WO1997045113A1

WO1997045113A1 - Nsaids for regulating the reproductive cycle of domesticated animals

Info

Publication number: WO1997045113A1
Application number: PCT/US1997/007568
Authority: WO
Inventors: Wolfgang Jochle
Original assignee: Sepracor Inc.
Priority date: 1996-05-30
Filing date: 1997-05-05
Publication date: 1997-12-04
Also published as: AU2827797A

Abstract

Compositions and methods for regulating the reproductive cycle of domesticated animals using nonsteroidal anti-inflammatory drugs (NSAIDs). Further, the present invention relates to compositions and methods for controlling and managing the breeding of domesticated animals, especially pigs, by synchronizing their reproductive cycles by administering an effective amount of an NSAID compound, preferably ketoprofen. Optionally, one or several additional active ingredients such as a hormone or steroid may be administered in conjunction with the NSAID compound in order to synchronize the reproductive cycle, especially estrus and/or ovulations.

Description

NSAIDS FOR REGULATING THE REPRODUCTIVE CYCLE OF DOMESTICATED ANIMALS

l. Field of the Invention

The present invention relates to compositions and methods for regulating the reproductive cycle of domesticated animals using nonsteroidal anti-inflammatory drugs (NSAIDs) . Further, the present invention relates to compositions and methods for controlling and managing the breeding of domesticated animals, especially pigs, by synchronizing their reproductive cycles with special emphasis on synchronizing the occurrence of estrus and the timing of ovulation.

2. Background of the Invention

2.1. Controlling the Reproductive Cycle of Domesticated Animals

Control and synchronization of the reproductive cycles in domesticated animals has long been recognized as a desirable goal in livestock management. Control of the reproductive cycle is desirable for a number of reasons. For example, it facilitates simultaneous breeding of large numbers of animals at a predetermined time, preferably using artificial insemination. Breeding seasons can be shifted to a more desirable time, and selected animals may be bred at a preferred time.

A number of approaches have been used to control the reproductive cycle of domesticated animals. For example, rudimentary physical methods include manipulating the animals' exposure to light to advance the length of the breeding season, controlling the temperature of the animals surroundings, exposing animals to specific or non-specific sounds, and physical stimulation of the animals' reproductive organs. One method simply involves keeping the males separated from the female herds and introducing the males only at a selected time. This method is somewhat effective in synchronizing cycling, especially when done prior to the breeding season. By far, the most important method of reproductive control involves chemicals. Control of the reproductive cycle of domesticated animals using a variety of chemicals has been widespread for many years. Originally, naturally occurring compounds were the only ones available to control reproductive functions. Now, such natural compounds are increasingly being replaced by identical or modified molecules of synthetic origin, as is the case of progestins for progesterone, or by natural hormones which are more readily available and less expensive.

Typically, in controlling the reproductive functions of domesticated animals, chemicals are used to either:

(A) mimic the cyclic corpus luteum (CL) phase, when endogenous progesterone levels are high; or

(B) mimic the luteolytic effect of endogenous Prostaglandin F (PGF) which blocks endogenous progesterone production. Modern methods of reproduction control usually combine both approaches, typically employing method (A) followed by method (B) . Method A usually involves administration of progesterone via intravaginal devices for 7 to 15 days (cattle, sheep, goat, deer, horses) or feeding orally active progestins like chlormadinone acetate and melengestrol (cattle, sheep, goat, horses) or allyl trenbolone (horses, pig) . Removable ear implants containing norgestomet are also used in cattle.

Method B involves the use of PGF analogs. In many domesticated animals such as cattle, sheep, goat and horses, the cyclic CL is sensitive to the luteolytic effects of exogenous PGF analogs from Day 5 or 6 of the 20-day estrous cycle. This allows synchronization, with a single treatment, of about 65 to 75% of all animals in a herd of normally cycling animals. Two PGF injections, 12-14 days apart, synchronize effectively all treated animals. Using (A) for 7 or 8 days and (B) on Day-2 after progestin removal has been shown to provide synchronization of estrus and ovulation in cattle. However, in pigs, it is known that the cyclic CLs are not responsive to PGF until Days 12 or 13 of their 18 to 20-day cycle. Therefore, administration of PGF cannot effectively be used to synchronize estrus in pigs, unlike virtually all other species of domesticated animals.

For pigs, 40 years of effort to achieve control of cycles and ovulations have met with limited success. From 1960 to 1975, administration of progestin, at doses later identified as being too low, caused often irreversible damage (permanent ovarian cyst formation) to the reproduction system of the gilt or sow.

Since the improper dosages were identified, higher doses have been used successfully. For example, studies have shown that altrenogest is useful in the synchronization of oestrus in gilts. F. Martinat-Botte, "Synchronization of Oestrus in Gilts With Altrenogest: Effects on Ovulation rate and Foetal Survival", Animal Reproduction Science , 39 (1995), pp. 267-74. One of the few products that has been developed commercially is allyl trenbolone (Hoechst/Roussel-Uclaf) . However, this product has been approved only in a few countries. For example, allyl trenbolone is approved in the United States for use in horses, which are not being used as food producing animals. Even in those countries in which it is approved, it is likely not to be reapproved due to the controversy surrounding its potential mutagenic and/or carcinogenic characteristics. Further more, allyl trenbolone is very expensive and must be fed daily for 15 to 18 days. Hence an alternative treatment is needed.

Pigs in which the CL life span has been prolonged, as with the establishment of pregnancies, have been shown to be responsive to PGF. Prolongation of the CL life span in early pregnancies in sows/gilts, i.e., the prevention of luteolysis, is caused by large quantities of estrogens produced by developing embryos from about Days 10 to 14 after conception. This has led to the demonstration that groups can be synchronized by breeding all animals by a boar when in heat and, following conception of the last animals, a single treatment of PGF to abort all sows or gilts simultaneously. Since these animals will revert to cyclic functions equally synchronized, they can be inseminated together. However, this method is economically unattractive, time consuming, wasteful and offensive to many people.

