WO2001011986A1

WO2001011986A1 - Animal feed containing hydrolyzable calcitriol derivatives

Info

Publication number: WO2001011986A1
Application number: PCT/US2000/014884
Authority: WO
Inventors: Hector F. Deluca
Original assignee: Wisconsin Alumni Research Foundation
Priority date: 1999-08-17
Filing date: 2000-05-31
Publication date: 2001-02-22
Also published as: AU5305600A

Abstract

An animal feed containing a hydrolyzable calcitriol derivative as the active ingredient. The hydrolyzable calcitriol causes improved utilization of phosphorus, calcium, potassium, magnesium, zinc, iron and manganese in animal feed so as to minimize, or perhaps eliminate, the need for supplemental quantities of these minerals in an animal diet. In addition, low phosphorus containing feeds reduce the polluting effects on the environment since less phosphorus is excreted in the animal's feces which are then spread on agricultural land.

Description

ANIMAL FEED CONTAINING HYDROLYZABLE CALCITRIOL DERIVATIVES

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with United States Government support awarded by the National Institutes of Health (NIH), Grant #DK- 14881.

The United States Government has certain rights in this invention.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention relates to an animal feed containing a hydrolyzable calcitriol derivative as an active ingredient. The use of a hydrolyzable calcitriol derivative in animal feed causes the absorption of plant minerals, such as calcium and phosphate, from feed allowing minerals bound in the form of phytate to be utilized by animals. These hydrolyzable calcitriol derivatives also increase utilization of minerals available from inorganic sources in the animal's diet. This allows for a severe reduction of, and possibly the complete elimination of, supplemental inorganic mineral additives as an ingredient in animal feed. Up to 80% of the phosphorus (P) present in plant foods and feeds exists as a complex of phytic acid (myoinositol hexaphosphate), hereinafter referred to as phytate. Phytate may structurally be illustrated by the following formula and represents the primary source of P that is potentially available from cereals in the diet:

The P in phytate cannot be totally digested by simple-stomached animals, including humans, and it therefore passes through the gastrointestinal (GI) tract and is excreted in the feces. In animal nutrition, this is accounted for in diet formulation whereby 1.5 to 2.0% of an inorganic phosphate source is supplemented to meet the animal's minimal P requirement. Addition of inorganic P to poultry, swine, companion animal, and fish diets is expensive. It is often stated that supplemental inorganic P for these species is the third most expensive dietary ingredient, after energy and protein. An animal's body requires P for formation of bones and teeth, for phospholipid (cell membrane structure) and nucleic acid (RNA, DNA) synthesis, for synthesis of ATP and other high-energy P compounds, and for proper acid-base balance in the body. Roughly 85% of the body P is in the skeleton. Dry bone is comprised of 50% organic matrix (protein in the form of collagen, glycoproteins, non- collagen proteins, and lipids) and 50% inorganic material (mostly a Ca-P salt, i.e., hydroxyapatite).

Supplemental inorganic P is provided to animal diets in one of three feedgrade forms; dicalcium phosphate (18.5% P), monocalcium phosphate (21.5% P) or deflorinated phosphate (18.0% P). The combined total market for these products is estimated to be 675 million dollars per year in the U.S., Canada, Mexico, Western Europe and Japan. If one were to include South America, Eastern Europe, Asia, Africa, China, India, and Southeast Asia, (where market data are difficult to obtain), the total market for feed-grade phosphates could easily be expected to exceed 1 billion dollars annually. In North America, 50% of feed-grade phosphate consumed is used for poultry feeding. It has been discovered that a hydrolyzable calcitriol derivative may be readily incorporated into animal feed due to its relatively high solubility in oil, and that use of a calcitriol derivative having a substituent hydrolyzable in vivo to convert the derivative to lα,25-dihydroxyvitamin D3 or to a lα,25-dihydroxyvitamin D3 analog as an ingredient in animal feed would severely reduce the need for supplemental inorganic P in animal feed.

Phytate complexes in plant foods and feeds (eg., cereal grains and by-products, beans) also bind cations such as calcium, potassium, magnesium, zinc, iron and manganese (Erdman, 1979) illustrated schematically as follows:

A feed additive such as the hydrolyzable calcitriol derivatives as disclosed herein that causes the increased utilization of P from both organic and inorganic sources should also increase utilization of these other elements from both organic and inorganic sources as well. The present invention has established that a hydrolyzable calcitriol derivative increases the utilization of not only P from both organic and inorganic sources but also zinc, iron, calcium, potassium, magnesium, and manganese from both organic and inorganic sources. Thus, because these trace elements are always added in supplemental form to diets for swine, poultry and companion animals (e.g. as feed-grade ZnO or ZnSO₄Η2θ; FeSO₄Η2θ; MnO or MnSO₄Η2θ) use of a hydrolyzable calcitriol derivative would lower, or perhaps eliminate, the need for supplemental quantities of these mineral salts in a practical-type grain-oilseed meal diet. By reducing or eliminating the inorganic P supplement and the supplement of trace mineral salts, the remaining diet would contain more usable energy. Thus, grain-oilseed meal diets generally contain about 3,200 kcal metabolizable energy per kilogram of diet, and mineral salts supply no metabolizable energy. Removal of the unneeded minerals and substitution with grain would therefore increase the usable energy in the diet.

Currently, phytase is being used in much of Europe and Asia to reduce P pollution. The use level is 600 units per kilogram diet, but this level was selected because of cost of the enzyme and not because 600 units will maximize phytate utilization. However, use of a hydrolyzable calcitriol derivative in accordance with the present invention would also reduce the need to feed expensive levels of phytase.

Animal producers are forced to feed high P diets because of the phytate content of diets. This increases P in the excreta waste products (both feces and urine). Excess P from animal, as well as human waste, is generally spread on the soil, where a portion of it gets washed into ground water and then into ponds, streams, rivers, lakes and oceans. Too much P in water stimulates growth of algae, and algae take up considerable oxygen. This robs marine life of the oxygen they need to grow, reproduce and thrive.

