.g., 3 days, 7 days, 30 days, and so forth. This below-detailed example describes usefuland preferred carprofen release rates from implants containing carprofen, and also defines 20 doses associated therewith.

The input rate of carprofen into the canine body which would provide a steady Stateplasma carprofen concentration of 10 pg/mL was calculated using the following équationfrom Gibaldi, M. and D. Perrier, “Pharmacokinetics", 2nd ed. Drugs and the PharmaceuticalSciences, ed. J. Swarbrick, Vol. 15,1982, New York: Marcel Dekker, Inc.: -66- c. 011213

Ro_

Cl where Css is the steady State plasma carprofen concentration, Ro is the input rate, and Cl isthe systemic clearance. The systemic clearance was estimated from the pharmacokineticsobserved after oral administration of 2 mg/lb carprofen to dogs, using the équation:

Cl =

FD

AUC where F is the bioavailability (assumed to be 1 for an implant), D is the oral dose, and AUCis the average area under the plasma carprofen concentration vs. time curve, extrapolated toinfinity. A clearance of Cl = 5 mL/hr per Ib body weight was obtained. Using this clearance,and a target Css of 10 pg/mL, a carprofen release rate Ro of 50 pg/lb/hr was catculated,which upon multiplication by 24 hr/day gives a daily input rate of 1.2 mg/lb/day. A total doseof 3.6 mg/lb or 8.4 mg/lb or 36 mg/lb would be required for 3,7, or 30 day therapy,respectively. In summary, an implant containing 8.4 mg carprofen per Ib body weight,releasing carprofen at a rate of 50 pg per Ib of body weight per hr, maintains a plasmacarprofen concentration of 10 pg/mL for 7 days. This will maintain 80% COX-2 inhibition for7 days.

To obtain the implant dose and carprofen reiease rate required to maintain a plasmacarprofen concentration of 2 pg/mL (50% COX-2 inhibition), the dose and release rate for a10 pg/mL target are linearly adjusted by multiplication by 0.2. Thus for a target plasmacarprofen concentration of 2 pg/mL, a carprofen release rate of 10 pg/lb/hr is needed, or0.24 mg/lb/day. A total dose of 0.72 mg/lb or 1.68 mg/lb or 7.2 mg/lb would be required for3, 7, or 30 day therapy, respectively,

To obtain the implant dose and carprofen release rate required to maintain a plasmacarprofen concentration of 20 pg/mL (90% COX-2 inhibition), the dose and release rate for a10 -pg/mL target are linearly adjusted by multiplication by 2. Thus for a target plasmacarprofen concentration of 20 pg/mL, a carprofen release rate of 100 pg/lb/hr is needed, or2.4 mg/lb/day. A total dose of 7.2 mg/lb or 16.8 mg/lb or 72 mg/lb would be required for 3, 7, or 30 day therapy, respectively.

Useful carprofen implants of the présent invention release carprofen into the caninebody at a rate of 0.24 mg/lb/day or greater.

Preferred carprofen implants of this invention release carprofen into the canine bodyat a rate of 0.24 to 1.2 mg/lb/day. -67- 011213

More preferred carprofen implants of this invention release carprofen into thecanine body at a rate of 1.2 to 2.4 mg/lb/day.

Useful carprofen implants hâve a total carprofen dose up to 2 gm, limited by the sizeof an implant which can be reasonably administered to a dog. Of course, more than oneimplant may be administered at the same time. EXAMPLE 7