One can mimic the estrogen effect of early pregnancy by injecting or otherwise administering daily estradiol or orally effective estrogens like allyl estradiol or mestranol, respectively. However, many modes of administration such as injections are too labor intensive to be practical on a large scale. Likewise, using estrogens as top dressing or in premixes with feed will likely not receive approval from governmental agencies due to its environmental impact. Control of the estrous cycle and ovulation in domesticated animals is a prerequisite for the use of widespread artificial insemination. Consequently, as a result of the absence of a effective and practical method of synchronizing the reproductive cycle of pigs, artificial insemination in pigs is used presently on a small to medium scale only.

Likewise, control of the estrus cycle and ovulation is also a prerequisite for a variety of animal husbandry management techniques such as the desirable "in and out" management technique useful in the breeding of pigs. In "in and out" management, a group of gilts (young female pigs who have not weaned their first litter) are bred at the same time. The group of gilts enter a farrowing facility together where they give birth to their litters. The litters are weaned together and all of the animals are subsequently removed from the facility at the same time. The facility is then cleaned and disinfected for the next group of animals. The reproductive cycles of the gilts (properly referred to as "sows" after their first pregnancy) are synchronized and bred together again and the cycle is started again. Such groups of females can be managed together over their entire productive lifespan. The size of the groups of animals is dependent upon the physical limitations of the farrowing facilities. If the method is supplemented by controlled parturitions, litters can be adjusted by size and material capabilities (cross-fostering) . Such a management technique provides an efficient and predictable method of animal husbandry, especially for pigs.

There remains a need for an effective and practical method of synchronizing the reproductive cycle of domestic animals, especially pigs. To date, efforts have focused on a search for hormones that would control the estrus and ovulation in domestic animals, especially with pigs. There has been a lack of research on substances that may control such hormones. This lack of research is due, in part, to controversies such as those surrounding the synthetic antigonadotropin compound called methallibure, wherein embryotoxic and teratogenic properties ultimately resulted in withdrawal from the market in many countries.

Due to physiological limitations unique to pigs, there are additional concerns when the domesticated animal sought to be regulated is the pig. In particular, pigs are known to have a relatively rapid metabolism; further, pigs are known to be prone to gastrointestinal toxicity, such as ulcers. Accordingly, any chemical methods of regulating the reproductive cycle of pigs must be suitable in view of these physical limitations.

2.2. Nonsteroidal Anti-infli*"^«"'^»t-orv Drugs Most non-steroidal anti-inflammatory drugs ("NSAIDs") are chiral molecules due to the presence of an asymmetric carbon. For instance, the 2-arylpropionic acid derivatives contain a chiral center at the alpha-carbon. Examples of NSAIDS include almioprofen, benoxaprofen, carprofen, cicloprofen, fenoprofen, flurbiprofen, fluxoxaprofen, ibuprofen, ketoprofen, ketorolac, loxoprofen, naproxen, priprofen, suprofen, tiaprofenic acid, azapropazone, bumadizone, oxyphenbutazone, clidanac, etodolac and sulindac. Typically, these drugs are marketed as their CT/ 97/07568

racemates, i.e., equal mixture of optical isomers or enantiomers.

Many organic compounds exist in optically active forms, i.e., they have the ability to rotate the plane of plane-polarized light. In describing an optically active compound, the prefixes D and L or R and S are used to denote the absolute configuration of the molecule about its chiral center(s). The prefixes d and 1 or (+) and (-) are employed to designate the direction of rotation of plane-polarized light by the compound, with (-) or 1 meaning that the compound is levorotatory. A compound prefixed with (+) or d is dextrorotatory. For a given chemical structure, these compounds, called stereoisomers, are identical except that they are mirror images of one another. A specific stereoisomer may also be referred to as an enantiomer, and a mixture of such isomers is often called an enantiomeric or racemic mixture.

Stereochemical purity is of importance in the field of pharmaceuticals, where many of the most prescribed drugs exhibit chirality. A case in point is provided by the L-form of the beta-adrenergic blocking agent, propranolol, which is known to be 100 times more potent than the D-enantiomer. Furthermore, optical purity is important since certain isomers may actually be deleterious rather than simply inert. For example, it has been suggested that the D- enantiomer of thalidomide is a safe and effective sedative when prescribed for the control of morning sickness during pregnancy, while the corresponding L-enantiomer has been believed to be a potent teratogen. Ketoprofen, which is illustrated as compound (I) , is a NSAID that is known to inhibit the biosynthesis of prostaglandins by inhibiting the cyclooxygenase enzyme, which is ubiquitous in mammalian tissues.

Ketoprofen

The enantiomers of ketoprofen are disclosed in Yamaguchi et al., Nippon Yakurigaku Zasshi . 90: 295-302 (1987). This reference states that the s-enantiomers of 2-arylpropionic acids have 15-300 times higher prostaglandin synthetase inhibitory activities than the R-enantiomers in the rat. Additionally, the S-enantiomer of ketoprofen is disclosed in U.S. Pat. NOS. 4,868,214, 4,962,124, and 4,927,854. Each of these patents alleges that the analgesic activity of ketoprofen resides exclusively in the S(+) enantiomers. The enantiomers of ketoprofen are also disclosed in Abas et al., J . Pharmacol . Exp. Ther . , 240: 637-641 (1987). This reference states that R-ketoprofen is metabolically converted to S-ketoprofen in the rabbit.

Furthermore, Caldwell et al., Biochem . Pharmacol . 37: 105-114 (1988) state that the interconversion of R-2- arylpropionic acids to S-2-arylpropionic acids is a phenomenon that has been suggested to occur for a variety of 2-arylpropionic acids. Caldwell et al. also teach that the combination of chiral inversion and stereoselective metabolism provides for a more rapid clearance of the R- enantiomers of 2-arylpropionic acids. Additionally, Caldwell et al. allege that "at best, the R-isomers function as prodrugs for the therapeutically active S-forms" when the racemic drug is administered and thus add to both the therapeutic and toxic effects of the active S-enantiomers. This reference further contends that "at worst, the R- enantiomers are undesirable impurities in the active drug" causing difficulties due to non-stereoselective toxicity. Thus the reference alleges that the use of only the S-isomers should provide safer and more effective use of this class of drugs.