In many parts of Europe and Asia, P pollution has become such a problem and concern that penalties in the form of stiff financial fines are imposed on livestock producers who spread too much P-laden manure on the soils. Because of this, much of Europe now uses a microbial phytase product (BASF), even though this product (which hydro lyzes phytate) is very expensive, in fact too expensive to be cost effective (at 600 units/kg diet) as a feed additive in the U.S. at the present time. Many U.S. soils are being described as "P saturated", thus resulting in a greater concentration of P in soil leachates. High-P water leachate in areas such as the Chesapeake Bay has been blamed for excessive algae growth and increased fish kills in bay waters (Ward, 1993). In Europe, the feed industry group FEFANA issued a position paper in 1991 entitled "Improvement of the Environment". They proposed that P in manure from livestock production should be reduced by 30% (Ward, 1993). The limits of P that can be applied to soils in Europe have been discussed by Schwarz (1994). Accordingly, it is estimated that use of a hydrolyzable calcitriol derivative that is active in increasing phosphorus utilization in accordance with the present invention, could cut the P content of animal waste products by up to 80%.

Initial work focused on use of 1,25 dihydroxycholecalciferol (1,25- (OH)2U3) in the absence or presence of 1200 units of microbial phytase (BASF). Edwards (1993) showed that l,25-(OH)₂D₃ is effective in improving P utilization from phytate-bound P, and Biehl et al (1995) confirmed his results. Moreover, both studies showed that l,25-(OH)2D3 works additively with microbial phytase in releasing P from dietary phytate complexes. It seems likely that l,25-(OH)2D3 exerts is effects in two ways: (a) the 1,25 compound likely increases the activity of intestinal phytases or phosphatases that hydrolyze phytate (Pileggi et al, 1955; Maddaiah et al, 1964) and (b) the 1,25 compound is known to stimulate phosphate transport (Tanka and DeLuca, 1974), facilitating transport of P from GI tract to plasma and hence bone.

Under normal dietary circumstances, cholecalciferol (vitamin D3) that is added to a diet gets absorbed from the GI tract and is transported via blood to the liver where the liver enzyme 25-hydroxylase acts on the compound to form 25-OH D3. This compound is the normal blood metabolite of vitamin D3. A small portion of 25-OH D3 undergoes a further hydroxylation step in the kidney, at the 1-α position, resulting in the calciotropic hormone l ,25-(OH)₂D3. Because l,25-(OH)2D3 is expensive to synthesize and because oral 25-OH D3 is not the active form in phosphate absorption, it was proposed that 1-α-OH D₃ would be an effective compound for increasing phosphate utilization. It has been discovered that lα-hydroxylated vitamin D compounds and particularly 1- α-OH D3 will be absorbed from the GI tract and then be transported to the liver where 25-hydroxylase would act upon it to bring about synthesis of 1,25-dihydroxylated compounds and particularly l,25-(OH)2D3. A portion of these compounds would then be transported back to the GI tract where they would activate intestinal calcium and phosphate absorption. The net effect would be an increased utilization of P (also Zn, Fe, Mn, K, Mg, and Ca) from the phytate complex as well as from the inorganic P supplement itself.

This invention relates to hydrolyzable calcitriol derivatives defined herein as biologically active vitamin D compounds modified with hydrolyzable groups at one or more of the 1 , 3 and 25 carbon positions, such as esters of lα,25-dihydroxyvitamin D3 or esters of 1,25- dihydroxyvitamin D3 analogs, and their use to regulate over time the function of l,25(OH)2D3 (or of l,25(OH)2 D3 analogs) to increase utilization of P (also Zn, K, Mg, Fe, Mn, and Ca) from organic sources such as the phytate complex as well as from inorganic sources such as inorganic mineral supplements commonly added to animal diets. The active hydrolyzable calcitriol derivatives useful in the present invention have also been found to have higher solubility in the oils typically found in animal feed than calcitriol derivatives that are hydroxylated in the 1 , 3 and/ or 25 carbon positions. Such high solubility advantageously allows for ease of formulation and less degradation of the active, which otherwise might be caused by ultraviolet light or oxygen.

An important consideration is that when given by mouth or by injection, large amounts of l,25(OH)2D3 or of l,25(OH)2D3 analogs are available to the tissues initially but little is left after 2-4 hours due to metabolism and excretions. A process whereby calcitriol or a calcitriol analog can be made available in vivo more slowly and more continuously would avoid peaks and valleys in its availability thereby providing an effective in vivo level of the compound over a more prolonged period of time and also avoiding or substantially reducing episodes of hypercalcemia that often result from the sudden availability of excessive amounts of the substance.

The present invention provides a method for modulating and regulating the in vivo activity of biologically active vitamin D compounds, such as calcitriol or analogs of calcitriol. More specifically, this invention provides modified vitamin D compounds that are highly soluble in the oils of animal feed and which also exhibit a desirable and highly advantageous pattern of biological activity in vivo, namely, the more gradual onset and more prolonged duration of activity. As a consequence of such advantageous properties, these compounds represent novel agents for increasing utilization of P, Zn, Fe, Mn, K, Mg and Ca from organic sources such as the phytate complex as well as from inorganic sources such as inorganic mineral supplements commonly added to animal diets.

Structurally, the key feature of the modified vitamin D compounds having these desirable attributes is that they are derivatives of lα,25- dihydroxyvitamin D3, or derivatives of lα,25-dihydroxyvitamin D3 analogs, in which a hydrolyzable group is attached to the hydroxy group at carbon 25 and, optionally, to any other of the hydroxy groups present in the molecule, such as at carbon 1 and/ or 3. Depending on various structural factors — e.g. the type, size, structural complexity — of the attached group, these derivatives hydrolyze to lα,25-dihydroxyvitamin D3, or to a lα,25-dihydroxyvitamin D3 analog, at different rates in vivo, thus providing for the "slow release" of the biologically active vitamin D compound (i.e. 1,25-dihydroxyvitamin D3, or an analog thereof) in the animal's body. The "slow release" in vivo activity profiles of such compounds can, of course, be further modulated by the use of mixtures of hydrolyzable calcitriol derivatives (e.g. mixtures of different hydrolyzable derivatives of 1,25-dihydroxyvitamin D3, or different hydrolyzable derivatives of 1,25- dihydroxyvitamm D3 analogs) or the use of mixtures consisting of one or more hydrolyzable vitamin D derivative together with hydroxylated vitamin D compounds.

