SK148199A3 - 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

Technical field

The present invention relates to the treatment of pain and inflammation in dogs with anti-inflammatory drugs which are non-steroidal anti-inflammatory drugsNSAIDs, and in particular with those which have a reduced incidence of adverse gastrointestinal side effects, since these side effects are widespread and are widespread potentially a serious problem in dogs.

Dogs, especially older dogs, are inflammatory processes such as

BACKGROUND OF THE INVENTION

As one skilled in the art, e.g. veterinarians, well known canines, i.e. It is very susceptible to chronic degenerative joint disease. Due to the very large number of dogs that are kept as pets or for utility purposes, such as guard dogs and guide dogs for the blind, there is a continuing effort to find pharmaceutical preparations that prevent or completely stop the progression of such inflammatory disease processes in dogs or at least alleviate symptoms. inflammation such as pain and swelling. One group of these pharmaceutical formulations that has been extensively investigated for anti-inflammatory and analgesic use in humans and currently in dogs is the group of non-steroidal anti-inflammatory drugs (NSAIDs). Over the past decade, this type of anti-inflammatory drug has been extensively investigated and has now been discovered and developed for use in humans by new improved formulations of this type.

However, the use of NSAIDs in dogs has been more limited, e.g. only two of these NSAIDs have been approved by the Food and Drug Administration, Committee on Veterinary Medicine (FDA / CVM), for use in dogs in the United States, i. j. ARQUEL®, meclofenamic acid, and RIMADYL®, carprofen. Therefore, veterinary medicine has less experience and knowledge about the safety and efficacy of NSAIDs in dogs. For example, the most common indication for treating NSAIDs in veterinary medicine is degenerative joint disease (DJD), which in dogs is often the result of a variety of developmental diseases, e.g. hip dysplasia and osteochondrosis, as well as traumatic joint injuries. In addition to treating chronic pain and inflammation, NSAIDs in dogs are also useful for the treatment of acute pain after surgery and for the treatment of clinical symptoms associated with osteoarthritis.

This requirement for the treatment of dogs with NSAIDs, coupled with the lack of any NSAIDs approved for this purpose, resulted in a significant unclassified use of human-approved NSAIDs in dogs, sometimes with adverse consequences. Veterinary medicine literature is full of publications on gastrointestinal bleeding, perforation and peritonitis in dogs in conjunction with NSAIDs that have been approved for use in humans such as indomethacin, naproxen, aspirin, ibuprofen and phenylbutazone. Although these gastrointestinal adverse reactions also affect human patients, dogs often receive inappropriately high doses because of lack of correct dosage information and dogs have an innate high susceptibility to these gastrointestinal adverse reactions. Accordingly, there is an urgent need for safe yet effective NSAIDs for the treatment of pain and inflammation in dogs.

While the search for safe and effective NSAIDs in dog therapy must address the possibility of serious adverse gastrointestinal reactions, other adverse reactions include toxic effects on the kidney and liver. However, the most serious of adverse reactions are gastrointestinal effects, such as single or multiple ulcerations, including gastrointestinal, gastric, duodenal or small and large intestine bleeding. These adverse reactions are usually weak, but can often be severe and occasionally even life-threatening. Indeed, the therapeutic index using NSAIDs in dogs can be so low that such treatment can be contraindicated.

The term therapeutic index is often generally defined as the ratio of LD ₅₀ to ED 50 of a drug and indicates how selective a drug is to exert its desired effect. However, as used herein, the term therapeutic index corresponds more to the definition used in the veterinary field, which is the ratio of the maximum dose tolerated by the animal to the minimum dose effective in the animal. In the present invention, the term animal refers, of course, to dogs. The maximum tolerated dose in a particular canine patient will typically be determined by a variety of assays and techniques. For example, gastrointestinal bleeding can be detected by tests generally used to detect occult bleeding in stool samples, while endoscopy can be used to detect ulcerations or perforations. When an animal is euthanized as part of a study, autopsy may also provide valuable information.

Therefore, it has been hitherto expected in the art that each NSAIDs particularly suited to the treatment of dogs will have a low therapeutic index. It has always been hoped that the therapeutic index will not be so low that the drug becomes unacceptable for use in dogs. Thus, an important aspect of the present invention was the surprising discovery that the anti-inflammatory agents described herein have an extremely high therapeutic index when used to treat pain and inflammation in dogs, and further that this unexpected property of said anti-inflammatory agents is quite unique, actually excluding all other NSAIDs. .

In recent years, a significant body of knowledge has accumulated in terms of the mechanism of action by which NSAIDs achieve their anti-inflammatory treatment outcomes and whether they also cause serious gastrointestinal adverse reactions. Although much of this knowledge has been gathered with respect to the mechanism of NSAIDs in humans, it is largely applicable to explain the mechanism of action in dogs, although there is apparently some species specificity, as detailed below. With respect to the therapeutic efficacy of NSAIDs, it has long been known that the mechanism by which NSAIDs reduce inflammation is the ability to disrupt the arachidonic acid cascade that leads to endogenous production of prostaglandins, thromboxanes and leukotrienes. These lipid compounds are collectively called eicosanoids because they are commonly derived from C2o ^- polyunsaturated fatty acids, the eicosenoic acids, the most abundant of which is arachidonic acid. Arachidonic acid, cis-Δ ⁵ , cis-Δ ⁸ , cis-Δ ¹¹ , cis-Δ ¹⁴ eicosatetraenoic acid, is a major precursor of many prostaglandins and leukotrienes, which are mediators of inflammation.

In the first stage of the arachidonic acid cascade, it is released as a result of tissue-specific stimuli through hormones or proteases or membrane disruption, and involves the action of a specific phospholipase A ₂ . The result is free arachidonate, which is treated in the second stage of the cascade by a bifunctional enzyme prostaglandin peroxoperoxide synthase, also called prostaglandin-H-synthase (hereinafter PGH-synthase), the first activity being cyclooxygenase and the second activity resulting in a reduction of two electrons. Most NSAIDs act as an inhibitor of the cyclooxygenase activity of PGH-synthase, thereby blocking the production of various prostaglandins, which are locally acting hormones, which perform their functions through binding to specific cell receptors. Prostaglandins are very effective, but they also catabolize rapidly. Some of these prostaglandins are mediators of the inflammatory process, but some also have a protective function of the gastrointestinal tract. Blockage of the production of these beneficial prostaglandins is one of the major factors contributing to adverse gastrointestinal reactions that occur with the use of NSAIDs. Accordingly, there continues to be a search for pharmaceutical compositions which, although acting as cyclooxygenase inhibitors, have, due to some other mode of action or otherwise, a substantially reduced incidence of gastrointestinal adverse reactions and resulting side effects.

More recently, it has been discovered that cyclooxygenase (COX) has two isozymes, a constitutive enzyme (COX-1) and an inducible enzyme (COX-2), which have different systems in different systems, in humans and indeed in all other mammalian species studied. activity. The identification of the COX-2 isozyme has previously led to the conclusion that it could be responsible for the production of prostaglandins exclusively or primarily at sites of inflammation. It has now been shown that this is the case, the selective inhibition of the COX-2 isozyme reduces inflammation without the side effects of gastrointestinal toxicity. COX-1 and COX-2 are 60% homologous, have similar K _m values and the same binding sites on arachidonic acid, but COX-2 adopts a wider range of substrates than COX-1.

Another metabolic pathway leads from the above arachidonate to the formation of leukotrienes through the action of lipoxygenase. Some of these leukotrienes are also mediators of inflammation, and therefore great efforts have been made to find pharmaceutical preparations that are double inhibitors of both cyclooxygenase and lipoxygenase.

The specific NSAID that has been used to treat canine inflammatory diseases, and the only one of the two approved to date for use in the United States by the Food and Drug Administration, the Committee on Veterinary Medicine (FDA / CVM), is the above carprofen. Carprofen, racemic 6-chloro-α-methylcarbazole-2-acetic acid, belongs to the arylpropionic acid family of NSAIDs. Other members of this group are eg.

benoxaprofen, cycloprofen, phenoprofen, flurbiprofen, furaprofen, indoprofen, ketoprofen, pirprofen and surprofen. While these compounds are structurally closely related, they may, however, have different anti-inflammatory and other biological properties. For example, carprofen has been shown to be a relatively weak cyclooxygenase inhibitor, but it has also been shown to significantly reduce pain and swelling and other signs of inflammation in humans and in various animal models. These carprofen evaluations are described in the technical literature, which is partially listed and discussed below. Carprofen has also been shown to be inactive in rats with respect to lipoxygenase and is not likely to block the production of leukotrienes. Although it appears that the mode of action of carprofen is still unknown, it has been shown to be to some extent active against phospholipase Ä ₂ .

The current state of the art, as shown in the conclusions of the references discussed below, is largely disorganized in attempts to explain in dogs the mechanism of action of carprofen by which carprofen is capable of having good anti-inflammatory activity, while exhibiting small undesirable gastrointestinal and other reactions. In the prior art, carprofen is characterized as having little to no activity as a cyclooxygenase inhibitor and it has been concluded that it must therefore act by some completely different mechanism.

As mentioned above, it has recently been reported that there are isozymes, constitutive COX-1 and inducible COX-2, with different roles in protecting the gastrointestinal mucosa and mediating inflammation. This naturally led to the investigation of compounds and the search for any compound which, when used in dogs as well as in other animals and humans, could only inhibit the inducible COX-2 isozyme, i. would be a selective COX-2 inhibitor. These investigations mainly concerned the evaluation of the inhibitory activity of the enantiomers of various NSAIDs, including in particular ketoprofen, ketorolac and flurbiprofen. Taking these particular

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NSAIDs were investigated for the difference in inhibitory potential in terms of one enantiomer versus the other and also the inhibitory potential when examined for each enantiomer separately against COX-1 and compared with the inhibitory potential against COX-2. The results of these investigations showed that in all three NSAIDs the enantiomers were equally effective against both COX-1 and COX-2. Thus, neither the R- nor the S-enantiomer of either of these NSAIDs showed selectivity for COX-1 or COX-2. While there were indeed differences in efficacy between enantiomers, with the S-enantiomer being in any case more effective in inhibiting COX-1 versus COX-2, each enantiomer showed the same inhibitory potential, i.e., the same. no enantiomer was able to distinguish between the two isozymes.

Therefore, as a result of this prior art research, the state of the art is such that neither carprofen nor any other drug from classical NSAIDs having a carboxylic acid group was considered a selective COX-2 inhibitor in dogs or any other animal species. These conclusions have been strengthened by revealing the state of the art with respect to the conformation of the sequenced structures of the COX-1 isozyme and the COX-2 isozyme when coupled to various inhibitors at the level of their basic functional molecular configuration. As a result of these published studies, it is now learned in the art that inhibitors containing a carboxylic acid group such as carprofen are substantially unable to be selective COX-2 inhibitors. Until now, only compounds containing a sulfonyl group and the compound nabumetone, naphthalenyl-2-butanone have been reported as selective inhibitors of the COX-2 isozyme.

Recently, studies using various animal species have been conducted in the art in connection with the above discovery of the constitutive COX-1 and inducible COX-2 isozyme to investigate whether there is any stereoselective inhibition specific to one or more of said species. These studies of the individual activity of R- and Senantiomers of certain NSAIDs to inhibit COX-1 versus COX-2 in different animal species revealed that there was a consistent difference in the efficacy of the R- and S-enantiomers in all chiral NSAIDs examined. However, these studies also reported that neither the R- or S-enantiomer of any of the investigated chiral NSAIDs was species-specific selective for COX-1 versus COX-2. For example, it has been shown that while the S-enantiomer is e.g. three times more potent than the R-enantiomer with respect to both COX-1 and COX-2 isozymes in a given species, and with respect to these isozymes, the S-enantiomer also shows an effective inhibition of both COX-1 and COX-2 isozymes, i. The S-enantiomer does not show selective inhibition of COX-2 in a given species.

In view of the prior art described above, especially when the animal species of interest were dogs, it was quite unexpected that, according to the present invention, carprofen was found to be a surprisingly potent inhibitor of the dog's COX-2 isozyme and further selective COX-2 inhibitor in the dog. dog. Moreover, the selectivity of carprofen over COX-2 is more than twice that of virtually all other NSAIDs, including NSAIDs containing a carboxylic acid group and reportedly COX-2 selective NSAIDs containing a sulfonyl group. The selection of carprofen as an excellent selective inhibitor of COX-2 isozyme in dogs from all other NSAIDs is directed not only against the current state of the art, but is also an unexpected discovery in terms of surprising results. The fact that all of the other NSAIDs evaluated here have been approved for administration to humans in the United States, including those that are now commercially available, adds confidence in the accuracy of these conclusions. Carprofen dominates all other NSAIDs evaluated here, including those that have been approved for human use or for evaluation in clinical trials.

In view of the prior art described above, especially when the animal species of interest were dogs, it was further unexpected that, according to the present invention, the carprofen Senantiomer was found to be a highly selective inhibitor of the COX-2 isozyme versus the COX-1 isozyme in the dog. significantly higher than any other NSAIDs or Senantiomers thereof including NSAIDs containing a carboxylic acid group and allegedly COX-2 selective NSAIDs containing a sulfonyl group. The selection of the S-enantiomer of carprofen as an excellent and selective inhibitor of COX-2 isozyme in dogs, which simultaneously exhibits little or no adverse gastrointestinal or other reactions in dogs, from all other NSAIDs, is directed not only against the current state of the art but also a rather unexpected discovery in terms of surprising results. The fact that all of the other NSAIDs evaluated here have been approved for administration to people in the United States, including those that are now commercially available, adds confidence in the accuracy of these conclusions.

In view of the prior art described above, especially when the animal species of interest were dogs, it was further unexpected that the present invention has found that the carprofen Senantiomer has surprisingly improved anti-inflammatory levels due to its highly selective COX-2 isozyme inhibitor. , analgesic and antipyretic activity compared to all other NSAIDs having a carboxylic acid group or their S-enantiomers, and also has a surprisingly low degree of undesirable gastrointestinal or other reactions compared to all other NSAIDs including a carboxylic acid group or their S-enantiomers .

As noted, carprofen belongs to the arylpropionic acid family of NSAIDs, and is also a member of a subgroup of these compounds, which are substituted carbazolacetic acids.

These compounds and their use as anti-inflammatory, analgesic, and anti-rheumatic agents are described in U.S. Patent 3,896,145. The anti-inflammatory activity of carprofen in dogs has been investigated by McKellar et al. (and published in Pharmacokinetics, Tolerance and Serum Thromboxane Inhibitor 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 both carprofen and racemic carprofen in dogs were further investigated by McKellar et al. (and published in Stereospecific Pharmacodynamics and Pharmacokinetics of Carprofen in the Dog, J. Vet. Pharmacol. Therap., 17, 447-454, 1994). In the publication of the research results, the authors concluded that: the mode of action of CPF [carprofen] remains unknown. ... CPF administered as a racemic mixture or as either (-) (R) - or (+) (S) - enantiomer did not significantly inhibit the formation of TxB ₂ in blood or PGE ₂ and 12-HETE in the inflammatory exudate, suggesting that that it does not act as a conventional NSAID.

It is known that the main mode of action of most NSAIDs is the inhibition of the cyclooxygenase enzyme in the formation of inflammatory prostaglandins from arachidonic acid.

... a large number of mediators now known to be involved in inflammatory processes provide many possible targets for anti-inflammatory drugs and CPF is likely to have its main activity targeting one or more mediators that have not yet been identified .

Thus, these experts concluded that the mode of action of carprofen cannot be primarily attributed to the inhibition of cyclooxygenase and, in fact, the lack of activity of carprofen against cyclooxygenase, lipoxygenase and phospholipase suggests that its main mode of action may be a mechanism other than eicosanoid inhibition. These findings and conclusions pointed other experts in the art away from the surprising discovery of the present invention that carprofen administered to dogs exhibits almost complete inhibition of PGE 2 synthesis, as shown below in the example below.

Another study of carprofen in dogs was published by Holtsinger et al. (in The Therapeutic Efficacy of Carprofen (Rimadyl-V ™) in 209 Clinical Cases of the Canine Degenerative Joint Disease, V.C.O.T., 5, 140-4, 1992). The authors have suggested from in vitro studies that carprofen can exert its anti-inflammatory effect at least in part by inhibiting neutrophil migration and that it could explain how carprofen can be as effective as indomethacin as an anti-inflammatory agent and still have an ulcerogenic potential that is 16 times less . However, the authors have expressed many objections to their conclusions and thus have failed to aim in the direction of the discovery on which the present invention is based.

A study of the use of carprofen for the treatment of osteoarthritis in dogs was published by Vassaur et al. (in Randomized, controlled trial of efficacy of carprofen, and nonsteroidal anti-inflammatory drug, in the treatment of ostearthritis in dogs, J. Am. Vet. Med. Assoc., 206 (6), 807-811, 1995). These scientists were also unable to explain the activity of carprofen. On the one hand, they concluded that prostaglandins protect the gastrointestinal mucosa and that carprofen, like other NSAIDs, inhibits prostaglandin synthetase and blocks prostaglandin biosynthesis. But this conclusion was inconsistent with the outcome of their study, which found that carprofen had little or no harmful effects on the gastrointestinal mucosa of dogs.

A study of the use of carprofen for the treatment of acute post-operative pain in dogs was published by Lascelles et al. (in Postoperative analgesic and sedative effects of carprofen and pethidine in dogs, Veterinary Record, 134, 187-191, 1994). Similarly, these scientists were equally embarrassed as to how carprofen works, on the one hand they noted that carprofen ...

at therapeutic doses, it appears to be a weak inhibitor of prostaglandin synthetase (or cyclooxygenase), the enzyme responsible for the synthesis of inflammatory mediators produced by tissue damage, while admitting, on the other hand, that the study nevertheless proved to be a good analgesic for both acute and chronic pain .

A much earlier study that compared the biological activities of indomethacin with those of the spatial isomers and carprofen racemate in humans was performed by Gaut et al. (and published in Stereoisomeric Relationships Among AntiInflammatory Activity, Inhibition of Platelet Aggregation, and Inhibition of Prostaglandin Synthetase, Prostaglandins, Vol. 10, No. 1, July 1975). The study concluded that carprofen racemate, unlike indomethacin, had no effect on platelet aggregation and thus should not increase bleeding time at doses that are anti-inflammatory active. The data from this study also suggest that the carprofen racemate and the (S) isomer are more specific in anti-arthritic activity and are less ulcerogenic than indomethacin.

Part of the results of the prostaglandin research presented in 1994 at the 9 ^th International Conference on Prostaglandins and Related Compounds focused on COX-2 selectivity. Lecture at the conference presented by Battistini et al. entitled COX-1 and COX-2: Toward the Development of More Selective NSAIDs and published in DNA & P, 7 (8), October 1994, contained comparative data obtained on a large number of cell types including human, mouse or rat using various stimuli such as is published in technical literature reviewed and cited by Battistini et al. Data demonstrating IC 50 values against COX-1 and COX-2 for a number of anti-inflammatory compounds including carprofen were presented and the COX-2 / COX-1 ratio was used to determine COX-2 selectivity. In inverting the ratios published by Battistini et al. so as to be consistent with the ratios used herein to facilitate comparison, most COX-2 selective compounds have ratios of 1428.57 to 50,000.00.

The IC 50 (μΜ) values of carprofen for both COX-1 and COX-2 isozymes have been shown to be quite identical (10.96) to give a ratio of 1.00, clearly demonstrating that carprofen is not

Values cited by Battistini published by Akarasareenont et al. (in Relative Potency of Nonsteroid Anti-Inflammatory Drugs As Inhibitors of Cyclooxygenase-1 or Cyclo-oxygenase-2, Br. J. Pharmacol, Preceedings Supp., No. 183P, 5-7, January by the group of Mitchell et al. nonsteroidal antiinflammatory drugs as is COX-2 selective, et al

1994) and the same (Selectivity of Inhibitors of Constitutional and Inducible Cyclooxygenase, Pre. Natl. Acad. Sci. USA, 90, 11693-7, 1993). IC ₅₀ (gg / ml) (n = 9) inhibition of COX-1 by carprofen (derived from bovine aortic endothelial cells) and COX-2 (derived from lipopolysaccharide-stimulated J774.2 macrophages) were 3 ± 0.41 and 3 ± 1, respectively. , 72, respectively, to give a ratio of 1.00. This is in complete contrast to data for several compounds that have been shown to have 1000 to 4000 times greater selectivity for COX-2. They noted in the publication that none of the selective COX-2 inhibitors described is a carboxylic acid, such as the vast majority of existing NSAIDs, including carprofen. Indeed, all COX-2 selective inhibitors have a sulfonyl group in the molecule which is incorporated into its 1,2-benzothiazine-1,1-dioxide ring structure in the case of meloxicam. If this correlation is valid, the publication speculates, then the Arg 150 residue of the COX protein would not be an essential binding site for selective COX-2 inhibitors. Arg is essential for COX activity because it binds to the terminal carboxylic acid group of arachidonic acid and is therefore most likely a binding site for the carboxylic acid functional group of most existing NSAIDs. Accordingly, it would be expected that an inhibitor that is selective for COX-2 would bind to a site that is unique to the protein structure of the COX-2 isozyme and not to a site that is common to the protein structures of the COX-1 isozymes. and COX-2.