Similarly, it has been generalized that the pharmacokinetics of the enantiomers of 2-arylpropionic acids are different due, at least in part, to the unidirectional metabolic inversion of the R to the S enantiomer. However, it has been found that this interconversion depends on the particular compound and the particular species in which it is administered. Jamali, Eur. J. Drug Metabolism Pharmco 13(1): 1-9 (1988) .

Most recently, U.S. Pat. No. 5,331,000 has disclosed and claimed methods of using R(-) ketoprofen in humans to elicit an analgesic or antipyretic effect while reducing gastrointestinal (GI) toxicity.

Details of the mechanisms of some components of the reproductive cycle of domestic animals, including pigs, have been studied using an NSAID as a research tool. For example, indomethacin (a PG synthetase inhibitor) has been used to inhibit certain aspects of CL function in the estrous cycle of female pigs. See e .g. , B. Akinlosotu et al. "Prostaglandin E_j Counteracts the Effects of PGF₂ in Indomethacin Treated Cycling Gilts", Prostaglandins , 55 (1988) pp. 81-93; R. Kraeling et al., "Corpus Lutem Function After Indomethacin Treatment During the Estrous Cycle and

Following Hysterectomy in the Gilt", Biology of Reproduction , 25, pp. 511-18) (1981) . In addition, it has been proposed that certain NSAIDs may have some utility in preventing premature labor in domestic animals. See , W. Jochle and D. Lamond Control of Reproductive Functions in Domestic Animals , pl22 (1980) . However, in the span of over a decade and a half, the applicant is unaware of the use of any NSAIDS to control the estrous cycle of pigs or to breed pigs.

The use of ketoprofen in the domesticated animal industry has been very limited. Ketoprofen has been approved in the United States only for administration by injection as an anti-inflammatory to treat arthritis in horses. In Europe, ketoprofen has been marketed for oral administration as an anti-inflammatory for dogs, cats and horses. However, the oral administration of ketoprofen is limited to only 8 days due to gastrointestinal toxicity and related problems. In fact, it has been known for some time that NSAIDs, such as ketoprofen, have a variety of adverse side effects, including gastrointestinal toxicity in many domesticated animals such as dogs, cats and pigs. In general, the use of NSAIDs such as ketoprofen has been avoided in the domesticated animal industry.

In sum, there is a great need for a safe, effective, inexpensive and practical non-hormonal means for enhancing the breeding of domestic animals, particularly pigs. Drug intervention to date has been hampered by one or more problems such as toxicity, efficacy, cost, environmental and animal right concerns.

3. βiimmwyγ of the Invention

The present invention is directed towards methods for regulating the reproductive cycles of domesticated animals which comprises administering an effective amount of an NSAID or an optically pure isomer thereof.

The present invention is further directed to methods for controlling and managing the breeding of domestic animals which comprises synchronizing the reproductive cycle of the animals by administering an effective amount of an NSAID or an optically pure isomer thereof, and subsequently impregnating the animal, preferably by artificial insemination. The present invention is still further directed to methods for regulating the reproductive cycles of domestic animals, preferably pigs, which comprises administering an effective amount of racemic ketoprofen or an optically pure enantiomer thereof. The present invention is also directed to methods for controlling and managing the breeding of domestic animals, including pigs, which comprises synchronizing the reproductive cycle of the animals by administering an effective amount of racemic ketoprofen or an optically pure enantiomer thereof and subsequently impregnating the pigs, preferably by artificial insemination. The present invention encompasses methods for controlling and managing the breeding of sows and gilts which comprises synchronizing the reproductive cycle of the sow or gilt by administering an effective amount of the R(-) ketoprofen substantially free of its S(+) stereoisomer. The present invention also encompasses compositions and methods for controlling and managing the breeding of sows and gilts which comprises synchronizing the reproductive cycle of the sow or gilt by administering an effective amount of S(+) ketoprofen substantially free of its R(-) stereoisomer.

Without being limited by any theory, applicant believes that the NSAIDs of the present invention affect the estrus and ovulation of sows and gilts by preventing luteolysis in their normal cycle. Thus, the invention encompasses a method of preventing luteolysis in sows and gilts which comprises administering racemic ketoprofen or an optically pure enantiomer thereof.

The present invention is also directed towards compositions for regulating the reproductive cycle of domesticated animals which comprise an effective amount of an NSAID or an optically pure isomer thereof, and a physiologically acceptable carrier. The NSAID may also be used in a feed composition or top dressing.

The present invention is further directed towards pharmaceutical compositions for regulating the reproductive cycle of domesticated animals which comprise an effective amount of racemic ketoprofen or an optically pure enantiomer thereof and a pharmaceutically acceptable carrier.

The present invention is still further directed towards compositions for regulating the reproductive cycle of domesticated animals which comprise an effective amount of the R(-) or S(+) stereoisomer of ketoprofen in a vehicle suitable for consumption by the animal, i.e., a feed component. The feed component may optionally contain an attractant, i .e . , a substance or substances that are palatable to the particular animal in question.

4. Detailed Description of the Invention It has been discovered that NSAIDS can employed be aε agents to control reproductive function in domesticated animals. Surprisingly, it has been discovered that ketoprofen is particularly useful for control of reproduction in pigs i.e., sows and gilts. Racemic ketoprofen and its enantiomers each provide a unique advantage for use in pigs. Without being limited by theory, it is believed that by inhibiting endogenous formation of PGF₂ (in the uterus of domesticated animals) for a long enough period of time,

NSAIDS, preferably ketoprofen and its stereoisomers, prevent luteolysis in normally cycling female animals, i.e., the demise of the cyclic corpora lutea (or corpus luteum) . Administration simultaneously to groups of sows or gilts for a desired period of time results in synchronized estrus and ovulation following the withdrawal of the NSAIDS.