Resolution of (S)-6-chloro-a-methyl-carbazole-2-acetic acid A solution of 4.3 g of (R) - a-methylbenzylamine in 20 mL of acetone was added to asolution of 9.7 g of partially resoived 6-chloro-a-methyl-carbazole-2-acetic acid (recoveredfrom filtration of a previous résolution of the racemate). After standing at room températurefor 24 hrs, the mixture was filtered and the filter cake was washed with cold acetone to yieldafter drying 7.3 g. Following two additional recrystallizations from acetone, 1.9 g of (S)- 6-chloro-a-methyl-carbazole-2-acetic acid (R)- α-methylbenzylamine sait, [a],,22 - 13.6° wasobtained. Further recrystallizations from acetone did not change the rotation. The sait wasdissolved in 50 mL of warm acetone and the solution after filtration was poured into 500 mLof dilute hydrochloric acid. Following filtration and drying, 1.4 g was obtained, which uponcrystallization from chloroform gave 0.9 g. of (S)- 6-chloro-a-methyl-carbazole-2-acetic acid,m.p. 198° - 201° , ία]η22 + 53.2°, (c 1.33, CH3OH). EXAMPLE 8

Species specificity of COX-2 selectivity: activity in members of the species Canis familians .(dogs) compared to activity in members of the species Rattus norvegicus (white rats) and inHomo sapiens (humans)

The very high degree of COX-2 selectivity exhibited by carprofen in dogs hasalready been amply demonstrated in Example 1. Equally surprising was the discovery thatthis sélective inhibition of the COX-2 enzyme appears to be an activity which is spécifie tothe species Canis familians, and not shared by other species. This discovery was based onthe évaluation of the inhibitory activity of racemic carprofen in members of the speciesRattus norvegicus (white rats) and in members of the species Homo sapiens (humans).

In vivo cyclo-oxygenase selectivity was evaluated in rats by the method of Griffithsétal., described in Agents &amp; Actions, 32, (1991), 313-320. The COX-2 inhibitory activity wasevaluated in accordance with the effect of racemic carprofen on prostaglandin PGE2production as measured in the synovial fluid of the rat. Synovial fluid is secreted by thesynovial membrane and is contained in joint cavities. During joint inflammation, COX-2 isinduced in joint tissues and prostaglandin products accumulate in the synovial fluid. The -68- 011213 OOX-1 inhibitory activity was evaluated in accordance with the effect of racemic carprofenon prostaglandin PGE2 production as measured in the mucosal lining of the rat stomach,which contains significant amounts of the constitutive COX-1 isozyme. Inhibition of thisstomach isozyme results in adverse gastrointestinal side effects. The COX-1 ED50 was 6.4 5 mg/kg, while the COX-2 ED50 was 0.63 mg/kg. These results indicate that in rats, there isonly 10-fold selectivity for the COX-2 isozyme by racemic carprofen.

For humans, the COX-2 inhibitory activity was evaluated in accordance with theeffect of racemic carprofen on levels of COX-2 in human umbilical vein endothélial cells(HUVEC) stimulated by IL-1 and phorbol myristate acetate (PMA) in accordance with the 10 method of Habib et al. described in J. Biol. Chem., 268, 23448-23454, 1993. Theseendothélial cells under the stimulation of interleukin-1 (IL-1 ) and PMA are most likely tocontain significant amounts of the inducible COX-2 isozyme. The COX-1 inhibitory activitywas evaluated in accordance with the effect of racemic carprofen on levels of COX-1 asmeasured by a human washed platelets (HWP) TXB2 biochemical assay, in accordance with 15 the procedures of Grossman, ef al. described in Inflamm. Res., 44, 253-257, 1995. Theseplatelets are most likely to contain significant amounts of the constitutive COX-1 isozyme.The HUVEC (COX-2) IC50 (μΜ) was 1.20, while the HWP TXB2 (COX-1) IC50 (μΜ) was 0.77.These results indicate that in humans, there is no selectivity for the COX-2 isozyme byracemic carprofen. 20 EXAMPLE 9

Tablet formulation of (S)- 6-chloro-a-methyl-carbazole-2-acetic acid

Tablet Formulation

Inaredients Weiaht Der Tablet ( S)- 6-chloro-a-methy l-carbazole-2-acetic acid 25.00 mg Lactose, Ü.S.P. 64.50 mg Com Starch 10.00 mg | Magnésium Stéarate 0.50 mg 25 -69- 011213 EXAMPLE 10