An even more specific interpretation of the molecular interactions of classical NSAIDs and COX-2 selective inhibitors with the protein structure of the enzyme PGH synthase, more commonly known as the cyclooxygenase enzyme, was published by Kurumbail et al. (in Structural Basis for Saline Inhibition of Cyclo-oxygenase-2 by Anti-Inflammatory Agents, Nature, 384, 644-648, December 1996). This interpretation is based on published ligand-bound mouse COX-2 structures and complexes with flurbiprofen, indomethacin and SC-558, a selective COX-2 inhibitor that has a phenylsulfonamide group but does not have a carboxylic acid group as found at 3.0 resolution. up to 2,5 A.

The present publication suggests the expectation that human and murine COX-2 enzymes are very similar because they are 87% identical and have a strictly conserved sequence at the cyclooxygenase active site. Flurbiprofen, a competitive slow-binding inhibitor for both COX-1 and COX-2, binds in the long hydrophobic channel and does not release the substrate into the cyclooxygenase active site. SC-558 is a diaryl heterocyclic inhibitor with a central ring of pyrazole and a sulfonamide group attached to one of the two aryl rings. In COX-2, the channel that leads from the membrane to the cyclooxygenase active site branches at the SC-558 binding site, one branch forming a cavity that receives the bromophenyl ring from SC-558, while the other branch forms a pocket that is actually in the structure of COX-1 is inaccessible, but which accommodates the entire phenylsulfonamide group in COX-2. This pocket is more accessible in COX-2 because isoleucine is substituted with valine having a larger side chain at position 523. The sulfonamide group's access to the new pocket in COX-2 is facilitated by further converting isoleucine to valine at position 434, forming the molecular gateway, that spreads through a new hydrophilic pocket. Finally, at position 513, histidine in COX-1 is replaced by arginine in COX-2, and the superposition of the two enzymes indicates that the imidazole ring of histidine in COX-1 does not propagate sufficiently to directly interact with the sulfonamide group SC-558 as in the case of s. arginine with the enzyme COX-2. The present publication notes that in each of the three cases described above, the COX-2 inhibitory profile is dramatically altered by a single amino acid mutation.

The present publication continues to conclude that the primary determinant of COX-2 salinity in the diaryl heterocyclic class of inhibitors, including SC-558, seems likely to be a phenylsulfonamide group. However, the absence of the carboxyl group is also significant. Arginine at position 120 with its guanidine group is one of the few charged residues in the cyclooxygenase hydrophobic channel and stabilizes the carboxylate of classical NSAIDs, such as flurbiprofen, through charge-charge interaction. The lack of this carboxyl group in SC-558 is probably also an important component of its COX-2 selectivity. This conclusion is supported by the results of attempts to improve its ability against COX-2 by incorporating an acidic group on the pyrazole diaryl heterocyclic structure, which has consistently led to low selectivity.

The present publication includes a first example of a membrane protein that has been successfully studied as a target in a structure-based drug design and outlines the state of the art in terms of structure / activity relationships of NSAIDs to cyclooxygenase isozymes and their resulting anti-inflammatory activity against their unwanted gastrointestinal reactions. In the context of this prior art, the present invention will be seen as a completely unexpected development that provides a surprisingly optimal efficacy / safety profile for treating inflammation in dogs.

The possibility of enantioselective inhibition of the COX-2 isozyme has been investigated and published by Carabaza et al. (in Stereoselective Inhibition of Inducible Cyclooxygenase by Chiral Nonsteroidal Antiinflammatory Drugs, J. Clin. Pharmacol., 36, 505-512, 1996). Stereoselective inhibition of COX-2 by ketoprofen, flurbiprofen and ketorolac was studied in three different in vitro systems and the results were compared with COX-1 inhibition in three parallel in vitro models. It was found that both isozymes were inhibited by the S-enantiomers of all three NSAIDs on an equally effective basis, but that the R-enantiomers of all three NSAIDs inhibited both isozymes with significantly and consistently less potency. In other words, all three Renantiomers showed the same potency in inhibiting both COX-1 and COX-2, but their degree of potency was much lower than that of the corresponding S-enantiomers in all cases. The term significant degree of enantioselectivity in this published study refers only to R- versus S- and does not refer to the selectivity of COX-2 uniquely exhibited by the S-enantiomer of carprofen according to the present invention.

SUMMARY OF THE INVENTION

According to the present invention, there is provided a method of treating or preventing pain and inflammatory processes and diseases associated with inducible cyclooxygenase-2 (COX-2) activity in a member of the Canis familiaris species in need thereof, while reducing or eliminating the unwanted side effects associated with simultaneous inhibition of constitutive cyclooxygenase-1 (COX-1) activity by selectively inhibiting COX-2 activity relative to COX-1 activity, wherein the ratio of selectivity of COX-2: COX-1 inhibition activity is at least 3: 1 based on ex vivo inhibitory levels in whole blood measured at a dose providing> 80% inhibition of COX-2 and preferably> 90% inhibition of COX-2, the method comprising administering to said member of the Canis familiaris species for treating pain and inflammation according to the above mentioned therapeutically effective amount of anti-inflammatory COX-2. a selective inhibitory compound comprising a member selected independently from the group of anti-inflammatory compounds consisting essentially of salicylic acid derivatives, p-aminophenol derivatives, indole acetic acid and indene acetic acid, heteroaryl acetic acid, arylpropionic acid, anthranilic acid, enolic acid and alkanones.

Also within the scope of the present invention is the practice of the above-described method of treating or preventing pain and inflammatory diseases and processes by administering more than one member selected from the aforementioned class of anti-inflammatory compounds. The suitability of any such combination of anti-inflammatory active agents is likely to be based on obtaining a favorable balance of the pharmaceutical properties of the individual agents involved. For example, the rate of onset of action may be counterbalanced by a prolonged therapeutic half-life, or the tendency to build up large cellular stores in certain tissues may be counterbalanced by a higher rate of plasma protein binding. It is intended that all such combinations are within the scope of the present invention.

The present invention provides any anti-inflammatory COX-2 selective inhibitor that meets the above described limitations. Of these selective inhibitory compounds, a subset of carprofen compounds useful in the treatment or prevention of pain and inflammatory processes and diseases in a member of the species Canis familiaris of formula (I) is preferred:

where A is hydroxyl, C 1 -C 4 alkoxy, amino, hydroxyamino, C 1 -C 2 monoalkylamino, C 1 -C 2 dialkylamino, X and Y are independently H or C 1 -C 2 alkyl and n is 1 or 2

R ⁶ is halogen, alkyl having 1 to 3 carbon atoms, trifluoromethyl or nitro,

R ⁹ is H, an alkyl group having 1 to 2 carbon atoms, a phenyl group or a phenylalkyl group having 1 to 2 carbon atoms, wherein the phenyl is optionally monosubstituted with fluorine or chlorine, -C (= O) -R, wherein R is an alkyl group containing 1 to 2 carbon atoms or a phenyl group, optionally monosubstituted with fluorine or chlorine, or -C (= O) -O- ¹ , wherein R ¹ is an alkyl group having 1 to 2 carbon atoms, where X and Y are different, their (- (R) - and (+) (S) -enantiomers and all pharmaceutically acceptable forms of salts, prodrugs and metabolites thereof, which are therapeutically active for the treatment or prevention of pain and inflammation. When the inhibitor of formula (I) exists as the (-) - (R) - and (+) - (S) enantiomers, the (+) (S) -enantiomer alone is provided according to the present invention or when both enantiomers are present together, it is provided a racemic or non-racemic mixture thereof.

The present invention further provides, in a preferred aspect of the above-described method of treating or preventing pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, administering said dog to treat pain and inflammation of a therapeutically effective amount of an anti-inflammatory COX-2 selective inhibitory compound. wherein the in vitro potency of IC 50 against COX-2 in said dog is at least 30-fold, preferably at least 40-fold, more preferably at least 50-fold, even more preferably at least 60-fold, and even more preferably at least 80-fold, and most preferably at least 100-fold greater than its in vitro IC50 activity against COX-1 in said species, said inhibitor being a member selected from the group of anti-inflammatory compounds consisting essentially of salicylic acid derivatives, p-aminophenol derivatives, indole acetic acid and indene acetic acid, hetero acids arylacetic acid, arylpropionic acid, anthranilic acid, enolic acid and alkanones.

The present invention still further provides, in another preferred aspect of the above-described method for treating or preventing pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, administering said dog to treat pain and inflammation of a therapeutically effective amount of an anti-inflammatory COX-2 selective inhibitory. a compound that selectively inhibits substantially only inducible COX-2 without substantially inhibiting the corresponding constitutive COX-1.

Further provided are the methods described above, wherein said therapeutically effective amount of an anti-inflammatory compound of formula (I) as defined, and in particular said (+) (S) -enantiomer of 6-chloro-N-methyl-9H-carbazole-2-acetic acid is systemically administered to said member of Canis familiaris, said systemic administration comprising: 1) injecting or infusing into suitable body tissues or cavities of a pharmaceutical composition comprising said inhibitor in a suitable liquid form for administration of said inhibitor by systemic administration that is intraarterial, intra- or transdermal , subcutaneously, intramuscularly, intraspinally, intrathecally, or intravenously, wherein said inhibitor is: a) contained in the solution as a solute, b) contained in the discontinuous phase of the emulsion or the discontinuous phase of the inverse emulsion that is inverted for injection or infusion, said emulsions comprising suitable emulsifiers; or c) included in suspension as a suspended solid in colloidal or microparticle form, said suspension comprising suitable suspending agents, 2) injection or infusion into suitable body tissues or cavities of a pharmaceutical composition comprising said inhibitor in a suitable liquid form to serve as a depot for administration of said an inhibitor of systemic administration, wherein said formulation provides a supply of said inhibitor and thus delayed, sustained and / or controlled release of said inhibitor for systemic distribution, 3) instillation, inhalation or insufflation into suitable body tissues or cavities of a pharmaceutical formulation containing said inhibitor in a suitable solid form for administration of said inhibitor, wherein said inhibitor is: a) contained in a solid implant formulation that is embedded in suitable body tissues or cavities, said formulation providing a delayed, controlling and / or controlling the release of said inhibitor,

(b) contained in a particulate composition that is inhaled into the lungs; or (c) contained in a particulate composition that is introduced into suitable body tissues or cavities, wherein said composition optionally provides delayed, sustained and / or controlled release of said inhibitor; 4) ingesting a pharmaceutical composition comprising said inhibitor in a suitable solid or liquid form for oral administration of said inhibitor, wherein said inhibitor is: a) contained in a solid dosage form, or b) contained in a liquid dosage form. Suppositories can be considered a special type of implant because they contain starting materials that are solid at room temperature but melt at body temperature and slowly release the active ingredient to saturate the surrounding body tissues where the active ingredient is absorbed and transported by reaches systemic administration. Dosage forms are also contemplated that allow transdermal and mucosal administration to achieve systemic administration, including, but not limited to, transdermal patch technology.

Further provided is a method for the treatment or prevention of pain and inflammation as described above, comprising ingesting or administering a solid oral dosage form selected from the group consisting of delayed release oral tablets, capsules, capsules, lozenges, lozenges and multiparticulates, enteric tablets and capsules. release and absorption in the stomach, to facilitate administration distal from the stomach in the dog, sustained release oral tablets, capsules and microparticles that provide systemic administration of the active ingredient in a controlled manner for 10 hours, fast-dissolving tablets, encapsulated solutions, oral paste, granular form incorporated or for mixing into the feed of a treated dog, and a chewable form in which said active ingredient is consumed together with an acceptable feed or may alternatively be administered by leaching from a portion of the non-digested feed during chewing treated dog. For use of the above-described dosage forms, microencapsulated formulations of the active ingredient are also included, which can then be incorporated into a tablet, capsule or other finished formulation. Still further provided is a method comprising ingesting a liquid oral dosage form selected from the group consisting of a solution, suspension, emulsion, inverse emulsion, elixir, extract, tincture and concentrate, optionally added to the drinking water of the treated dog. Each of these liquid dosage forms, when formulated according to methods known in the art, can either be administered directly to the treated dog or added to the drinking water of the treated dog. On the other hand, the concentrated liquid form is formulated to be first added to a given amount of water from which an aliquot may be taken for administration directly to the dog or added to the drinking water of the dog.

Still further provided are the methods described above, wherein said therapeutically effective amount of an anti-inflammatory compound of formula (I) as defined is administered locally to the site of inflammation to said member of Canis familiaris. Still further provided is a method of topical administration, said topical administration comprising: 1) injecting or infusing a local inflammatory site of a pharmaceutical composition comprising said inhibitor in a suitable liquid form for administering said inhibitor by topical administration that is intraarterial, intraarticular, intrachondral, intracostal, intracystic, intra- or transdermal, intrafascicular, intraligamentous, intramedullary, intramuscular, intraneural, intraosteal, intrapelvic, intrapericardial, intraspinal, intrasternal, intrasynovial, intratarsal or intrathecal, including the components which provide for said delayed / sustained release a local inflammatory site, wherein said inhibitor is contained: a) in solution as a solute, b) in the discontinuous phase of the emulsion or in the discontinuous phase of the inverse emulsion that is inverted or (c) in suspension as a suspended solid in colloidal or microparticle form, said suspension comprising suitable suspending agents; (2) injecting or infusing a pharmaceutical composition comprising said inhibitor in a suitable liquid form; so as to serve as a depot for administering said inhibitor to said local inflammatory site, said composition providing a supply of said inhibitor and thus delayed, sustained and / or controlled release of said inhibitor to said local inflammatory site, or 3) instillation by inhalation or insufflation of the pharmaceutical a composition comprising said inhibitor in a suitable solid form for administering said inhibitor to said local inflammatory site, wherein said inhibitor is comprised of: a) a solid implant preparation that is inserted into said topically b) in a particulate composition that is inhaled to a local inflammatory site comprising the lungs, or c) in a particulate composition, wherein said composition optionally provides delayed, sustained and / or controlled release of said inhibitor to said local inflammatory site; is introduced into a local inflammatory site, wherein said composition optionally provides delayed, sustained and / or controlled release of said inhibitor to said local inflammatory site. Other specific dosage forms for topical administration are also within the scope of the present invention. For example, formulations for application to the skin, preferably with increased absorption through mechanical treatment of the formulation into the skin, such as friction, can be used to deliver the active ingredient of the inhibitor to a local area, such as an inflamed joint in need of such treatment. These preparations may be in the form of gels, lotions, balsams, ointments and other formulations designed for topical application.

Still further provided are the methods described above, wherein a therapeutically effective amount of an anti-inflammatory inhibitor is administered to said member of Canis familiaris in an amount expressed in mg per kg body weight of said dog 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 0.5 mg / kg to about 10.0 mg / kg / day, and most preferably from about 0, 5 mg / kg to about 8.0 mg / kg / day. Administration of 6-chloro-α-methyl acid 24

9H-carbazole-2-acetic is typically provided at a dosage of about 4.0 mg / kg / day.

In addition, the present invention provides a pharmaceutical composition for treating or preventing pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, the composition comprising a pharmaceutically acceptable carrier together with a therapeutically effective amount for treating pain and inflammation of an anti-inflammatory selective cyclooxygenase inhibitor. -2 (COX-2), wherein the ratio of selectivity of COX-2 to COX-1 is at least 3: 1 based on ex vivo inhibitory levels in whole blood measured at a dose providing> 80% inhibition of COX-2 and preferably> 90% inhibition of COX -2. In a preferred aspect of said pharmaceutical composition for the treatment or prevention of pain and inflammatory processes and diseases, the therapeutically effective anti-inflammatory COX-2 selective inhibitor has an IC ₅₀ potency of at least 30-fold, preferably at least 40-fold, more preferably at least 50-fold. even more preferably at least 60-fold and even more preferably at least 80-fold and most preferably at least 100-fold greater than the in vitro potency of the IC 50 against cyclooxygenase-1 (COX-1), said inhibitor being a member selected from the group of anti-inflammatory compounds consisting essentially of salicylic acid derivatives, p-aminophenol derivatives, indole acetic acid and indene acetic acid, heteroarylacetic acid, arylpropionic acid, anthranilic acid, enolic acid and alkanones.

Further provided is a pharmaceutical composition as described above, wherein said inhibitor is a member selected from the group consisting of arylpropionic acids and still further said inhibitor is a compound of formula (I) as defined above. Further provided is a pharmaceutical composition as described above, wherein the in vitro potency of IC ₅₀ against cyclooxygenase-2 (COX-2) of the inhibitory compound of formula (I) is at least 100-fold greater than its in vitro potency of IC ₅₀ against cyclooxygenase-1 (COX-1). ), wherein one of X and Y is H and the other is a methyl group, and when both of the resulting enantiomers occur, the (+) (S) -enantiomer is present in an amount of at least 75%. In particular, there is provided a pharmaceutical composition as described above, wherein for formula (I), for R ² , n = 1, one of X and Y is H and the other is methyl and A is hydroxyl, alkoxy containing 1 to 2 carbon atoms or amino R ⁶ is chlorine or trifluoromethyl and R ⁹ is H, methyl, acetyl, benzoyl or acetyloxy, and when the resulting enantiomers are present together, the (+) (S) -enantiomer is present in an amount of at least 85%, preferably at least 90%, more preferably at least 95% and most preferably at least 99%.

Still further provided is a pharmaceutical composition as described above, wherein said inhibitor comprises 6-chloro-α-methyl-9H-carbazole-2-acetic acid and when the two resulting enantiomers are present together, the (+) (S) enantiomer is present in an amount of at least 85%. %, preferably at least 90%, more preferably at least 95% and most preferably at least 99%. In particular, there is provided a pharmaceutical composition as described above, wherein said inhibitor is comprised entirely of the (+) (S) -enantiomer of the acid

6-chloro-a-methyl-9H-carbazole-2-acetic acid.

Also provided is a pharmaceutical composition as described above, wherein a therapeutically effective amount of the anti-inflammatory inhibitor present is sufficient, in the context of the dosing regimen and the parameters used for administration, to provide the treated dog with an amount of said inhibitor expressed in mg per kg body weight of said dog daily. 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 0.5 mg / kg to about 10, 0 mg / kg / day and most preferably from about 0.5 mg / kg to about 8.0 mg / kg / day. Administration of 6-chloro-α-methyl-9H-carbazole-2-acetic acid is typically provided by a dosage of about 4.0 mg / kg / day.

In yet another preferred aspect of the pharmaceutical composition of the present invention there is provided an anti-inflammatory COX-2 selective inhibitor that selectively inhibits substantially only inducible COX-2 without substantially inhibiting the corresponding constitutive COX-1. In a particularly preferred embodiment, it comprises a pharmaceutically acceptable carrier together with a therapeutically effective amount for the treatment of pain and inflammation of an anti-inflammatory COX-2 selective inhibitor comprising a compound of formula (I) wherein for R ² , n = 1, one of X and Y is H; the other is methyl, and A is hydroxyl, C 1 -C 2 alkoxy or amino, R ⁶ is chloro or trifluoromethyl and R ⁹ is H, methyl, acetyl, benzoyl or methoxycarbonyl, and (+) The (S) -enantiomer is present in an amount of at least 99%. Said inhibitor consists essentially of the (+) (S) -enantiomer of 6-chloro-α-methyl-9 H -carbazole-2-acetic acid. Preferably, said pharmaceutical composition comprises a therapeutically effective amount of said inhibitor sufficient in the context of the dosage regimen and the parameters used to administer, to treat the treated dog, an amount of said inhibitor expressed in mg per kg body weight of said dog per day. kg to about 8.0 mg / kg / day.

Still further provided is the above-described pharmaceutical composition in a dosage form that can provide the desired therapeutically effective amount of an anti-inflammatory selective COX-2 inhibitor for the treatment of pain and inflammation in a suitable regimen. However, many of the pharmaceutical preparations described above and below are intended to act for a long time, i. they provide inhibitory activity for more than a few hours or one day, and provide this activity for several days to a week or more. Examples of such long acting pharmaceutical formulations are, in particular, implants and depots, and some of them are intended to provide inhibitory activity for up to a month or more. The required therapeutically effective amount of the inhibitor required to treat or prevent pain and inflammation, expressed in mg per kg body weight of said member of the Canis familiaris species daily, ranges from about 0.01 mg / kg to about 20.0 mg / kg / day. day, preferably from about 0.1 mg / kg to about 12.0 mg / kg / day, more preferably from about 0.5 mg / kg to about 10.0 mg / kg / day, and most preferably from about 0.5 mg / kg / day. kg to about 8.0 mg / kg / day. Administration of 6-chloro-α-methyl-9H-carbazole-2-acetic acid is typically provided at a dosage of about 4.0 mg / kg / day.

