JPH0329372B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a livestock or poultry food composition containing 1α-hydroxyvitamin _D3 and a calcium salt. It is known that 1α-hydroxyvitamin D compounds, which also contain a 25-hydroxy group, have advantageous biochemical properties that make them quite useful in therapy. That is, they are more rapidly transgenic and are cleared from the system more rapidly than the corresponding 1α-unsubstituted compounds, and are therefore more likely to induce vitamin toxicity than normal vitamin D compounds, which are only slowly cleared from the system. gender becomes smaller. Additionally, the hydroxylated derivatives are often effective in alleviating symptoms of vitamin D deficiency that apparently do not respond to conventional vitamin treatments. Such 1α-hydroxyvitamin D compounds can be synthesized by techniques similar to those used for the synthesis of the corresponding 1α-unsubstituted derivatives, especially 1α,3β-dihydroxysteroids of the cholestane series.
- Can be produced by photochemical decomposition of dienes using ultraviolet irradiation. The inventors have surprisingly discovered that 1α-hydroxyvitamin D compounds and 1α-hydroxy-
It has been discovered that 9,10-dihydrotachysterol exhibits significant activity upon oral administration. 1α-Hydroxyvitamin _D3 is remarkable in this regard. This is 1α,25-dihydroxyvitamin D ₃
This is completely unexpected given the disclosures made to date. Previous disclosures have shown that oral dosing of this dihydroxyvitamin has very low activity (as determined by e.g. anti-rickets activity measurements).
and indicate that parenteral administration of dihydroxyvitamins is necessary to achieve effective therapeutic results. In view of the similarity in the properties of the biological activity of the compounds in other respects, 1α
Hydroxyvitamin D compounds are generally expected to exhibit similar general behavior to the corresponding dihydroxyvitamins. However, the oral Administered 1α-hydroxyvitamin _D3 and 1α,25-dihydroxyvitamin
The following table showing the effect of D ₃ (0.1 μg/Kg, intragastric intubation) shows that 1α-hydroxyvitamin D ₃ demonstrated excellent activity upon oral administration, whereas orally administered 1α,25-dihydroxyvitamin _D3 was relatively inactive and did not significantly alter serum calcium levels when compared to controls. It shows. This table also shows that the metabolic changes induced by 1α-hydroxyvitamin _D3 are relatively short-lived;
The results show that serum calcium levels in 1α-hydroxyvitamin D3 _- treated rats become very close to that of control rats within 24 hours after vitamin administration. This means that 1α-hydroxyvitamin
This ensures that D ₃ is rapidly removed from the system and therefore has no possibility of producing undesirable vitamin toxicity side effects.

Table: The oral activity of 1α-hydroxyvitamin _D3 and its ease of administration make this compound of very important therapeutic value with a wide range of applications, and the known parenteral administrable
This considerably enhances the utility of this compound compared to 1α,25-dihydroxyvitamin D derivatives. The effect of the 1α-hydroxyvitamin D ₃ of the present invention is further enhanced by combining it with a calcium salt (eg lactate, phosphate, gluconate or hypophosphite). The activity of the compositions of 1α-hydroxyvitamin D ₃ and calcium salts according to the invention when administered orally makes them valuable in veterinary applications. For example, said compounds can be incorporated into animal and poultry feeds for prophylactic purposes, and thus according to the invention livestock or poultry feeds containing 1α-hydroxyvitamin D ₃ and calcium salts. A composition is provided. 1α-Hydroxyvitamin _D3 has the effect of stimulating calcium absorption in the intestine, which is beneficial in the treatment or prevention of hypocalcemia in animals such as rickets. It has been found that intestinal calcium uptake is enhanced by including calcium salts in animal feed.
