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1. WO1984000549 - METHODE DE PREVENTION DE LA FIEVRE VITULAIRE CHEZ LE BETAIL A LAIT

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Description

Methed of Preventing Milk Fever
in Dairy Cattle

Technical Field
This invention relates to a method for preventing milk fever (parturient paresis) in dairy cattle.
More particularly this invention relates to a method for preventing milk fever disease in dairy cattle which comprises adrainistering to the cattle vitamin D derivatives which are characterized by the presence of a hydroxyl group of at least one of the C-1 and C-25 positions in the vitamin D molecule and where the C-24 position carries a rretabolically stable blocking group.
Background Art
Parturient paresis, or milk fever disease, occurs with substantial frequency in high-producing dairy cattle generally beg-uining with the third and later lactations. The disease involves a hypocalcemda and a hypopnosphatemia at the time of parturition or shortly thereafter, resulting in the animal becoπring imrobilized and flacid. This phenomemon has been clearly related, to the lew blood calcium resulting from the foπration of milk at the time of parturition. However, many aniirals do not suffer the disease and are able to provide sufficient calcium in their bleed from intestinal and bone sc-urces to meet the irπrediate derπands of new milk formation. The reason for the difference among individual animals remains unknown. However, several irethcds of preventing the disease have been suggested and tried. These are:
(1) calcium infusions, which is the cαrrnon irethed of
treatment of the disorder;
(2) the administration of large amounts of vitamin D,
which will prevent the hypocalcemic response; or (3) conditioning of animals with either a low calcium
diet or an acidic diet.
Unfortunately, large amounts of ordinary vitemin D have not been successful primarly because it can only be given once, and often vitamin D intoxication or calcification of organs (soft tissue) becomes a major concern. Calcium infusions are excellent teeatirents, but it is not practical ly possible to prevent the disease by calcium infusion alone, since it is not possible to forecast when milk fever will result from calving. Lew calcium conditioning of the animal is not desirable since it is ciuring the non-lactating or dry periods that cows are able to replace the bone calcium which had been depleted by previous lactation. Use of acid diets tends to produce an acidosis that results in other problems. Therefore, none of these t reatments has provided a ccmplete solution to the prevention of parturient paresis.
The discovery of active vitamin D metabolites and their analogs has led to the development of more effective methods for treating parturient paresis. Thus, it has been shown that 25-hydroxyvitamin D3, a metabolite of vitamin D, given in relatively large amounts, provides substantial protection against the disease (U.S. Patent 3 ,646,203) . Simi larly, much lower doses of 1α-hydroxyvitamin D3 or 1,25-dihydroxyvitamin D3 have also been shown to be effective in preventing the disease (U.S. Patents 3,879,548 and 4,110,446) , and the use of a mixture of 1α-hydroxyvitamin D3 and 25-hydroxyvitamin D3 has recently been proposed as a method for providing very marked protection against milk fever disease (U.S. Patent 4 ,338 ,312) . Disclosure of Invention
During the course of investigations of milk fever disease, it was found that the cows that care down with the disease have above normal average blood levels of 24,25-dihydroxyvitamin D3, a metabolite of vitamin D. This observation, suggested a possible causal relationship between circulating 24 ,25-dihydroxyvitjamin D3 and the disease and led to further studies in which this metabolite was administered to cows prior to parturition.
A herd of dairy cattle at third lactation or better was divided randomly into two groups of 28 cows each. The non-treated group (control group) received an inbramuscular Injection of corn oil without vitarnin D compound dissolved in it, whereas the treated group received the same dose of oil containing 24,25-dibydroxyvitamin D3 (4 mg/animal) . The results of this experiment are shown in Table 1.



It is evident from the data presented in Table 1 that the dairy cattle receiving 24,25-dihydroxyvitamin D3 had a dramatically higher incidence of milk fever (about 50%) than the control group (14% milk fever incidence). It is evident, therefore, that the presence of 24-hydroxylated vitamin D increased the incidence of milk fever, and it appears to predispose the animal to the disease. Re-examination of the plasma levels of 24,25-dihydroxyvitamin D3 in animals that ultimately came down with milk fever disease show (see Table 2) that these animals have higher than normal levels of 24,25-dihydroxyvitamin D3 prior to the onset of milk fever.


The indicated tirnes represent the time of calving as 0 and other times are days before and after calving.

Days

The foregoing results clearly establish a correlation between 24,25-dibydroxy vitamin D3 and milk fever disease, and indicate that the accumulation of this vitamin D metabolite in the animal is undesirable in the sense that it contributes to a higher incidence of the disease and appears to predispose the animal to it.
It is to be noted that all of the txeatments heretofore proposed as the most effective means of preventing milk fever disease, i.e. the use of vitamin D derivatives such as
25-hydrσxyvitamin D3, 1α,25-dihydroxyvitamin D3 or 1α- hydrαxyvitamin D3, involve the administration ccmpounds that are non-hydroxylated at C-24. The data presented in Table 1 and Table 2 lead to the conclusion that the absence of
24-hydroxylation is an important characteristic of an effective anti-milk fever agent. However, all of these vitamin D derivatives can serve, either directly or
indirectly, as substrates for enzymatic 24-hydroxylation in vivo in the animals and can give rise to the undesired 24,25-dihydroxy side chain structure (e.g. by the reactions:
25-hydroxyvitamin D3 → 24,25-dihydroxyvitamin D3; 1α,