In particular, there is provided a pharmaceutical composition as described above, wherein said therapeutically effective amount for treating pain and inflammation of an anti-inflammatory inhibitor is provided in a form suitable for systemic administration to said member of Canis familiaris, said pharmaceutical composition comprising said inhibitor in a suitable liquid form for administration. inhibitor by: 1) injection or infusion, which is intraarterial, intra- or transdermal, subcutaneous, intramuscular, intraspinal, intrathecal or intravenous, wherein said inhibitor: a) is contained in the solution as a solute, b) is contained in the discontinuous phase of the emulsion or in the discontinuous phase of an inverse emulsion that is inverted for injection or infusion, said emulsions comprising suitable emulsifiers, or c) is included in the suspension as a suspended solid in colloidal or microparticle form, said suspension containing an emulsion containing a suitable emulsifier. 2) injections or infusions into suitable body tissues or cavities to serve as a depot, said composition providing a supply of said inhibitor for systemic distribution, 3) instillation, inhalation or insufflation into suitable body tissues or cavities of said pharmaceutical composition in a suitable solid form, wherein said inhibitor: a) is comprised in a solid implant formulation that provides delayed, sustained and / or controlled release of said inhibitor, b) is contained in a composition comprising particles that is inhaled into the lungs, or c) is comprised in a particulate composition that is introduced into suitable body tissues or cavities, said composition optionally providing delayed, sustained and / or controlled release of said inhibitor, or 4) ingesting said pharmaceutical composition in a suitable solid or liquid. orally administered by said inhibitor, wherein said inhibitor: a) is contained in a solid dosage form, or b) is contained in a liquid dosage form.

In a particularly preferred aspect of the above pharmaceutical formulations that provide and / or controlled release of the COX-2 selective inhibitor described delayed are included sustained anti-inflammatory all such dosage forms that achieve > 80% inhibition of COX-2 isozyme activity and result in plasma a concentration of said inhibitor of at least 10 gg / ml for at least 4 hours, preferably at least 8 hours, more preferably for at least 12 hours, even more preferably for at least 16 hours and even more preferably for at least 20 hours and most preferably for at least 24 hours. Advantageously, the above-described dosage forms that achieve> 80% inhibition of COX-2 isozyme activity and result in plasma concentrations of said inhibitor of at least 15 gg / mL are included for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, even more preferably at least 20 hours. hours, and most preferably at least 24 hours. More preferably, the above-described dosage forms that achieve> 90% inhibition of COX-2 isozyme activity and result in plasma concentrations of said inhibitor of at least 20 pg / ml are at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.

Particular dosage forms of the above-described pharmaceutical preparations include suppositories as a special type of implant containing starting materials that are solid at room temperature but melt at body temperature and slowly release the active ingredient to saturate the surrounding body tissues where the active ingredient is absorbed and transported to achieve systemic administration, solid oral dosage forms selected from the group consisting of delayed release oral tablets, capsules, capsules, lozenges, lozenges and multiparticulates, gastro-resistant tablets and capsules to prevent release and absorption in the stomach, to facilitating administration distally from the stomach in a dog, sustained release oral tablets, capsules and microparticles that provide systemic administration of the active ingredient in a controlled manner for up to 24 hours, fast-dissolving tablets, encapsulated solutions, oral paste, gra a null form incorporated into or mixed with the treated dog's feed, and a chewable form in which said inhibitor inhibitor ingredient is consumed together with an acceptable feed or may alternatively be administered by leaching from a portion of the feed that is not digested during chewing by the treated dog; oral dosage forms selected from the group consisting of solutions, suspensions, emulsions, inverse emulsions, elixirs, extracts, elixirs and concentrates, optionally added to the drinking water of the treated dog. Each of these liquid dosage forms, when formulated according to methods known in the art, can either be administered directly to the treated dog or added to the drinking water of the treated dog. On the other hand, the concentrated liquid form is formulated to be first added to a given amount of water from which an aliquot can be taken for administration directly to the dog or added to the drinking water of the dog.

Further provided is a pharmaceutical composition as described above, wherein said therapeutically effective amount for treating pain and inflammation of said anti-inflammatory inhibitor is provided in a dosage form suitable for topical administration to an inflammatory site to said member of Canis familiaris, said pharmaceutical composition comprising said inhibitor in a suitable liquid form. administering said inhibitor via: 1) injection or infusion into a local inflammatory site that is intraarterial, intraarticular, intrachondral, intracostal, intracystic, intra- or transdermal, intrafascicular, intraligamentous, intramedullary, intramuscular, intranasal, intraneural, intraneural, ophthalmic administration, intraosteal, intrapelvic, intrapericardial, intraspinal, intrasternal, intrasynovial, intratarsal or intrathecal, including components that provide delayed, controlled and / or sustained release of said inhibitor to said local inflammatory site, wherein said inhibitor is contained in: a) as a solute, b) in the discontinuous phase of the emulsion or in the discontinuous phase of the inverse emulsion that is inverted for injection or infusion, said emulsions comprising suitable emulsifiers, or c) in suspension as a suspended solid in colloidal or microparticle form, said suspension comprising suitable suspending agents, or 2) injections or infusions to serve as a depot for administering said inhibitor to said local inflammatory site, said composition providing a supply of said inhibitor and thus delayed, sustained and / or controlled release and wherein said composition also comprises components that ensure that said inhibitor is predominantly active locally, with little systemic transfer activity, or wherein said pharmaceutical composition comprises said inhibitor in a suitable solid form for administration of said inhibitor. 3) instillation, inhalation or insufflation to said local inflammatory site, wherein said inhibitor is comprised of: a) in a solid implant composition inserted into said local inflammatory site, said composition optionally providing delayed, persistent and / or controlled releasing said inhibitor to said local inflammatory site, b) in a particulate preparation that is inhaled to a local inflammatory site comprising the lungs, or c) in a particulate preparation that is introduced into a local inflammatory site, said the composition comprises components which ensure that said inhibitor is primarily active locally, with no significant activity for the transmission system, and optionally provides delayed-, sustained-, and / or controlled-release of said inhibitor into said local site of inflammation.

According to the present invention, combinations of one or more other therapeutically active formulations are provided together with the active ingredients for the treatment of pain and inflammation that form the above-described pharmaceutical formulations of the present invention. When the joint is seriously inflamed and simultaneously infected with microorganisms, e.g. bacteria, fungi, protozoa, viruses and the like, it will be desirable to administer the active ingredient of the present invention in combination with one or more antibiotics, antifungal, antiprotozoal, antiviral or similar therapeutic agents. Further, the active ingredient of the present invention may be administered not only in combination with other NSAIDs, but also in combination with inhibitors of other inflammatory mediators comprising one or more members selected from the group consisting essentially of classes of these inhibitors and examples thereof including antagonχ antagonists. -receptor, ínχ and B2 kinin receptor antagonists, prostaglandin inhibitors such as PGD-, PGF-, PGI2- and PGE receptor antagonists, thromboxane A2 (TXA2-) inhibitors, 5- and 12-lipoxygenase inhibitors, LTC4-, LTD4 leukotriene inhibitors / LTE4- and LTB ₄ -, PAF receptor antagonists, gold in the form of an aurothio group with various hydrophilic groups, immunosupres32. Active preparations, e.g., < tb > ______________________________________ < tb > cyclosporin, azathioprine and methotrexate, anti-inflammatory glucocorticoids, penicillamine, hydroxychloroquine, anti-gout agents, e.g. colchicine, xanthine oxidase inhibitors, e.g. allopurinol, and uricosuric drugs, e.g. probenecid, sulfinpyrazone and benzbromarone. It is further provided that the anti-inflammatory compositions of the present invention are administered in combination with therapeutic agents for treating disease states, syndromes and symptoms found in older dogs, comprising one or more members selected from the group consisting essentially of therapeutic agents and conditions treated. which include cognitive therapeutics to counteract the loss and deterioration of memory, antihypertensives and other cardiovascular drugs intended to compensate for the effects of atherosclerosis, including hypertension, ischemic heart disease including angina, stroke and myocardial infarction, selected from diuretics, vasodilators, vasodilators, β-adrenergic receptor antagonists such as propranolol, angiotensin II converting enzyme inhibitors (ACE inhibitors) such as enalapril used to treat geriatric dogs with mitral insufficiency, and enalapril alone and in combination with neutral endopeptidase inhibitors, angiotensin II receptor antagonists such as losartan, renin inhibitors, calcium channel blockers such as nifedipine, sympatholytics such as methyldopa, and ₂ -adrenergic agonists such as clonidine adrenergic antagonists , such as prazosin, and HMG-CoA reductase (antihypercholesterolemic) inhibitors, such as lovastatin or atorvastatin, and further include antineoplastic agents, particularly antimitotics including vincaalkaloids such as vinblastine and vincristine, secretory analogs and systemic growth hormone anesthetics, growth hormone analgesics, , and H ₂ -receptor antagonists and other gastroprotective agents. Still further, the possibility is provided that the above combinations of therapeutic agents are used for treatment

· Acute conditions in dogs, including bacterial infections occurring concurrently with degenerative joint disease and for the treatment of chronic conditions in dogs when. the regimen used for this purpose comprises administering the anti-inflammatory compositions of the present invention in combination with other drugs commonly used to treat chronic conditions including osteoarthritis, formulating the anti-inflammatory compositions of the present invention with one or more other therapeutic agents forming the intended combination into a suitable dosage form comprising all medicaments forming the combination, including when the different drugs have different half-lives, by means of the controlled release forms of said drugs with different release times, to achieve a relatively uniform dosage, the food-mixing formulation in which the said drugs used in the combination occur together in an additive in said feed preparation. According to the present invention there is further provided a co-administration wherein the combination of drugs is accomplished by co-administering said drugs to be combined, including co-administration via different dosage forms and modes of administration, using combinations according to different but regular and continuous dosing schedules. maintain the desired plasma levels of said drugs used in the treated dog, even if the individual drugs forming said combination are not administered simultaneously to said dog

It is also contemplated that, according to the present invention, a kit suitable for commercial use for the therapeutic treatment or prevention of pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, the kit comprising a suitable container may be provided. in the form of an outer shell and an inner container removably contained therein, said container containing a suitable dosage form of the active ingredient comprising a COX-2 selective inhibitor, wherein the ratio of selectivity of COX-2 to COX-1 is at least 3: 1 based on ex vivo inhibitory whole blood levels measured at a dose providing 80% inhibition of COX-2, preferably> 90% inhibition of COX-2 as described elsewhere herein, wherein said container is accompanied by printed instructional and information material that may be attached to said container, enclosed in said container or exhibited as integral no part of said container, said instructional and information material, and explicitly notifies the reader that said active ingredient, when administered to a treated dog, effectively inhibits COX-2 induced at an existing or expected site of pain and inflammation in said dog, wherein the COX-2 selectivity ratio wherein the COX-1 is at least 3: 1 based on the ex vivo inhibitory levels in whole blood measured at a dose providing> 80% inhibition of COX-2, thereby treating or preventing said pain and inflammation that would otherwise result. In a preferred embodiment of said container suitable for commercial use, said instructional and information material declares and explicitly informs the reader that said active ingredient, when administered to a treated dog, effectively provides complete inhibition of COX-2 induced in said dog, thereby treating or preventing pain and inflammation, but also that the active ingredient thus administered to said dog causes substantially no inhibition of constitutive COX-1 in said dog, thereby avoiding undesirable gastrointestinal and other adverse effects resulting from significant inhibition of said COX-1 in most dogs.

It is also contemplated that, according to the present invention, a kit of the type described immediately above comprising a suitable container as described is further provided, said container containing an oral dosage form of a compound of formula (I) which may be in the form of a chewable or edible oral tablet. , a unit dose package called a unit dose sachet, a suspension formed from a unit dose sachet, an oral suspension powder or an oral suspension per se, which does not exhibit an adverse effect on the digestion if said container is accompanied by the described printed instructional and information material. it does not express or imply that the oral form may be taken with or without food.

It is an object of the present invention to find a solution to the serious problem faced by the veterinary field of the decade, the need for effective but safe anti-inflammatory treatment in dogs. The severity and rigidity of this problem stems from the fact that potentially all anti-inflammatory agents, especially NSAIDs, which have been tested for use in dogs have, together, unacceptable and sometimes dangerous adverse reactions in dogs that greatly limit their use. The most common and most severe of these adverse reactions is disruption and irritation of the gastrointestinal mucosa, leading to ulceration, bleeding and possibly perforation and peritonitis. These adverse reactions are mediated by the inhibition of various prostaglandins by NSAIDs with inhibitors, resulting in limited blood supply to the protective gastrointestinal mucosa, whose total mass is gradually severely reduced and the protective function is reduced.

Gastric damage by NSAIDs is caused by at least two different mechanisms. Local irritation with orally administered NSAIDs permits back diffusion of the acid into the gastric mucosa, causing tissue damage. Parenteral administration, on the other hand, can damage and bleed, which is correlated to the biosynthesis of gastric prostaglandins that fulfill the cytoprotective function of the gastric mucosa. These prostaglandins, in particular PGI2 and PGE2, inhibit acid secretion in the stomach, increase blood flow through the mucosa and promote secretion of cytoprotective mucus in the intestine. Other undesirable side effects of NSAIDs also involve causing inhibition of platelet function, prolongation of gestation or spontaneous labor, changes in renal function and hypersensitivity reactions.

All of the above-described adverse side effects of NSAIDs are likely to be related to the blockade of the synthesis of endogenous prostaglandins. Thus, there is a substantial interest in the discovery of NSAIDs that do not result in systemic induction of these undesirable side effects. Dogs are not only particularly susceptible to inflammatory processes and diseases such as degenerative joint disease, but are also particularly sensitive to the complications of adverse gastrointestinal reactions that follow.

The term dog, dogs, as used herein, refers to any member of the Canis familiaris species having a large number of different breeds. While laboratory determination of biological activity can be performed using a particular breed, it is contemplated that the inhibitory compounds of the present invention will be useful for treating pain and inflammation in each of these numerous breeds.

In its broadest aspect, it is a surprising discovery that a small group of anti-inflammatory agents, the best example of which is carprofen, 6-chloro-armethyl-9 H -carbazole-2-acetic acid, has a high degree of selectivity for canine cyclooxygenase-2 (COX-2). ) and that this selectivity is unique in a large class of anti-inflammatory preparations having a carboxyl group and a carboxyalkylaryl group having 1 to 4 carbon atoms and / or a heteroaryl group, to which the group of carprofen compounds also belongs. This unique and unexpected selectivity in dogs has far-reaching implications for the safe and effective treatment of dogs suffering from any of a number of inflammatory processes and diseases.

In the art of treating inflammatory processes and diseases, in particular dogs, it has now been accepted that a cascade of endogenous reactions that form various prostaglandin compounds in the body, beginning with arachidonic acid, continues with an essential enzyme called cyclooxygenase. This enzyme has been found to exist in two isozyme forms that are separate and act independently, isozyme constitutive cyclooxygenase-1 (COX-1) and isozyme cyclooxygenase-2 (COX-2). The COX-1 isozyme is responsible in dogs for the production of prostaglandins that have important functions in the stomach, intestines, kidneys and platelets, some of which are protective in nature, particularly with respect to the gastrointestinal mucosa. The COX-2 isozyme is responsible in dogs for the production of prostaglandins such as PGE ₂ , which mediates acute and chronic inflammation between neutrophils, macrophages, endothelial cells and fibroblasts, lipopolysaccharides (LPS), various cytokines, e.g. IL-1 and TNF, by growth factors, e.g. EGF and PDGF, and many other agents. For example, the COX-2 isozyme can be detected by immunological transmission in mononuclear cells of pleural exudate after carrageenan-induced pleuritis in a rat model.

Finally, it is the object of the art to disclose compounds that are capable of inhibiting the activity of COX-2 isozyme in dogs with 100% efficacy, while simultaneously inhibiting the activity of COX-1 isozyme with 0% efficacy. In practice, this involves searching for compounds that selectively inhibit the COX-2 isozyme, i. Compounds which provide optimal anti-inflammatory treatment with a relatively high degree of COX-2 inhibition at a dose that also produces minimal undesirable side effects with a relatively low degree of COX-1 isozyme inhibition. Until the disclosure of the present invention described herein, the only compounds that have been shown to exhibit COX-2 selectivity are those having a sulfonyl or sulfonamide group rather than a carboxyl group, as is characteristic of the vast majority of classical NSAIDs. These observations have led to the idea that the Arg 150 residue of COX-1 is not an essential binding site for COX-2 selective inhibitors, while the Arg 150 residue is essential for COX-1 activity because it binds the terminal carboxylic acid group of arachidonic acid and analogously function carboxylic acid of the above classical NSAIDs. It would be expected that COX-2 selective inhibitors would bind to some unique features of the canine COX-2 isozyme rather than a feature that is common to the canine COX-1 and COX-2 isozymes.

Accordingly, the present invention provides a method of selectively inhibiting inducible cyclooxygenase-2 (COX-2) relative to the inhibition of the corresponding constitutive cyclooxygenase-1 (COX-1) and thereby treating or preventing pain and inflammatory processes and diseases associated therewith in a member of Canis familiaris. in need of such treatment, wherein the ratio of COX-2 to COX-1 selectivity is at least 3: 1 based on in vivo and ex vivo whole blood inhibition levels measured at a dose or dose range providing> 90% inhibition of COX-2, including administration said dog having a therapeutically effective amount of an anti-inflammatory compound for treating pain and inflammation according to the above limitations.

The present invention further provides a method as described above for treating or preventing pain and inflammatory processes and diseases in a member of the species Canis familiaris, wherein said anti-inflammatory COX-2 selective inhibitor comprises a compound of formula (I):

I

L_yJ n

where A is hydroxyl, C 1 -C 4 alkoxy, amino, hydroxyamino, C 1 -C 2 monoalkylamino, C 1 -C 2 dialkylamino, X and Y are independently H or C 1 -C 2 alkyl carbon, and n is 1 or 2,

R ⁶ is halogen, alkyl having 1 to 3 carbon atoms, trifluoromethyl or nitro,

R ⁹ is H, an alkyl group having 1 to 2 carbon atoms, a phenyl group or a phenylalkyl group having 1 to 2 carbon atoms, wherein the phenyl is optionally monosubstituted with fluorine or chlorine, -C (= O) -R, wherein R is an alkyl group containing 1 to 2 carbon atoms or a phenyl group, optionally monosubstituted with fluorine or chlorine, or -C (= O) -OR ¹ , wherein R ¹ is an alkyl group containing 1 to 2 carbon atoms, where X and Y are different, their (-) - (R) - and (+) (S) -enantiomers, and all pharmaceutically acceptable forms of salts, prodrugs and metabolites thereof, which are therapeutically active for the treatment or prevention of pain and inflammation. When the inhibitor of formula (I) exists as the (-) - (R) - and (+) (S) -enantiomers, the (+) (S) enantiomer alone is provided according to the present invention or when both enantiomers are present together, they are provided racemic or non-racemic mixture.

It is quite surprising that the preferred carprofen group of compounds characterized by the functionality of α-methylacetic acid has many times greater selectivity for COX-2 in dogs than any of the classic carboxyl-containing NSAIDs, and many times greater selectivity for COX-2 in dogs such as any of the sulfonyl and sulfonamide-containing NSAIDs considered highly COX-2 selective compounds in the art. This aspect of the present invention is encompassed in a method of treating or preventing inflammatory processes and diseases comprising administering to a dog an anti-inflammatory therapeutically effective amount of a canine cyclooxygenase-2 (COX-2) inhibitor having an in vitro potency of IC ₅₀ of COX-2 in said dog of at least 50 times. greater than its in vitro potency of an IC 50 against canine cyclooxygenase-1 (COX-1) in said dog, said inhibitor being a member selected from the group of anti-inflammatory compounds consisting essentially of salicylic acid derivatives, p-aminophenol derivatives, indole acetic acid and indene acetic acid, heteroaryl acetic acid, arylpropionic acid, anthranilic acid, enolic acid and alkanones, and the member is selected in particular from the group consisting of anti-inflammatory preparations having a carboxyl group and a carboxyalkylaryl group having 1 to 4 carbon atoms and / or a heteroaryl group. In particular, said inhibitor is a member selected from the group consisting of a class of arylpropionic acids of non-steroidal anti-inflammatory drugs.

Carprofen and a group of carprofen derivatives preferably used in the methods and compositions of the present invention may be prepared according to synthetic methods well known to the skilled organic chemist. For example, compounds of formula (I) wherein R ⁶ is halogen, C 1 -C 3 alkyl, trifluoromethyl or nitro and wherein R ⁹ is H or methyl may be prepared by reacting (1) a phenylhydrazine in which the phenyl moiety has a phenyl moiety the desired R ⁶ substitution and the α-nitrogen of the hydrazine has the desired R ⁹ substitution, with (2) cyclohexanone having the desired R ² substitution. The resulting 1,2,3,4-tetrahydrocarbazole is then aromatized to form the desired carbazole of formula (I). The aromatization may be carried out using (1) a flavoring agent, e.g. p-chloranil, o-chloranil, 2,3-dichloro-5,6-dicyanobenzoquinone (DDQ), sulfur, palladium on carbon or lead oxide, in the presence of (2) a solvent such as xylene, benzene, toluene, quinoline, dimethylsulfoxide (DMSO) and dimethylformamide (DMF), (3) at a temperature ranging from room temperature to the reflux temperature of the reaction mixture, preferably at reflux temperature.