This avenue to reproductive cycle control is prerequisite for the routine use of artificial insemination as well as for a variety of animal husbandry management techniques, and is needed for pigs since conventional methods of reproductive cycle control used in most other species of domesticated animals either do not work in pigs, for example administration of prostaglandins, or are too expensive or not available, as with the administration of progestins.

4.1. Definitions

In order to more clearly illustrate the present invention, the following definitions are provided, which definitions are by no means limiting and which are consistent with common usage in the art.

The term "domesticated animal" as used herein means any of various animals domesticated by man so as to live and breed in a tame condition. Such animals include but are not limited to horse, sheep, pig, cattle, goat, buffalo and deer. The term "breeding" means any natural or artificial method of impregnating domesticated animals. The term "optically pure" as used herein means about 90% or more of the total weight of the composition is the optical isomer (or enantiomer) in question.

The terms "pharmaceutically acceptable salts" or "a pharmaceutically acceptable salt thereof" refer to salts prepared from pharmaceutically acceptable non-toxic acids or bases including inorganic acids and bases and organic acids and bases. Since the compound of the present invention is acidic, salts may be prepared from pharmaceutically acceptable non-toxic bases including inorganic and organic bases. Suitable pharmaceutically acceptable base addition salts for the compound of the present invention include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from lysine, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine.

4.2. Compositions

NSAID compounds or their stereoiεomers can be employed in the present invention to regulate the reproductive cycles of domesticated animals, most preferably pigs. The NSAID compounds can be used to control and manage the breeding of domesticated animals, including pigs, by synchronizing the reproductive cycle of the animals by administering an effective amount of such compounds.

It is contemplated that any NSAID compound or isomer thereof may be employed in thi_* present invention. Examples of suitable NSAIDs include but are not limited to almioprofen, benoxaprofen, carprofen, cicloprofen, banamine, fenoprofen, flurboprofen, fluxoxaprofen, ibuprofen, ketoprofen, ketoralac, loxoprofen, naproxen, priprofen, suprofen, tiaprofenic acid, azapropazone, bumadizone, oxyphenbutazone, clidanac, etodolac and sulindac. These compounds and their methods of preparation are well known in the art. Similarly, the enantiomers of these known compounds may be obtained using conventional techniques for resolution of racemates or other enantiomer mixtures (see, for example, "Stereochemistry of Carbon Compounds" by E.L. Eliel (McGraw Hill 1962) for optically active resolving agents, and Lochmuller, CH. et al., J. Chromatography 1975, Vol. 113, No. 3 pp. 283-302), or asymmetric synthesis. The racemic mixture of ketoprofen (i.e., a 1:1 mixture of the two enantiomers) possesses analgesic and antipyretic activity; however, this racemic mixture while offering the expectation of efficacy, may cause adverse effects in domestic animals. Utilizing the substantially optically pure R(-) isomer of ketoprofen results in clear dose related definitions of efficacy, diminished adverse effects, and accordingly, an improved therapeutic index.

However, it has been discovered that certain NSAIDs are more desirable than others for particular domesticated animals. For example, ketoprofen and its enantiomers each provide a unique advantage for use in pigs. Thus, the user can choose that which best suits their needs and resources. In one embodiment of the present invention, it is desirable to employ ketoprofen to regulate the reproductive cycle of pigs; in another the stereoisomers can be used to exploit their distinguishable biological profiles.

In an embodiment of the invention, the ketoprofen may be employed either as a racemic mixture or more preferably either the R(-) or S(+) enantiomer may be employed to take advantage of the particular benefits associated with each. More specifically, because pigs are prone to stomach ulcers, it is most desirable to use the R(-) ketoprofen enantiomer substantially free of its S(+) enantiomer. Alternatively, the S(+) ketoprofen enantiomer substantially free of its R(-) enantiomer can be used, if S(+) ketoprofen enantiomer is enteric coated such that it is released in the small intestines only. It is believed that the R(-) ketoprofen is metabolized, after ingestion by the pig, to form S(+) ketoprofen, which is potent in inhibiting PGF_2α. The term "adverse effects" includes, but is not limited to gastrointestinal, renal and hepatic toxicities, leukopenia, increases in bleeding times due to, e.g. , thrombocytopenia, and prolongation of gestation. The term "gastrointestinal toxicities" includes but is not limited to gastric and intestinal ulcerationε and erosions. The term "renal toxicities" includes but is not limited to such conditions as papillary necrosis and chronic interstitial nephritis.

The term "substantially free of its S(+) isomer" as used herein means that the composition contains at least 90% by weight of R(-) ketoprofen and 10% by weight or less of the corresponding S(+) ketoprofen. In a preferred embodiment the term "substantially free of the S(+) stereoisomer" means that the composition contains at least 95% by weight of R(-) ketoprofen and 1% or less of the corresponding S(+) ketoprofen. In the most preferred embodiment, the term "substantially free of its S(+) stereoisomer" as used herein means that the composition contains greater than 99% by weight of R(-) ketoprofen and less than 1% of the corresponding S(+) ketoprofen. These percentages are based upon the total amount of ketoprofen present in the composition. The terms "substantially optically pure R(-) isomer of ketoprofen" or "substantially optically pure R(-) ketoprofen" and "optically pure R(-) ketoprofen" or "optically pure R(-) isomer of ketoprofen" are also encompassed by the above-described amounts. Likewise, the term "substantially free of its R(-) isomer" is used in a similar fashion in that described above for the S(+) isomer.

When employing the racemic mixture of ketoprofen in the present invention, it may be administered with an optional enteric coating in order to protect against gastrointestinal toxicity. If either S(+) ketoprofen or R(-) ketoprofen is employed alone, it is believed that a lower dosage of either compound may be used as compared to the dosage required for a racemic mixture of R and s ketoprofen. Employing the R(-) ketoprofen alone may reduce any adverse physical effects such gastrointestinal toxicity. As noted above, if the S(+) isomer is employed alone, it is preferably enteric coated to prevent gastrointestinal toxicity.

