CA1041828A - Composition of matter and process - Google Patents

Abstract

ABSTRACT OF THE DISCLOSURE
The administration of 25-hydroxycholecalciferol, 24,25- dihydroxycholecalciferol, 25,26-dihydroxycholecal-ciferol, 1,25-dihydroxycholecalciferol, 25-hydroxydihydro-tachysterol3, 25-hydroxyergocalciferol, 1-.alpha.-hydroxychole-calciferol, and their acylates to hens increases the thick-ness of the egg shells. Compositions and methods are provided.

Description

~014B

~, T~!E BACK~,ROUND
The poultry industry has recently made significant advances in dealing with many of its problems. However, one problem area which has met with little success is the effort to decrease the incidence of broken hens' eggs. Approxi-mately 5-8~ of eggs laid by hens are prematurely broken with a loss to egg producers of up to ~100 million dollars or more per year. Generally, ~he reason for the premature breakage appears to be insufficient calcification of the egg shell, a condition which produces an egg shell which is too thin to withstand forces exerted upon it. It is well known that Vitamin D3 affects the shell thickness of hens' eggs. However, Vitamin D3 has little or no effect on the thickness of egg shells from some hens. Furthermore, the-activity of Vitamin D3 for increasing shell thickness reachesa plateau which does not show an increase even at extremely high dosages of Vitamin D3. ~ ;
Recently an active metabolite of Vitamin D3 has been -;solated. This metabolite, the 25-hydroxy derivative oF - ~-` ~ 20 Vitamin D3, generically known as 25-hydroxycholecalciferol, ~ and referred to hereafter as 25-HCC, has been shown to be -s more active than Vitamin D3 in the rat line rickets cure ' assay and in the rickets prevention assay in young chicks. -` -However, no particular tests using 25-HCC in mature 25 egg-laying chickens or other species are disclosed in the~
prior art. Perhaps of even greater significance, only problems dealing with bone calcification such as rickets '---and osteomalacia, or general membrane calcium transport, A
5uch as cattle milk fever and hypoparathyroidism in humans -" , . .
' 30 have been dealt with by the art. ~

. , .

-2- ~
,' , `

", ~a~zf~

The increasing of hen egg shell lhickness dnd lhc c~Iring or preventing of bone conditions such as rickets and osteomalacia proceed by different physiological mechanisIns of action. The substance which is responsible for increasing shell thickness is calcium carbonate. The substance depos-ited which aids in treating the various bone diseases is calcium hydroxyapatite, a complex mineral-like salt of phosphorus, oxygen, and calcium which is chemically unre-lated to carbonate. Furthermore, the organic matrices in ; lO which each of these substances is deposited differ radically ; from each other. Egg shell organic matrix is comprised of noncellular arnorphous sheets of protein. The bone organic matrix is formed, living tissue capable of reproducing itself.
.
BRIEF SUMMARY OF THE INVENTION
~, , .
It has been found that 25-HCC and related compounds o be described hereinafter have the capacity to increase the thickness of hens' egg shel!s. When administered to -hens, these compounds increase the thickness of shells fron a substantial number of hens whose egg shells exhibit a . ~ . , , thickness of less than or equal to 0.013 inch, a thickness which is considered to be critical since most breakage ~ -occurs in egg shells with a thickness less than 0.013 inch.
Therefore, in accordance with this invention, a feed 2~ formulation for increasing the thickness of hens' egg shells ;s disclosed comprising a chicken feed in combination with .. ~ .
~¦~ an egg shell thickening effective but non-toxic quantity of -~ a compound selected from the group consisting of

-3-,~ .

", ,.: , , .~ O

3o~

~0418Zf~

OH ~ OH

¦I CH2 R-O R' ~ OA
(a) (b) ., .

~ OH

Ir .~ 15 RO R-O/ aoRa (c) (d) . ~-~
where R=H, or acyl where acyl is an acyl radical of normal or Isomerized alkane carboxylic acid of from 2 to about 8 carbon atoms and R', R " , and R " ' can be H, OH, or O acyl where;n acyl is defined as above, but that at least two o~ - -., . . - , ~ 5 R', R " , and R " ' must be hydrogen and that if R', R " , or :
.~J R" ' is OH then R must be H, and if R', R " , or R" ' is O
acyl, acyl defined as above, then R must be the same acyl, . and Ra ;5 selected from hydrogen or acyl as defined above . . and when Ra Is hydrogen, R is hydrogen and when Ra ;5 acyl, 30 R is the same acyl. ::-, ~ ........ `

,........................... . .