It is important to stress that the critical structural feature of the vitamin derivatives identified above is the presence of a hydrolyzable group attached to the hydroxy group at carbon 25 of the molecule. The presence of a hydrolyzable group at that position imparts on the resulting derivatives the desirable "slow- release" biological activity profile mentioned above. Other hydroxy functions occurring in the molecule (e.g. hydroxy functions at carbons 1 or 3) may be present as free hydroxy groups, or one or more of them may also be derivatived with a hydrolyzable group. The fact that the introduction of a hydrolyzable group at carbon 25 of the vitamin D molecule markedly modulates the in vivo biological activity pattern of the resulting derivative was not appreciated previously. The realization of the importance of this specific modification, and the demonstration of its marked and highly beneficial biological effects form the basis of this invention.

The "hydrolyzable group" present in the above-mentioned derivatives is preferably an acyl group, i.e. a group of the type Q^CO-, where Q¹ represents hydrogen or a hydrocarbon radical of from 1 to 18 carbons that may be straight chain, cyclic, branched, saturated or unsaturated. Thus, for example, the hydrocarbon radical may be a straight chain or branched alkyl group, or a straight chain or branched alkenyl group with one or more double bonds, or it may be an optionally substituted cycloalkyl or cycloalkenyl group, or an aromatic group, such as substituted or unsubstituted phenyl, benzyl or naphthyl. Especially preferred acyl groups are alkanoyl or alkenoyl groups, of which some typical examples are formyl, acetyl, propanoyl, hexanoyl, isobutyryl, 2- butenoyl, palmitoyl or oleoyl. Another suitable type of hydrolyzable group is the hydrocarbyloxycarbonyl group, i.e. a group of the type Q²-O-CO-, where Q² is a Ci to Ciβ hydrocarbon radical as defined above. Exemplary of such hydrocarbon radicals are methyl, ethyl, propyl, and higher straight chain or branched alkyl and alkenyl radicals, as well as aromatic hydrocarbon radicals such as phenyl or benzoyl.

Among the modified vitamin D compounds having the desirable in vivo bioactivity profile indicated above, an especially important and preferred class are certain acyl ester calcitriol derivatives characterized by the following general structure:

where X¹ and X², independently represent hydrogen or an acyl group, and where X³ represents an acyl group as previously defined. Two other very important groups of modified vitamin D compounds are the corresponding acyl esters of the calcitriol side chain homologs, i.e. where the side chain is extended by up to 5 additional carbon groups at the 22, 23 or 24 carbon positions, and the acyl derivatives of the 19-nor- l ,25- dihydroxyvitamin D analogs, i.e. where the methylene group at carbon 19 is replaced by two hydrogen atoms. The present invention, therefore, provides a series of modified vitamin D compounds that are useful to increase utilization of P, Zn, Fe, Mn, K, Mg and Ca in animal diets. More specifically, the preferred method comprises the incorporation of an effective amount of one or more of the above-indicated acyl ester derivatives of lα,25-dihydroxyvitamin D3 or of the corresponding side chain homologated and/ or 19-nor derivatives of l ,25-dihydroxyvitamin D3 analogs in animal diets.

The above compounds may be administered alone or in combination with other acceptable agents. Dosages of from not less than about 2μg/kg diet to not more than about 30μg/kg diet of the individual compound per se, or in combinations, are generally effective. This method has the distinct advantage that it will increase utilization of P, Zn, Fe, Mn, K, Mg and Ca by animals due to the bioconversion of these compounds to calcitriol and similar analogs thereof which have been proven to be effective for such purpose. Further, these compounds advantageously will be less likely to cause hypercalcemia or hypocalcemia then the underivatized compounds even if the compound is administered continuously on a daily basis, as long as the appropriate compounds and dosages are used, it being understood that the compounds and the dosage levels will be adjusted dependent upon the response of the subject as monitored by methods known to those skilled in the art. Finally, these compounds allow for ease of formulation while at the same time limiting degradation caused by UV light and O because of their high solubility in animal feed oils. In summary, the potential benefits of the present invention include (1) reduction in or possible elimination of the need for inorganic P supplements for animal (including fish) diets; (2) reduction in P pollution of the environment; (3) reduction or possible elimination of the need for supplemental Zn, Mn, K, Mg, Ca and Fe in animal diets; and (4) reduction of the quantity of phytase needed for maximal P utilization from feeds. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT The present invention provides an animal feed containing a hydrolyzable calcitriol derivative as the active ingredient to accomplish the improved results disclosed herein. The use of a hydrolyzable calcitriol derivative in animal feeds removes minerals such as calcium and phosphate from the intestine allowing the phytate to solubilize making the minerals (preferably P and Ca) contained in the phytate complexes bioavailable for animals. These hydrolyzable calcitriol derivatives also increase utilization of minerals (especially P and Ca) from inorganic sources in the diet thus further reducing the need for supplemental inorganic minerals. This results in a feed composition having a severe reduction of, and possibly the complete elimination of, supplemental inorganic minerals such as calcium and phosphorus as an ingredient. The present invention provides novel modified vitamin D compounds as the active ingredient which are hydrolyzable in vivo to calcitriol, or analogs of calcitriol, over a period of time following administration, and as a consequence regulate the in vivo availability of active calcitriol, or analogs of calcitriol, thereby also modulating their activity profile in vivo. The term "activity profile" refers to the biological response over time of vitamin D compounds such as calcitriol or analogs of calcitriol. Individual modified compounds, or mixtures of such compounds, can be administered to "fine tune" a desired time course of response.