Compounds of formula (I) which are acids, ie, where A is a hydroxyl group and salts of these acids with bases, can be converted to amides of formula (I) wherein A is amino, hydroxyamino, monoalkylamino of 1 to 2 carbon atoms, dialkylamino a group containing 1 to 2 carbon atoms, by (1) the formation of the corresponding acid chloride by treatment with phosphorus pentachloride (PCl5) followed by (2) reaction with the appropriate amine reactant to give the desired amine in the presence of pyridine or triethylamine equivalent , which serves as the basis for the transfer of the proton, thereby removing the H ⁺ C1 formed. The same acid chlorides that are formed in step (1) can be reacted with an appropriate alkanol to give esters of formula (I) wherein A is C 1 -C 4 alkoxy. It is also desirable to carry out this reaction in the presence of a base, such as pyridine, which can neutralize the H ⁺ Cl formed so that it does not interfere with any acid-sensitive alkanol reactant.

The above-described synthetic approaches for preparing a preferred carprofen group of compounds used in the methods and compositions of the present invention are described in detail in U.S. Pat. No. 3,896,145, which is incorporated by reference in its entirety.

When X and Y are different in the definition of R ² , then there is a chiral (asymmetric) carbon atom. The racemic mixture of the (R) - and (S) -enantiomers is the result of a 50:50 mixture of the two enantiomers. According to the present invention, it has been discovered that the (S) -enantiomer of a preferred carprofen group of compounds of formula (I) having a chiral carbon is an enantiomer having a surprising degree of unexpected canine COX-2 selectivity, a unique biological activity that virtually none of the classical NSAIDs having a carboxylic acid group, and in particular not their S-enantiomers. Therefore, the (S) -enantiomer of a preferred carprofen group of compounds of formula (I) having a chiral carbon has a surprisingly reduced degree of adverse gastrointestinal and other reactions in dogs compared to potentially all other NSAIDs having a carboxylic acid group, and particularly compared to their S-enantiomers. Thus, it would also be quite unexpected that the S-enantiomer of carprofen, because it is a very highly selective inhibitor of canine COX-2 isozyme, surprisingly improved the degree of anti-inflammatory, analgesic and antipyretic activity in dogs compared to potentially all other NSAIDs characterized by a carboxylic acid group.

One particularly preferred embodiment of the present invention is the use of only the (S) -enantiomer of carprofen, 6-chloro-methyl-9H-carbazol-2-acetic acid, as an active ingredient or medicament in the methods and compositions of the present invention. However, other embodiments are contemplated to be within the scope of this preferred group of the present invention. For example, non-racemic mixtures of the (R) - and (S) enantiomers may be used, in which case the (S) -enantiomer is present in an amount of at least 85%, preferably at least 90%, more preferably at least 95% and most preferably at least 99%. Since the (R) and (S) -enantiomers are identical in terms of molecular weight, density and the like, it is necessary to establish some basis for the above percentages. In other words, the percentages may be by weight, by volume, by chemical equivalents and the like. The reason for including the above amounts of the (R) -enantiomer may be quite practical, since it is not desirable to remove absolutely every last trace of the (R) -enantiomer from the racemic mixture. This may also be due to reasons relating to the overall useful biological properties.

It will also be appreciated by those skilled in the art that dosing ranges given elsewhere herein for a preferred class of carprofen compounds are described with respect to the racemic 50:50 mixture of enantiomers where a chiral compound is included. This is largely a matter of agreement. When the active ingredient used as the therapeutic composition comprises a mixture and nantiomers other than a 50:50 mixture or when the therapeutic composition contains substantially 100% of the (-) (R) - or (+) (S) -enantiomer alone, the skilled artisan is able to calculate the desired the actual dose amount directly, simply by multiplying said dose amount by a factor that reflects the ratio of the amount of enantiomer used to the amount present in said dose relative to the 50:50 mixture of enantiomers. Thus, when a dose of 4 mg / kg / day is indicated for a 50:50 racemic mixture, the corresponding dosage amount when using substantially 100% of the (+) (S) -enantiomer is one-half of said amount, i. 2 mg / kg / day.

Since the pharmaceutical compositions of the present invention comprising a member of a preferred group of carprofen compounds envisage the use of both racemic mixtures containing the 50% (S) -enantiomer and non-racemic mixtures with about 99% or less of the (S) -enantiomer together with less than 50% of the (R) enantiomer , the racemates of the carprofen group of the compounds of formula (I) having a chiral carbon must be distinguished into optically active isomers. This can be readily accomplished using techniques and techniques known in the art. For example, some racemic mixtures may be precipitated as eutectics after they have been resolved. However, it is usually preferred to use chemical procedures for resolution, depending on which diastereomers are formed from the racemic mixture by an optically active resolving agent. For example, optically active bases, e.g. D-α-methylbenzylamine, which can react with a carboxyl group. The diastereomers thus formed are then separated by selective crystallization and converted to the corresponding optical isomer.

Included within the scope of the present invention are all anti-inflammatory therapeutically active and pharmaceutically acceptable forms of salts, prodrugs and metabolites of the preferred carprofen group of compounds used in the present invention. This includes in particular their acid addition salts, wherein A is defined as anything other than a hydroxy group formed by treating compounds of formula (I) with pharmaceutically acceptable organic and inorganic acids, e.g. hydrogen halides such as hydrogen chloride, hydrogen bromide, hydrogen iodide, other mineral acids and their corresponding salts such as sulfate, nitrate, phosphate and the like, and alkyl and monoarylsulfonates such as ethanesulfonate, toluenesulfonate and benzenesulfonate, and other organic acids and their corresponding salts such as acetate, tartrate, maleate, succinate, citrate, benzoate, salicylate, ascorbate and the like.

When A is defined as a hydroxy group in a preferred carprofen group of the compounds used in the present invention, their salts may be formed by treatment with pharmaceutically acceptable bases. Examples of these bases are alkali metal hydroxides, including potassium hydroxide, sodium hydroxide and lithium hydroxide, alkaline earth metal hydroxides such as barium hydroxide and calcium hydroxide, alkali metal alkoxides, e.g. potassium ethanolate and sodium propanolate, and various organic bases such as piperidine, diethanolamine, and N-methylglutamine. Also included are aluminum salts of the compounds of formula (I).

In addition to the use of the various salt forms of the compounds of formula (I) described above, the scope of the present invention includes the use as an active ingredient of all analgesic and anti-inflammatory therapeutically active and pharmaceutically acceptable prodrugs and metabolites of the above compounds. In particular, it includes those derivatives wherein R ⁹ is defined as a C 1 -C 2 alkyl group, especially a methyl, phenyl or C 1 -C 2 phenylalkyl group, especially a benzyl group, wherein the phenyl is optionally monosubstituted by fluorine or chlorine, in particular 4-fluorophenyl, -C (= O) -R, wherein R is C 1 -C 2 alkyl or phenyl, especially acetyl and benzoyl, wherein the phenyl is optionally monosubstituted with fluorine or chlorine, or -C 1-6 alkyl; JOR ¹ , wherein R ¹ is an alkyl group containing 1 to 2 carbon atoms, in particular acetyloxy. These N-groups are readily cleaved during the metabolism of the compounds of formula (I), forming the desired prodrugs from these particular derivatives.

The present invention has been described in the paragraphs immediately above regarding a preferred class of carprofen compounds of formula (I). However, the present invention contemplates a broader scope with respect to providing anti-inflammatory COX-2 selective inhibitors. As noted above, such COX-2 selective inhibitors include those whose COX-2 selectivity to COX-1 selectivity ratio is at least 3: 1 based on in vivo and ex vivo total blood inhibitory levels measured at a dose or dose range providing> 80 % inhibition of COX-2. In accordance with the pharmacokinetic (PK) data described below, it has been shown that there is a relationship between a pharmacological or toxic response in dogs to an anti-inflammatory COX-2 selective inhibitor of the present invention and an achievable concentration of said inhibitors, e.g. in the blood or plasma of said dogs. For immediate exposure to the present invention, said pharmacological response in dogs is herein viewed as an anti-inflammatory response resulting from the inhibition of the COX-2 isozyme, whereas said toxic reaction in dogs is seen as an adverse side effect, e.g. gastrointestinal as a result of inhibition of the COX-1 isozyme. Consequently, the concentration of the inhibitory drug in the system of circulation of said dogs is related to the concentration of the inhibitory drug at its site of action.

As is well known, the most important pharmacokinetic parameters are bioavailability, the proportion of said drug that is absorbed into the systemic circulation, the volume of distribution, related to the amount of space in the body that is able to contain the drug, and clearance related to the body's ability to exclude medicine. According to the present invention, the ratio of selectivity of COX-2 to COX-1, which must be at least 3: 1, is determined by ex vivo measurement of the percent (%) inhibition of each of said isozymes in total dog blood. Briefly, the ex vivo measurement procedure performed comprises initially administering a test compound of a selective inhibitor at a predetermined total dose level, e.g. 4 mg / kg (2 mg / lb), pre-selected dog. The dose is administered according to a predetermined dosage regimen, e.g. once daily (sid), twice daily (bid), and the like, after total blood samples were taken from each test animal. Assays for the activity of COX-1 and COX-2 is based on stimulation of said blood with either a calcium ionophore for thromboxane _B2 (COX-1) or lipopolysaccharide (LPS) for prostaglandin _E2 (COX-2). These procedures are further described in more detail below in the description of the examples.

COX-2 selectivity is an essential property of the anti-inflammatory inhibitors useful in the present invention and is desirably at a ratio of at least 3: 1 compared to inhibition of COX-1 activity. For a given compound, it is not sufficient to simply have a selectivity ratio of 3: 1 since the compound could have the desired selectivity ratio at some point over its total dose range, but would fail to provide an overall adequate inhibition of COX-2 activity. Thus, it is further desirable that an anti-inflammatory inhibitor that is within the scope of the present invention also have to provide> 80% inhibition of COX-2 activity and preferably> 90% inhibition of COX-2 activity. This is considered to be the minimum degree of inhibition adequate to ensure satisfactory pharmacological activity with respect to the treatment and prevention of pain and inflammation.

Further, it is insufficient for a given compound to simply have a 3: 1 selectivity ratio at a given dose and provide £80% inhibition of COX-2 activity, preferably> 90% inhibition of COX-2 activity at some other dose. Preferably, both criteria must be met at the same level of exposure and at the same time after dosing. Therefore, it is also desirable that the 3: 1 selectivity ratio occurs at a dose concentration or dose concentration range that also provides 8080% inhibition of COX-2 activity.

In order to demonstrate the way in which the above-described limitations will be applied to a potential COX-2 selective inhibitor, the following example is given. Said candidate is evaluated according to the procedures described above for measuring COX-1 and COX-2 inhibitory activity in whole blood samples at a total dose of 4.4 mg / kg (2 mg / lb) administered sid, the activity levels being determined at 2, 4 and 12 hours after administration. When the data points for COX-1, COX-2, and untreated samples were plotted against inhibition (%) versus time, the COX-1 curve was found to decrease quite steeply from 10-15% inhibition at 2 hours to no inhibition observed in 12 hours. On the other hand, the COX-2 curve was found to decrease only slightly over the same time, decreasing from about 95% inhibition at 2 hours to about 90% inhibition at 12 hours.

Thus, it should be noted that at a dose of 4.4 mg / kg (2 mg / lb) administered s.i.d. the potential inhibitor fulfilled (1) a selectivity ratio of at least 3: 1 and (2) a limitation of> 80% inhibition of COX-2 required for the anti-inflammatory COX-2 selective inhibitor to fall within the scope of the present invention. Because the data from both graphs were compared to the same dose of 4.4 mg / kg (2 mg / lb) at the same time of 12 hours, the third limitation was also met, the first two restrictions being met for the same dose or range dose.

Because of the large number of different dosage amounts of said compound contemplated, similar determinations can be made and graphs drawn from the resulting data. However, the pooled results are demonstrated by graphing the% inhibition of COX-1 and COX-2 against dose or plasma concentration rather than time. The curve representing COX-2 data appears first and steeply increases, starting at the last dose concentration that produces identifiable% inhibition, and as the dose approaches higher concentrations, the curve reaches an inhibition level of> 80% and then becomes asymptotic. The COX-1 data curve appears later and rises less steeply than the COX-2 data curve and also becomes asymptotic with significantly less% inhibition. A contemplated compound is within the scope of the present invention when a line parallel to the y-axis and capturing both curves at the same dose concentration results in data points that satisfy both limitations, a 3: 1 ratio and> 80% inhibition.

The field of the present invention with respect to these anti-inflammatory COX-2 selective inhibitors included herein may be assessed or expressed in various terms, although substantially identical to those used above. For example, it is contemplated that the present invention provides, in particular, a method of treating or preventing pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, the method comprising administering to said dog for treating pain and inflammation a therapeutically effective amount of an anti-inflammatory cyclooxygenase inhibitor. 2 (COX-2), for which the in vitro potency of IC ₅₀ in said dog is at least 30 times greater, preferably at least 40 times greater, more preferably at least 50 times greater, even more preferably at least 60 times greater, and even more preferably at least 80 times greater - and at least 100 times greater than its in vitro potency of an IC 50 against cyclooxygenase-1 (COX-1) in said dog, wherein said inhibitor is a member selected from the group of anti-inflammatory compounds consisting essentially of acid derivatives salicylic acid, p-aminophenol derivatives, indole acetic acids and the like denoacetic acid, hetaryl acetic acid, arylpropionic acid, anthranilic acid, enolic acid and alkanones.

More specifically, according to the methods of the present invention described above, there is provided a method of treating or preventing pain and inflammatory processes and diseases as described above, wherein said in vitro potency of the IC 50 against the cyclooxygenase-2 (COX-2) inhibitor compound of Formula (I) is at least 100 times greater. as disclosed by the in vitro potency of IC 50 against cyclooxygenase-1 (COX-1), wherein one of X and Y is H and the other is a methyl group, and when both resulting enantiomers occur together, the (+) (S) enantiomer is present in an amount of at least 75%. In particular, there is provided a process as described above wherein for formula (I), for R ² , n = 1, one of X and Y is H and the other is methyl, and A is hydroxyl, C 1 -C 2 alkoxy or amino, R ⁶ is halogen, in particular chloro or trifluoromethyl and R ⁹ is H, methyl, acetyl, benzoyl or methoxycarbonyl, and when the two resulting enantiomers are present together, the (+) (S) -enantiomer is present in an amount of at least 85%, preferably at least 90%, more preferably at least 95% and most preferably at least 99%.

Still further provided are the methods described above, wherein said inhibitor comprises 6-chloro-α-methyl-9H-carbazole-2-acetic acid and when the two resulting enantiomers are present together, the (+) (S) -enantiomer is present in an amount at least 85%, preferably at least 90%, more preferably at least 95% and most preferably at least 99%. In particular, there is provided a process as described above, wherein said inhibitor is composed entirely of the (+) (S) -enantiomer of 6-chloro-or-methyl-9H-carbazole-2-acetic acid.

The present invention may also be described as providing a method that selectively inhibits substantially only inducible cyclooxygenase-2 (COX-2) without substantially inhibiting the corresponding constitutive cyclooxygenase-1 (COX-1), thereby treating or preventing pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, the method comprising administering to said dog for treating pain and inflammation a therapeutically effective amount of an anti-inflammatory compound of formula (I) above wherein R ² , X, Y, n, and R ⁶ and R ⁹ are as defined, including (-) (R) and (+) (S) -enantiomers, and all anti-inflammatory therapeutically active and pharmaceutically acceptable forms of salts, prodrugs and metabolites of the above compounds. Here again, when the inhibitor of formula (I) exists as the (-) (R) - and (+) (S) -enantiomers, the (+) (S) -enantiomer itself is provided according to the present invention or when both enantiomers are present together, provided a racemic or non-racemic mixture thereof.

To some extent substantially parallel to its other above descriptions, the present invention may be further described as comprising the above method that selectively inhibits substantially only inducible cyclooxygenase-2 (COX-2), wherein said inhibitor comprises carbazole-2-acetic. and wherein when both of the resulting enantiomers are present in the 6-chloro-N-methyl-9H acid, the (+) (S) -enantiomer is present in an amount of at least 85%, preferably at least 90%, more preferably at least 95% and most preferably at least 99%. In particular, there is provided a process as described above, wherein said inhibitor is composed entirely of the (+) (S) -enantiomer of 6-chloro-α-methyl-9H-carbazole-2-acetic acid.

When the compounds of formula (I) or their enantiomers or salts thereof are to be used as active ingredients in the methods and compositions of the present invention, they can be incorporated into standard pharmaceutical dosage forms. For example, they are useful when administered in systemic or topical, oral or parenteral applications, and for this purpose they are combined with conventional pharmaceutical excipients, diluents and adjuvants, e.g. organic and inorganic inert carrier materials such as water, gelatin, lactose, starch, magnesium stearate, talc, vegetable oils, acacia, polyalkylene glycols, and the like. These pharmaceutical preparations can be used in solid form, e.g. as tablets, lozenges, suppositories, capsules, and in particular in combination with a tasty portion of a dog-friendly feed or as a food additive or can be administered in liquid form, e.g. such as solutions, suspensions, standard and inverse emulsions and elixirs. Pharmaceutical excipients and adjuvants which may be added include preservatives, antioxidants, antimicrobial agents and other stabilizers, wetting agents, emulsifiers and suspending agents, and anti-caking compounds, fragrances and colorants, preparations to improve compressibility, or to induce delayed, persistent and / or or controlled release of the active ingredient and various salts to alter the osmotic pressure of the pharmaceutical composition or to act as buffers. Particular dosage forms that have been used successfully include 5% mixed micellar solution of carprofen for intravenous injections and 3% digestible paste and oral tablets at doses of 25 mg, 75 mg and 100 mg.

In the methods and compositions of the present invention, particularly those wherein the inhibitor comprises 6-chloro-α-methyl 9 H -carbazol-2-acetic acid and both resulting enantiomers co-exist, a non-racemic mixture is a preferred embodiment. In particular, in such preferred non-racemic mixtures, it is desirable to have the (+) (S) -enantiomer present in an amount of at least 85%, preferably at least 90%, more preferably at least 95%, and most preferably at least 99%. Thus, in such non-racemic mixtures, the (+) (S) -enantiomer will be the predominant component, not only because it is significantly more potent than the (-) (R) -enantiomer in inhibiting cyclooxygenase-2 (COX-2) but also because it is highly selective for inhibition of cyclooxygenase-2 (COX-2) as compared to cyclooxygenase-1 (COX-1). Corresponding minor amounts of the (-) (R) -enantiomer, i. less than 15%, less than 10%, and less than 5% are optionally included when it is considered desirable to balance the inhibitory properties of the cyclooxygenase or other enzyme. When the amount of the (-) (R) -enantiomer present is less than 5% and less than 1%, the reason for inclusion will usually reflect the practicality of the method used to distinguish the enantiomers. When this process is time-consuming or expended, it is often desirable from a practical point of view to simply allow this minor portion of the (-) (R) enantiomer to be transferred to the final product of the non-racemic mixture.

The anti-inflammatory inhibitors of formula (I) of the present invention can be administered to a systemically treated dog as a pharmaceutical composition in a suitable liquid form by injection or infusion. There are a large number of sites and organ systems in the body of the dog that allow a properly formulated pharmaceutical product to penetrate the entire body and all organ systems of the treated dog once injected or infused. Injection is a single dose of the pharmaceutical composition usually injected into a selected tissue using a syringe. The most common types of injections are intramuscular, intravenous and subcutaneous. In contrast, infusion is the gradual introduction of the pharmaceutical composition into the selected tissue. The most common type of infusion is intravenous. Other types of injection or infusion include intraarterial, intra- or transdermal (including subcutaneous) or intraspinal, especially intrathecal. In liquid pharmaceutical preparations, the anti-inflammatory inhibitor may be included in the solution as a solute. However, the most common and most preferred type of such formulation requires a salt inhibitor which has reasonably good water solubility. Water (or saline) is by far the most preferred solvent for these formulations. Occasionally, supersaturated solutions may be used, but these pose a stability problem that makes them impractical for daily use.

If it is not possible to obtain a form of a compound of formula (I) having the necessary degree of water solubility, as can sometimes happen, it is within the ability of the skilled person to prepare an emulsion, a dispersion of small droplets of one liquid, discontinuous or internal , continuous or external phase with which it is immiscible. The two fluids are maintained in an emulsified state using emulsifiers that are pharmaceutically acceptable. Thus, if the anti-inflammatory inhibitor is a water-insoluble oil, it may be administered in an emulsion in which it is a discontinuous phase. Although the inhibitor is water soluble but can dissolve in a water-immiscible solvent, an emulsion may be used. While the inhibitor will most commonly be used as the discontinuous or internal phase of what is called an oil-in-water emulsion, it may also be used as the discontinuous or internal phase of the inverse emulsion, commonly referred to as water-in-oil. In this case, the anti-inflammatory inhibitor is water soluble and can be administered as a simple aqueous solution. However, inverse emulsions are inverted when injected or infused into an aqueous medium, such as blood, and provide the advantage in this aqueous medium that they provide a faster and more efficient dispersion of the inhibitor than would be achieved using an aqueous solution. Inverse emulsions are prepared using suitable pharmaceutically acceptable emulsifiers well known in the art. When the anti-inflammatory inhibitor has limited solubility in water, it may also be administered as a suspended solid in colloidal form or in microparticle form in a suspension prepared using suitable pharmaceutically acceptable suspending agents. Suspended solids containing the inhibitor may also be formulated as delayed, sustained and / or controlled release formulations.