Capsule formulation of (S)- 6-chloro-a-methyl-carbazole-2-acetic acid

Capsule Formulation ingrédients Weiaht nerCaosuie (S)- 6-chloro-a-methyl-carbazo!e-2-ace.tic acid Lactose, Ü.S.P. Corn Siarch, U.S.P. Talc, U.S.P. 50 mg 124 mg 30 mg 5 mg Total Weight 210 mg EXAMPLE 11

Parentéral formulation of (S)- 6-chloro-a-methyi-carbazole-2-acetic acid

Parentéral Formulation

Ingrédients oer 1 cc airiDule Weiaht oer Ameute ( S)- 6-chloro-a-methy l-carbazole-2-acetic acid 10,2 mg Methyl Paraben, U.S.P. 1.8 mg Propyl Paraben, U.S.P. 0.2 mg Sodium Hydroxide, U.S.P. q.s. ph 9.0 mg Water for Injection, U.S.P. q.s. ad 1.0 cc

Claims (3)

1. 70 011213 WHAT ÎS CLAIMED 15:

   1. A pharmaceutical composition for treating or preventing inflammatoryprocesses and diseases as in Claims^, 3 and ^further comprising wherein said inhibitorycompound is used in combination with one or more therapeutically active agents underthe folio wing conditions. A. where a joint has become seriously inflammed as well as infected at the sametime by bacteria, fungi, protozoa, and/or virus, said inhibitory compound is administered incombination with one or more antibiotic. antifungal, antiprotozoal, and/or antiviral therapeuticagents; B. where a muiti-fold treatment of pain and inflammation is desired. said inhibitorycompound is administered in combination with inhibitors of other mediators of inflammation,comprising one or more members independently selected from the group consistingessentially of:

   1. NSAIDs; 2. H, -receptor antagonists; 3. kinin-B·, - and B2-receptor antagonists; 4. prostaglandin inhibitors selected from the group consisting of PGD-, PGF-PG!2 -, and PGE-receptor antagonists; 5. thromboxane A2 (TXA2-) inhibitors; 6. 5-and 12-lipoxygenase inhibitors; 7. leukotriene LTC4 -, LTD4/LTE4 -, and LTB4 -inhibitors;
2. 8. PAF-receptor antagonists; 9. gold in the form of an aurothio group together with one or more hydrophilic groups; 10. immunosuppressive agents selected from the group consisting ofcyclosporine, azathioprine, and methotrexate; 11. anti-inflammatory glucocorticoids; 71 011213 12. peniciilamine; 13. hydroxychloroquine; 14. anti-gout agents inciuding coichicine: xanthine oxidase inhibitors includingallopurinol; and uricosuric agents selected from probenecid, sulfinpyrazone, andbenzbromarone; C. where older dogs are being treated for disease conditions, syndromes andsymptoms found in gériatrie dogs, said inhibitory compound is administered in combinationwith one or more members independently selected from the group consisting essentially of: 1. cognitive therapeutics.to counteract memory loss and impairment; 2. anti-hypertensives and other cardiovascular drugs intended to offset theconséquences of atheroscierosis, hypertension, myocardial ischemia, angina, congestiveheart faiiure, and myocardial infarction, selected from the group consisting of: a. diuretics; b. vasodilators; c. β-adrenergic receptor antagonists; d. angiotensin-ll converting enzyme inhibitors (ACE-inhibitors), alone oroptionsily together with neutral endopeptidase inhibitors; e. angiotensin 11 receptor antagonists; f. renin inhibitors; g. calcium channel blockers; h. sympatholytic agents; i. a2-adrenergic agonists; j. α-adrenergic receptor antagonists; and k. HMG-CoA-reductase inhibitors (anti-hypercholester-olemics); 3. antineoplastic agents selected from: a. antimitotic drugs selected from: i. vinca alkaloids selected from: [1] Vinblastine, and [2] vincristine; 4. growth hormone secretagogues; 5. strong analgésies; 6. local and systemic anesthetics; and 7. H2 -receptor antagonists, proton pump inhibitors, and other gastroprotective agents. 011213 u