As used herein the term "vitamin D compound" encompasses compounds which have the C-ring, D-ring and 3β-hydroxycyclohexane A- ring of vitamin D interconnected by the 5,7 diene double bond system of vitamin D together with any side chain attached to the D-ring. In other words, the vitamin D compounds encompassed herein include those having a "vitamin D nucleus" comprising substituted or unsubstituted A-, C-, and D-rings interconnected by a 5, 7 diene double bond system typical of vitamin D together with a side chain attached to the D-ring. As used herein the term "modified vitamin D compound" encompasses the hydrolyzable calcitriol derivatives referred to herein, and is broadly used to specify any vitamin D compound in which one or more of the hydroxy functions present in such a compound are modified by derivatization with a hydrolyzable group. A "hydrolyzable group" is a hydroxy-modifying group that can be hydrolyzed in vivo, so as to regenerate the free hydroxy functions. In the context of this disclosure, the term hydrolyzable group preferably includes acyl and hydrocarbyloxycarbonyl groups, i.e. groups of the type Q^CO- and Q²-O-CO, respectively, where Q¹ and Q² have the meaning defined earlier.

Structurally, the modified vitamin D compounds encompassed may be represented by the formula I:

where R⁵ and R⁶ each represent hydrogen, or taken together R⁵ and R⁶ represent a methylene group.

The side chain group R in the above-shown structure represents a steroid side chain of the structure below: WO 01 /l 1986 PCT/USOO/14884

- 14 -

where the stereochemical center (corresponding to C-20 in steroid numbering) may have the R or S configuration, (i.e. either the natural configuration about carbon 20 or the opposite unnatural configuration), and where Z is selected from Y, -OY, -CH₂OY, -C ≡ CY and -CH = CHY, where the double bond may have the cis or trans geometry, and where Y is selected from a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, where R¹ is selected from hydrogen, OX⁴, fluoro, trifluoromethyl, and Ci-5- alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R² is selected from hydrogen, fluoro, trifluoromethyl and Ci-5 alkyl, which may be straight- chain or branched, and optionally, bear a hydroxy substituent, and where R³ and R⁴, independently represent trifluoromethyl or C1-5 alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R¹ and R², taken together, represent an oxo group, or an alkylidene group, =CR²R², or =CR²R³, or the group -(CH2)_P-, where p is an integer from 2 to 5, and where R³ and R⁴, taken together, represent the group -(CH2)_q-, where q is an integer from 2 to 5. In the above-shown structures X¹, X² and X⁴ independently represent hydrogen, or a hydrolyzable group such as an acyl group or a hydrocarbyloxycarbonyl group, and X³ represents a hydrolyzable group such as an acyl group or a hydrocarbyloxycarbonyl group, as previously defined herein. Some specific examples of such modified vitamin D compounds include calcitriol derivatives such as: lα,25(OH)₂-D₃- l ,3,25-Triacetate where X¹=X²=X³=CH₃CO (structure Ila below); lα,25(OH)₂-D₃- l ,3,25-Trihexanoate where X¹=X²=X³=CH₃(CH₂)4CO (structure lib below); lα,25(OH)₂-D₃- 1 ,3,25-Trinonanoate where X¹=X²=X³=CH₃(CH₂)7CO (structure lie below); and lα,25(OH) -D₃-25-Acetate where X =X²=H and X³=CH₃CO (structure lid below).

a. Xⁱ = X² = X³ = CH3CO b. χι = X² = X³ = CH₃(CH₂)₄CO c. Xⁱ = X² = X³ = CH₃(CH₂)₇CO d. Xⁱ = X² = H; X³ = CH3CO

The above modified vitamin D compounds may be prepared by any of numerous known processes from known starting materials. In particular, the preparation of mono, di and tri-esters of lα,25-(OH)2-D3 is disclosed in copending U.S. patent application Serial No. 09/043,509 filed on March 23, 1998 the description of which is specifically incorporated herein by reference. As used in the description and in the claims, the term hydroxy- protecting group signifies any group commonly used for the temporary protection of hydroxy functions, such as for example, alkoxycarbonyl, acyl, alkylsilyl, and alkoxyalkyl groups, and a protected hydroxy group is a hydroxy function derivatized by such a protecting group. Alkoxycarbonyl protecting groups are groupings such as methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The term "acyl" has been previously defined herein, but preferably specifically includes, in the context of a protecting group, an alkanoyl group of 1 to 6 carbons, in all of its isomeric forms, or a carboxyalkanoyl group of 1 to 6 carbons, such as an oxalyl, amlonyl, succinyl, glutaryl group, or an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl substituted benzoyl group. The word "alkyl" as used in the description or the claims, denotes a straight- chain or branched alkyl radical of 1 to 10 carbons, in all its isomeric forms. Alkoxyalkyl protecting groups are groupings such as methoxymethyl, ethoxyethyl, methoxyethoxymethyl, or tetrahydrofuranyl and tetrahydropyranyl. Preferred alkylsilyl protecting groups are trimethylsilyl, triethylsilyl, t-butyldimethylsilyl, and analogous alkylated silyl radicals.

The above hydrolyzable calcitriol derivatives may be administered alone to animals in an edible carrier or in combination with other feed additive agents. The above hydrolyzable calcitriol derivatives or combinations thereof can be readily administered as a top dressing, or by mixing them directly into animal feed, or separately from the feed as a supplement in an edible carrier to be later mixed with the feed, by separate oral dosage, by injection or by transdermal means or in combination with other growth related edible compounds, in doses of from about 2μg/kg diet to about 30μg/kg diet, the proportions of each of the compounds in the combination being dependent upon the particular problem being addressed and the degree of response desired, are generally effective to practice the present invention. In poultry, amounts in excess of about lOμg/kg of diet of hydrolyzable calcitriol derivatives, are generally unnecessary to achieve the desired results, may result in hypercalcemia, and may not be an economically sound practice. It should be understood that the specific dosage in any given case will be adjusted in accordance with the specific compounds, condition of the animal and the other relevant facts that may modify the activity of the effective ingredient or the response of the animal, as is well known by those skilled in the art.

If administered separately from the animal feed as a supplement, dosage forms of the various compounds can be prepared by combining them with non-toxic acceptable edible carriers to make either immediate release or slow release formulations, as is well known in the art. Such edible carriers may be either solid or liquid such as, for example, corn starch, lactose, sucrose, soy flakes, peanut oil, olive oil, sesame oil and propylene glycol (and used as a supplement or top dressing on feed). The dosage forms may also contain adjuvants, such as preserving, stabilizing, wetting or emulsifying agents, solution promoters, etc. They may also contain other therapeutically valuable substances.