25-dihydroxyvitamin D3 → 1α ,24,25-trihydroxyvitamin D3;

Iα-hydroxyvitamin D3 → 1α,25-dihydroxyvitamin D3→ 1α,

24,25-trihydroxyvitamin D3) . The occurence, in vivo, of 24-hydroxylation according to the reactions shown, is well established and, hence, the vitamin D compounds now used for the treatment of milk fever disease will unavoidably yield the 24,25-dihydroxy metabolites. This is also true for compounds of the vitamin D2 series, which are known to undergo an analogous sequence of hydroxylation reactions. A further complicating feature is that for any given anirral the rates of these in vivo 24-hydroxylations are unknown and unpredictable, being dependent on a number of physiological parameters (e.g. Ca and phosphate status, other hormones, etc.) , and hence, the effectiveness of these compounds to combat milk fever may be affected in an unpredictable fashion to the extent that they contribute, by in vivo metabolism to the undesired
24,25-dihydroxy metabolite pool.
Based upon the observation and results described hereinbefore it is clear that successful prophylaxsis against milk fever disease can be achieved by administrating to dairy cattle a vitamin D derivative which retains the strucrtural features known to be important to such activity, namely, hydroxylation at at least one of C-1 and C-25 positions, but which cannot be readily hydroxylated in vivo at the C-24 position.
Suitable compounds for this purpose are, therefore, analogs of the vitamin D3 or vitamin D2 series characterized by the structure


wherein A and B are hydrogen, or taken together form a double bond, wherein X is fluoro, hydrogen or methyl, and where R1 and R2 are selected from hydrogen and hydroxy except that at least one of R1 and R2 must be hydroxy. The hydroxy group at carbon 3 and/or the hydroxy groups at carbons 1 and/or 25 may also be acylated, e.g. be present as the acetyl, propionyl, benzoyl or other conventional acyl derivatives known in the art, such acylated derivatives also being suitable and effective as anti-milk fever agents.
The above compounds are characterized by the presence of a hydroxy group(s) at C-1 and/or 25, which confer high anti-milk fever potency, and a blocking group at carbon 24 in the form of a 24-fluoro, or 24,24-difluoro substituent, the function of which is to prevent the introduction of a
24-hydroxy group during in vivo metabolism.
Specific examples of such compounds are:
24,24-difluoro-25-hydroxyvitamin D3
24,24-difluoro-1α,25-dihydroxyvitamin D3
24,24-difluoro-1α,-hydroxyvitamin D3
24-fluoro-25-hydroxyvitamin D3
24-fluoro-1α,25-dihydroxyvitamin D3
24-fluoro-1α-hydroxyvitamin D2
24-fluoro-25-hydroxyvitamin D2
The fluoro substituents in the C-24 position are known to block in vivo hydroxylation and therefore such compounds cannot readily give rise to the 24,25-dihydroxylated forms of vitamin D which in the normal metabolic course in vivo result from the metabolism of any 25-hydroxylated vitemin D compound, however, the above named C-24-fluoro analogs are at least as effective as the corresponding compounds absent the fluoro blocking group at the C-24 position.
Such 24-blocked vitamin D analogs are readily available by synthetic processes known in the art, e.g. as described in United States Letters Patent Nos. 4,305,880, 4,196,133 and 4,229,357. In addition, 1-hydroxylated analogs are also available by combination of chemical and enzymological synthesis as described, for example, in United States Letters Patent Nos. 4,201,881 and 4,226,788. Since the C-24-blocked compounds set forth above are capable of preventing the detrimental accumulation of the 24,25-dihydroxylated vitamin D metabolites in vivo while retaining the desirable vitamin D-like and milk fever combatting characteristics they are eminently suitable for purposes of the present invention and are the preferred compounds for such purpose.
The present invention is practiced by administering any of the 24-blocked vitamin D compounds shown above to cows prior to parturition. Advantageously, the ccmpounds are administered about 3 to 8 days prior to the expected calving date and may be given as a single dose, or, if preferred, as irultiple doses over a period of days. Dosage amounts depend on the compound being administered, with 24-blocked-25-hydroxyvitamin D derivatives generally being given in doses of 1-10 mg per animal, while in the case of the corresponding 24-blocked 1α-hydroxylated compounds, dosages of 0.1-1 mg per animal are effective.
The administration of a combination of the active
24-blocked vitamin D compounds is also possible and effective. Such combinations most advantageously involve the co-adminis tration of a 25-hydroxyvitamin D compound and a 1-hydroxy vitamin D analog. For example, the administration of a
24-blocked-25-hydroxyvitamin D compound together with the corresponding 24-blccked-1α-hydroxyvitamin D compound in a ratio preferably adjusted so as to provide the 25-hydroxy cαiipσund in about 5-10 fold excess over the 1α-hydroxylated material in a highly effective method of treatment.
Any conventional route of administraton may be used. For example, the compounds, dissolved in an innocuous oil conventionally used in veterinary practice, such as corn oil, sesame oil, propylene glycol, etc. , can be administered by an Intramuscular or subcutaneous injection, or may be applied as a solution in a solvent such as dimethylsulfoxide for transcutaneσus absorption. Alternatively, the compounds can be administered after compounding with other materials, as a bolus, or in encapsulated form or, again with suitable adjuvants, or in a suitable solvent, as a top dressing for grains or other dietary components fed to the animals.
Illustrative examples of specific compounds which are effective in preventing milk fever disease and of protocols for their adirinistration are set forth below.
1. 24-blocked-25-hydroxyvitamin D
24,24-fluoro-25-hydroxyvitamin D3 dissolved in
corn oil is given by intramuscular injection
to high-producing cows which are characterized
by having a propensity toward milk fever
disease in an amount sufficient to supply each
animal with about 4 mg of the compound. The
injection is made five days prior to the
expected calving date and is effective in
preventing milk fever disease.
2. 24-blocked 1α-hydroxyvitamin D
24,24-difluoro-1α,25-dihydroxyvitamin D3 dissolved
in corn oil, when given to cows having a tendency
toward milk fever, by intramuscular injection in an amount to provide each animal with about 0.4 mg
five days prior to calving is effective to prevent
milk fever.