   2, A pharmaceuticai composition for treating or preventing pain and inflammatoryprocesses and diseases associated with the activity of inducibie cyclo-oxygenase-2 (COX-2)in a member of the species Canis familiaris in need of such treatment, whiie at the same timereducing or eiiminating undesirable side effects associated with simultaneous inhibition of theactivity of constitutive cyc!o-oxygenase-1 (COX-1 ), comprising: A. a therapeuiically effective amount for treating pain and inflammation, of an anti-inflammatory sélective COX-2 inhibitory compound which selectively inhibits COX-2 activitywith reference to COX-1 activity, wherein the selectivity ratio of COX-2 : COX-1 activityinhibition is at least 3 : 1 based on ex vivo inhibition levels in whole blood measured at a dosegiving > 80% COX-2 inhibition, comprising an anti-infiammatory sélective COX-2 inhibitorycompound comprising a compound of the formula:

   wherein: X t c LŸ_J R2 is· o 11 C-A n where A is hydroxy, (C, - C4)alkoxy, amino, hydroxyamino, mono-iC, -C2)aikylamino, di-(C, -C2)alkylamino; X and Y are independently H or (C, - C2)alkyl; and n is 1 or 2; R6 is halogen, (C.| - C3)alkyI, trifiuoromethyi, or nitro; R9 is H; (Ct - C2)aikyl; phenyl or phenyl-(C, - C2)alkyl, where phenyl is optionaliy mono-substituted by fiuoro or chioro; -C(=O)-R, where R is (C, - C2)alkyt or phenyl.optionaliy mono-substituted by fiuoro or chioro; or -C(=O)-O-R’ . where R1 is (C, -C2)alkyl; where X and Y are different, the (-)(/?) and (+)(S) enantiomers thereof; and ailpharmaceutically acceptable sait forms, prodrugs and métabolites thereof which arethe râpe uticaily active for treating or preventing pain and inflammation; and 73 011213 B. a pharmaceutically acceptable carrier therefor. 3· A pharmaceutical composition according to ClaimjL wherein said anti-inflammatorysélective COX-2 inhibitory compound is carprofen, 6-chloro-a-methyl-9/-/-carbazole-2-aceticacid. ψ. A pharmaceutical composition according to Claiml wherein said anti-inflammatorysélective COX-2 inhibitory compound is comprised entirely of (S)-enantiomer of carprofen, 6-chloro-a-methyi-9/-/-carbazole-2-acetic acid. 5”. A pharmaceutical composition as in Claimsi, 3. or further comprising whereinsaid anti-inflammatory sélective COX-2 inhibitory compound is provided in a dosage formsuitable for systemic administration by: A. injection or infusion in suitable liquid form which is intraarterial, intra- ortransdermal, subcutaneous, intramuscular, intraspinai, intrathecal, or intravenous, whereinsaid inhibitory compound: 1. is contained in solution as a soluté; 2. is contained in the discontinuous phase of an émulsion, or thediscontinuous phase of an inverse émulsion which inverts upon injection or infusion, saidémulsions containing suitable emuisifying agents; or . 3. is contained in a suspension as a suspended solid in colloïdal ormicroparticulate form, said suspension containing suitable suspending agents; B. injection or infusion into suitable body tissues or cavities as a depot, wherein saidcomposition provides storage of said inhibitor and thereafter delayéd-, sustained-, and/orcontrolled-release of said inhibitory compound for systemic distribution; C. instillation, inhalation or insufflation into suitable body tissues or cavities insuitable solid form, where said inhibitory compound: 1. is contained in a solid implant composition providing delayed-, sustained-,and/or controlled-release of said inhibitory compound; 2. is contained in a particulate composition to be inhaled into the lungs; or 3. is contained in a particulate composition to be blown into said suitable bodytissues or cavities, wherein said composition optionally provides delayed-, sustained-, and/orcontrolled-release of said inhibitory compound; or 01 121 3 7l D. ingestion in suitable soiid or liquid form for pérorai delivery of said inhibitorycompound, where said inhibitory compound: 1. is contained in a soiid dosage form; or 2. is contained in a liquid dosage form.