The NSAID compounds can be administered to the domesticated animals using a variety of conventional methods. For example, the NSAID compounds may be employed in pharmaceutical compositions suitable for oral or parenteral (including subcutaneous, transdermal, intramuscular and intravenous) administration. Likewise, the NSAID compounds may be administered in the form of vaginal or rectal suppositories. The most preferred mode of administration is orally in the form of a premix which is administered as a top dressing to the animal's normal feed.

The pharmaceutical compositions of the present invention comprise NSAIDs as the active ingredient, or a pharmaceutically acceptable salt thereof, and may also contain a pharmaceutically acceptable carrier, and optionally, other therapeutic ingredients.

The compositions of the present invention can be in the form of liquid or solid preparations. Suitable liquid preparations include suspensions or solutions in aqueous or non-aqueous liquids, oil-in-water emulsions, water-in-oil emulsions, elixirs, and aerosols. Suitable solid preparations include powders, capsules, cachets, tablets and pellets. Carriers for such solid preparations include starches, sugars, macrocrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, and the like. Oral solid preparations (such as powders, capsules, tablets, cachets, chewable tablets or pellets) are preferred.

The most preferred oral solid preparation is a premix wherein the NSAID is mixed into a compatible food component such as dextrose, fructose or lactose. Optionally, the solid preparations may contain or be employed in conjunction with a attractant compound in order to overcome any aversions due to undesirable tastes or odors of the NSAIDs. Artificial sow's milk is a preferred attractant. Other attractants or additives are known in the art. Examples of suitable additives include but are not limited to dextrose, fructose or lactose which have a favorable color and odor.

Such liquid and solid preparations may be prepared by any of the methods of pharmacy, but all methods include the step of bringing into association the active ingredient with the,carrier which constitutes one or more necessary ingredients. In general, the compositions are prepared by uniformly and intimately admixing the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.

For example, a tablet may be prepared by compression or molding, optionally, with one or more accessory ingredients. Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with a binder, lubricant, inert diluent, surface active or dispersing agent. Molded tablets may be made by molding, in a suitable machine, a mixture of the powdered compound moistened with an inert liquid diluent.

Desirably, regardless of its form, each administration contains from about 10 mg to about 1000 mg of the NSAID active ingredient. Most preferably, each administration contains either one of three dosages, about 10 mg, about 100 mg and about 250 mg of the active ingredient and is administered more than once a day. The dosage per administration is about 0.1 mg/kg to about 2.5 mg/kg by weight of the treated animal, and preferably 1.0 mg/kg to about 2.0 mg/kg. Preferably, the NSAID compound is formulated into a pharmaceutically acceptable carrier and is administered orally in conjunction with the animal's feed. The NSAID compounds can be administered in conjunction with the animal feed using any of a variety of techniques. Conventional animal feed comprises substances such as alfalfa, brome hay, corn, soybean oil meal, meat scrap, fish meal, bone meal and the like, as well as vitamin, mineral and other supplements. As mentioned above, it is most preferred if the NSAID compound is formulated into a solid preparation as a premix and administered as a top dressing to the animals feed. Solid preparations can also be admixed with the feed prior to introducing the feed to the animals so as to create a blend or of feed and NSAID compound. The solid preparation may also be blended with one or more of the feed ingredients or a small portion of all of the ingredients, and then the initial blend is uniformly blended with the balance of the feed material. Any suitable mixing technique employed in the animal feed manufacture art may be employed.

Further, the NSAID may be formulated into a liquid preparation and applied to a solid feed so as to coat the feed particles. Alternatively, such liquid suspension may be blended with one or more of the feed ingredients or a small portion of all of the ingredients, and then the initial blend is uniformly blended with the balance of the feed material. Any suitable mixing technique employed in the animal feed manufacture art may be employed. Additionally, such liquid preparation can be blended with a liquid feed preparation or the animals' water supply.

4.3. Methods

The present invention also relates to methods for controlling and managing the breeding of domestic animals which comprises synchronizing the reproductive cycle of the animals which comprise administering an effective amount of an NSAID or its stereoisomer. Upon synchronization of the reproductive cycle, the animals are bred either naturally or artificially, preferably by artificial insemination. The particular NSAID compound, its amount and the frequency of its administration can vary widely depending of the particular type of livestock and as well as the individual characteristics of each animal. It is well within the purview of the εkilled artiεan to determine the appropriate dosage for a given animal. In a one embodiment of the invention, an NSAID compound is employed to regulate the reproductive cycle of domesticated animals. It is suggested as an example that oral administration to the domesticated animals range from about once a week to about 4 times daily, preferably 3 times a week to about 3 times daily, most preferably once or twice per day. Preferably, the total daily dosage of the NSAID administered will range from about 50 mg to about 5000 mg, more preferably from about 75 mg to about 2500 mg, and most preferably from about 100 mg to about 1000 mg of the NSAID compound, depending upon the compound used. Preferably, the dosage per administration is about 1 mg/kg to about 2 mg/kg by weight of the treated animal.

In another embodiment of the present invention, the control and management of the breeding of domestic animals by synchronizing the reproductive cycle of the animal, especially estrus and ovulations, is achieved by administering both an effective amount of an NSAID or its stereoisomer and an effective amount of one or more active compounds such as steroids or hormones or combinations thereof.