3. 24 -blocked-25 -hydroxyvitamin D in combination with 24- blocked-1α-hydroxyvitamin D
24,24-difluoro-25 -hydroxyvitamin D3 and 24,24- difluoro-1α,25-dihydroxyvitamin D3 dissolved in
corn oil in the ratio of 10:1 are given by
inteamuscular injection to cows characterized by a high incidence of milk fever disease five days prior to calving. Sufficient of the solution is given to provide each animal with about 4 mg and 0.4 mg
respectively of the 24-blocked-25-hydroxyvitamin D3 and the 24- blocιked-1α,25-dihydroxyvitamin D3 com
pounds. The admixture of the compounds is effective in preventing milk fever.
When the compounds are to, be administered through the rredium of a top dressing for feed the compound or mixture of compounds to be administered in that fashion is first dispersed in an edible or innocuous liquid carrier such as peanut oil, olive oil, corn oil, cottonseed oil and the like and in that form is sprayed on about 2 to 3 pounds of feed which is to be presented to the animal.
The dispersion in the carrier is readily accomplished by first dissolving the compound or admixture in ether, then thoroughly dispersing that solution in the carrier liquid followed by evaporation of the ether. The concentration of the compound in the carrier liquid is not critical and it has been found that as much as 16 mg of the 24-blocked-25-hydroxyvitamin D can be readily dispersed in 5 ml of oil. In any event the concentration should be great eriough that the feed to which it is applied does not become excessively oily. Also sufficient of the oil dispersed compound should be applied to the 2-3 pounds of feed so that an animal will receive from about 0.1 mg to 1 mg of the 24-blocked 1α-hydroxyvitamin D compound or from about 1 mg to 10 mg of the 24-blocked-25-hydroxyvitamin D compound whether such compounds are administered alone or in admixtaure.

Alternatively, the liquid dispersion of the compound can be absorbed on a starch or sugar base, e.g. corn starch, sucrose, lactose, fructose, and then such concentrate mixed into or sprinkled upon 1-3 pounds of feed, the amount of concentrate in all cases being sufficient to contain the dosage level specified above.
When the compounds are to be administered as a bolus the specific compounds are first dispersed in an innocuous oil or other liquid (propylene glycol) as described above and the dispersion is absorbed on an edible or inert but ingestible substrate, e.g. starches or sugars, and are either packed into gelatin capsules for administration, or combined with other inert materials which will permit and enhance compaction into a tablet form as is well known in the art. Generally each bolus, whether in capsule or tablet form, should contain a dosage level of one of the 24-blocked compounds, or admixture thereof in the ranges set forth above.
Also, and as set forth hereinbefore the dispersion of the one or more of the specified compounds can be administered directly as an intramuscular injection.
It is to be understood that both the cis- and transforms of the 24-blocked vitamin D compounds can be utilized effectively for the purposes specified herein. Moreover, vitamin D compounds blocked at the 24-position with
substituents other than those specifically shown can also be used. For example, the blocking group can be others of the halogen series than fluorine and can also be 24-dialkylated cxirpounds such as the dimethyl compounds. IN all cases the blocked compounds should be characterized by metabolic stability and by vitamin D activity.
In all cases with the administration of the 24-blocked vitarnin D compounds in accordance with the present invention, if calving does not occur wi-thin the expected time after first administration of the compound, generally five days, a repeat injection of the compound may be given. In fact, a third dose of the compound may be given if after an additional five days the cow has not calved.
It is to be appreciated that the dosages specified above are not necessarily critical. Dosages can, of course, vary with the size of the cow to which it is being administered. In any event sufficient of the material must be given in all cases to accomplish the desired end of preventing milk fever disease. Administration of amounts in excess of such amounts should be avoided as economically unsound.