~0418Z~

A further aspect of this invention is the process for increasing the shell thickness of eggs from hens comprising - feeding the hens an egg shell thickening chicken feed which comprises a chicken feed in combination with an egg shell thickening effective but non-toxic quantity of a compound selected from the group of compounds disclosed above with reference to composition components except that 25 hydroxy cholecalciferol and its acylates are omitted.
` DETA I LED DESCR I PT I ON OF THE I NVENT I ON
The compounds employed in the compositions and uses of `~ those compositions are prepared by methods known to the art.
The unacylated compounds of the invention have been prepared in the following references:
-, Formula A ~ . :
25-HCC - R=R ' =R " --R ' " =H
~'- U.S. Patent 3,565,924 . 24, 25 - Dihydroxycholecalciferol - R=R'=R" ~=H, `~
.:` . R"=OH ~ . - . `
` U.S. Patent 3,715,~74 ~0 25,26 - Dihydroxycholecalciferol - R=R' =R " =H, R" '=OH
, ' ~ Biochemistry 9:24:4776-4780, 1970 ~ la,25 - Dihydroxycholecalciferol - R=RI'=R' I ' =H, .~ . ` R ' -OH
Biochemistry 10:14:2799-2804, 1971 .
Formula B
- Hydroxydihydrotachysterol 3 - R=H
Federation Proceedings 29:2:687, 1970 Formula C -` 30 25 - Hydroxyergocalciferol, R=H, Biochemistry 8:9:3515-3519, 1969 Formu1a D
: la - Hydroxycholecalciferol, R=H
J. Am. Chem. Soc. 95:8, 2748 (197~).
,, ~.he acylated compounds of this invention can be prepared ' ~,~, -5-"

, . , " .
, ; -~

",... . .. . ..

10~
by taking any of the above unacylated compounds and reactiny it with the acid anhydride or chloride of the two to eight carbon atom hydrocarbon carboxylic acid desired according to the method disclosed in Example 16 of Babcock and Campbell, Belgian Patent No. 747,492. Illustrated reactants and respective products are the following: -25-hydroxycholecalciferol treated with acetic anhydride gives 25-hydroxy-cholecalciferol ~-acetate 25-hydroxycholecalciferol treated with butyryl chloride gives 25-hydroxy-cholecalciFerol 3-butyrate ,~ .
25-hydroxycholecalciferol treated with .; octanoyl chloride gives 25-hydroxy-15 cholecalciferol 3-octanoate 24,25-dihydroxycholecalciferol treated with : acetic anhydride gives 24,25-dlhydroxy-cholecalciferol 3,24-diacetate 24,25-dihydroxycholecalciferol treated with . 20 isobutyryl chloride gives 24,25-dihydroxy- : .
cholecalcifero~ 3,24-diisobutyrate .- ~`
-~ . 25,26-dihydroxycholecalciferol treated with - :
propionic anhydride gives 25,26-dihy-.~ droxycholecalciferol 3,26-dipropionate -, 25 1a~25-dihydroxycholecalciferol treated with ., , pentanoic anhydride gives 1.25-dihydroxy-cholecalciferol 1,3-dipentanoate 25-hydroxydihydrotachysterol3 treated with acetic anhydride gives 25-hydroxydihy- -drotachysterol ~-acetate i 25-hydroxyergocalciferol treated with - -`J, ' ' acetic anhydride gives 25-hydroxyergo- `
cal c i ferol-3-acetate 1a-hydroxycholecalciferol treated with ..
- propionyl chloride gives cholecalciferol , lJ3-dipropionate ~ .
': It is preferred to use the hydrated forms of.thé metabo- .-~: - lite5 whenever poss;ble. For example, the hydrate of 25-HCC
Is preferred over the.non-hydrate of 25-HCC~ The method of 40 preparation is dTsclosed in Belgian Patent No. 747,492, ~, . . .
.

, ,~ . .