While systemic administration is most often accomplished by fluid injection or infusion, there are many situations in which it is preferable or even necessary to administer an anti-inflammatory inhibitor as a solid. Systemic administration of the solids is accomplished by instillation, inhalation, or insufflation of the pharmaceutical composition in a suitable solid form containing the inhibitor. Installing the inhibitor may require incorporation of the solid implant preparation into suitable body tissues or cavities. The implant may comprise a matrix of biocompatible and biodegradable material in which the solid inhibitor particles are dispersed, or in which droplets or isolated drops of the liquid inhibitor are optionally retained. Desirably, the matrix is decomposed and completely absorbed by the body. The matrix formulation is also preferably selected to provide delayed, sustained and / or controlled release of the inhibitor over an extended period of time, even up to several months.

A number of dosage forms described herein may be formulated to provide delayed, sustained and / or controlled release of the active ingredient from said dosage form. In a particularly preferred aspect of the pharmaceutical compositions of the present invention that provide delayed, sustained and / or controlled release of the active ingredient of an anti-inflammatory selective COX-2 inhibitor, all such orally administered dosage forms that achieve> 80% inhibition of COX-2 isozyme activity are included; result in plasma concentrations of said inhibitor of at least 10 µg / ml for at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, even more preferably at least 16 hours, and even more preferably at least 20 hours, and most preferably about 24 hours. Advantageously, the above-described dosage forms that achieve> 80% inhibition of COX-2 isozyme activity and result in plasma concentrations of said inhibitor of at least 15 μς / πιΐ are at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, even more preferably at least 20 hours, and most preferably about 24 hours. More preferably, the above-described dosage forms that achieve> 90% inhibition of COX-2 isozyme activity and result in plasma concentrations of said inhibitor of at least 20 gg / ml are at least 4 hours, preferably at least 8 hours, more preferably at least 12 hours, even more preferably at least 20 hours, and most preferably about 24 hours.

Therefore, a useful controlled release dosage form of the carprofen of the present invention is one that maintains a carprofen plasma level greater than 2 gg / ml most of the day after a single oral dose of 4.4 mg / kg (2 mg / lb). Preferred oral controlled release dosage forms of carprofen according to the present invention are those that maintain a plasma concentration of carprofen greater than 2 µg / ml for a period of time longer than that during which a comparable plasma level of the immediate release dosage form of carprofen is maintained when indicated. immediate release dosage forms and controlled release dosage forms administered at the same dose, e.g. 4.4, 3.96, 3.52 or 3.08 mg / kg (2, 1.8,

1.6 or 1.4 mg / lb). For example, the preferred 4.4 mg / kg (2 mg / lb) oral controlled release dosage forms of the present invention maintain a carprofen plasma concentration of greater than 10 µg / ml for greater than 10.5 hours.

Immediate release carprofen dosage forms containing doses of 3,96, 3,52 or 3,08 mg / kg (1,8, 1,6 or

1.4 mg / lb) maintain a plasma concentration of carprofen greater than 10 µg / ml for 9.5 hours, 8.5 hours and 7.5 hours, respectively. Preferably, 3.96 mg / kg (1.8 mg / lb) oral controlled release dosage forms maintain a plasma concentration of carprofen of greater than 10 µg / ml for greater than 9.5 hours. Similarly, maintaining thresholds at doses of 3.52 mg / kg (1.6 mg / lb) and 3.08 mg / kg (1.4 mg / lb) lasts 8.5 hours and 7.5 hours, respectively. Practical characteristics for preferred oral dosage forms for the controlled release of carprofen at doses higher than

4.4 mg / kg (2 mg / lb) or less than 3.08 mg / kg (1.4 mg / lb) can be calculated on the basis of linear pharmacokinetics, the controlled forms are able to maintain (2 mg / lb / day) is simultaneously an oral dose according to the like. More preferably, the oral carprofen release dosage forms are those that maintain a plasma concentration of carprofen greater than 10 gg / ml for a period of time longer than or equal to the period observed after the immediate release carprofen dosage form is administered at any higher dose. Thus, these controlled release dosage forms of carprofen maintain at least 80% inhibition of COX-2 in canine blood over a period of time longer than that achieved by the immediate release dosage form at a higher dose.

Most preferred oral drug release of carprofen are those that have plasma levels of carprofen above about 10 gg / ml for a period of time longer than or equal to the period observed after the immediate release formulation of carprofen.

4.4 mg / kg (2 mg / lb) (10.5 hours) when said oral controlled release dosage forms of carprofen are administered at a dose of less than 4.4 mg / kg (2 mg / lb). The performance of the 4.4 mg / kg (2 mg / lb) oral carprofen immediate release dosage form is taken as a baseline standard for the purpose of this comparison, since the 4.4 mg / kg / day recommended and accepted efficacy of the present invention as described herein .

The term implant always refers to a solid pharmaceutical composition containing an anti-inflammatory inhibitor, while a depot usually means a liquid pharmaceutical composition containing an anti-inflammatory inhibitor that is stored in any suitable body tissue or cavity to form a reservoir or reservoir that slowly migrates to surrounding tissues and organs. systemically expand. However, these distinctions are not always strictly adhered to in the art, and it is therefore believed that liquid implants and solid depots, and even mixed solid and liquid forms, are included within the scope of the present invention. Suppositories can be viewed as a type of implant because they contain starting materials that are solid at room temperature but melt at body temperature and slowly release the active ingredient to saturate the surrounding body tissues where the active ingredient is absorbed and transported by systemic administration is achieved.

Systemic administration may also be accomplished by inhalation or insufflation of the powder, i. a particulate preparation comprising an inhibitor. For example, an inhibitor in powder form may be inhaled into the lungs using conventional particulate spray device. The inhibitor as a particulate preparation may also be administered by insufflation, i. introduced or otherwise dispersed in suitable body tissues or cavities by simple dusting or by using conventional particulate spray device. These particulate formulations may also be formulated to provide delayed, sustained and / or controlled release of the anti-inflammatory inhibitor in accordance with well-known principles and materials.

Other means of systemic administration that can use the inhibitors of the present invention in either liquid or solid form include the transdermal, intranasal, and ophthalmic routes. In particular, transdermal patches prepared according to well-known drug delivery technology can be prepared and applied to the skin of a treated dog, whereby the active ingredient, due to its formulated soluble properties, migrates across the epidermis and into the dermal layers of the dog skin. distribution of the active ingredient over the desired extended period of time. Also included are implants that are positioned below the epidermal layer of the skin, i. between the epidermis and the skin dermis of the treated dog. Such an implant is formulated according to the well-known principles and materials commonly used in this drug delivery technology and can be prepared to provide delayed, sustained and / or controlled release of the active ingredient into the systemic circulation of the dog. These subepidermal (subcuticular) implants are as easy to introduce and are effective as transdermal patches for drug delivery, but do not have the disadvantage that they will degrade, be damaged or accidentally removed as a result of being exposed to the dog's upper skin.

Pharmaceutical formulations of special types suitable for oral administration to dogs may also be proposed. Pharmaceutical preparations suitable for oral administration, i. oral ingestion or oral administration may be liquid or solid. Preferred oral dosage forms for systemic administration are solids, e.g. palatable oral preparations such as fast dissolving tasty wafers, tablets, capsules, caplets, health candies, lozenges and the like, and liquid substances, e.g. solutions, suspensions, emulsions, elixirs, elixirs, and the like. Useful pharmaceutical formulations of special types suitable for oral administration to dogs include, but are not limited to, items such as oral paste administered at the root of the treated dog's tongue, granular form for administration by mixing in dog food, and chewable form where the active ingredient is consumed together. with an acceptable feed or chewable form which can administer the active ingredient by leaching from the portion of the feed that is not digested during chewing to the treated dogs. As is known in the art, the formulation of such acceptable formulations takes into account the dog's behavior with regard to the extent of chewing of the dosage form to be used and the resulting dosage level.

With respect to other routes of administration and corresponding dosage forms described herein, dosage forms intended for oral administration are also suitably formulated to provide controlled, sustained and / or delayed release of the active ingredient. Typically, this includes oral delayed release tablets, capsules and microparticles, as well as enteric tablets and capsules that prevent release and absorption of the active ingredient in the dog's stomach and facilitate intestinal administration distal to the dog's stomach, i. in the dog's intestines. Other typical oral dosage forms include sustained release oral tablets, capsules and microparticles that provide systemic administration of the active ingredient in a controlled manner over an extended period of time, e.g. for 24 hours. Where rapid administration of the active ingredient is desired or desirable, a controlled release oral dosage form can be prepared in the form of a fast-dissolving tablet, which will preferably also include highly soluble salt forms of the active ingredient.

The present description of the dosage forms contemplated within the scope of the present invention has categorized these forms, to a large extent from convenience, into local and systemic administration forms as well as solid and liquid forms. However, these resolutions are quite arbitrary and are not to be taken in any way limiting the scope of the present invention in terms of administration routes and dosage forms. For example, the description has already made it clear that some routes of administration, although seemingly local, may also have systemic effects or consequences. The line between liquid and solid dosage forms outlined herein may also be unclear in common practice. For example, a suitable oral dosage form for use in the present invention includes encapsulated solutions, mixed solid and liquid formulations. Microemulsion formulations, also in the field of the present invention, can also be characterized as mixed solid and liquid dosage forms.

The anti-inflammatory inhibitor may also be administered topically to the site of inflammation of the treated dog. Topical administration against systemic administration means a more focused method against a more generalized method of administering a pharmaceutical composition comprising an anti-inflammatory inhibitor to a dog suffering from pain and inflammation. However, the use of depots and implants as well as delayed, sustained and / or controlled release formulations tends to obscure these distinctions. Therefore, the above-described liquid and solid pharmaceutical formulations containing an anti-inflammatory inhibitor can also be used for topical administration, but with an emphasis on selecting ingredients for said formulations which tend to promote absorption of the inhibitor into local tissue at the site of administration but also tend to prevent infiltration and migration of the inhibitor into distant and different tissues and subsequent systemic transfer.

Topical administration is directed to suitable tissues and body cavities into which an anti-inflammatory inhibitor can be injected, infused, implanted, deposited, inserted, instilled or insufflated. Such administration may include, but is not limited to, intraarterial, intraarticular, intrachondral, intracostal, intracystic, intra- or transdermal, intrafascicular, intraligamentous, intramedullary, intramuscular, intranasal, intraneural, intraocular, i. j. ophthalmic administration, intraosteal, intrapelvic, intrapericardial, intraspinal, intrasternal, intrasynovial, intratarsal or intrathecal or intravenous.

Pharmaceutical formulations in liquid form containing an inhibitor provide the advantage of allowing fluid to be injected into or close to the site of inflammation. This is particularly the case where the joint is inflamed or degenerative joint disease. By injecting the inhibitor directly into the joint, a high concentration of inhibitor can be achieved in a short time, which not only substantially increases the inhibitor's access to the site of inflammation and hence the therapeutic activity of the inhibitor, but also minimizes the occurrence of unexpected adverse reactions that might otherwise occur. The result is a high local inhibitor concentration with a correspondingly low systemic transferred concentration.

Injectable pharmaceutical formulations containing an inhibitor can also be made, wherein the pharmaceutical formulation is in a delayed, sustained and / or controlled release form. The formulations of the recognized formulation may be solids, semi-solids, gels, or other fluid / solid combinations in which a disintegrating matrix or series of coatings is used to ensure continuous release of the inhibitor at a predetermined rate or variable rate, if desired. Prolonged-release and long-acting terms as well as other terms are used to describe these formulations. All of them use different combinations of biodegradable polymers, e.g. various cellulose-based polymers, and natural materials, e.g.

magnesium stearate to achieve slow release of the inhibitor contained in the pharmaceutical compositions may be injected topically when suitably liquid or suspendable, or may be administered by other means if they are of a more solid nature.

A therapeutically effective amount for the treatment of pain and inflammation of the inhibitory compounds of formula (I) is administered to the treated dog in an amount expressed as milligrams per kilogram of body weight of said dog per day: mg / kg / day. The term daily as used herein should not be construed as necessarily requiring that each particular dosage form be administered to the treated dog on a daily basis. The term daily is merely an indication of the smallest satisfactory, but relative, period of time that is used as part of the total unit for measuring the dose of the anti-inflammatory inhibitor administered. The dose, i. the therapeutically effective amount for treating pain and inflammation of an inhibitor is usually in the range of about 0.01 mg / kg to about 20.0 corn starch and / or uniform in the matrix. These mg / kg / day, preferably from about 0.1 mg / kg to about 12.0 mg / kg / day, more preferably from about 0.5 mg / kg to about 10.0 mg / kg / day, and most preferably from about 0.5 mg / kg to about 8.0 mg / kg / day. For example, a 50 lb dog has 23 kg (1 kg = 2.2 lb) and thus will be most preferably treated with about 10 mg to about 180 mg of the therapeutic composition per day. The partial amounts are not significant and the dosage will be appropriately rounded to a number that corresponds to the unit dosage amounts normally available; when the dosage form is e.g. an injectable liquid, a preferred dosage amount can be achieved more accurately. On the other hand, when the dosage form is e.g. oral tablet, a greater approximation of the preferred dose will have to be made. Thus, a 10 mg dose approaches a halved 25 mg tablet and a 180 mg dose approaches the use of a 100 mg tablet together with a 75 mg tablet or three 25 mg tablets, as these are typical dosage amounts in oral tablets. As one skilled in the art will recognize, when the most commonly used dosage form is an oral tablet and a large number of dogs are treated with a daily regimen, additional convenience is achieved through the use of a dispenser containing all available dosage amounts of said tablets, e.g. 25 mg, 75 mg and 100 mg tablets. Thus, virtually any preferred dosage amount can be approximated using a combination of said tablets and / or halves thereof.

It is not only necessary for the skilled artisan, such as a veterinarian, to determine the preferred route of administration and the appropriate dosage form and amount, but also to determine the dosage regimen, i. frequency of doses. Generally speaking, it is most likely that the choice will be between once a day (s.i.d.) and twice a day (b.i.d.), and that the former will provide faster and more thorough treatment, while the latter will provide less thorough but more durable treatment. However, this generalization does not take into account such important variables as the specific type of pain or inflammation, the specific therapeutic agent and its pharmacokinetics, and the specific patient (dog) involved in the treatment. With the approved product on the market, much of this information is already provided due to the results of clinical trials conducted during approval. In other cases, this information may be obtained directly according to the teachings and instructions contained in the immediate descriptions perceived in the light of the knowledge and experience of the practitioner. The results obtained may also be correlated with data from corresponding evaluations of the approved product in the same tests.

The above dosage amount ranges, which are also given elsewhere herein, are for racemic mixtures of compounds of formula (I) having a chiral carbon or for individual compounds of formula (I) where no chiral carbon atom is present. As will be appreciated by the skilled artisan, i. the anti-inflammatory therapeutically effective amount will vary with the practicing veterinarian or a person with advanced animal health experience when using a non-racemic mixture of the compounds of formula (I). For example, when 85% of the mixture is the (S) enantiomer, the necessary dose is usually reduced. These considerations are based on the assumed equal potency and the fact that the (S) -enantiomer is significantly more active than the (R) -enantiomer. However, other differences, the degree of difference between the activities of the two enantiomers, in particular the difference in pharmacokinetics between the enantiomers, must also be considered when determining the proper dosage. For example, it has been found that there is a significant difference in clearance rate between the (-) (R) and (+) (S) enantiomers. This again affects the calculation of the amount of active compound to be administered. The practitioner will normally determine this on a case-by-case basis, as is common practice in the art, such as established methods by which the data necessary for auxiliary calculations can be obtained.

Typical dosage forms and amounts will include (1) intravenous administration of carprofen at a dose of 4.0 mg / kg / day by body weight injected into the right v. cephalica, (2) oral carprofen at a dose of 4.0 mg / kg / day by body weight as a toothpaste applied to the root of the tongue, administered one hour before feeding, and (3) oral carprofen at a dose of 4.0 mg / kg / day by body weight as 25 mg, 75 mg and 100 mg tablet preparations, applied to the root of the treated dog's tongue, administered one hour before feeding.

The active ingredients of the present invention may also be combined with other therapeutically active ingredients, which will be readily apparent to one skilled in the art, which will normally be determined by the circumstances in which the therapeutic composition of the present invention is administered. For example, when the joint is seriously inflamed and simultaneously infected with microorganisms, e.g. bacteria, fungi, protozoa, viruses and the like, it will be desirable to administer the active ingredient of the present invention in combination with one or more antibiotics, antifungal, antiprotozoal, antiviral or similar therapeutic agents. The active ingredient of the present invention may be administered not only in combination with other NSAIDs of the type described in greater detail herein but also in combination with inhibitors of other inflammatory mediators to provide multiple inflammatory inhibitory activity. Other classes of these inhibitors and examples thereof include e.g. one or more members selected from the group consisting essentially of classes of these inhibitors and examples thereof, including H 1 -receptor antagonists, B 1 and B ₂ kinin receptor antagonists, prostaglandin inhibitors such as PGD-, PGF-, PGI _2- and PGE, thromboxane A ₂ (TXA2-) inhibitors, lipoxygenase 5- and 12- inhibitors, LTC ₄ , LTD4 / LTE4- and LTB4- leukotriene inhibitors, PAF receptor antagonists, gold in the form of aurothio group with various hydrophilic groups, immunosuppressive agents, e.g. cyclosporin, azathioprine and methotrexate, anti-inflammatory glucocorticoids, penicillamine, hydroxychloroquine, anti-gout agents, e.g. colchicine, xanthine oxidase inhibitors, e.g. allopurinol, and uricosuric drugs, e.g. probenecid, sulfinpyrazone and benzbromarone.

Because inflammation is most prevalent among geriatric dogs, those skilled in the art will appreciate that the anti-inflammatory compositions of the present invention can also be administered in combination with therapeutic agents for treating disease states, syndromes and symptoms, often found in older dogs. Such therapeutic compositions and conditions in which they are used include, e.g. cognitive therapeutics to counteract memory loss and deterioration. Another large group of such therapeutic agents include antihypertensives and other cardiovascular drugs designed to compensate for hypertension, ischemic heart disease including angina, stasis heart failure and myocardial infarction, e.g. diuretics, vasodilators such as hydralazine, β-adrenergic receptor antagonists such as propranolol, angiotensin II converting enzyme inhibitors (ACIA inhibitors) such as enalapril used for the treatment of geriatric dogs with mitral insufficiency inhibition alone and enalaprilide antagonists angiotensin II receptor such as losartan, renin inhibitors, calcium channel blockers such as sympatholytics such as methyldopa, α 2 -adrenergic such as clonidine, α-adrenergic antagonists such as prazosin, and HMG-CoA reductase ester (antihyperperololesterol) such as lovastatin.

Still other classes of such therapeutic agents include antineoplastic agents, particularly antimitotics, including vincaalkaloids such as vinblastine and vincristine, to treat various cancers, therapeutic agents to treat renal failure, anti-obesity drugs to treat psoV excess weight problems, anti-parasitic drugs nifedipine, an agonist, a receptor, both endo- and ectoparasites, which typically attack dogs and itching medications to treat different types of pruritus in dogs.

Other types of drugs that can be used in combination with the anti-inflammatory agents of the present invention include growth hormone secretagogues, potent analgesics, local and systemic anesthetics and H2-receptor antagonists, and other gastroprotective agents. Those skilled in the art will recognize that some of the above combinations of therapeutic agents are most commonly used to treat various acute conditions in dogs, e.g. bacterial infections concomitant with degenerative joint disease. But these experts have the same, if not more, interest in treating chronic conditions in dogs.

According to the regimen used for this purpose, it is contemplated that the anti-inflammatory compositions of the present invention will be administered in combination with other drugs commonly used to treat chronic conditions such as osteoarthritis. It is also contemplated that administration in combinations will be understood as a number of different forms of course and will still be within the scope of the present invention. For example, the anti-inflammatory compositions of the present invention can be simply formulated with one or more other therapeutic agents that form the intended combination into a suitable dosage form, such as an oral tablet, containing all the medicaments forming the combination. The varying half-lives of different drugs may be adapted to those skilled in the art of preparing formulations through the controlled-release forms of said drugs with different release times so as to achieve a relatively uniform dosage. The dosage form used for compounding can also be prepared according to well-known principles of formulation technology, wherein the drugs used in combination simply co-exist in the additive in the feed composition. The present invention also contemplates co-administration in which the combination of drugs is accomplished by the simultaneous administration of the drugs to be combined. This co-administration may even be through different dosage forms and modes of administration. The present invention further contemplates the use of such combinations according to different but regular and continuous dosing schedules that maintain the desired plasma levels of the drugs used in the treated dog, even if the individual drugs forming the combination are not administered simultaneously to said dog. The design and administration of all such combinations is routine in the art.