Suitable steroids and hormones include but are not limited to polypeptides; polypeptides of hypothalamic origin such as FSH-RH (FSH-releaser hormone) , LH-RH (LH-releaser hormone) , GN-RH (nona- or decapeptideε) , and GH-RH (thyrotropin and growth hormone releaser hormones) ; protein hormones such as a) pituitary hormones such as FSH (follicle stimulating hormone) ; and LH (lutein^zing hormone) , b) hormones from the feo-maternal complex such as eCG (PMSG- pregnant mare serum gonadotropin) and hCG (human chronic gonadotropin) ; Steroid hormones and their analogues such as a) estrogens and their esters such as estradiol, estrone, estriol and the like, estrogen steroid derivatives such as ethinyl estradiol and mestranol, non-steroidal analogues such aε diethylstilbestrol (DES) and hexestrol, b) androgens and their esters such aε testosterone, testosterone propionate and the like, androgen derivatives such as methyl testoεterone, c) progestins such as progesterone, methoxyprogesterone acetate (MAP) , megeβtrol acetate (MA) , melengestrol acetate (MGA) , chlormadinone acetate (CAP) , delmadinone acetate (DMA) , fluorogestone acetate (FGA) , northandrolone (NBA or Norgestomet) , norethisterone acetate (NET acetate) , Oxolven, and proligestone, d) corticosteroids derivatives such aε glucocorticoids; prostaglandins and their analogues such as PGF_j,,, PGF^-THAM, RS-9390 (Prostalene) , ICI 81,008, ICI 80,996 (Cloprostenol) .

Preferred steroids and hormones include but are not limited to estrogens, hypothalamic releasing hormones, prostaglandins and gonadotropins. More preferred steroids and hormones include but are not limited to orally active estrogens, luteolylic prostaglandins, GnRH or its analogs and gonadotropins of feto-placental origin. Most preferred steroids and hormones include but are not limited to ethinyl estradiol and menεtranol, PGF^ and its analogs such as cloprostenol, GnRH or its analogs such as burerelin, deslorelin or D-Phe⁶-LHRH and eCG (PMSG) and hCG.

In thiε embodiment of the invention, the steroid or hormone is administered either simultaneously, before or after the administration of the NSAID compound. In a preferred embodiment of the invention, the steroid or hormone iε administered simultaneously, e.g., administering ketoprofen simultaneously with orally active estrogens or progestinε. In another embodiment of the present invention, the steroids and/or hormones are adminiεtered in a predetermined schedule and sequence. For example, PGF^ or its analogs are administered 20 to 30 hours, preferably about 24 hours; after administration of the ketoprofen. Likewise, eCG and a GnRH analog or hCG is administered at about 24 hours and a second time at about 75 hours to about 80 hours after administration of the ketoprofen. This schedule is believed to allow for a fixed time of breeding an entire treated population of gilts or sows. The particular steroid or hormone and NSAID compound, aε well aε the amount and frequency of their administration can vary widely, depending of the particular type of livestock and as well as the individual characteristics of each animal. It is well within the purview of the skilled artisan to determine the appropriate dosage of each for a given animal.

It is suggested aε an example that oral administration to sows or gilts range from about once a week to about 4 times daily, preferably 3 times a week to about 3 times daily, most preferably once or twice per day. The daily dosage of the NSAID administered will range from about 50 mg to about 5000 mg, preferably from about 75 mg to about 2500 mg, and more preferably from about 100 mg to about 1000 mg, of the NSAID compound. The dosage of the hormone or steroid administered will range from about 50 mg to about 5000 mg, preferably from about 75 mg to about 2500 mg, more preferably from about 100 mg to about 1000 mg. Preferably, the dosage per administration of these compounds is about 1 mg/kg to about 2 mg/kg by weight of the treated animal.

In a more preferred embodiment of the invention, racemic ketoprofen, its optically pure stereoisomer or a pharmaceutically acceptable salt thereof is selected as the NSAID compound for controlling and managing the breeding of pigs. In a most preferred embodiment R(+) or S(-) ketoprofen substantially free the corresponding enantiomer are εelected as the NSAID compound.

It is suggested as an example that oral administration to sows or gilts range from about once a week to about 4 times daily, preferably 3 times a week to about 3 times daily, most preferably once or twice per day. The dosage of the ketoprofen administered will range from about 50 mg to about 5000 mg, preferably from about 75 mg to about 2500 mg, and more preferably from about 100 mgto about 1000 mg of the ketoprofen compound. In another preferred embodiment of the present invention, the ketoprofen iε administered in conjunction with a steroid or hormone. Preferred steroids and hormones include but are not limited to estrogens, hypothalamic releasing hormones, prostaglandins and gonadotropins. More preferred steroids and hormones include but are not limited to orally active estrogens, luteolylic prostaglandins, GnRH or its analogs and gonadotropins of feto-placental origin. Most preferred steroids and hormones include but are not limited to ethinyl estradiol and menstranol, PGF^ and its analogs such as cloprostenol, GnRH or itε analogs such as burerelin deslorelin or D-Phe⁶-LHRH and eCG (PMSG) and hCG. The dosage of the hormone or steroid administered will range from about 50 mg to about 5000 mg, preferably from about 75 mg to about 2500 mg, and more preferably from about 100 mg to about 1000 mg. Preferably, the dosage per administration of these compounds is about 1 mg/kg to about 2 mg/kg by weight of the treated animal.

In any given group or population of gilts or sows, it is very likely that the individual animals are at various stages of the reproductive cycle. Therefore, the treatments described above should be administered for at least about 12 days. Preferably, the treatments are administered for about 14 to about 18 days. Using the methods of the present invention, synchronization of the reproductive cycle typically occurs in about 2 to about 4 days after withdrawal of the NSAID compound. Breeding should be performed shortly thereafter. Preferably breeding occurε within 2-4 dayε after withdrawal of the NSAID compound or at a predetermined time of about 24 to about 42 hourε after adminiεtration of GnRH or hCG, or other hormones if they are used. The preferred mode of breeding is artificial insemination during the synchronized estrus or at a fixed time schedule; however, any conventional method of inseminating domestic animals including natural breeding iε contemplated as being useful in the present invention. 5. Examples

The invention is further defined by reference to the following examples describing the preparation of certain compositions of the present invention, as well as their 5 utility. It will be apparent to those skilled in the art that may modifications, both to materials and methods, may be practiced without departing from the purpose and interest of this invention.