10~18;~

Example 8.
. The chicken feed into which the compounds are combined is any standard diet fed an egg-lay;ng hen. An example of such a diet is given below: .
Layer Formulation (Fed after 20 ~eeks Ingredients of age) . (Pounds) Corn meal, No. 2 667.5 10 yellow, ground Soybean meal . 100 50% protein Fish meal 15 65~ protein Meat and bone scraps 75 50% protein Corn distillers . 50 ~- -. dried so1ubles ~ Alfalfa meal 15 -~ 20 17% protein ~ ~ Limestone, ground 70 :~ Salt, jodized 2.5 - :
- DL-Methionine 0.5 ... . . .
.. ~ Vitamin premix 5 ~ .:
~ ~ _ _ _ _ _ _ _ ~ _ _ .
Calculated Composition . ~- .
ProteinJ 4 - ~ ~ 17.2 Calcium, % . : 3 4 : The calcium level for egg-iaying hens is well known and : Important for the calcification of egg shells. Generally ':~ 30 from about 2.5 to about 5-5S calcium~ calculated as weight of calcTum per weight of feed formulation is required, ~`~ preferably from about 3.0 to about 4.0~. When levets of ,, ~ calcium at the upper end of the ~range are employed, a diet ~, ~ ' , . . .
s~ -7- ~

,; ~ . .
, , ,, , , . , ~," ", .... . .. . .. . . .

~014B

~0418Zf~
with increased energy and protein concentration can be used.
Prior to combination of the active compound with the feed, it is preferable to coat the compound to prevent degradation. This is particularly desirable when employing the non-hydrated form of the compound. Any of the standard vitamin coatings can be employed. For example, the crystal-line compound can be coated with a solid antioxidant in an amount sufficient only to coat the surface of the crystals with the substance and then drying the crystals as in U.S.
Patent No. 3,110,783, or a solid or liquid composition con-taining the compounds can be stabilized by addition of standard antioxidants such as a tocopherol, propyl gallate, -butylated hydroxy anisole, and butylated hydroxy toluene.
The active compound can be combined with the chicken ~' 15 feed formulation as the pure compound or in combination - -~ with other feed components, in its crystalline form, in --~ solution, or in suspension, as long as the compound is uni-~ formly present throughout the feed compositions. ` ~ -.. ...
-~ , The compound should be present in the feed formulation in egg shell thickening but non-toxic quantities. As much ' compound can be used as desired as long as toxic effects - ~
f~: are not observed. The preferred quantity of compound is i~- that minimum amount which induces the desired egg shell -~
" thickening results. Quantities of 25-HCC of from about 5 ~9 to about 20 ~g./lb. of feed formulation have induced egg shell thickening results as shown i-n Example 1.
The feed formulation of this invention is fed to hens ~ ~
- on a normal dietary intake and desired egg shell thickening` -, ~' is observed.
The following examples are illustratlve of the inven- ~-,,s ,. , ~ , '. . .
~,~ -8- ` `

, ~

~014B
lO9~ Zf~

tion and are not to be construed as lHIliting the invention.
Example 1 150 Hyline strain SCWL hens fed with a diet including a commercially available form of stabilized Vitamin D3 and producing eggs with shells varying in thickness from less than 0.011 to greater than 0.015 inch, are evenly distributed among six treatment groups so thàt each treatment group con-` tained all representative values of shell thickness. To a basal non-Vitamin-D supplemented feed formulation is added daily either 25-HCC or Vitamin D3. This diet is then fed to hens. The daily formulation was done to avoid vitamin degradation. Shell changes and rate of production are determined weekly. The chickens are on this schedule for .. . . . . .
ten weeks.
lS The results from this experiment show that shell thicknesses increased after administration of 25-HC~ or Vitamin D3 from 0.001 to 0.003 inch. The increase in shell thickness of eggs frcm hens treated with 25-HCC was at , least as good as from hens treated with Vitamin D3.- Egg 20 production appears to be unchanged after treatment with ~ -' 25-HCC.
Tables-l and ll present data with regard to improving ;
~ shell thickness in eggs frorn a given number of hens after .,?S'`' ~, ' , ' adminlstration of 25-HCC or Vitamin D3.~ -~-, . , ~
~ , :
,~ . , :, , ~'','.' ',. . - ' ~:
,,~-, , - ~ - :

',??, ~

~;, ' , , ':