The stimulatory effect of 1α-hydroxyvitamin _D3 on calcium uptake in the intestine and on the movement of calcium from the bones are both several times greater than in the case of natural vitamin _D3 . Therefore, adding 1α-hydroxyvitamin _D3 to the feed may have an adverse effect if the calcium content in the feed is insufficient, as the serum calcium concentration is increased by mobilization of calcium from the bones. . Therefore, it would be beneficial to provide an animal feed composition that includes calcium salts along with 1α-hydroxyvitamin _D3 . Animal feed according to the foregoing embodiments of the invention may be prepared by adding the vitamin compounds as described above in a conventional manner, e.g. to a solid feed such as animal cake or to a granular mineral supplement (e.g. flour, corn flour, soybean flour, limestone, essential minerals, etc.). It can be manufactured by mixing with feed supplements such as supplements containing ingredients such as trace element sources and other vitamins. The vitamins mentioned above may be applied in any form that allows their homogeneous distribution in the feed. For example, the vitamin compound can be applied dissolved in water or a suitable organic solvent (eg ethanol), after which the solvent can be removed from the feed if desired. Furthermore, antioxidants such as ascorbic acid, butylated hydroxyanisole or hydroquinone may also be added to improve the storage stability of the vitamin-containing feed. Said vitamins may also be applied in solid form (eg crystalline solids) and are preferably mixed intimately with the feed to ensure homogeneous distribution. The solid vitamin may be first coated with an antioxidant, such as those mentioned above, or adsorbed onto a substrate such as ground limestone or chalk by known methods, if desired, to enhance its stability. Good too. Furthermore, in certain applications it may be advantageous to use the vitamin compounds mentioned as a component of preparations that would otherwise be nothing more than ordinary multivitamin preparations. Typical supplements for preventive purposes are 0.1 to 2.0 μg per pound of supplement.
(~0.2 to 4.5 μg/Kg) of 1α-hydroxyvitamin _D3 . This supplement is suitable for livestock such as dairy cows and pigs from 10 to 20 lbs (22 to 44
kg)/animal/day. An important application of animal feeds containing 1α-hydroxyvitamin D ₃ and calcium salts is in the prevention of hypocalcemia in livestock, such as cows, especially cows, at or near the calving period. 1α−
Hydroxyvitamin _D3 is particularly valuable in this regard. This is because the high activity and low toxicity of this compound makes it possible to administer it in low doses and prophylactically over long periods of time to groups of animals, including for example animals without a history of hypocalcemia. be. This is in contrast to the traditional use of vitamin D compounds in this field. Because of the high drug doses required when using compounds such as vitamin _D3 , for economic reasons among others, it has traditionally been customary to administer said vitamins only to animals with a previous history of hypocalcemia. Because it was hot. Yet another application of the vitamin-containing feed according to the above-described aspect of the invention is in the prevention or treatment of milk fever in dairy cows. That is, said feed is given to cows before calving for preventive purposes;
1α-Hydroxyvitamin _D3 content is 10-200μg
(for example, 15 to 100 μg)/day. Feed compositions for poultry according to the above-described aspects of the invention can generally be manufactured using the technical procedures described for animal feeds above. Composition containing 1α-hydroxyvitamin _D3 and calcium salts, with typical vitamin levels between 0.2 and 12 μg
(for example 1-8 μg)/Kg of feed. The composition may be any conventional poultry feed composition, such as grain flour, soybean flour, fish meal, corn flour, meat and bone scraps, Distillas solubilus,
It may be added to a composition consisting of one or more ingredients selected from essential trace element sources and other vitamins. It has been found that administration of a poultry feed composition according to the invention to poultry at the time of laying reduces the tendency of the poultry to produce soft shell eggs. The feed composition for this application contains a source of calcium, such as ground limestone or oyster shells, to aid mineralization of the eggshells, and the calcium level of the feed desirably ranges from about 2.5 to 5.5% by weight; Preferably 3-4
%. The invention further encompasses poultry feed compositions containing 1α-hydroxyvitamin _D3 , for example in a content of 0.2 to 12 micrograms of vitamin per kg of feed, preferably 1 to 8 micrograms. Applications of the novel 1α-hydroxyvitamin _D3 in animals include the prevention of hypocalcemia in livestock such as cattle, especially cows at or near calving. This is because the high activity and low toxicity of this compound makes it possible to administer it prophylactically at low doses over long periods of time to groups of animals, including for example animals without a history of hypocalcemia. be. This is in contrast to the traditional use of vitamin D compounds in this field. This is because, due to the high doses required when using compounds such as vitamin _D3 , it has traditionally been customary, especially for economic reasons, to administer this vitamin only to animals with a history of hypocalcemia. This is because it was hot. Furthermore, it has been found that administering an effective dose of 1α-hydroxyvitamin D ₃ together with a calcium salt to laying poultry is effective in reducing the tendency of the poultry to produce soft shell eggs. The invention will be further illustrated by the following detailed references and examples. All temperatures are in degrees Celsius. Reference Example 1 (a) Cholesta-1,4,6-trien-3-one Cholesterol (19.3 g) and dichlorodicyanoquinone (38 g) were mixed with dry dioxane (500 g).
ml) under reflux for 22 hours. The mixture was then cooled, filtered and the liquid was evaporated to dryness. The residue was chromatographed on alumina and eluted with benzene/hexane followed by benzene to give the title trienone as a pale oil (11.5 g).