The present invention also relates to an animal feed composition and method of compounding an animal feed utilizing a hydrolyzable calcitriol derivative or a combination of hydrolyzable calcitriol derivatives to lower and/ or eliminate supplementing the diet with P, Zn, Fe, Mn, Mg, K and Ca in the animal feed. The hydrolyzable vitamin D compounds suitable for this use have been previously described herein. The amount of a phosphorus supplement (18.5%P) that may be incorporated with the feed may be reduced to 0% to about 0.9% on a dry weight basis. This is a significant reduction from the normal amount of phosphorus supplement incorporated in animal feed compositions of about 1.5% to about 2.5%. This beneficial reduction in phosphorus is a direct result of the incorporation of a hydrolyzable calcitriol derivative in the animal feed. The animal feed may be any protein-containing organic meal normally employed to meet the dietary requirements of animals. Many of such protein-containing meals are typically primarily composed of corn, soybean meal or a corn/ soybean meal mix. For example, typical commercially available products fed to fowl include Egg Maker Complete, a poultry feed product of Land O' Lakes AG Services, as well as Country Game 8B Turkey Grower a product of Agwa, Inc. Both of these commercially available products are typical examples of animal feeds with which a hydrolyzable calcitriol derivative may be incorporated to reduce or eliminate the amount of supplemental phosphorus, zinc, manganese, calcium, potassium, magnesium, and iron intake required in such compositions. Thus, any type of protein-containing organic meal may be utilized as the base mix to which the hydrolyzable calcitriol derivatives, and reduced supplemental phosphorus, zinc, manganese, calcium, potassium, magnesium and iron amounts of the present invention may be incorporated. The present invention is applicable to the diet of numerous animals, which herein is defined as including mammals, fowl and fish. In particular, the diet may be employed with commercially significant mammals such as swine e.g. pigs, cattle e.g. cows, sheep, goats, laboratory rodents (rats, mice, hamsters and gerbils), fur-bearing animals such as mink and fox, and zoo animals such as monkeys and apes, as well as domestic mammals such as cats and dogs. Typical commercially significant fowl or poultry include chickens, turkeys, ducks, geese, pheasants and quail. Commercially farmed fish such as trout would also benefit from the diet disclosed herein. In a method of compounding feed for animals in accordance with the present invention, one or more hydrolyzable calcitriol derivative is incorporated with the animal feed in an amount of from about 2μg/kg feed to about 30μg/kg feed on a dry weight basis. The feed mixture is then fed as a mash or is formed into desired discrete shapes for further processing and packaging. In general, these discrete shapes may be pellets, blocks or briquettes formed by known extrusion and/ or compacting techniques. The particular processing technique utilized does not affect the performance of the hydrolyzable calcitriol derivative in the animal feed mixture. The present invention is more specifically described by the following examples, which are meant to be illustrative only.

EXAMPLE 1 This example illustrates the serum calcium response of rats over time to three compounds, namely, lα,25(OH)2D3-l ,3,25-triacetate, lα25(OH) D₃-l ,3-diacetate, and lα,25(OH)₂D₃ (unesterified).

In this biological test, rats were fed a calcium-containing (0.47% calcium), vitamin D-deficient diet for a period of 8 weeks to deplete them of vitamin D. They were then provided a single oral dose of 1,000 pmol or 1 nanomole of each of the compounds, and serum calcium was determined by bleeding the rats at the various times as shown in Table 1. Because calcium is present in the diet and hence their intestine, the rise in serum calcium largely represents intestinal calcium absorption. The results show clearly that the 1,3-diacetate and un-esterified calcitriol produce essentially the same time course of response, illustrating that acetylation of the C- l and C-3 hydroxy groups does not significantly alter the biological response, presumably because the acyl groups at these positions are removed rapidly, e.g. by digestive enzymes. In marked contrast, the triacetate did not begin to show a response until 12 to 18 hours post-dose, peaking at 24 hours. Thus, the 25-acetate group probably remained intact over a more prolonged period and was then slowly hydrolyzed inside the body. Thus, the triacetate clearly delays utilization of the calcitriol indicating that the in vivo activity profile of the parent calcitriol can be changed very markedly by acylation of the C-25- bydroxy group.

TABLE 1

(A) (B) (C)

Time Control l ,25(OH D₃ Diacetate Triacetate

0 Hours 4.3 ± 0.07

8 Hours 6.06 ± 0.89 5.42 ± 0.90 4.06 ± 0.38

12 Hours 7.39 + 0.10 6.50 ± 0.13 4.70 ± 0.12

18 Hours 6.97 + 0.51 6.24 ± 0.51 5.91 ± 0.30

24 Hours 6.45 ± 0.50 5.98 ± 0.43 6.7 ± 0.54

48 Hours 4.43 ± 0.18 5.59 ± 0.80 5.11 ± 0.48 5.10 ± 0.43

11 data = Mean ± S.D.

(A) from (B), N.S. diff.

(A) from (C), at 8 hrs, 12 hrs, p<0.001

EXAMPLE 2

This example illustrates the serum calcium response of rats over time to two compounds, namely, lα,25(OH)2D3 (unesterified) and lα,25(OH)₂D3-25-acetate, administered by three different methods, namely, orally (oral), intramuscularly (I.M.), and subcutaneously (Sub. Cu.).

In this test, 1 nanomole of each compound was given to vitamin D deficient rats fed a 0.47% calcium, 0.3% P diet in 0.1 ml propylene glycol 95%/ 5% ethanol. There were at least 4 rats per group. Serum calcium was determined at the various times shown in Table 2 following a single WO 01/11986 PCT/TJSOO/14884

21

dose given by the indicated route. The results are in accord with those of Example 1. Regardless of route of administration l,25(OH)2D3-25-acetate shows a more gradual onset of in vivo activity and a delayed peak of activity. Thus, the 25-monoacetate clearly delays utilization of the calcitriol confirming that the presence of a C-25-O-acyl group has a pronounced effect on the activity pattern and time course of response of a biologically active vitamin D compound.