   6. A pharmaceutical composition according to Claim 5 wherein said dosage formscomprise one or more members selected independently from the group consisting essentiallyof suppositories; soiid pérorai dosage forms selected from the group consisting of delayed-release tablets, capsules, caplets, lozenges, troches, and muitiparticulates; enteric-coatedtablets and capsules which prevent release and absorption in the stomach of said memberbeing treated to faciiitate delivery distal to the stomach of said member; sustained-reiease oraltablets, capsules and microparticulates which provide systemic delivery of said inhibitor in acontrolled manner over at least a 10-hour period; a chewable or ingestible oral tablet: a unitdose packet sachet, a suspension made from said unit dose packet sachet, a powder for oralsuspension, or an oral suspension perse; a fast-dissolving tablet; encapsulated solutions: anoral paste; à granular form incorporated in or to be incorporated in said member's food; and apalatable chewable form in which said inhibitor is consumed along with said palatabiechewable form, or is deiivered by ieaching from said chew, which is not consumed. duringmastication by said member being treated; iiquid pérorai dosage forms selected from thegroup consisting of solutions, suspensions, émulsions, inverse émulsions, élixirs, extracts,tinctures, and concentrâtes; and the above-recited soiid dosage forms contamingmicroencapsulated formulations of the active ingrédient, which is incorporated into said soiiddosage form.

   7 . A pharmaceutical composition as in Claim 6 comprising an oral controlled releasecarprofen dosage form able to maintain plasma carprofen levels above approximately 10pg/mLfor a period of time greater than 10.5 hours, when administered at a dose of about 2mg/lb or less.

   3, A pharmaceutical composition as in Claims2, 5, or 4-further comprising said anti-infiammatory sélective COX-2 inhibitory compound in combination with one or more othertherapeutically active agents independently selected from the group consisting essentially of: A. anti-infectious agents comprising one or more antibiotic, antifungal,antiprotozoal, or antiviral therapeutic agents; 011213 75* B. inhibitors of other mediators of inflammation, comprising one or moremembers independently selected from the group consisting essentially of:

   1. NSAlDs; 2. H, -receptor antagonists; 3. kinin-B, - and B2 -receptor antagonists; 4. prostaglandin inhibitors selected from the group consisting of PGD-, PGF-PGl2 -, and PGE-receptor antagonists; 5. thromboxane A2 (TXA2-) inhibitors; 6. 5- and 12-iipoxygenase inhibitors; 7. ieukotriene LTC« -, LTD4/LTE4 -, and LTB4 -inhibitors;