This solidified when left to stand. The physical properties of this material were appropriate. (b) 1α,2α-epoxy collector-4,6-dien-3-one The trienone (1 g) from (a) above was dissolved in ethanol (50 mg) at 0° with 10% aqueous sodium hydroxide (0.25 ml) and 30% _{aqueous H2O2} (2.5ml)
Processed with. The mixture was kept at 5° overnight, then the resulting epoxide was separated, washed with aqueous alcohol and dried to give the title compound (0.86 mg). Recrystallization from ethanol gave colorless needle crystals mp 107-109°. (c) 1α,3β-Dihydroxycholest-5-ene To a stirred solution of liquid ammonia (80 ml) containing ammonium chloride (0.5 g) and metallic lithium (0.2 g) in dry tetrahydrofuran (50 ml) was added dry tetrahydrofuran (25 ml). ) in which a deoxygenated solution of the epoxide (4.3 g) from (b) above was added dropwise. When the blue color disappears,
The steroid addition was stopped and more lithium (0.2g) and ammonium chloride (1g) were added followed by more epoxide solution.
This series of operations was repeated until all the steroid had been added. At this point, additional pieces of lithium (0.2g, 0.8g total) were added, then more ammonium chloride (8g total)
g) was added. Most of the ammonia was then evaporated and the remaining mixture was poured into ice water and extracted with chloroform. The chloroform was concentrated to give a brown gummy material. This was chromatographed on ammonium oxide (160g). Elution with ethyl acetate/benzene gave the 1α,3β-diol as a glass-like material. This crystallized quickly upon addition of ethanol. Recrystallization from aqueous ethanol yields the title compound (1.7 g) mp161.5
~163° was obtained. Actual value C80.40, H11.39%,
Calculated value ( _C27H46O2 ) _C80.54 , H11.52 _% . Reference Example 2 (a) 1α-Hydroxycholester-4,6-dien-3-one Epoxydienone from Reference Example 1(b) (130 mg)
was treated with zinc dust (1 g) with stirring in ethanol (10 ml) and then 3 drops of acetic acid were added.
The mixture was then filtered and the liquid was concentrated to dryness. Chromatography on silica gel gave cholesta-1,4,6-trien-3-one, which could be recovered and recycled, and then the title compound. λmax3600, 3400, 1675, 1625 and 1590
cm ^-1 . δ6.15 (2 protons S, H6, H7), 5.73
(1 proton singlet, H4), δ4.15 (1 proton, thin multiplet, H1). (b) 1α,3β-dihydroxycholest-5-ene Hydroxydienone (0.6 g) from (a)
A solution in tetrahydrofuran (2 ml) and pyridine (2 ml) was dissolved in hexamethyldine lazan (1.5 ml) and trimethylchlorosilane (0.6 ml).
ml) to its trimethylsilyl ether. This crude trimethylsilyl ether was dissolved in tetrahydrofuran (10
ml) and this solution was added dropwise to a stirred solution of lithium metal (approximately 200 mg) in liquid ammonia (20 ml). After a few minutes aluminum chloride (2g) was added and the mixture was stirred. Additional lithium metal (approximately 100mg)
added. The solution was stirred again. Additional ammonium chloride was then added and the mixture was poured into cold water. The product was isolated by extraction with ether and methylene dichloride, followed by column chromatography to give the title compound. When crystallized from ethanol, its mp was 158-161°. After recrystallization, its MP was 161.5-163°. [α] _D (CHCl ₃ ) −38°. This material is identical to the product of Reference Example 1(c), and upon hydrogenation it produces a 1α, identical in all respects to the standard sample.
