Note: Descriptions are shown in the official language in which they were submitted.
WO9~3/15608 ~ U ~ PCT/U~90/02933
Use of epostane to enhance egg laying .n turkeys.
Fleld of .he I~ven~ion
; The inven~ion relates co a process of ennancing eg~ laYing in
turkevs us.ng epostane. I
Infor~a~ion Disclosure S~atemen~
Epos.ane is the Uni~ed Staees Adopted Name (1988 US~ and the
USP Dic~ionary of Drug ~ames, 1961-1987 Cumulative List) for (4~
17~)-4.5-epoxy-3,17-dihydroxy-4,17-dLme~hylandros~-2-ene-2-carboni-
trLle ha~ing the s~ructural formula
C
~ .... C:~
.`;C ~,J Formula I
"~
(represanelng th~ onol eorm) an~ haviA~ ucLli~ a~ an Lncer~eptLve
' (pregnaney disrupein~) agenc.
Chriscians0n U.S. Pat. ~,160,027 lssu~d July 3, 1979 describes
epostane as the produe~ Oe part (f) of E~PLE 1, thac is, ~l~, 5~-
epoxy-17~-hydroxy-4,17-dime~hyl-3-oxoandrostane-2~-carbonicrile
having the struetural formula
C~. OH
C~3 ~ --c-~
.~C., /\~ /~/~
o ~J Formula Il
.i ~3
i
:. : .: .. .. ... : . : , . : . : :: , . . :; : ::
W O 90/l560~ 2 0 ~ 3 9 ~ ~ PCT/US90/02933
-2~
which represen~s ~he keto forrn of epostane, The pacent shows the
interceptive utilit~ of epos~ane in the rat and ~he monkev.
Epos ane has been st~cied for e~fects on fertility in numerous
ma~malian species and the published resules of ~he studies conseituee
a bibliography which in March of 1989 nu~bered 66 references, none of
which relates to any avian species,
Three of the references for which the bibliography contalns
ahstracts relaee to ovulation. De Paolo (J. Endocrinol., vol.
118~1), pp. 53 68, 1~88, arld Snyder et al. (Proc. Soc. i`~'~?. Biol,
.~ed., voL 1-7~3), pp. 238 247i, 1984) show lnhiblcion of ovulation in
the ra~ with epostane and Webb ~. Reprod. F~.rtJI., vol 79(t), rr.
231-240, 1987) shows increase in ovulacion race in che ewe wieh
epostane,
The onl~ prior ar~ known co applicane relaeing to use of
epostnno in an n~L~In ~pocios ~Iro 3S ~ollows~ (1) nn unpub1ir,hQd
repor~ showin~ closo r~l3~d ~loar~s~ o pla~m~ pro~Q~3ron~3 nnd
estrncliol concenerntLorls Lrl tho chick~rl eherow:Lth. Til~3 r~Qre L9 1n
tho forrn ~ a lote~3~ dlle~t~ US~ 19, 1~85 Eroln ilo~i3L J. ~h~lrp of 'i~l-le
A~r l a~ll tlO~rA ~ d i~ 3 ~ ~ AI'~ h ~ ln~ e L'~ R~ 3 n r(~ 3 R ~
Mlcllothian, ScocL~Rcl co i~. LoL30n o~ Sc~rlLng ~ln~hrop Gro~p Lcd. 1n
Guildford, Englancl, throu~ whom epostane for the work had boen
supp1ied; (ii~ 3 publlc.3eLGn entLtled STERLIhG DRUG I~C. PRESENTATIO~
TO E'INANCIAL COMMUN~T~' dated October 23, 1986 ln a part cntitlcd
EPOSTA~E a~ pages 79-80 states at page 80 that "[t¦he endocrine
25 effects ~of eposeane] on egg produceion in poulery are also being
pursued" and ehat "[t]his lateer indication is of particular impor-
tance in the broiler industry". The first scatemenc merely sets
rorth a goai and does no~ identify a species, The second s~atement
identifies the chicken as a species "of particular impo~tance" and
does not men~ion the turkey, Accordingly the presently described
invention is not described or made obvious by the publication,
Moreover, as shown below epostane was found not effective in enhanc-
ing egg laying in the chicken.