   8. PAF-receptor antagonists; 9. gold in the form of an aurothio group together with one or more hydrophiiic oroups; 10. immunosuppressive agents selected from the group consisting ofcyclosporine, azathioprine, and methotrexate;· 11. anti-inflammatory glucocorticoids; 12. peniciliamine; 13. hydroxychloroquine; 14. anti-gout agents inciuding colchicine; xanthine oxidase inhibitors includingallopurinol; and uricosuric agents selected from probenecid, sulfinpyrazone, andbenzbromarone; C. therapeutic agents for the treatment of gériatrie dogs comprising one or moremembers independently selected from the group consisting essentially of: 1. cognitive therapeutics to counteract memory loss and impairment; 2. anti-hypertensives and other cardiovascuiar drugs intended to offset theconséquences of atherosclerosis, hypertension, myocardial ischemia, angina, congestiveheart faiiure, and myocardial infarction, selected from the group consisting of: a. diuretics; b. vasodilators; c. β-adrenergic receptor antagonists; d. angiotensin-ll converting enzyme inhibitors (ACE-inhibitors), alone oroptionally together with neutral endopeptidase inhibitors; e. angiotensin II receptor antagonists; f. renin inhibitors; 76 011213 g. calcium channel blockers; h. sympatholytic agents; i. a2-adrenergic agonists; j. α-adrenergic receptor antagoniste; and k. HMG-CoA-reductase inhibitors (anti-hypercholester-olemics); 3. antineoplastic agents selected from: a. antimitotic drugs selected from:i. vinca alkaloids selected from: [1] Vinblastine, and [2] vincristine; 4. growth hormone secretagogues; 5. strong analgésies; 6. local and systemic anesthetics; and 7. H2 -receptor antagonists, proton pump inhibitors, and other gastroprotective agents. Ç[, A package suitable for use in commerce for the therapeutic treatment orprévention of pain and inflammation processes and diseases in a member of the speciesCanis famiiïaris in need of such treatment, comprising: A. a suitable container optionally in the form of an outer package and an innercontainer removably housed therein; B. a suitable dosage form, enclosed in said container, of an anti-inflammatorysélective COX-2 inhibitory compound of the formula:

   wherein: R2 is- x t •c I _Y_J o li C-A n 77 011213 where A is hydroxy, (C, - C4)alkoxy, amino, hydroxyamino, mono-(C, -C2)alkylamino, di-ÇC, -C2)alkylamino; one of X and Y is H and the other is (C, - C2 )alkyl; and n is 1 or 2; R6 is halogen, (C, - C3)alkyi, trifluoromethyi, or nitro; R9 is H; (C, - C2)alkyl; phenyl or phenyl-(C, - C2)alkyI, where phenyi is optionally mono- substituted by fluoro or chioro; -C(=O)-R, where R is (C, - CJalkyl or phenyl,optionally mono-substituted by fluoro or chioro; or -C(=O)-O-R’ , where R' is (C, -C2)alkyl; wherein (+)(S) enantiomer is présent in amount of at least 75%; and ail pharmaceuticallyacceptable sait forms, prodrugs and métabolites thereof which are therapeuticaily active fortreating or preventing pain and inflammation; and C. printed instructions! and informational material associated with said container,which is attached to said container, enclosed in said container, or displayed as an intégral partof said container, said instructional and informational material stating in words which convey toa reader thereof of ordinary skiil in the art that said compound of Formula (!) comprising atherapeutic agent contained in said package, yvhen administered to said member of thespecies Canis famiiiaris to be treated, effectively inhibits cyclo-oxygenase-2 (COX-2) inducedat an existing or expected site of pain and inflammation in said dog, thereby treating orpreventing said pain and inflammation which would otherwise resuit therefrom. while at thesame tïme reducing or eliminating undesirable side effects associated with simultaneousinhibition of the activity of constitutive cyclo-oxygenase-1 (COX-1) by selectively inhibitingCOX-2 activity with référencé to COX-1 activity, wherein the selectivity ratio of COX-2 ; COX-1activity inhibition is at least 3 ; 1 based on ex vivo inhibition levels in whole blood measured ata dose giving > 80% COX-2 inhibition. lo.. A package according to Claim wherein said anti-infiammatory sélective COX-2inhibitory compound of Formula (I) comprises carprofen, 6-chloro-a-methyl-9H-carbazole-2-aceticacid.
3. 11. A package according to Claim wherein said anti-inflammatory sélective COX-2inhibitory compound is comprised entirely of (S)-enantiomer of carprofen, 6-chloro-a-methyl-9H-carbazole-2-acetic acid.