A sample of 3β-dihydroxy-5α-cholestane was obtained. Reference example 3 (a) 1α,3β-dibenzoyloxycholest-5
-Ene 1α,3β-dihydroxycholest-5-ene (1.2g) in pyridine (10ml) containing dimethylaminopyridine (20mg) was treated with benzoyl chloride (5ml). After standing overnight at room temperature, the reaction mixture was poured into water and the product was extracted with ether, washed with dilute aqueous hydrochloric acid, saturated sodium bicarbonate solution and water. Evaporation of the ether part yields dibenzoate (1.6
g) mp147-150° was obtained. When recrystallized from ethanol, the product had a melting point of 151-153°. [α] _D +24°. Analysis calculation value (C ₄₁ H ₅₄ O ₄ ) C80.61%, H8.91%, actual value
C80.43, H8.74%. (b) 1α,3β-dibenzoyloxycholester-5,
A solution of the dibenzoate described in 7-Diene (a) (0.58g) in hexane (10ml) was treated with dibromodimethylhydantoin (0.15g) and heated under reflux for 25 minutes. After cooling, the mixture was filtered and the liquid was concentrated to a pale oil. This oil was dissolved in dry xylene (3ml) and this was dissolved in trimethylphosphite (0.4ml) in xylene (5ml).
ml) dropwise to the refluxing solution. Heating under reflux was continued for 1.75 hours. After this time the solvent was removed under reduced pressure and the residue was crystallized from acetone/methanol to give the title compound. After recrystallization from ethanol/acetone,
This product had a melting point of 161-162°.
[α] _D −8°. Analysis calculation value _{( C41H52O4} )C80.88% _,
H8.61%, actual value C80.69%, H8.66%. (c) 1α,3β-dihydroxy collector-5,7-
Ethanol (30ml) containing diene KOH (0.6mg)
and the dibenzoate (300 mg) from (b) dissolved in water (0.5 ml) and kept at 80° for 0.5 h under an argon atmosphere. The reaction mixture was then cooled, diluted with water and extracted with ether.
Evaporation of the ether extract gave the title compound as a crystalline solid. Recrystallization from methanol gave a product with mp 155-158°. λmax (ethanol) 263 (7700), 272
(11000), 282 (11900), 295 (7000) nm This product (95 mg) in deoxygenated ether (200 ml) was dissolved in a mixture of toluene (24 ml) and CS ( ₄ ml) per methanol for 12 min. Irradiation was carried out using a 200 watt Hanobia lamp surrounded by a liquid solution. This cold solution is
Transferred to an argon-filled flask and removed the ether at 0°. The residue was dissolved in deoxygenated absolute alcohol (8 ml) and
Heated under reflux for 1.5 hours. Biological assays performed using vitamin D-deficient chicks revealed that the 1α-hydroxyvitamin _D3 [λmax264
(19000)] is characterized by a very rapid onset of physiological activity (within 3 hours), which has been tentatively shown to be 1α, 25
- It has only been observed for natural products characterized as dihydroxyvitamin _D2 . Reference example 4 Irradiation of 1α,3β-diacetoxycholester-5,7-diene 50 mg of 1α,3β-diacetoxycholester-5,
7-diene (mp 118-119°, prepared by reacting 1α,3β-dihydroxycholester-5,7-diene with acetic anhydride using a method similar to that of Reference Example 3(a)). Irradiated for 11 minutes in deoxygenated ether (200 ml). The ultraviolet absorption spectrum of this mixture showed the desired absorption increase in the 220-268 nm range and decrease in the 268-295 nm range. It was essentially homogeneous on silica gel (CHCl ₃ ), but separated into two distinct spots on 1% AgNO ₃ -silica gel-chloroform.