The methods and compositions of the present invention are useful for treating or preventing pain and / or inflammation in dogs. Pain, which is a more or less localized feeling of discomfort, distress or distress, is the result of stimulation of specialized nerve endings and may occur or be viewed as independent and inflammatory independent. For example, chronic pain and pain induced and / or associated with surgery, e.g. perioperative and postoperative pain is initially little associated with inflammation. Opioid analgesics are effective in the treatment of postoperative pain but do not affect inflammation. However, NSAIDs may be superior to these opioid analgesics in the treatment of some forms of postoperative pain and are particularly effective in situations where inflammation has induced pain sensitivity to normally non-painful mechanical or chemical stimuli. While NSAIDs inhibit the biosynthesis and release of prostaglandins, which are inflammatory mediators, there are data suggesting that the analgesic effect of NSAIDs may occur through a mechanism other than inhibiting prostaglandin synthesis.

Thus, the present invention has been described in terms of usefulness in the treatment and prevention of pain and inflammation, since these most commonly occur together in tissue injury and disease processes and conditions mediated by COX-2. However, it is not intended to be complicated to associate pain and inflammation with respect to the applicability of the present invention in their treatment and prevention, and must be viewed in the context of the present description as being the subject, either individually and separately or together in combination, compositions and other aspects of the present invention.

The inflammatory process itself can be triggered for a variety of causes, including infectious agents, ischemia, antigen-antibody interactions, and thermal or other physical injury. The response to each of these causes varies characteristically, but they all have a lot in common. Clinical symptoms include erythema, edema, sensitivity to touch and pain. Three distinct phases can be recognized, but each phase is mediated by a different mechanism. The first acute transient phase includes local vasodilation and increased capillary permeability, the second, delayed subacute phase includes leukocyte and phagocytic cell infiltration, and the third, chronic proliferative phase includes tissue degeneration and fibrosis. NSAIDs, as a therapeutic class of anti-inflammatory agents, have been shown to inhibit the enzymatic production and release of prostaglandins involved in the pathogenesis of inflammation and fever. However, NSAIDs do not inhibit the formation of eicosanoids, such as leukotrienes, which also contribute to inflammation, nor interfere with the emergence of numerous other mediators of inflammation.

According to the present invention, it has been found that a group of carprofen compounds of formula (I), and in particular carprofen alone, and hence the (S) -enantiomer of carprofen, the only NSAIDs having a carboxylic acid group, has surprising and unexpectedly high selectivity for the COX-2 isozyme. While this particular enzyme is an important mediator of inflammation, there are many other important mediators of inflammation that either have no interaction with NSAIDs or no well-known association with the effect of NSAIDs. These mediators include several groups of leukocytes, cellular adhesion molecules, soluble mediators, as well

C5a, PAF and leukotriene B1, cytokines such as IL-1 and TNF, growth factors such as GM-CSF and TGF-β, histamine, bradykinin and 5-HT. While the compounds of formula (I) are shown to be unequivocal COX-2 inhibitors, there is no intention to bind to any particular mechanism of action by which the compounds of formula (I) may exert their anti-inflammatory activity.

Indeed, it has been pointed out above that the anti-inflammatory action of carprofen and other compounds of formula (I) is not fully understood and that it has hitherto been considered that the actual mode of action may involve neutrophils, also known as polymorphonuclear leukocytes. PAF stimulates aggregation, release of leukotrienes and lysosomal enzymes, and the formation of superoxides, all of which promote inflammation, in these cells.

The uniqueness of carprofen and the compounds of formula (I) from NSAIDs has been generally presented. Although the compounds of formula (I) are clearly NSAIDs, they cannot easily be placed in any recognized NSAIDs chemical classification:

salicylic acid aspirin p-aminophenols acetaminophen indole / indene acetic acid indomethacin, sulindac, etodolac heteroarylacetic acid tolmetin, diclofenac, ketorolac arylpropionic acid ibuprofen, naproxen, flurbiprofen, ketoprofen anthranilic acid mefenamic acid, acid meclofenamic enolic acid oxicams, e.g. piroxicam, tenoxicam pyrazolidinediones phenylbutazone alkanones nabumetone

Carprofen and the compounds of formula (I), _and when they are propionic acids, do not belong to the subgroup of arylpropionic acids, since the carbazole group of carprofen is a heteroaryl and not an aryl group. Carprophenes do not belong to the heteroaryl acetic acid subgroup because carprophenes are propionic acids and not acetic acids. Carprofen cannot be placed in any of the other subgroups without breaking the basis of classification. The only NSAID approved for the treatment of humans that is recognized to be selectively active on human COX-2 is from the list above, nabumetone, which is not acid at all but 2-butanone. Although the acidic metabolite is active, only a small proportion of this metabolite is COX-2 selective in dogs.

Examples of numerous classical NSAIDs from this broad class are the compounds listed in the table below, which gives the common name and IUPAC name for each compound and its structure. All of the listed compounds were printed by the USP Dictionary of USAN and International Drug Names, (C.A. FLEEGER, ed., United States Pharamcopeial Convention, Inc. Rockville, MD, 1995). The USAN (United States Adopted Names) program provides simple and usable non-patented drug names, and the naming process begins when the drug enters the clinical trial stage. The name and structure of carprofen are given at the beginning of the table to facilitate comparison.

Table 1

GENERAL NAME .y · · ΛΚ> ·,. · .., -. NAME BY IUPAC STRUCTURE ......- * · · carprofen acid 6-chloro-α-methyl-9H-carbazole-2-acetic acid > 6-5 / 0 cr alclofenac acid 3-chloro-4- (2-propenyloxy) benzeneacetic acid Cl aspirin acid 2- (acetyloxy) benzoic acid " o u- _v o diflunisal acid · ............ 2 ', 4'-difluoro-4-hydroxy-3-. blphenylcarboxyl. ·· .'_ » 0 - ^H ° F

T

. . ·. ''; »T / .1 GENERAL NAME ....... · NAME BY IUPAC ·%. . * · -.:½. · VAt ·: ' STRUCTURE diclofenac acid 2 - [(2,6-dichloro-phenyl) amino] benzeneacetic acid 1 rR o ^{ϋ o ==} ^ 0 I Etodolak acid 1,8-diethyl-1,3,4,9-tetrahydropyrano [3,4-b] indole-1-acetic acid about felbinac acid [1,1'-biphenyl] -4-acetic acid · acid flufenamic acid 2 - {[3- (trifluoromethyl) phenyl] amino} benzoic acid O ^ OH Flunixin meglumine (Banamine®) ¹ ( acid 2 - {[2-methyl-3- (trifluoromethyl) phenyl] amino) -3-pyridinecarboxylic acid with 1-deoxy-1- (methylamino) -Dlucitol (1: 1) γ- ( ^CF ϊΡ α OH) 1 Flurbiprofen acid 2-fluoro-4-a-methyl- [1,1'-biphenyl] -4-acetic acid · F

i is a trademark approved for use outside of the United States

------- r-T, r. - GENERAL NAME \ ^J NAME BY IUPAC structure ζ ^ η ·: Ι ... . *> {w. 41 ibuprofen acid α-methyl-4- (2-methylpropyl) benzeneacetic acid AJ 1 llonidap 6-chloro-5-fluoro _m-2, 3-dihydro- (hydroxy-2-t_J.enylmetylen) -2-oxo-lH-indole-l-carboxamide ΟγΝΗ, I lndomet_acfn acid 1- (4-Chloro-benzoyl) -5-methoxy- 2-methyl-1H-indole-3-acetic acid JRO '· - 0 OH Indoprofen acids 4- (1,3-Dihydro-1-oxo-2H-indol-2-yl) -benzeneacetic acid CO-Q-Y », ⁰ I Isoxepafc and and acid. 6,11-dihydro-11-oxodibenz [b] oxepine-2-acetic acid I - r Ketoprofen (Ketofen®) ^{1 1} acid 3-benzoyl-a-methyl-benzeneacetic acid ⁰ 1 s ^ ^h ° of ÝÝVt

\

GENERAL NAME OF NAME BY IUPAC STRUCTURE Ί j 1 J · · .Λ Meclofenamic acid (Arquel®) ¹ acid 2 - [(2,6-dichloro-3-) methylphenyl) amino] benzoic acid ° V ° H _H ^C 'j δ acid mefenamic acid 2 - [(2,3-dimethylphenyl) amino] benzoic acid · - Meloxicam (Metacam) 4-hydroxy-2-methyl-N- (5-methyl- 2-t7Íazolyl) -2 H-l, 2- benzotJiazín-3-carboxamide 1 1 JB ^H o''o INabumeton and 6-MNA 4- (6-methoxy-2-naphthalenyl) -2-butanone, an agent metabolised in vivo to  active 6-methoxy-2-naphthylacetic acid (6-MNA), shown at right Sccnr naproxen acid 6-Methoxy-α-methyl-2-naphthaldhoacetate - acid niflumic acid 2- [3- (trifluoromethyl) aniline] nicotinic acid. 1 l about'

GENERAL NAME . · .. ·> 1 ...... · ... · * <* ·· · »* w ¢ 1 NAME BY IUPAC STRUCTURE Nimesulide (Sulidene®) ¹ 4'-nitro-2 H - fenoxymetansulfónanilid HjCSOjNH NO. NS-398 N- [2- (cyclohexyloxy) -4nitrofenyl] -methanesulfonamide Q ^ο γ ^ γ ' ^Ν ° ^ϊ H phenylbutazone 4-butyl-l, 2-diphenyl-3,5pyrazolidindión % —H I Piroxicam (Feldene®) ¹ 4-hydroxy-2-methyl-N-2-pyridinyl-2H-1,2-benzothiazole-3-carboxamide-1,1-dioxide * OH 0 p | J OX A 0 0 tenidap 5-Chloro-2,3-dihydro-3- (hydroxy-2-thienylmethylene) -2-oxo-1H-indole-1-carboxamide s ° γ-ΝΗ,

η5

The carprofen compound group, characterized by the functional α-methylacetic acid, is many times more COX-2 selective in dogs than any of the sulfonyl or sulfonamide-containing NSAIDs listed in the table above. To demonstrate this unexpected property, the COX-2 selectivity of carprofen, a compound of the present invention, and the COX-2 selectivity of certain selected compounds from the above table were compared. The results are illustrated in the working examples described below.

As described above, selectivity for COX-2 is usually determined as the ratio of inhibition of COX-1 to inhibition of COX-2 or vice versa. In the present disclosure, the ratio of COX-1 inhibition to COX-2 inhibition in dogs is used essentially for the sake of simplicity. Both inhibition values are the values of IC _50, which is, the more active the test compound, the lower the IC 50 value. In fact, this ratio reverses that when it is COX-1: COX-2 and the test compound is very selective for canine COX-2, the ratio will be a large number versus a number much less. Thus, the test compounds that are most selective for canine COX-2 will have the highest ratio number.

DETAILED DESCRIPTION OF THE INVENTION

Example 1

Comparative evaluation of canine cyclooxygenase-1 and -2 inhibition by carprofen and other NSAIDs

Dog COX-1 activity evaluation protocol

Test drugs were dissolved and diluted the day before the assay was performed in 0.1 mL DMSO, 9.9 mL Hanks Balanced Salt Solution (HBSS) and stored overnight at 4 ° C. On the day of the assay, citrate blood was collected from the donor dog, centrifuged at 190 xg for 25 minutes at room temperature, and the resulting platelet-rich plasma was then transferred to a new tube for further precedition. Plates were washed by centrifugation at 1500 xg for 10 minutes at room temperature. Plates were washed with plate buffer containing Hanks buffer (Ca free) with 0.2% bovine serum albumin (BSA) and 20 mM HEPES. Platelet counts were then adjusted to 1.5 x 10 ⁷ / ml and then 50 μ ml calcium ionophore (A23187) was added along with calcium chloride solution to 50 μΐ diluted test drug on the plates to give final concentrations of 1.7 μΜ A23187 and 1.26 mM Ca. Then, 100 μΐ of dog washed plates were added and samples were incubated at 37 ° C for 15 minutes, after which the reaction was stopped by adding 20 μΐ 77 mM EDTA. Plates were then spun at 2000 xg for 10 minutes at 4 ° C, and then 50 µΐ of the supernatant was assayed for thromboxane B ₂ (TXB ₂ ) by enzyme immunoassay (EIA). The amount of TXB ₂ in pg / ml was calculated using the standard line contained on each plate from which the percent inhibition of COX-1 and the IC ₅₀ value for the drugs tested can be calculated.

Protocol to evaluate canine COX-2 activity

The canine histiocytoma (macrophage-like) cell line from the American Type Culture Collection designated DH82 was used to introduce a protocol to evaluate the COX-2 inhibitory activity of various drugs tested. 10 µg / ml LPS was added to the flasks of these cells and then the tissue culture flasks were incubated overnight. For the COX-2 assay, the same test drug dilutions were used as described above in the COX-1 protocol, and prepared the day before the assay was performed. Cells were harvested from the culture flasks by wiping and then washed with Eagle's minimal medium (MOM) combined with 1% fetal bovine serum, spun at 1500 rpm for 2 minutes and adjusted to a concentration of 3.2 x 10 ⁵ cells / ml. To 50 μΐ of test drug dilution, 50 μΐ of arachidonic acid in MOM was added to give a 10 μΜ final concentration, and 100 μΐ of the cell suspension was also added to give a final concentration of 1.6 x 10 ⁵ cells / ml. Test suspensions were incubated for 1 hour, then centrifuged at 1000 rpm for 10 minutes at 4 ° C, and then 50 µL aliquots of each test drug sample were transferred to EIA plates. EIA was performed for prostaglandin E ₂ (PGE ₂ ) and the concentration of PGE ₂ was calculated using the standard line contained on each plate. From this data, the percent inhibition of COX-2 IC 50 value for test drugs can be calculated. Repeated investigations of COX-1 and COX-2 inhibition were conducted over several months. Averages were calculated from the results and one COX-1: COX-2 ratio was calculated. The data obtained, together with the number of tests performed for each test sample, are shown in the following table of values.

Table 2

- '•. • ji' - // NSALD '.. · ’· · <, ·, *, * .; γ ·· / \ ‘. Count test g · x i .. o I ^υ ° .. j. . · ·> ..... COX-2? I. IC _M μΜ ' : Íie0X-Tze, OX-2 ^ · λ?;. · · · .h ratio pjA Carprofen (racemate) 9 13.2 0102 129 Carprofen (S-) 3 6.71 0.0371 181 Carprofen (R- 4 > 25.0 5.97 > 19.04 Flufenamic acid 6 2.31 0.0475 48.6 nimesulide 6 2.15 0.0565 38.0 Niflumic acid 6 1.3 0.0464 22.2 Meclofenamic acid < ä 5 0737 0.0478 15.4 I Tolfenamic acid 4 0206 0.0137 15.0 I naproxen 3 7.8 0626 11.3 Mefena'ic acid 4 0403 0.0362 11.1 felbinac 3 2:54 0362 ™ i 6-MNA 6 28.3 4.21 6.72 NS-398 7 0.587 0137 4.28 flurbiprofen 4 0505 0123 4.10 diclofenac 3 0246 0.0778 3.16 Meloxicam 5 0891 0307 2.90 Fenylbutazo'n 5 > 10.0 3.79 ibuprofen 4 1.3 0391 2.63 tenidap 12 0469 0228 2.6 alclofenac 3 13.2 7:41 1.78 ilonidap 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 0223 0.585 0381 ketoprofen 5 0.0286 • 0123 0.232. indomethacin 6 0.0558 0.366 0152 aspirin 3 34.3 > 100 <0.343 vedaprofen •

Example 2

Determination of ex vivo inhibition of COX-1 and COX-2 activity by carprofen from canine whole blood

The aim of this study was to evaluate the inhibitory potency of carprofen against COX-1 and COX-2 activity using an ex vivo procedure in canine whole blood. Three dogs received a 10 mg / kg dose of racemic 6-chloro-α-methylcarbazole-2-acetic acid (carprofen) administered orally (po) in a capsule dosage form, three dogs received a dose of 2 mg / kg on the same basis, and three dogs were not treated. All dogs in the study were sampled 0 hours prior to dosing, followed by blood sampling at 1, 3, and 6 hours post-dose. Test tubes containing 2 μΐ of either (A) calcium ionophore A23187 giving a final concentration of 50 μΜ were prepared, stimulating thromboxane B2 (TXB2) production to determine COX-1 activity, or (B) lipopolysaccharide (LPS) to give a final concentration of 10 μg / ml, which stimulates the production of prostaglandin E2 (PGE2) to determine COX-2 activity. The test tubes used as controls contained vehicle and were not stimulated by the addition of any reagent. 500 μΐ blood samples were added to each of the test tubes described above and then incubated at 37 ° C for one hour for calcium ionophore-containing test tubes and overnight for LPS-containing test tubes. After incubation, 10 μΐ EDTA was added to give a final concentration of 0.3% to prevent plasma coagulation, which sometimes occurs after frozen plasma samples. The incubated samples were spun at 4 ° C and the resulting plasma sample of ~ 200 µΐ was collected and stored at -20 ° C in polypropylene 96-well plates. In order to determine the endpoints for this study, thawing was avoided to measure TXB2 production, and the PGE ₂ enzyme immunoassay kits (EIA) available from Cayman, using the principle of competitive binding of the indicator to the antibody and endpoint determination by colorimetry, were used. Plasma samples were diluted to approximate the range of the standard amount supplied in the diagnostic or research kit, ie 1/500 for TXB ₂ and 1/750 for PGE ₂ .

The data presented in Table 3 below demonstrate the percent inhibition of COX-1 and COX-2 activity relative to their values at 0 hours. Data are expressed as means of treatment groups in pg / ml TXB ₂ and PGE ₂ produced per sample. Plasma dilution was not mediated at these values.

The data in Table 3 shows that at a dose of 2 mg / kg, inhibition of COX-2 was significant at all time points. At 3 and 6 hours after dosing, a slight decrease in COX-2 inhibition was observed compared to data obtained at 10 mg / kg. The data in Table 3 also shows that at a dose of 2 mg / kg, there was no significant inhibition of COX-1 activity at any time point involved. This result was consistent with the observed excellent carprofen tolerance in dogs during the study. Data for the 10 mg / kg dose showed that at each time point there was complete inhibition of COX-2 activity and a very strong inhibition of COX-1 activity beginning at 1 hour and achieving stable levels over time points 3 and 6 hours. Thus, the data in Table 3 clearly demonstrates that at a dose of 2 mg / kg, the concentration of carprofen has good COX-2 selectivity. As the dose increased from 2 to 10 mg / kg, increasing inhibition of COX-1 activity became apparent.

Table 3

Inhibition of COX-1 activity - group diameters

TXB ₂ pg / ml / well Percent inhibition

Lesson 0 hrs 1 hour 3 hrs 6 hours 1 hour 3 hrs 6 hours untreated 102 134 77 65 0% 25% 36% 2 mg / kg 72 75 66 46 0% 8% 36% 10 mg / kg 51 31 6 7 39% 88% 86% inhibition activities COX-2 - group diameters PGE ₂ pg / ml / well Percentage of inhibition Lesson 0 hrs 1 hour 3 hrs 6 hours 1 hour 3 hrs 6 hours untreated 393 900 349 405 0% 11% 0% 2 mg / kg 511 33 98 190 94% 81% 63% 10 mg / kg 336 19 12 12 94% 96% 96%

COX inhibition is observed when the measured percent inhibition is greater than the percent measured in the untreated controls. The percent inhibition in the above table is calculated directly according to the following formula:

(PGE ₂ vt = 0) - (PGE ₂ vt = 1)% inhibition (1 hour) --------------------—-----, - - _x oooo (PGE ₂ vt = 0)

Example 3

Determination of ex vivo inhibition of COX-2 activity by carprofen from canine whole blood

This study followed the procedures described in the example but with some modifications described in more detail

Three dogs were dosed with 2 mg / kg of racemic 6-chloro-α-methylcarbazole-2-acetic (carprofen) above, below.

acid (carprofen) orally (po) as a tablet formulation at 0 hours, three dogs received 4 mg / kg on the same basis at 0 hours and three dogs were untreated. All dogs in the study were sampled at 0 hours prior to dosing, followed by blood sampling at 2 and 4 hours post-dose. Test tubes containing 2 μΐ of lipopolysaccharide (LPS) were prepared to give a final concentration of 10 µg / ml. Untreated test tubes were used as controls. 500 μΐ blood samples were added to each of the test tubes described above and then incubated at 37 ° C overnight. After incubation, 10 μΐ EDTA was added to give a final concentration of 0.3%. The incubated samples were spun at 4 ° C and the resulting plasma sample of ~ 200 µΐ was collected and stored at -20 ° C in polypropylene 96-well plates. Enzyme immunoassay kits (EIA) available from Cayman were used to measure PGE ₂ production, and the endpoint was determined by colorimetry. Plasma samples were diluted to 1/750 for PGE ₂ .