,,''~" ~

~ol~e u~- c 10~8;~3 a~
E
~_ r o u~ O :) O t~
o . . ~ ~ In ~ ~o ._ C~ ~ ~ t-- ~ . 0 0 .. _ . ~ . ' :, n ~ I
O ~
~~ Q lo C
~ E ~_) O ~) ~ C~
E ~' 1'1 z ~-- c ._ -' I 3 v) ~ . .
.` z . , O
t~
<n O ....
c . v I I ~ V --- .
~ ~ _ D ~-- . .
I-- E O ~ ~_ .~ _ ~ Z t~ ~ _ '~ CUo :.1, ~ c~ c ~ Q~ ' - , .' ' .
. .~ Z O O C
t cr V ~~ -- a,~
.~ ~ ~ _ C ~ C _ ,~, 0~ -- q) o ~ o ~ o -- Q~ O
0 ~ --I_ IJJ E ~ Q_ Cl _Q_ ~ c~_ ~ ~ O ~ O ~ O ~ ~ O~
" Z IL ~ ', t~ O .' ~ , , z~ ~
O D Q~ . ~ .
U~ _ ~
' ~ - _ ~,~ O O 1~ 0 0 0 .0 ~ C~J ., ' ':
~ ' .:
-$ - ~> .
.'~ . Q~ 0 ~ , , ~ ,~ c . , aO ~ ~ ~, ~ ~' ~ '. . .-:'.
z Q v ~ I T I T
t O - - Ir~U'~ ,r~ ~ '' -.,,,, , . ~ ,, .

J' ' ~ C~ , :"
O ~ ~ U~ ~O
''. ~' . ' ~ L , ' ~' "

.. . . . . .
/~, . .
- 1 0- .
'',', ' ' :
~i, ' , "

. V
~_ ~
C ~ E b'~ ~R ~ b~
a~ ~ o o u~ In O
I ~n > ~ C~J ~D ~o O
O J ~ ,~
~R-3 E
.~ ~
~ ._ .` Q~
~ >-_ C O u ~ ~_ I Q~
E c ~> $ r _ ~
Z E ~ ~ v z 3 t~ ~
.-~ z . . ' ,~, O
. v ., O O

IL-- I C h~--I~ I ~-- C .
~' ~7 I $ ~ _ c ~ a~ O 0 T Z ~ ~ ~ c . .
z ~
3 . _ u~ ~ .
.
oo~ C
~ z O o _ aCJ ~
.. , ~_ ~ ~ --v o a~ o -- ~ o ~ oo ~ _ ~ _ " _ " ~ _ ~, _ ~ : -~ 3 -: ~ O ~ ~0~,0~ ~ o~ o~
:,; IL z ~ ~ a.
' o _ . . ~, : . .- , -J ~ O O It~ ~
. ~ ~ : ~ ~:
.:~ ' 0 `~
- O : ~ :: `~ . ` .' , : ' .' ,',' , , ~ C
` ~ O E E
" o ~

:, ~JJ Q - -- -.- .
L

~'`, `, . `

, ~- , ,, ", , ` , ~, ~ '"' ` ' ' ~, ' -~01 4 B
" .

~O~l~Zf~
The results of Tables I and l l clearly delllonstrate ~he surprising superiority of 25-HCC over Vitamin D3 with regard to improving shell thicl~ness in eggs from a given number of hens. Where the egg shells are initially less than 0.013 inch, which may indicate an insufficient utilization of Vitamin D3 by the hen, the number of hens whose egg shell thicknesses increased with administration of 25-HCC was double that of the hens treated with Vitamin D3. Moreover, the dosages of 10 and 20 ~19. are on the Vitamin D9 activity plateau previously noted. Where the egg shells are initially greater than 0.013 inch, the improvement of shell thickness when using 25-HCC is even more pronounced. The number of - --hens whose shell thickness increased after administration c)r ' 25-HCC was approximately two and a half that of the hens 1~ treated with Vitamin D3. Since these were seemingly normal hens, the results fannot be explained by any theory now know n .
Al though 5 ~9. of 25-HCC was the smal lest quant i ty employed in this experiment, this does not imply that ., , : - , .
desired egg shell thickening results cannot be achieved with smaller quantities. Likewise quantities above 20 ~g. could ~-be equally or perhaps even more effective- without showing . ~-observable toxic effects.
Exampl e 2 1 l ~ ~ -: 25 A cal ci um source such as oyster shel i s, phys i cal l y separated from the chicken feed formulation, and impregnated wTth sufficient 25-HCC to provide 3 ~9. of 25-HCC per chicken ~-- :, ~ per day is fed to chickens. The chickens ingest the oyster ~ ~
.- -.' shell and absorb the 25-HCC from the digestive tract. De-30 sired egg shell thickening is observed in subsec~Jently laid eggs.
~,, - ' -12- ;