The lower spot corresponded to the R _f of the starting material. Less polar substances (approximately 20mg) are 262-272nm
It had a broad UV absorption band with a nearby "flat" maximum (small humps at 282 and 295 nm) and a minimum at 234 nm. This material contained crude previtamins. A small amount of this mixture was dissolved in hexane and its UV absorption spectrum was recorded (estimated concentration approximately 20 mg/). It was then treated with a solution of iodine in hexane so that the overall concentration of iodine was about 0.4 mg/d, and it was placed in scattered light for 45 minutes. The hexane solution was washed with dilute aqueous sodium thiosulfate and then with water;
It was dried and its UV absorption spectrum was rerecorded. It showed the characteristic absorption of taxerol derivatives (max 282 nm, shoulders 272, 292 nm) and this absorption was increased by a factor of 22. The entire crude previtamin was dissolved in deoxygenated isooctane (1.0 ml). The absorption at 262nm is
When the 30μ aliquot was diluted to 3ml, it was 0.39. The solution was then heated to about 75° under argon for a total of 2.25 hours, during which time the absorption at 262-265 nm increased to a maximum value of 0.54 (for a solution of the same concentration as above). As expected, this absorption initially increased quickly and then gradually as the equilibrium mixing state was approached. When this aliquot was treated with iodine in hexane as described above, it exhibited a characteristic absorption for taxerol, but the increase in absorption was only 0.43-0.47. The equilibrated mixture was essentially homogeneous on both silica gel and 1% AgNO ₃ silica gel (developed with chloroform). Approximately 12 mg of this mixture was mixed with deoxygenated methanol (1.0
ml) and deoxygenate this solution.
Treated with 1.5% methanolic KOH (0.5 ml) and kept at room temperature under argon for 1.5 h. Dilution with water and extraction with ether gave 1α,3β-diol.
This showed two very close main spots on silica gel (developed with 4% MeOH- _CHCl3 ). This less polar fraction (approximately 5 mg) has a maximum in UV absorption at 264 nm and 228 nm.
It showed a broad absorption with a minimum value at . this is
It was 1α-hydroxyvitamin _D3 . Treatment of aliquots with iodine as described above in hexane shifts the maximum to 270 nm;
This results from conversion to 56-transvitamin. The more polar fraction had a smooth absorption band in UV absorption with a maximum at 260 nm and a minimum at 235 nm. This was a previtamin. When this is treated with iodine as described above, 268
It gave a complex ultraviolet absorption spectrum with maxima at 276, 286, 298, 312 and 327 nm. Reference Example 5 1α-Hydroxyvitamin D ₃ In deoxygenated ether (200 ml), 135 mg of
1α,3β-diacetoxycholesta-5,7-diene (produced as in Reference Example 4)
Irradiate for 15 min, and remove the product with 1% _AgNO3.
- 68 mg of starting material (more polar fraction) when separated on silica gel (CHCl ₃ ) (preparative TLC)
and crude previtamin (54 mg, more non-polar fraction) were obtained. The previtamin thus obtained was heated at 75° for 2 hours in deoxygenated isooctane (15 ml) under argon. The resulting mixture of vitamins and previtamins was dissolved in methanol (4 ml) and the solution was treated with 1 ml of 2.5% methanolic KOH and kept at room temperature for 2 hours. Dilution with water and extraction with ether yielded vitamins and previtamin diols. Separation of this on silica gel (Preparative TLC) (8% MeOH- _CHCl3 ) yielded 13 mg of the vitamin (R _f 0.35) and 8 mg of the previtamin (R _f 0.31). When this vitamin is recrystallized from ether-pentane, fine colorless needle crystals mp132-133° (heating rate 1°/4 seconds), m.
p.128-129° (heating rate 1°/25 seconds) was obtained. Ultraviolet absorption (ether) λmax264nm (20200),
λmin299nm (10800). There is a 9% error in the absorbance value, but the λmax/λmin ratio is 1.87±10%.
[α] ²⁰゜_D (ether, C ~ 0.3%) + 26゜±2゜, [
α〕 ²⁰゜_D
× Product of extinction coefficient (at 264 nm) approximately 5.2 × 10 ⁵ ±
10%, νmax ( _CHCl3 ) 3700, 3500, 1600~1650,
1040cm ^-1 . NMR (d ₆ acetone) H ₆ + H ₇ , δ6.20
(At first glance, J = 11.5Hz) AB quartet, H ₁₉ δ4.92
and two thin 1-proton multiplets at δ5.37ppm. The provitamin (λmax 260 nm and λmin 232 nm) (11 mg, from two separate irradiations) was dissolved in deoxygenated isooctane (8 ml) and heated to 75° for 1.5 hours. Isolated by preparative TLC as before, an additional 4.6
mg of vitamins were obtained. Decomposition had occurred here, and there was actually no previtamin left. The analysis for 1α-hydroxyvitamin _D3 was
Actual value C80.6%, H11.04%, _calculated value _{( C27H44O2} )
C80.9%, H11.07%. Reference Example 6 (a) 1α-hydroxycholesterol bistrimethylsilyl ether 1α,3β-dihydroxycholest-5-ene (1.0g) and N-trimethylsilylimidazole
10 ml was added, and the mixture was heated and stirred at 90° under a nitrogen stream.