TABLE 2

Serum Calcium Response to l,25-(OH)₂D₃ and its 25-Acetate

Serum Calcium

(mg/ : 100 ml)

Administration

Compou Route Dav 1 Dav 3 Dav 6 Dav 10 nd

None (Control) 5.9±0.34 - 5.7±0.34 5.2±0.63

1 ,25- Oral 9.33±0.41 - 8.6±0.52 7.15±0.68 (OH)₂D₃

1 ,25- Oral

ΓOH)₂D₃ 7.70±0.11 8.87±0.6 8.54±0.15 8.83±0.71

Acetate

I.M. 8.10±0.24 9.17±0.53 9.77±0.3 -

Sub. 7.78±0.28 9.00±0.28 9.84±0.39 - Cu.

EXAMPLE 3 The following is a measure of the relative solubility of lα- hydroxyvitamin D3 and the lα-hydroxyvitamin D₃-3-acetate as prepared by the usual lα-hydroxylation methods originally described by Paaren et al.

Three solvents were used to test relatively solubility to determine which of these compounds might be more easily incorporated into the oils used for top dressing. Oils are triglycerides that have a polarity between dichloromethane and hexane. The relative solubility of the 3-acetate was compared with that of the free lα-hydroxyvitamin D3 (I0C-OH-D3) compound. The procedure was to place in the bottom of a test tube an excess of each of these compounds. That is, of the order of 20 mg. To each sample was added 1 ml of hexane. The sample was allowed to stand with frequent swirling in the order to ensure complete solubility equilibrium. An aliquot was taken of the solution and diluted in hexane for determination of absorbence at 265 nm. At a temperature of 25°C, then the solubility of lα-OH-D3 proved to be 300 μg/ml. Under the same conditions, the 3-acetate derivative had a solubility of 78 mg/ml. Thus, a solubility ratio was found of 260: 1 for the 3-acetate derivative versus the lα-OH-Ds.

Using the same procedure, the solubility at 25°C was tested in dichloromethane and in ethanol. The solubility of the I0C-OH-D3, in dichloromethane was found to be 370 μg/ml. For the 3-acetate, 1.3 mg/ml. The ratio of solubility in this solvent, therefore, was 3.5: 1 in favor of the 3-acetate. In ethanol the solubility was found to be 300 μg/ml, whereas the 3-acetate derivative was found to dissolve at 730 μg/ml. Thus, the acetate is twice as soluble as the free I0C-OH-D3 in ethanol. From these results, it is very clear that the 3-acetate provides markedly improved solubility into the organic solvents over that of the free sterol and, therefore, provides for an ease of incorporation into fatty components of diet or supplements. It is envisioned that the 3-acetate derivative could be readily incorporated into soy bean oil or cotton seed oil, arachis oil or safflower oil. Such a concentrate could then easily be incorporated into a feed supplement or feed. Because of the preferred solubility in oils, it is very likely that the 3-acetate would be much more stable in a supplement or a feed additive than the I0C-OH-D3. EXAMPLE 4 The Preparation of the lα-OH-D3 3-Acetate: The procedure used is described in U.S. Patent No. 4,313,942 (Example 1). The following modifications to this procedure were done: After workup of the lα-hydroxylated product, the solvent was evaporated under vacuum to dryness, and 3 ml of glacial acetic acid was added. The mixture was heated at 55-60°C for 30 minutes, and the reaction mixture worked up as described in the patent and dried over magnesium sulfate. After removal of the magnesium solvent, the product was dissolved in 6 ml of ethyl acetate and 20 mg of malaic anhydride was added to the solution. The reaction was monitored by following the products on HPLC using an analytical size silica gel column, equilibrated and developed in 1.7% isopropanol in hexane. When the 5,6-trans-isomer disappeared from the profile, the reaction was quenched with ice and 10% sodium hydroxide. The reaction mixture was stirred for 10 minutes, 150 ml of ether added and the ether transferred to a separatory funnel. The ether layer was washed with ice and 10% sodium hydroxide and then several times with water until a neutral pH was achieved. The solvent was dried over magnesium sulfate, filtered and taken to dryness. The product was further purified by dissolving it in 5% ethyl acetate in hexane and applied to a 3 X 30 column of lipid activated silica gel in 5% ethyl acetate hexane. The column was eluted batchwise with 5%, 10%, 20%, and 50% ethyl acetate in hexane. The product eluted at 20% ethyl acetate and hexane, leaving behind the 5,6-trans-isomer and other contaminants. 120 mg of the 3-acetyl lα-OH-D3 was obtained.

Claims

CLAIMS I claim:

1. A feed composition comprising: an animal feed; and a hydrolyzable calcitriol derivative.

2. The composition of claim 1 wherein said hydrolyzable calcitriol derivative has the structure:

where R⁵ and R⁶ each represent hydrogen, or taken together R⁵ and R⁶ represent a methylene group, and where R is represented by the structure:

where the stereochemical center at c^arbon 20 in the side chain may have the R or S configuration, and where Z is selected from Y, -OY, -CH2OY, -C ≡ CY and -CH = CHY, where the double bond may have the cis or trans stereochemical configuration, and where Y is selected from a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, where R¹ is selected from the group consisting of hydrogen, OX⁴, fluoro, trifluoromethyl, and Ci-5- alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R² is selected from the group consisting of hydrogen, fluoro, trifluoromethyl and C1-5 alkyl, which may be straight- chain or branched, and optionally, bear a hydroxy substituent, and where each of R³ and R⁴, is selected from trifluoromethyl and Ci-5 alkyl, which may be straight chain or branched and optionally bear a hydroxy substituent, and where R¹ and R², taken together, represent an oxo group, or an alkylidene group, =CR²R², =CR²R³, or the group -(CH2)_P-, where p is an integer from 2 to 5, and where R3 and R₄, taken together, represent the group -(CH2)q-, where q is an integer from 2 to 5, and where X¹, X², and X⁴ are each hydrogen, an acyl group, or a hydrocarbyloxycarbonyl group, and where X³ represents an acyl group or a hydrocarbyloxycarbonyl group.