0 5.1 Example 1 Preparation of R(-) Ketoprofen

The following is a description of the preparation of racemic ketoprofen by an enzymatic process. Included is a description of the synthesis of the water-soluble ester used (a three step procedure) , as well as the actual enzymatic 5 resolution, subsequent base hydrolysis, and the recovery of R(-) ketoprofen acid.

A. Synthesis of Ketoprofen Dimethylethanolamine Ester ₀ Racemic ketoprofen (0.5 moles) is added to thionyl chloride (1.0 moles) in a flask fitted with a drying tube. Dimethylformamide (0.25 mL) is added to the reaction mixture and the mixture was stirred and warmed until the ketoprofen dissolves and gas evolution commences. The heat is removed _ and the mixture is stirred at room temperature for 18 hours. The thionyl chloride is removed under reduced presεure and the oily residue of acid chloride slowly solidifies.

¹ The acid chloride iε dissolved in tetrahydrofuran (125 mL) and added to a solution of N,N-dimethylethanolamine ₀ (1.0 moles) in tetrahydrofuran (500 mL) cooled to 0° C in a flask equipped with a drying tube. After the addition, the reaction mixture is stirred at room temperature for 18 hours. A saturated aqueous solution of potasεium carbonate (500 mL) is added to the reaction mixture and the resulting organic _ layer is removed. The aqueous layer is extracted with diethyl ether (2 x 250 mL) and the organic layers are combined, washed with a saturated aqueous solution of sodium chloride, dried over potassium carbonate and the solvent removed under reduced pressure. The product is isolated as a colorless viscous oil.

B. Quarternization of the N,N-

Dimethylethanolamine

The resulting N,N-dimethylethanolamine ester is dissolved in diethyl ether (500 mL) and cooled to 0° C. A solution of dimethyl sulfate (0.36 moles) in diethyl ether

(500 mL) iε added to the cooled solution and the resulting solution was εtirred at room temperature for 18 hours. The resulting solid material is removed by filtration, washed with diethyl ether and dried under vacuum to yield the N,N,N- trimethylethanolammonium ester of ketoprofen (ketoprofen choline ester) as a white solid.

C. Enzymatic Transesterification of the Racemic Ketoprofen Choline Ester

The choline ester (0.36 moles) is dissolved in 0.2M sodium phosphate buffer (900 ml. pH 7.0). To this solution is added methanol (100 mL) and Protease type XXVII (3 gm) which is available commercially from sigma Chemical Co. The reaction is allowed to stir gently at room temperature for

24 hours. The reaction mixture is extracted with diethyl ether (2 x 250 mL) and the organic layer is reserved. The aqueous layer is adjusted to pH 2 by the addition of concentrated sulfuric acid and the resulting pressure and the volume is adjusted to 900 mL by the addition of 0.2M sodium phosphate buffer (pH 7.0). To thiε solution is added methanol (100 mL) and Proteaεe type XXVII (2 gm) . The reaction is allowed to stir gently at room temperature for

24 hours. The reaction mixture is extracted with diethyl ether (2 x 250 mL) and thiε organic layer is combined with the layer reserved from the first enzymatic reaction. The combined ether layers are dried over magnesium sulfate and the solvent removed under reduced preεεure to leave crude

R(-) ketoprofen methyl eεter which iε dried under vacuum. D. Preparation of R(-) Ketoprofen The crude ester is combined with ethanolic potassium hydroxide solution (pH 13) and the resulting mixture is stirred for 1 hour at room temperature. The reεulting εolution is adjusted to pH 2 by the addition of hydrochloric acid. The resulting mixture is extracted with diethyl ether and the combined ether solutionε are dried over magnesium sulfate and the solvent removed under reduced pressure to leave crude R(-) ketoprofen. The crude acid is recrystallized from diethyl ether to yield R(-) ketoprofen.

5.2. Example 2 Toxicity

The following is a description of a study of the effects of the isomers of ketoprofen in the guinea pig. Groups of 6-10 guinea pigs are dosed orally with either vehicle, racemic ketoprofen (20, 10, 5, 1 and 0.1 mg/kg), S(+) ketoprofen (20, 10, 5, 1 and 0.1 mg/kg). Within 24 hours after the dose, the animals are euthanized and groεs abnormalities are recorded in the GI tracts, with particular attention to the gastric mucosa of the stomach. Microerosions and redness (irritations) are noted, and the effects are compared between the treatment groups as described by Aberg & Larsεon (Acta Pharmacol. Toxicol. 28: 249-257, 1990) . Based on such observations, the R(-) isomer is seen to cause virtually no gastrointestinal irritation.

5.3. ffTTflT?** 3 Leukopenia

To test white-cell survival, an in vitro test method iε used, where a primary bone marrow cell culture is exposed to increasing concentrations of teεt compounds such as R(-) ketoprofen and S(+) ketoprofen. A known inducer of i leukopenia, such as thiouracil, is used as a positive control. The survival of the granulocytes is measured using conventional differential cell-counting methodology.

The risk for leukopenic effects of escalating concentrations of drugε in vitro is studied in groups of animals in which a mild granulocytopenia has initially been induced either by drugs such as thiouracil or chloramphenicol, or by radiation. Repeated white-cell counts are performed to monitor the development of leukopenia in the animals.

5.4. Example 4 Oral Formulations Of Ketoprofen

Typical oral formulations of racemic, S(+) or R(-) ketoprofen ("active ingredient") in the form of a capsule are illustrated below.

Oral Formulation

Capsuleε:

Formula Ouantitv per caosule in mσ

A B C

Active Ingredient 10.0 100.0 200.0

Lactose 88.5 148.5 48.5

Titanium Dioxide 0.5 0.5 0.5

Magnesium Stearate 1.0 1.0 1.0

Compresεion Weight 100.0 250.00 250.00

5.5. Example 5 Feed Composition (Or Top Dressing For Use With Feed)

An example of a premixed powder top dressing is illustrated below. Alternatively, the premix can be added to a feed component and presented aε a feed composition to the animal. The amounts are provided in milligrams.