After cooling, hexane was added and the mixture was washed twice with saturated brine. After drying over anhydrous sodium sulfate, it was filtered and concentrated. The obtained crude product was purified with 30 g of silica gel column chromatography, and the yield was 1.26 g (yield:
93%) of the desired product was obtained. As spectrum is
546 showed M ⁺ . (b) 1α,3β-bistrimethylsilyloxycholester-5,7-diene 1α-hydroxycholesterol bistrimethylsilyl ether (0.55 g) in hexane (10
ml) solution was treated with dibromomethylhydantoin (0.15g) and heated under reflux for 25 minutes.
After cooling, the filtrate was concentrated. The resulting residue was dissolved in dry xylene (3ml) and this solution was added dropwise to a refluxing solution of trimethylphosphite (0.4ml) in xylene (5ml). Heated at reflux for 1.75 hours after which time the solvent was removed under reduced pressure.
Careful purification was performed using 30 g of silica gel column chromatography to obtain 0.23 g (42% yield) of the target compound. Mass spectrum showed M ⁺ of 544. (c) 1α-Hydroxyvitamin D _{3bistrimethylsilyl} ether 1α,3β-bistrimethylsilyloxycholester-5,7-diene (230mg) deoxygenated with benzene (500ml) and ethanol (100ml)
dissolved in This solution was irradiated for 19 minutes using a 200 W Hanobia lamp surrounded by a Vycor filter. The reaction temperature was 18°C. This solution was then heated to reflux for 3.5 hours.
After completion of the reaction, the reaction solution was concentrated, and the resulting crude product was purified by silica gel thin layer chromatography (developed twice with hexane-benzene solvent) to obtain 85 mg of 1α-hydroxyvitamin D ₃ bistrimethylsilyl ether. I got it. The ultraviolet absorption (EtOH) was λmax×265 nm and λmin 228 nm. Mass spectrum showed M ⁺ at 544. (d) 1α-hydroxyvitamin D ₃ 1α-hydroxyvitamin D ₃ bistrimethylsilyl ether (50 mg) was dissolved in anhydrous tetrahydrofuran (5 ml), tetra-n-butylammonium fluoride (100 mg) was added, and under a nitrogen stream,
The mixture was treated in a cool, dark place for 5 hours. Water was added, extracted with ethyl acetate, and washed twice with saturated brine. It was dried over anhydrous sodium sulfate and overconcentrated. The obtained crude product was purified by silica gel thin layer chromatography (developed twice with a benzene-ethyl acetate solvent) to obtain 29 mg of the desired product. The melting point is
141-142℃, ultraviolet absorption (EtOH),
λmax was 265 nm and λmin was 228 nm. Mass spectrum shows M ⁺ at 400, 382, 364, 251,
A peak was also observed at 134. Example 1 Poultry Feed Composition 40 μg of 1α-hydroxyvitamin D ₃ is dissolved in ethanol (100-500 ml) and the resulting solution is slurried with 2 Kg of crushed limestone. The ethanol is then removed under reduced pressure while stirring the slurry, and the resulting vitamin-containing solid is added to the poultry feed at a rate of 20 g/Kg of feed. Example 2 Enriched Feed Supplement for Dairy Cattle, Pigs and Other Livestock 50 μg of 1α-hydroxyvitamin D ₃ is dissolved in 500 ml of ethanol and the resulting solution is slurried with 500 g of powdered Dolomite limestone. The slurry is then stirred under reduced pressure to remove the ethanol and the resulting vitamin-containing solids are mixed with 100 lbs of standard granular mineral supplement (45.5 Kg).

Claims (1)

[Claims] 1. A livestock or poultry feed composition containing 1α-hydroxyvitamin D ₃ and a calcium salt, wherein the content of 1α-hydroxyvitamin D ₃ is in the range of 0.2 μg to 12 μg per 1 kg of feed.