3. The composition of claim 1 wherein said hydrolyzable calcitriol derivative has the structure:

where X¹ and X² independently represent hydrogen, an acyl group or a hydrocarbyloxycarbonyl group, and where X³ is an acyl group or a hydrocarbyloxycarbonyl group

4. The composition of claim 3 wherein the acyl group is a Ci to Ciβ alkanoyl group.

5. The composition of claim 3 wherein X³ is an acyl group selected from the group consisting of formyl, acetyl, propanoyl and butanoyl.

6. The composition of claim 3 wherein X¹ and X² both represent hydrogen.

7. The composition of claim 6 wherein X³ represents a Ci to Cι₈ alkanoyl group.

8. The composition of claim 1 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-1,3,25-Triacetate.

9. The composition of claim 1 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trihexanoate.

10. The composition of claim 1 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trinonanoate.

11. The composition of claim 1 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D₃-25-Acetate.

12. The composition of claim 1 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Formate.

13. The composition of claim 1 wherein said animal feed includes phytate containing one or more mineral selected from the group consisting of phosphorus, calcium, zinc, iron, manganese, potassium and magnesium.

14. The composition of claim 1 wherein said animal feed includes an inorganic mineral supplement containing one or more mineral selected from the group consisting of phosphorus, calcium, zinc, iron, manganese, potassium and magnesium.

15. The composition of claim 1 wherein the concentration of said hydrolyzable calcitriol derivative in said animal feed is from about 2μg/kg to about 30μg/kg of feed on a dry weight basis.

16. The composition of claim 1 wherein said animal feed has an inorganic phosphorus content of less than about 0.9% by weight.

17. The composition of claim 1 wherein said animal feed is selected from the group consisting of poultry feed, swine feed, cattle feed, and fish feed.

18. A feed supplement composition, comprising: a hydrolyzable calcitriol derivative; and an edible carrier for said derivative.

19. The composition of claim 18 wherein said hydrolyzable calcitriol derivative has the structure:

where the stereochemical center at carbon 20 in the side chain may have the R or S configuration, and where Z is selected from Y, -OY, -CH2OY, -C ≡ CY and -CH = CHY, where the double bond may have the cis or trans stereochemical configuration, and where Y is selected from a radical of the structure:

_(CH₂)_m

where m and n, independently, represent the integers from 0 to 5, where R¹ is selected from the group consisting of hydrogen, OX⁴, fluoro, trifluoromethyl, and Ci-5- alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R² is selected from the group consisting of hydrogen, fluoro, trifluoromethyl and Ci-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy substituent, and where each of R³ and R⁴, is selected from trifluoromethyl and Ci-5 alkyl, which may be straight chain or branched and optionally bear a hydroxy substituent, and where R¹ and R², taken together, represent an oxo group, or an alkylidene group, =CR²R², =CR R³, or the group -(CH2)p-, where p is an integer from 2 to 5, and where R3 and R₄, taken together, represent the group -(CH2)q-, where q is an integer from 2 to 5, and where X¹, X², and X⁴ are each hydrogen, an acyl group, or a hydrocarbyloxycarbonyl group, and where X³ represents an acyl group or a hydrocarbyloxycarbonyl group.

20. The composition of claim 18 wherein said hydrolyzable calcitriol derivative has the structure:

21. The composition of claim 20 wherein the acyl group is a Ci to Cis alkanoyl group.

22. The composition of claim 20 wherein X³ is an acyl group selected from the group consisting of formyl, acetyl, propanoyl and butanoyl.

23. The composition of claim 20 wherein X¹ and X² both represent hydrogen.

24. The composition of claim 23 wherein X³ represents a Ci to ds alkanoyl group.

25. The composition of claim 18 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-1,3,25-Triacetate.

26. The composition of claim 18 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trihexanoate.

27. The composition of claim 18 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trinonanoate.

28. The composition of claim 18 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Acetate.

29. The composition of claim 18 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Formate.

30. A method of minimizing dietary requirements of phosphorus in animals comprising the steps of: feeding a diet containing phosphorus to an animal; and feeding with said diet an effective amount of a hydrolyzable calcitriol derivative for utilizing phosphorus in said diet.

31. The method of claim 30 wherein said hydrolyzable calcitriol derivative has the structure:

R¹ R² ■ R^J • (CH₂)_m — C- (CH₂)_n. c—^J ox 3³ \R⁴ where m and n, independently, represent the integers from 0 to 5, where R¹ is selected from the group consisting of hydrogen, OX⁴, fluoro, trifluoromethyl, and C1-5- alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R² is selected from the group consisting of hydrogen, fluoro, trifluoromethyl and Ci-5 alkyl, which may be straight-chain or branched, and optionally, bear a hydroxy substituent, and where each of R³ and R⁴, is selected from trifluoromethyl and Ci-5 alkyl, which may be straight chain or branched and optionally bear a hydroxy substituent, and where R¹ and R², taken together, represent an oxo group, or an alkylidene group, =CR²R², =CR²R³, or the group -(CH2)p-, where p is an integer from 2 to 5, and where R3 and R₄, taken together, represent the group -(CH2)_q-, where q is an integer from 2 to 5, and where X¹, X², and X⁴ are each hydrogen, an acyl group, or a hydrocarbyloxycarbonyl group, and where X³ represents an acyl group or a hydrocarbyloxycarbonyl group.

32. The method of claim 30 wherein said hydrolyzable calcitriol derivative has the structure:

where X¹ and X² independently represent hydrogen, an acyl group or a hydrocarbyloxycarbonyl group, and where X³ is an acyl group or a hydrocarbyloxycarbonyl group.

33. The method of claim 32 wherein the acyl group is a d to Cis alkanoyl group.

34. The method of claim 32 wherein X³ is an acyl group selected from the group consisting of formyl, acetyl, propanoyl and butanoyl.

35. The method of claim 32 wherein X¹ and X² both represent hydrogen.

36. The method of claim 35 wherein X³ represents a Ci to ds alkanoyl group.

37. The method of claim 30 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3- 1,3,25-Triacetate.

38. The method of claim 30 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trihexanoate.

39. The method of claim 30 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trinonanoate.

40. The method of claim 30 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Acetate.

41. The method of claim 30 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Formate.

42. The method of claim 30 wherein the phosphorus utilized is from phytate in said diet.

43. The method of claim 30 wherein the phosphorus utilized is from inorganic sources in said diet.

44. The method of claim 30 wherein said hydrolyzable calcitriol derivative is incorporated in said diet in an amount of from about 2μg/kg to about 30μg/kg of diet on a dry weight basis.