Plasma samples were also analyzed for total carprofen plasma concentration by HPLC with a Chromtech column (5 μπι, 100 x 4.6 mm, chiral AGP) and a mobile phase consisting of 10:90 (v / v) 2-propanol: 0.1M phosphate buffer pH 6.0 and detection by fluorescence (excitation 285 nm, emission 345 nm). Plasma samples (0.2 mL) were buffered with 0.05 M citric acid, pH 5.1, after addition of (S) -naproxen as an internal standard, and then extracted with 4: 1 (v / v) diethyl ether: dichloromethane. The ether layer was separated and then re-extracted with 0.005M Na ₂ CO ₃ , then the organic phase was removed by suction. The aqueous phase was buffered with 0.05M citric acid pH 5.1. And then extracted again with diethyl ether: dichloromethane. The ether was then transferred to clean tubes, evaporated under a stream of nitrogen, and the pellet was reconstituted in the mobile phase for HPLC for analysis.

The data presented in Table 4 below show PGE ₂ production (pg / ml / well), percent inhibition of COX-2 activity based on the values of each dog at 0 hour, and plasma exposure for individual dogs.

The data in Table 4 show that at 2 and 4 hours post-dose, a small difference was observed between doses, with a 4 mg / kg dose showing slightly greater inhibition of COX-2. Inhibition of COX-2 ex vivo is well correlated with plasma concentrations of carprofen.

Table 4

Inhibition of COX-2 activity and plasma concentrations of carprofen Dog no. dose (Mg / kg) PGE ₂ pg / ml / well percentage inhibition plasma concentration gg / ml 0 hrs 2 hrs 4 hours 2 hrs 4 hours 2 hrs 4 hours 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 directly according to the following formula:

(PGE ₂ tt = 0) - (PGE ₂ tt = 2)% inhibition (2 hours) = ------ «. I —-» - ---- w ------ τ --- r - χ 100 (PGE ₂ vt = 0)

Example 4

Determination of in vivo inhibition of COX-2 activity by carprofen in dogs in a model of carrageenan-induced inflammation

The aim of this study was to monitor the in vivo activity of COX-2 in a subcutaneously implanted chamber during induced inflammation. The COX-2 enzyme can be detected by Western analysis as early as 5 hours after carrageenan treatment (T. Kirchner et al., J. Pharmacol. Exp. Ther. 282, 1094-1101, 1997). In addition, COX-1 activity can be simultaneously determined by ex vivo methods as described in Example 2. Six Beagle dogs were surgically implanted with polyethylene golf balls with a whiffle approximately outside the diameter of about 4.2 cm below the skin just behind the shoulder blades. They recovered from surgery 1 month before being assigned to the experimental groups.

The experiment was performed in two replicates. On the day of the experiment, three dogs were dosed with 2 mg / kg carprofen for 30 minutes before the onset of inflammation and the other three dogs were used as controls. The hair around the ball was shaved and the area searched for a hole in the ball. This region was then labeled and sterilized with 2% iodine tincture. A needle was inserted into the opening and 1.5 cm ^{3 of} fluid, hereinafter referred to as exudate fluid (EF), was removed. After removal

1.5 cm ^{3 of} EF were added to the ball to induce an inflammatory process of 1.5 cm ^{3 of a} 0.33% solution of carrageenan in H ₂ O. EF samples were taken at 0, 5 and 24 hours after carrageenan injection. Blood samples were also collected so that whole blood COX-1 assays could be performed as described in Example 2.

EF samples had to be purified prior to the test as low levels of PGE ₂ were found in in vivo samples. This was done using 4 ml PGE ₂ affinity columns (Cayman Chomical). The column was washed first with 10 ml of 0.1 M phosphate buffer and then with 10 ml of water. EF was then diluted 1: 5 with 0.1 M phosphate buffer and added to the column. The column was then washed with 10 ml phosphate buffer and then 10 ml water. Finally, PGE _{2 was} removed from the column with 2.5 ml of 94% ethanol. The sample was evaporated under a stream of nitrogen and then diluted for PGE ₂ analysis using the Cayman EIA PGE ₂ (COX-2) kit as described in Examples 2 and 3.

Table 5 illustrates that the carrageenan induced an almost four-fold increase in ball ball PGE ₂ synthesis over background values. This increase was observed at 5 hours and persisted for at least 24 hours. Animals with a carprofen dose of 2 mg / kg showed almost complete inhibition of PGE ₂ synthesis, which was statistically significant at 5 hours and 24 hours, with p-values of 0.013 and 0.015, respectively. Ex vivo COX-1 data did not demonstrate inhibition of TXB ₂ at any of the time points tested. This suggests that carprofen inhibited COX-2 in vivo at the same time that it showed no effect on the ex vivo activity of COX-1.

Table 5

In vivo inhibition of COX-2 activity - group diameters dose (Mg / kg) PGE ₂ pg / ml / well Percentage of inhibition 0 hrs 5 hours 24 hours 5 hours 24 hours 0 3.4 17.1 13.0 0% 0% 2 6.5 1.9 3.2 71% 51%

Example 5

Calculation of the rate of oral carprofen controlled release dosage form and doses that provide 80% COX-2 inhibition in dogs over a longer period than the immediate release carprofen dosage form

A study was conducted to obtain data from which the usable, advantageous, preferred and most preferred oral controlled release carprofen dosage form for use in the present invention can be determined.

The plasma samples taken by the dog in Example 3 above were also tested for total plasma carprofen concentration by HPLC with a Chromtech column (5 µm, 100 x 4.6 mm, chiral AGP) and a mobile phase composed of 10:90 (v / v) 2 -propanol: 0.1M phosphate buffer pH 6.0 and detection by fluorescence (excitation 285 nm, emission 345 nm). Plasma samples (0.2 mL) were buffered with 0.05 M citric acid, pH 5.1, after addition of (S) -naproxen as an internal standard, and then extracted with 4: 1 (v / v) diethyl ether: dichloromethane. The ether layer was separated and then re-extracted with 0.005M Na 2 CO 3, then the organic phase was removed by suction. The aqueous phase was buffered with 0.05M citric acid pH 5.1. And then extracted again with diethyl ether: dichloromethane. The ether was then transferred to clean tubes, evaporated under a stream of nitrogen and the pellet reconstituted in the mobile phase for HPLC for analysis.

Plasma levels of carprofen were plotted against% COX-2 inhibition (from Example 3). Examination of this graph showed that 80% inhibition of COX-2 occurred at a plasma concentration of carprofen of approximately 10 gg / ml and that 50% inhibition of COX-2 occurred at a plasma concentration of carprofen of approximately 2 μg / ml and that 90% inhibition of COX-2 occurred at a plasma concentration of carprofen of approximately 20 pg / ml.

Following oral administration to dogs of immediate release carprofen formulations at a dose of 4.4 mg / kg (2 mg / lb), carprofen plasma concentrations above 10 µg / ml were approximately one hour. According to the present invention, they are 4.4 mg / kg / day (2 mg / lb / day) within the preferred dosage range of carprofen.

A useful controlled release dosage form of carprofen is one that maintains a plasma carprofen level of greater than 2 µg / ml most of the day after a single oral dose of 4.4 mg / kg (2 mg / lb).

Preferred controlled-release oral dosage forms of carprofen according to the present invention are those that maintain a plasma concentration of carprofen greater than 10 µg / ml for a longer period of time than that for which a comparable plasma level maintains the immediate release dosage form of carprofen, said carprofen dosage form. the immediate release dosage form of the controlled release carprofen is administered at the same dose, e.g. 4.4, 3.96, 3.52 or 3.08 mg / kg (2, 1.8,

1.6 or 1.4 mg / lb). For example, the preferred 4.4 mg / kg (2 mg / 1b) oral controlled release dosage forms of the invention maintain a carprofen plasma concentration of greater than 10 µg / ml for greater than 10.5 hours.

Immediate release carprofen dosage forms containing doses of 3,96, 3,52 or 3,08 mg / kg (1,8,

1.6 and 1.4 mg / lb) maintain a plasma concentration of carprofen greater than 10 µg / ml for 9.5 hours, 8.5 hours, and 7.5 hours, respectively.

Preferred 3.96 mg / kg (1.8 mg / lb) oral controlled release dosage forms of carprofen maintain a plasma concentration of carprofen greater than 10 µg / ml for greater than 9.5 hours. Similarly, the persistence of the thresholds at doses of 3.52 mg / kg (1.6 mg / lb) and 3.08 mg / kg (1.4 mg / lb) is 8.5 hours and 7.5 hours, respectively. Assuming ¹ linear pharmacokinetics, the effect characteristics for preferred oral controlled release carprofen dosage forms at doses greater than 4.4 mg / kg (2 mg / lb) or less than 3.08 mg / kg (1.4 mg) may similarly be calculated. / lb).

More preferably, the oral controlled release carprofen dosage forms are those that maintain a plasma concentration of carprofen greater than 10 µg / ml longer or as long as observed when the immediate release carprofen dosage form is administered at any higher dose. These controlled release dosage forms therefore maintain at least 80% inhibition of COX-2 in canine blood longer than the immediate release higher dose dosage form.

Most preferred oral formulations for the controlled release of carprofen are those that are capable of maintaining plasma carprofen levels greater than about 10 µg / ml longer or as long as observed with the administration of a 4.4 mg / kg (2 mg / lb) carprofen dosage form. immediate release (10.5 hours) when said oral controlled release dosage forms of carprofen are administered at a dose of less than 4.4 mg / kg (2 mg / lb). The efficacy of the immediate release oral formulation of carprofen immediate release 2 mg / kg (2 mg / lb) is considered a necessary standard for the purpose of this comparison since 4.4 mg / kg / day (2 mg / lb / day) is currently the recommended and received effective oral dose of the present invention as described herein.

As described below, the release rate for oral carprofen controlled release dosage forms that result in plasma carprofen concentrations in dogs of greater than 10 pg / ml is calculated. For ease of calculation, this calculated rate was a zero order rate, i.e., the calculated rate was a constant (i.e., zero order) rate for the controlled release formulations that release carprofen. It will be appreciated by those skilled in the art that practical dosage forms release only part of their drug release time in the zero order and then release at the first order or mixed order. To make the definition of carprofen delivery rate (or release rate) even more clear to one skilled in the art in the analysis below, a useful release rate was defined by the time 80% left the dosage form, regardless of the drug release mechanism used.

Zero-order release rates resulting in a simulated plasma concentration of carprofen greater than 10 gg / ml were determined according to the method of Zhou and Notary (see Zhou, M., and Re, N.). drugs for prolonged release formulations and validation of the method using simulated data, Biopharm. Drug Disp., 16, 319-331, 1995). Mean data resulting from oral administration of 4.4 mg / kg (2 mg / lb) of carprofen to dogs in an immediate release formulation were adjusted to

C = Ci (e ^{s 2t} - e- ⁵ *) where Ci, S2 and Si are parameters and ^t is time. During zero-order release, plasma concentrations of carprofen were simulated using the following equation:

C = (coCi / Dref) [(e ' ^s ? -1) / Si - (e ^s 2 * -1) / S ₂ ] where ko is the zero order release rate and D _re f is the reference dose. After the release is complete, the following equation is applicable:

C (Kocí / Dret) [[(1 - e ~ ^{p T} 1) / S _1] 2 ^t e ^p - [(1 - e ^{"T S} 2) / S _2] ^p e 2 ^e] wherein T = Dai / ko and D _CR is a controlled release dose.

Preferred oral dosage forms for the controlled release of carprofen

Table 6, shown below, demonstrates that the oral controlled release dosage forms of carprofen (dose of 4.4 mg / kg (2 mg / lb)) that release 80% of incorporated carprofen over time ranging from 1.6 to 19.2 hours have a capacity to induce plasma carprofen levels greater than 10 pg / ml for greater than 10.5 hours. Therefore, dosage forms for 4.4 mg / kg (2 mg / lb) are preferred.

Table 7, shown below, demonstrates that the oral controlled release dosage forms of carprofen (dose 3.96 mg / kg (1.8 mg / lb)) that release 80% of the incorporated carprofen over time ranging from 1.6 to 19, 2 hours, have a capacity to induce plasma carprofen levels greater than 10 gg / ml for greater than 9.5 hours. Therefore, dosage forms for 3.96 mg / kg (1.8 mg / lb) are preferred.

Table 8 demonstrates that oral controlled release carprofen dosage forms (dose 3.52 mg / kg (1.6 mg / lb)) that release 80% of incorporated carprofen over time ranging from 1.6 to 19.2 hours , have a capacity to induce plasma carprofen levels greater than 10 pg / ml longer than

8.5 hours. Therefore, dosage forms for 3.52 mg / kg (1.6 mg / lb) are preferred.

Table 9 demonstrates that the oral controlled release dosage forms of carprofen (dose 3.08 mg / kg (1.4 mg / lb)) that release 80% of the incorporated carprofen over time ranging from 1.6 to 19.2 hours , have a capacity to induce plasma carprofen levels greater than 10 gg / ml longer than

7.5 hours. Therefore, dosage forms for 3.08 mg / kg (1.4 mg / 1b) are preferred.

More preferably, oral dosage forms for the controlled release of carprofen

The controlled release carprofen dosage forms of the present invention are particularly useful as they are capable of maintaining plasma carprofen levels greater than 10 pg / ml longer than

10.5 hours, even when administered at doses lower than the preferred dose of 4.4 mg / kg (2 mg / lb) for effective use. Table 7 demonstrates that oral carprofen controlled release dosage forms (dose 3.96 mg / kg (1.8 mg / lb)) that release 80% of incorporated carprofen over time ranging from 4.8 to 19.2 hours , have a capacity to induce plasma carprofen levels greater than 10 pg / ml longer than

10.5 hours. These are more preferred dosage forms for a dose of 3.96 mg / kg (1.8 mg / lb).

Table 8 demonstrates that the oral controlled release dosage forms of carprofen (dose 3.52 mg / kg (1.6 mg / lb)) that release 80% of incorporated carprofen over time ranging from 4.8 to 19.2 hours , have a capacity to induce plasma carprofen levels greater than 10 pg / ml longer than

9.5 hours, i. longer than the time it can reach

3.96 mg / kg (1.8 mg / lb) immediate release dosage form. Thus, these are more preferred dosage forms for a dose of 3.52 mg / kg (1.6 mg / lb).

Table 9 demonstrates that the oral controlled release dosage forms of carprofen (dose 3.08 mg / kg (1.4 mg / lb)) that release 80% of incorporated carprofen over time ranging from 4.8 to 19.2 hours , have a capacity to induce a plasma carprofen level greater than 10 pg / ml for 8,5 hours or longer, ie longer than the time that it is able to reach the 3.96 mg / kg (1.8 mg / lb) immediate release dosage form. Thus, these are more preferred dosage forms for a dose of 3.08 mg / kg (1.4 mg / lb).

Most preferably, oral dosage forms for the controlled release of carprofen

The most preferred oral dosage forms for the controlled release of carprofen are those capable of maintaining a plasma carprofen level greater than about 10 pg / ml for a period of time or the same time as that observed with 4.4 mg / kg (2 mg / kg). 1b) an immediate release dosage form of carprofen (10.5 hours) when said oral controlled release dosage forms of carprofen are administered at a dose of less than 4.4 mg / kg (2 mg / lb). The efficacy of immediate release immediate release carprofen 4.4 mg / kg (2 mg / lb) is considered to be a necessary standard for comparison, since 4.4 mg / kg / day (2 mg / lb / day) is currently recommended an effective oral dose of the present invention. The data in Tables 6-9 were used to define the properties of the most preferred oral carprofen release dosage forms of the present invention.

For a dose of 3.52 mg / kg (1.6 mg / lb), the most preferred oral controlled release dosage forms are those that release 80% of the incorporated carprofen over a range of 6.4 to 19.2 hours. For a dose of 3.08 mg / kg (1.4 mg / lb), the most preferred oral dosage forms for the controlled release of carprofen are those that release 80% of the incorporated carprofen t

about 12.8 hours (10-14 hours).

Table 6

The number of hours, wherein the plasma concen exceed 10 gg / mL, after dosing ^with intermediaries qu ^ ° ^The forms for controlled release, which release rate ka (calculated), dose = 4.4 mg / kg (2 mg

speed zero order release (mg / lb / hour) Release time 80% carprofen from dosage form (hours) Time, Fri. . whose Jmatic ^K concentrate carprofen® ^aa * ^c 10 pg / ml others) __ —ΣΐΧ 0,084 19.2 0,100 16 1 Z 0,125 12.8 - 0,165 9.6 0,250 6.4 0,330 4.8 12 \ 1.0 1.6 \

Table 7

The number of hours, wherein the plasma carprofen concentrically _Potency exceed 10 gg / mL, after dosing mediocre T · vovúch forms of controlled release, where ^L s uvolňuľľ ^{ctvom? IEK?} Zia rate <vypoäítancu) dév'ka - 3.9E

speed zero-order release (mg / lb / hour) Release time of 80% carprofen from the dosage form (hours) The time during which the plasma concentration is Carprofen higher than 10 μς / πιΐ (hours) 0,075 19.2 14 0,090 16 14.5 0,113 12.8 14.5 0,150 9.6 13 ÔT225 6.4 11.5 0.3 4.8 11 0.9 1.6 10

Table 8

Number of hours in which the plasma concentration of carprofen is greater than ≥ 10 pg / ml, after administration by controlled release dosage forms that release carprofen at different rates (calculated), dose = 3.52 mg / kg (1.6 mg) / lb).

speed zero order release (mg / lb / hour) Release time of 80% carprofen from the dosage form (hours) The time during which the plasma concentration of carprofen is greater than 10 pg / ml (hours) 0, 067 19.2 11 0,080 16 13 0,100 12.8 12.5 0,135 9.6 11, 5 0.2 6.4 11 0.27 4.8 10 0.8 1.6 9

Table 9

Number of hours in which the plasma concentration of carprofen is greater than 10 pg / ml after dosing via controlled release dosage forms that release carprofen at different rates (calculated), dose = 3.08 mg / kg (1.4 mg / lb) ).

speed zero order release (mg / lb / hour) Release time of 80% carprofen from the dosage form (hours) The time during which the plasma concentration is Carprofen higher than 10 pg / ml (hours) 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 m 00 0.7 1.6 8

Example 6

Carprofen implants for dogs

Carprofen implants are suitable for the administration of carprofen over long periods of time, e.g. 3 days, 7 days, 30 days and so on. This example describes in detail the applicable and advantageous release rates of carprofen from carprofen-containing implants and also defines the dosages associated therewith.

The rate of entry of carprofen into the dog's body that would provide a steady state plasma concentration of carprofen of 10 gg / ml is calculated using the following equation from Gibaldi, M. and Perrier, D. (Pharmacokinetics, 2nd Ed., Drugs and the Pharmaceutical Sciences, eds. J. Swarbrick, Volume 15, New York: Marcel Dekker, Inc., 1982):

ro

Css = Cl

Where C _ss is the steady state plasma concentration of carprofen, Ro is the rate of entry and Cl is the systemic clearance. Systemic clearance is determined from the pharmacokinetics observed following oral administration of 4.4 mg / kg (2 mg / lb) of carprofen to dogs using the formula:

FD

Cl = _

AUC

Where F is the bioavailability (assumed to be 1 for the implant), D is the oral dose and AUC is the mean area under the plasma concentration of carprofen versus time, extrapolated to infinity.

A clearance of Cl = 5 ml / hour per pound of body weight was obtained. Using this clearance and a target Css of 10 gg / ml, a carprofen Ro release rate of 110 gg / kg / hour (50 pg / lb / hour) was calculated, giving an input rate of 2.64 mg / kg / day when multiplied by 24 hours per day. (1.2 mg / lb / day). Total dose of 7.92 mg / kg (3.6 mg / lb) or

18.48 mg / kg (8.4 mg / lb) or 79.2 mg / kg (36 mg / lb) will be required for treatment for 3, 7 or 30 days. In summary, the implant containing

18.48 mg carprofen per kilogram body weight (8.4 mg carprofen per pound body weight), releasing carprofen at 110 µg per kilogram body weight per hour (50 µg per pound body weight per hour), maintains a carprofen concentration of 10 µg / ml 7 days. This retains 80% inhibition of COX-2 for 7 days.

In order to obtain the implanted dose and carprofen release rate necessary to maintain the plasma concentration of carprofen 2 pg / ml (50% inhibition of COX-2), the dose and release rate for the target 10 µg / ml were linearly multiplied by 0.2. Thus, a carprofen release rate of 22 μg / kg / hour (10 pg / lb / hour) or 0.528 mg / kg / day (0.24 mg / lb / day) is required for a target plasma concentration of 2 µg / ml carprofen. A total dose of 1.584 mg / kg (0.72 mg / lb) or 3.696 mg / kg (1.68 mg / lb) or 15.84 mg / kg (7.2 mg / lb) will be required for a treatment duration of 3.7. or 30 days.

To obtain the implanted dose and carprofen release rate necessary to maintain a plasma concentration of carprofen of 20 μς / ιηΐ (90% COX-2 inhibition), the dose and release rate for the target 10 μg / ml were linearly multiplied by 2. Thus, for the target plasma concentration carprofen 20 pg / ml required carprofen release rate 220 μg / kg / hour (100 μg / lb / hour) or 5.28 mg / kg / day (2.4 mg / lb / day). A total dose of 15.84 mg / kg (7.2 mg / lb) or 36.96 mg / kg (16.8 mg / lb) or 158.4 mg / kg (72 mg / lb) will be required for a long 3 , 7, or 30 days.