,', :

;~ , , ", ,, , ,. ~ .. ,, , ,, ~

lO~l~s28 Examples 3 and 4 illustrate additional methods of administering the active compound to egg laying hens.
Examp!e 3 A quantity of 25-HCC sufficient to administer to a chicken about 3 ~9. of 25-HCC per day, assuming average water intake per day of the chicken, is metered into the chicken's water supply. To facilitate proper dispersion, the 25-HCC can be micronized if desired and dissolved in a surfactant such as polysorbate 80, and metered into the water supply. ~he chickens ingest their normal quantity of water '~ including the 25-HCC. Desired egg shell thickening is ob- ' served in subsequently laid eggs. ~
' Example 4 ' ' ~-` A quantity of 25-HCC sufficient to~provide a chicken ~ ' -- 15 with 60 days supply of 25-HCC is combined with an inert carrier such as a mineral oil and injected subcutaneously ~ -into the web of the wing. The 25-HCC is slowly released and absorbed. De~sired egg shell thickening is observed in~
A subsequently laid eggs. . ~ : ~ '' -, 20 Example 5 - - - - : -' In a similar manner as exemplified~in Examples 1, 2, -~
3, and 4, 24,25-dihydroxychole~alciferol, 25,26-dihydroxy-' ~ ; -'-cholecalcifèrol, 25-hydroxyergocalciferol, 25-hydroxydi- ' ,- hydrotachysterol3, la,25-dihydroxycholecalci~erol, and - -25 lo~hydroxycholecalcisFerol àre administered to egg laying ~ ~ ' ~' hens and results'similar to those of Examples 1, 2, 3, and ~ ~ - -' 4 are obta7ned. ' ' ''~ Examp!e 6 ~
s~ ' In a similar manner as exemplified in Examples 1, 2, 3, 0 and 4, 25-hydroxycholecalciferol 3-acetaté, 25-hydroxycho1e-,, , ., "
", '" , -: . . ,.
~ 13-? , , : , 3014~

~ ~041~2~
calciferol 3-butyrate, 25-hydroxycholecalciferol ~-octanoate, 24,25-dihydroxycholecalciferol 3,24-diacetate, 24,25-dihy-droxycholecalciferol 3,24-diisobutyrate, 25,26-dihydroxy-cholecalciferol 3,26-dipropionate, 1a,25-dihydroxycholecal-5 ciferol 1,3-dipentanoate, 25-hydroxydihydrotachysterol3 -- -: ~-acetate, 25-hydroxyergocalcife.rol 3-acetate, and 1~-chole-calcTferol-1,3-dipropionate are administered to egg-laying hens and results similar to those of xàmples 1, 2, 3, and

4 are obtained.
: 10 It should be noted that the parent molecules of the hydroxylated dihydrotachysterol and ergocalciferol metabo- .-. lites are not known for their egg shell thickening abilities.
. Consequently, any egg shell thickening effects should be .
; unexpected in their particular cases.
., . :
. 15 ~hen employing an acylate the dosage should be related .~ to its unacylated parent molecule on a molar basis. i~or -~, example, approximately 1.6 ~g. of 24,25-dihydroxycholecal- ~ --ciferol 3,24-dioctanoate is equivalent to 1.0 ~9. 24,25-di~
. , hydroxycholecalciferol. . . -, ;~
"
-, , , . ' ' ~ .: ' . . ' - ' ~'':,--t i~ .- -'I ' - . , .. .
l, ; . ' ' - :',' .

.
.. . .

,, . .

Claims (3)

?HE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A chicken feed composition for egg-laying hens comprising in combination with a chicken feed an effective but non-toxic egg shell thickening amount of a compound selected from 1.alpha.-hydroxycholecalciferol and its acylates, said acyl group having two to eight carbon atoms, inclusive, said chicken feed including from about 2.5 to about 5.5% by weight calcium.

2. A composition in accordance with claim 1 wherein there is from about 5 µg. to about 20 µg. of the compound per pound of feed.

3. A composition in accordance with claim 2 wherein the compound is 1.alpha.-hydroxycholecalciferol.