45. The method of claim 30 wherein said hydrolyzable calcitriol derivative is fed as a top dressing on said diet.

46. A method of enhancing mineral utilization from phytate complexes in animal feed, comprising the steps of: providing an animal feed comprising a diet containing phytate that binds a desirable cation; incorporating with said diet an effective amount of a hydrolyzable calcitriol derivative to form a feed mixture; and feeding said feed mixture to an animal.

47. The method of claim 46 wherein said hydrolyzable calcitriol derivative has the structure:

where m and n, independently, represent the integers from 0 to 5, where R¹ is selected from the group consisting of hydrogen, OX⁴, fluoro, trifluoromethyl, and Ci-5- alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R² is selected from the group consisting of hydrogen, fluoro, trifluoromethyl and Ci-5 alkyl, which may be straight- chain or branched, and optionally, bear a hydroxy substituent, and where each of R³ and R⁴, is selected from trifluoromethyl and Ci-5 alkyl, which may be straight chain or branched and optionally bear a hydroxy substituent, and where R¹ and R², taken together, represent an oxo group, or an alkylidene group, =CR²R², =CR²R³, or the group -(CH2)_P-, where p is an integer from 2 to 5, and where R3 and R₄, taken together, represent the group -(CH2)_q-, where q is an integer from 2 to 5, and where X¹, X², and X⁴ are each hydrogen, an acyl group, or a hydrocarbyloxycarbonyl group, and where X³ represents an acyl group or a hydrocarbyloxycarbonyl group.

48. The method of claim 46 wherein said hydrolyzable calcitriol derivative has the structure:

49. The method of claim 48 wherein the acyl group is a Ci to ds alkanoyl group.

50. The method of claim 48 wherein X³ is an acyl group selected from the group consisting of formyl, acetyl, propanoyl and butanoyl.

51. The method of claim 48 wherein X¹ and X² both represent hydrogen.

52. The method of claim 51 wherein X³ represents a Ci to Ciβ alkanoyl group.

53. The method of claim 46 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3- 1,3,25-Triacetate.

54. The method of claim 46 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trihexanoate.

55. The method of claim 46 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trinonanoate.

56. The method of claim 46 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Acetate.

57. The method of claim 46 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Formate.

58. The method of claim 46 wherein said hydrolyzable calcitriol derivative is incorporated in said diet in an amount of from about 2μg/kg to about 30μg/kg of diet on a dry weight basis.

59. The method of claim 46 wherein said hydrolyzable calcitriol derivative is fed as a top dressing on said diet.

60. The method of claim 46 wherein said cation is a mineral selected from the group consisting of phosphorus, calcium, zinc, iron, magnesium, potassium and manganese.

61. A method of enhancing mineral utilization from inorganic mineral supplements in animal feed, comprising the steps of: providing an animal feed comprising a diet containing an inorganic mineral supplement; incorporating with said diet an effective amount of a hydrolyzable calcitriol derivative to form a feed mixture; and feeding said feed mixture to an animal.

62. The method of claim 61 wherein said inorganic mineral supplement contains a mineral selected from the group consisting of calcium, potassium, magnesium, zinc, iron, manganese and phosphorus.

63. The method of claim 61 wherein said hydrolyzable calcitriol derivative has the structure:

where the stereochemical center at carbon 20 in the side chain may have the R or S configuration, and where Z is selected from Y, -OY, -CH2OY, -C

≡ CY and -CH = CHY, where the double bond may have the cis or trans stereochemical configuration, and where Y is selected from a radical of the structure:

where m and n, independently, represent the integers from 0 to 5, where R¹ is selected from the group consisting of hydrogen, OX⁴, fluoro, trifluoromethyl, and Ci-5- alkyl, which may be straight chain or branched and, optionally, bear a hydroxy substituent, and where R² is selected from the group consisting of hydrogen, fluoro, trifluoromethyl and Ci-5 alkyl, which may be straight- chain or branched, and optionally, bear a hydroxy substituent, and where each of R³ and R⁴, is selected from trifluoromethyl and Ci-5 alkyl, which may be straight chain or branched and optionally bear a hydroxy substituent, and where R¹ and R², taken together, represent an oxo group, or an alkylidene group, =CR²R², =CR²R³, or the group -(CH2) -, where p is an integer from 2 to 5, and where R3 and R₄, taken together, represent the group -(CH2)q-, where q is an integer from 2 to 5, and where X¹, X², and X⁴ are each hydrogen, an acyl group, or a hydrocarbyloxycarbonyl group, and where X³ represents an acyl group or a hydrocarbyloxycarbonyl group.

64. The method of claim 61 wherein said hydrolyzable calcitriol derivative has the structure:

65. The method of claim 64 wherein the acyl group is a Ci to Ciβ alkanoyl group.

66. The method of claim 64 wherein X³ is an acyl group selected from the group consisting of formyl, acetyl, propanoyl and butanoyl.

67. The method of claim 64 wherein X¹ and X² both represent hydrogen.

68. The method of claim 67 wherein X³ represents a Ci to ds alkanoyl group.

69. The method of claim 61 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-1,3,25-Triacetate.

70. The method of claim 61 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trihexanoate.

71. The method of claim 61 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-l,3,25-Trinonanoate.

72. The method of claim 61 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Acetate.

73. The method of claim 61 wherein the hydrolyzable calcitriol derivative is lα,25-dihydroxyvitamin D3-25-Formate.

74. The method of claim 61 wherein said hydrolyzable calcitriol derivative is incorporated in said diet in an amount of from about 2μg/kg to about 30μg/kg of diet on a dry weight basis.

75. The method of claim 61 wherein said hydrolyzable calcitriol derivative is fed as top dressing on said diet.

76. The method of claim 61 wherein said inorganic mineral supplement is phosphorus and is provided by adding monocalcium phosphate, dicalcium phosphate, defluorinated phosphate or mixtures thereof.