B

Active Ingredient 10.0 100.0 200.0

Inert Filler 88.8 112.5 137.5

Attractant 2.0 37.5 62.5

Total Weight 100.0 mg 250.0 mg 250.0 mg

5.6. Example 6 Efficacy Of NSAIDS In

Synchronizing The Reproductive Cycle Of Sows And Gilts A study is undertaken to determine the ability of

NSAIDs to synchronize the reproductive cycle of female pigs. This experiment is undertaken in two phases.

In the first phaεe, three groupε of giltε are formed and treated with an NSAID. Treatment is started on Day 10 of the eεtrous cycle for each group respectively.

Each group receives a different doεage of racemic ketoprofen, as a premix, in a top dressing on a small amount of food twice daily before their morning and evening feeding. An optimum dosage is selected from the three dosages tested. The different dosages are 1.0 mg/kg, 2.0 mg/kg and 3.0 mg/kg by body weight of the treated animal. Clinical observations are made only daily each morning for: a) vulva swelling and reddening, b) changes in behavior, c) degree of restlessness, d) response to back pressure, and e) eliciting a standing reflex. The optimum dose is determined as the minimum dose which prevents eεtrus during the treatment period and causes a synchronized appearance of estrus 2 to 4 days after the last ketoprofen adminiεtration, without causing clinically detectably side effects.

After selecting an optimum dosage, a second phase is undertaken in which another three groups of gilts are selected and treated with the optimum dosage of NSAID. The treatments are provided twice a day for 14 days, commencing on Day 5, 10 and 15 of the estrous cycle for each group respectively.

Progesterone blood concentrations are taken prior to the εtart of the treatment and alεo on Dayε 5, 10 and 14 of the treatment, aε well aε on Days 1, 2, 3, 5, and 10 after the treatment. Additionally, daily observations of the estrus are made, including observations of the intensity of estrous symptoms such aε scores. In 50% of all the treatment groups, synchronization of ovulation is attempted using PMSG/GnRH treatment schedule and 50% are allowed to ovulate spontaneously. All animals are inseminated twice during the observed estrus or in accordance with the PMSG/GnRH treatment regimen. All animals are sacrificed after 35 days after breeding. The number of pregnancies, of corpora lutea and of normal and abnormal embryos are all determined and evaluated to determine the extent of synchronization of the reproductive cycle achieved. It may be apparent to those skilled in the art that modifications and variations of the present invention are possible, in light of the above discloεure. It is understood that such modifications are within the spirit and scope of the invention, which is limited and defined only by the appended claims.

Claims

What is claimed is:

1. A method for regulating the reproductive cycle of domesticated animals which comprising administering to a domesticated animal an effective amount of a nonsteroidal anti-inflammatory drug, its optically pure isomer, or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein the domesticated animal is a gilt or sow.

3. The method of claim 1, wherein the nonsteroidal anti-inflammatory drug is racemic ketoprofen.

4. The method of claim 1, wherein the nonsteroidal anti-inflammatory drug is R(-) ketoprofen which iε substantially free of its S(+) stereoisomer.

5. The method of claim 1, wherein the nonsteroidal anti-inflammatory drug is S(+) ketoprofen which is substantially free of its R(-) stereoisomer.

6. The method of claim 1, which further comprises administering an effective amount of a steroid or hormone.

7. The method of claim 1 wherein the nonsteroidal anti-inflammatory drug is administered for about 12 to about 18 days.

8. The method of claim 7 wherein the nonsteroidal anti-inflammatory drug is administered once or twice per day.

9. The method of claim 1, wherein the amount of nonsteroidal anti-inflammatory drug administered is from about 10 mg to about 5000 mg per day.

10. The method of claim 6, wherein the steroid or hormone is an estrogen, hypothalamic releasing hormone, prostaglandin or gonadotropin or mixtures thereof.

11. A method for controlling the breeding of a domesticated animal compriεing:

(a) synchronizing the estrus cycle of said animals by administering to the animal an effective amount of ketoprofen, an optically pure isomer thereof or a pharmaceutically acceptable salt thereof; and

(b) breeding said animal.

12. The method of claim 11, wherein the domesticated animal is a gilt or sow.

13. The method of claim 11, wherein racemic ketoprofen is administered in step (a) .

14. The method of claim 13, wherein R(-) ketoprofen substantially free of its S(+) stereoisomer is administered in step (a) .

15. The method of claim 13, wherein S(+) ketoprofen substantially free of its R(-) stereoisomer is administered in step (a) .

16. The method of claim 11, which further comprises administering an effective amount of a steroid or hormone.

17. The method of claim 16 wherein said steroid or hormone is administered concurrently with or after the administration of ketoprofen or itε iεomerε, and before breeding.

18. The method of claim 16, wherein the steroid or hormone is estrogen, hypothalamic releasing hormone, prostaglandin or gonadotropin or mixtures thereof.

19. The method of claim 11, wherein ketoprofen is administered for about 12 to about 18 days.

20. The method of claim 19, wherein said administration is once or twice per day.

21. The method of claim 11, wherein the amount of nonsteroidal anti-inflammatory drug administered is from about 10 mg to about 5000 rag per day.

22. A composition for regulating the reproductive cycle of domesticated animals which comprises:

(a) an effective amount of ketoprofen, an optically pure isomer thereof, or a pharmaceutically acceptable salt thereof; and (b) an acceptable carrier.

23. The composition of claim 22, which further compriseε an effective amount of a steroid or hormone.

24. The composition of claim 23, wherein the steroid or hormone iε eεtrogen, hypothalamic releasing hormone, ^: prostaglandin or gonadotropin or mixtures thereof.

25. A feed composition for domesticated animals which comprises:

(a) a feed component; and

(b) an effective amount of a nonsteroidal anti-inflammatory drug, its optically pure isomer or a pharmaceutically acceptable salt thereof.

26. The feed composition of claim 25 which further comprises an attractant.

27. The feed composition of claim 26, wherein said composition is a top dresεing in the form of powder.