Useful carprofen implants of the present invention release carprofen into the body of a dog at a rate of 0.528 mg / kg / day (0.24 mg / lb / day) or higher.

Preferred carprofen implants of the present invention release carprofen into the dog's body at a rate of 0.528 to 2.64 mg / kg / day (0.24 to 1.2 mg / lb / day).

Most preferably, the carprofen implants of the present invention release carprofen into the body of the dog at a rate of 2.64 to 5.28 mg / kg / day (1.2 to 2.4 mg / lb / day).

Applicable carprofen implants have a total carprofen dose of up to 2 g, limited by the size of the implant that can be appropriately given to the dog. However, more than one implant may be administered simultaneously.

Example 7

Cleavage of (S) -6-chloro-α-methylcarbazole-2-acetic acid

A solution of (R) -α-methylbenzylamine (4.3 g) in acetone (20 ml) was added to a solution of partially cleaved 6-chloro-methylcarbazole-2-acetic acid (9.7 g) (obtained from the filtrate after the previous step). resolution of the racemate). After standing at room temperature for 24 hours, the mixture was filtered and the filter cake was washed with cold acetone and dried to give 7.3 g. After two further recrystallizations from acetone, 1.9 g of (R) -amethylbenzylamine salt of (S) -6-chloro-α-methylcarbazole-2-acetic acid was obtained, [α] ²² D -13.6 °. Further recrystallizations from acetone did not change the rotation. The salt was dissolved in warm acetone (50 mL) and the solution was poured into dilute hydrochloric acid (500 mL) after filtration. After filtration and drying, 1.4 g was obtained, which after crystallization from chloroform gave 0.9 g of (S) -6-chloro-α-methylcarbazole-2-acetic acid, mp 198 ° -201 °, [α] _η ²² + 53.2 °, (c 1.33, CH ₃ OH).

Example 8

Species specificity of COX-2 selectivity, activity in members of Canis familiaris (dogs) compared to activity in members of Rattus norvegicus (white rats) and Homo sapiens (humans)

The very high degree of COX-2 selectivity exhibited by carprofen in dogs has already been extensively demonstrated in Example 1. It has also been surprising that this selective inhibition of COX-2 has been shown to be activity specific to Canis familiaris and other species do not show it. The discovery was based on the evaluation of the inhibitory activity of racemic carprofen in members of Rattus norvegicus (white rats) and in members of Homo sapiens (humans).

In rats, cyclooxygenase selectivity in vivo was evaluated by the method of Griffitns et al. (Agents & Actions, 32, 313-320, 1991). COX-2 inhibitory activity was evaluated by the effect of racemic carprofen on prostaglandin PGE ₂ formation, as measured in rat synovial fluid. The synovial fluid is secreted by the synovial membrane and is contained within the joint cavity. During inflammation of the joints, COX-2 is induced in the joint tissues and prostaglandins accumulate in the synovial fluid. The inhibitory activity of COX-1 was evaluated by the effect of racemic carprofen on the production of prostaglandin PGE ₂ as measured in the mucosal lining of the rat stomach containing a significant amount of constitutive COX-1 isozyme. Inhibition of this gastric isozyme results in adverse gastrointestinal side effects. COX-1 ED ₅ o is 6.4 mg / kg, while COX-2 ED ₅ o is 0.63 mg / kg. These results show that racemic carprofen is only 10-fold more selective for the COX-2 isozyme in rats.

In humans, COX-2 inhibitory activity was evaluated by the effect of racemic carprofen on COX-2 levels in human umbilical vein endothelial cells (HUVEC) stimulated by IL-1 and phorbol myristate acetate (PMA) according to the method of Habib et al. (J. Biol. Chom., 268,23448-23454, 1993). These endothelial cells most likely contain significant amounts of inducible COX-2 isozyme when stimulated with interleukin-1 (IL-1) and PMA. COX-1 inhibitory activity was evaluated by the effect of racemic carprofen on COX-1 levels, as measured by the human washed plate (HWP) biochemical assay for TXB2, following the procedure of Grossman et al. (Inflamm. Res., 44, 253-237, 1995). Most likely, these plates contain significant amounts of constitutive COX-1 isozyme. HUVEC (COX-2) IC ₅₀ (μΜ) is 1.20, while HWP TXB ₂ (COX-1) IC ₅₀ (μΜ) is 0.77. These results show that racemic carprofen is not selective for the COX-2 isozyme in humans.

Example 9

Formulation of (S) -6-chloro-α-methyl-carbazole-2-acetic acid into tablets

Formulation into tablets

ingredients Weight in tablet (S) -6-chloro-α-methyl- carbazole-2-acetic acid 25,00 mg Lactose, U.S.P. 64.50 mg Maize starch 10,00 mg Magnesium stearate 0.50 mg

Example 10

Formulation of (S) -6-chloro-α-methyl-carbazole-2-acetic acid into capsules

Capsule formulation

ingredients Weight in capsule (S) -6-chloro-α-methyl- carbazole-2-acetic acid 50 mg Lactose, U.S.P. 124 mg Corn starch, U.S.P. 30 mg Talc, U.S.P. 5 mg total weight 210 mg

100

Example 11

Parenteral formulation of (S) -6-chloro-α-methylcarbazole-2-acetic acid

Parenteral formulation

Ingredients in a 1 cm ^J ampoule Weight in ampoule (S) -6-chloro-α-methyl- carbazole-2-acetic acid 10.2 mg Metylparaben, U.S.P. 1.8 mg Propylparaben, U.S.P. 0.2 mg Sodium hydroxide, U.S.P.q.s.ph 9.0 mg Water for Injections, U.S.P.q.s.ad 1,0 cm ^J

-101-

Claims (14)

PATENT CLAIMS Use of an anti-inflammatory COX-2 selective inhibitory compound comprising a compound of formula I wherein A is a hydroxyl group, an alkoxy group having 1 to 4 carbon atoms, an amino group, a hydroxyamino group, a monoalkylamino group having 1 to 2 carbon atoms, a dialkylamino group having 1 to 2 carbon atoms in each alkyl moiety; X and Y are independently hydrogen or alkyl having 1 to 2 carbon atoms; and n is 1 or 2; R is halogen, (C 1 -C 3) alkyl, trifluoromethyl or nitro, R ⁹ is hydrogen, C 1 -C 2 alkyl, phenyl or phenylalkyl containing 1 to 2 carbon atoms in the alkyl moiety, wherein the phenyl is optionally monosubstituted with fluoro or chloro; -C (= O) -R, wherein R is an alkyl group containing 1 to 2 carbon atoms or a phenyl group optionally monosubstituted with fluorine or a β-alkyl group; · · · · · · · · · · · -102chlórom; or -C (= O) -OR ¹ wherein R ¹ is a C ^1-2 alkyl group where X and Y are different, its (-) (R) and (+) (S) enantiomers and all pharmaceutically acceptable forms of salts, prodrugs and metabolites which are therapeutically active in the treatment or prevention of pain and inflammation, for the manufacture of a medicament for the treatment or prevention of pain and inflammatory processes and diseases associated with inducible cyclooxygenase-2 (COX-2) activity in a member of Canis familiaris in need of such treatment, while at the same time reducing or eliminating unwanted side effects associated with the simultaneous inhibition of constitutive cyclooxygenase-1 (COX-1) activity by selectively inhibiting COX-2 activity relative to COX-1 activity, wherein the ratio of selectivity to inhibiting COX- 2: COX-1 is at least 3: 1, based on the e. V vivo inhibition level in whole blood measured at a dose leading to a COX-2 inhibition greater than equal to 80%. The use according to claim 1, wherein the anti-inflammatory selective COX-2 inhibitory compound is carprofen, 6-chloro-α-methyl-9H-carbazole-2-acetic acid. Use according to claim 1, wherein the anti-inflammatory selective COX-2 inhibitory compound is composed exclusively of the (S) -enantiomer of carprofen, 6-chloro-α-methyl-9H-carbazole-2-acetic acid. The use of any one of claims 1, 2 or 3, wherein the inhibitory compound is used in combination with one or more other therapeutically active agents under the following conditions: 103 A. When the joint is severely inflamed and also infected with bacteria, fungi, protozoa and / or viruses, the inhibitor compound is administered in combination with one or more antibiotics, antifungal, antiprotozoal and / or antiviral therapeutics, B. When multiple treatments for pain and inflammation are desired, the inhibitory compound is administered in combination with inhibitors of other inflammatory mediators comprising one or more members independently selected from the group consisting essentially of: 1 2 3 4th 5th 6th 7 8, 9. inhibitors NSAIDs, H 1 -receptor antagonists, B 1 and B ₂ kinin receptor antagonists, prostaglandin inhibitors selected from the group consisting of PGD-, PGF-, PGI ₂ - and PGE antagonists, thromboxane A ₂ (TXA ₂ -) inhibitors, 5- and a-inhibitors 12-lipoxygenase, LTC4-, LTD4 / LTE4- and LTB4- leukotriene inhibitors, PAF receptor antagonists, gold in the form of an aurothio group together with one or more hydrophilic groups, 10. immunosuppressive agents selected from the group consisting of cyclosporin, azathioprine and methotrexate; 11. anti-inflammatory glucocorticoids, penicillamine, hydroxychloroquine, anti-gout agents including colchicine, xanthine oxidase, including allopurinol, and uricosuric drugs selected from probenecid, sulfinpyrazone and benzbromarone; C. when older dogs are treated for disease states, syndromes and symptoms found in geriatric dogs, the inhibitory compound 104 is administered in combination with one or more members independently selected from the group consisting essentially of: 1. cognitive therapeutics to counteract memory loss and deterioration; 2. antihypertensives and other cardiovascular medicinal products intended to compensate for the effects of atherosclerosis, hypertension, ischemic heart disease, angina, stasis failure and myocardial infarction, selected from the group consisting of: a. diuretics, b. vasodilators, c. β-adrenergic receptor antagonists, d. angiotensin II converting enzyme inhibitors (ACE inhibitors), alone or optionally together with neutral endopeptidase inhibitors, e. angiotensin II receptor antagonists, f. renin inhibitors, g. calcium channel blockers, h. sympatholytic i. <X 2 -adrenergic agonists, j. α-adrenergic receptor antagonists, and k. HMG-CoA reductase inhibitors (antihypercholesterolomics), 3. antineoplastic preparations selected from: a. antimitotics selected from: I. Vincaalkaloids selected from: [1] vinblastine and [2] vincristine, 4. secretory analogues of growth hormone; 5. strong analgesics, 6. local and systemic anesthetics; and 7. H2-receptor antagonists, proton-1 inhibitors; pumps and other gastroprotective agents. 105 A pharmaceutical composition for the treatment or prevention of pain and inflammatory processes and diseases associated with inducible cyclooxygenase-2 (COX-2) activity in a member of the Canis familiaris species in need thereof, while reducing or eliminating adverse side effects associated with simultaneous by inhibiting constitutive cyclooxygenase-1 (COX-1) activity, characterized in that the preparation comprises: A. A therapeutically effective amount for treating pain and inflammation of an anti-inflammatory COX-2 selective inhibitory compound that selectively inhibits COX-2 activity relative to COX-1 activity, wherein the ratio of COX-2: COX-1 selectivity inhibition is at least 3: 1 based on ex vivo inhibitory levels in whole blood measured at a dose leading to an inhibition of COX-2 of greater than or equal to 80%, including an anti-inflammatory COX-2 selective inhibitory compound comprising a compound of formula (I): where: R ² is n where A is hydroxyl, C 1 -C 4 alkoxy, amino, hydroxyamino, C 1 -C 2 monoalkylamino, C 1 -C 2 dialkylamino, X and Y are independently H or C 1 -C 2 alkyl carbon, and n is 1 or 2, 106 R ⁶ is halogen, alkyl having 1 to 3 carbon atoms, trifluoromethyl or nitro, R ^{8 9} is H, an alkyl group having 1 to 2 carbon atoms, a phenyl group or a phenylalkyl group having 1 to 2 carbon atoms, wherein the phenyl is optionally monosubstituted by fluorine or chlorine, -C (= O) -R, wherein R is an alkyl group containing 1 to 2 carbon atoms or a phenyl group, optionally monosubstituted with fluorine or chlorine, or -C (= O) -O-R ¹ , wherein R ¹ is an alkyl group containing 1 to 2 carbon atoms, where X and Y are different, their (-) (R) and (+) (S) -enantiomers, and all pharmaceutically acceptable forms of salts, prodrugs and metabolites thereof, that are therapeutically active for the treatment or prevention of pain and inflammation, and B. A pharmaceutically acceptable carrier for the above compounds. The pharmaceutical composition of claim 5, wherein the anti-inflammatory COX-2 selective inhibitory compound is carprofen, 6-chloro-α-methyl-9H-carbazole-2-acetic acid. The pharmaceutical composition of claim 5, wherein the anti-inflammatory COX-2 selective inhibitory compound is composed exclusively of the (S) -enantiomer of carprofen, 6-chloro-α-methyl-9H-carbazole-2-acetic acid. Pharmaceutical preparation according to claims 5, 6 or 7, characterized in that the anti-inflammatory COX-2 selective inhibitory compound is provided in a dosage form suitable for systemic administration by means of: A. Injection or infusion in a suitable liquid form, which is intraarterial, intra- or transdermal, subcutaneous, 107 intramuscular, intraspinal, intrathecal or intravenous, wherein the inhibitory compound: 1. is contained in the solution as a solute; 2. is contained in the discontinuous phase of the emulsion or in the discontinuous phase of the inverse emulsion which is inverted for injection or infusion, the emulsions containing suitable emulsifiers, or 3. is contained in the suspension as a suspended solid in colloidal or microparticle form, the suspension containing suitable suspending agents; B. Injection or infusion into suitable body tissues or cavities to serve as a depot, wherein the preparation provides a stock of the inhibitory compound for systemic distribution, C. Instillation, inhalation or insufflation into appropriate body tissues or cavities in a suitable solid form, wherein the inhibitory compound: 1. Is contained in a solid implant preparation that provides delayed, sustained and / or controlled release of the inhibitory compound; (2) it is contained in a particulate preparation which is inhaled into the lungs; or 3. is contained in a particulate composition that is introduced into suitable body tissues or cavities, wherein the composition optionally provides for delayed, sustained and / or controlled release of the inhibitory compound; or D. Ingestion in a suitable solid or liquid form for oral administration of an inhibitory compound, wherein the inhibitory compound: (1) it is contained in a solid dosage form; or 2. it is contained in a liquid dosage form. · team. Pharmaceutical preparation according to claim 8, characterized in that the dosage forms comprise one 108 or more members selected independently from the group consisting essentially of suppositories, solid oral dosage forms selected from the group consisting of delayed release tablets, capsules, capsules, lozenges, lozenges and multiparticulates, enteric-coated tablets and capsules to prevent release; and absorption in the stomach of the treated dog to facilitate administration distal to the dog's stomach, sustained-release oral tablet, capsules and microparticles that provide systemic administration of the inhibitor in a controlled manner for at least 10 hours, chewable or edible oral tablets, unit dose powder bag, suspension made from said a unit dose sachet of powder, oral suspension powder or oral suspension per se, fast-dissolving tablets, encapsulated solutions, oral paste, granular form incorporated into or incorporated into dog feed, and a tasty chewable form in which the inhibitor is consumed with the tasty chewable form or can be administered by leaching from non-digested food during chewing by the treated dog, liquid oral dosage forms selected from the group consisting of solutions, suspensions, emulsions, inverse emulsions , elixirs, extracts, elixirs, and concentrates, and the aforementioned solid dosage forms comprising microencapsulated formulations of the active ingredient which are incorporated into the solid dosage form. A pharmaceutical composition according to claim 9 comprising an oral controlled release carprofen dosage form capable of maintaining plasma carprofen levels greater than 10 µg / ml for a period of time greater than 10.5 hours when administered at a dose of 4, 4 mg / kg (2 mg / lb) or less. Pharmaceutical preparation according to claims 5, 6 or 7, characterized in that it further comprises 109 an anti-inflammatory COX-2 selective inhibitory compound in combination with one or more other therapeutically active agents independently selected from the group consisting essentially of: A. Anti-infectious agents, including one or more antibiotics, antifungal, antiprotozoal or antiviral therapeutic agents, B. Inhibitors of other inflammatory mediators, including one or more members independently selected from the group consisting essentially of: 1 2 3 4th 5th 6th 7th 8. 9. inhibitors NSAIDs, H 1 receptor antagonists, B 1 and B ₂ kinin receptor antagonists, prostaglandin inhibitors selected from the group consisting of PGD-, PGF-, PGI _2- and PGE antagonists, thromboxane A ₂ (TXA2-) inhibitors, 5- and 12 inhibitors -lipoxygenase, inhibitors of leukotrienes LTC ₄ -, LTD4 / LTE4- and LTB ₄ -, PAF receptor antagonists, gold in the form of aurothio group together with one or more hydrophilic groups, 10. immunopressants selected from the group consisting of cyclosporin, azathioprine and methotrexate; 11. anti-inflammatory glucocorticoids, penicillamine, hydroxychloroquine, anti-gout agents including colchicine, xanthine oxidase, including allopurinol, and uricosuric drugs selected from probenecid, sulfinpyrazone and benzbromarone; 110 C. Therapeutic preparations for treating geriatric dogs comprising one or more members independently selected from the group consisting essentially of: 1. cognitive therapeutics to counteract memory loss and deterioration; 2. antihypertensives and other cardiovascular medicinal products intended to compensate for the effects of atherosclerosis, hypertension, ischemic heart disease, angina, stasis failure and myocardial infarction, selected from the group consisting of: a. diuretics, b. vasodilators, c. β-adrenergic receptor antagonists, d. angiotensin II converting enzyme inhibitors (ACIA inhibitors), alone or optionally together with neutral endopeptidase inhibitors, e. angiotensin II receptor antagonists, f. renin inhibitors, g. calcium channel blockers, h. sympatholytic i. <X2-adrenergic agonists, j. α-adrenergic receptor antagonists a k. HMG-CoA reductase (antihypercholesterolemic) inhibitors 3. antineoplastic preparations selected from: a. antimitotics selected from: I. Vincaalkaloids selected from: [1] vinblastine and [2] vincristine, 4. secretory analogues of growth hormone; 5. strong analgesics, 6. local and systemic anesthetics, and lll 7. H2-receptor antagonists, proton pump inhibitors and other gastroprotective agents. A kit suitable for commercial use for the therapeutic treatment or prevention of pain and inflammatory processes and diseases in a member of the Canis familiaris species in need thereof, comprising: A. A suitable container, optionally in the form of an outer container and an inner container removably contained therein, B. A suitable dosage form enclosed in a container of an anti-inflammatory COX-2 selective inhibitory compound of formula (I) o C-A X -C I _Y_I where: R ² is n wherein A is hydroxyl, C 1 -C 4 alkoxy, amino, hydroxyamino, C 1 -C 2 monoalkylamino, C 1 -C 2 dialkylamino, one of X and Y is H and the other is C 1 -C 2 alkyl, and is 1 or 2 R ⁶ is halogen, alkyl having 1 to 3 carbon atoms, trifluoromethyl or nitro, R ⁹ is H, an alkyl group having 1 to 2 carbon atoms, a phenyl group or a phenylalkyl group having 1 to 2 carbon atoms 112 carbon atoms wherein phenyl is optionally monosubstituted with fluorine or chlorine, -C (= O) -R, wherein R is an alkyl group containing 1 to 2 carbon atoms or phenyl, optionally monosubstituted with fluorine or chlorine, or -C (OO) -OR ¹ , wherein R ¹ is an alkyl group containing 1 to 2 carbon atoms, wherein the (+) (S) -enantiomer is present in an amount of at least 75% and all pharmaceutically acceptable forms of salts, prodrugs and metabolites thereof; are therapeutically active to treat or prevent pain and inflammation; and C. the printed instructional and information material associated with the container that is attached to the container is enclosed in the container or is exposed as an integral part of the container, the instructional and information material announcing and explicitly notifying the reader, a person skilled in the art that a compound of formula (I) ) containing a therapeutic composition contained in the kit, when administered to a treated member of the Canis familiaris, effectively inhibits cyclooxygenase-2 (COX-2) induced at an existing or expected pain and inflammation site in a dog, thereby treating or preventing pain and inflammation that would otherwise result from this, while at the same time reducing or eliminating the undesirable side effects associated with the simultaneous inhibition of constitutive cyclooxygenase-1 (COX-1) activity by selectively inhibiting COX-2 activity relative to COX-1 activity, wherein the COX-2 selectivity ratio The COX-1 inhibition activity is at least 3: 1 based on ex vivo whole blood inhibitory levels measured at a dose resulting in a COX-2 inhibition greater than or equal to 80%. Kit according to claim 12, characterized in that the anti-inflammatory selective COX-2 inhibitor compound of formula (I) comprises carprofen, 6-chloro-α-methyl-9 H -carbazole-2-acetic acid. 113 Kit according to claim 12, characterized in that the anti-inflammatory selective COX-2 inhibitory compound is composed exclusively of the (S) -enantiomer of carprofen, 6-chloro-α-methyl-9H-carbazole-2-acetic.