Broodiness in the turkey hen i5 a physiological condition
characterized by aggressive territorlal n~s~ pro~ection and lncr~ase
nesting beh~vior with cessa~lon o~ egg production, whlch eogether
with costs of additlonal labor and acilieies needed to maintain
W O~30/1560X 2 0 5 3 9 0 g PCT/US90/02933
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broody hens result in significant economic loss in the turkey
indus~ry. The presently described and claimed invention fills a need
for a solution to the egg production aspect of ~he proble~ in the
~urkey industry.
S SU~ARY OF THE INVE~TION
The Lnvention is the me;hod of enhancing egg laying in the
curkey which comprises adminiseering eo a curkey hen an amounc of
eposeane sufElclene eo Lncrease sLgnlflcantly ehe number of eggs laid
durln~ the ~gg l~yLng c~ycle of s~ld turk~ h~n.
D~TAIL~ ~ESCRIP'rTO~ OF ~IE INV~,~TION
The egg laying cycle in the eurkey hen is aboue six months in
duraeion. Near che end of the cycle egg productlon declines. At ehe
end o che cyclc eho hen moles. E~8 productlon C0~.9~ flnd rises
agaln ~s th~ n~w c~cl~ ensues, A hon rlormally produces 75-~0 c~g3
por cycle, thus one ~g every 2.0-2.4 dnys. A9 9t~t~d nbovo broodl-
noss i5 A100 nn import~ e nctor Qf~ceLn~ ~,g produeeloF~ ln eurkoyhons. ~ccordLn~ly ~n ~rryLn~ o~le ~:ho 1nvonelon ~ c~ oE opos~no
on nctors whlch r~lneo eo b~oodlnoss ~nd o~s~s prod~celvley n9 w~
egg productLon ies~lf w~re nlso studL~d.
Any amounc Oe epostana suffLclene co eEect a signiricanc
incr~ase in thc numbor of eggs laid during the o~g laying cycle can
be usod, The preeerr~d dose is in the ranQe of 0,1-10 mg./kg, per
day. The epostane can be prepared for administration in any pharma-
ceutically acceptable oral or paren~eral dosage form. The oraldosage form can be solid or liquid and thus granùles, capsule,
tablet, solution, suspension or emulsion. The parenteral dosage form
can be solution, suspension or e~ulsion. An ethanol-cottonseed oil
vehicle is preferred. An oral dosage form is preferred. Gelatin
: 30 capsules can be used, but the preferred oral dosage form is granules
which may be mixed into or top-dressed onto the feed. Sustained
release dosage for~s can be used. The daily dose can be given for as
few as one day or as many as all the days of the egg laying cycle.
Daily doses for one day and five weeks (weeks 3~7) of the cycle werc
used ln th~ tes~s descrlb~d bclow.
In a first t~st of ~h~ inv~lleion th~ cffeces o~ a singl~ 0,
1.25, 2,5, 5 or 10 mg,/kg, oral or parenteral dose of epostane on
W 0 90/15608 2 0 ~ 3 9 0 ~ Pcr/us90/02933
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blood plasma concencracion of progesterone, estradiol, corticosterone
and prolactin and on eg~ production in the curkey h~n were deter-
~ined. One hundred hen (110) 52-week old firse cycle turkey hens
were divided randomly inco 10 groups of 11 hens each for the test.
The oral doses of epostane were given in gelatin capsules. For
parenteral ad~inistracion the eposeane was dissolved in echanol-
cot~onseed oil (10:90) at a concencration of 20 mg./ml. and given
subcutaneously. 8100d samples were taken one w~ek b~Eore modicaeion
and at 6, 12, 24 nncl ~-a hours posem~dlcation. Anal,sLci of the Eour
blood plasma variables was done by radloLmmunoassay. Egg produceion
was measured for 12 days before medication and 3 days postmedication,
whereupon the hens were sacrificed. Daca were analy~ecl by analysis
of variance.
Unlike the cffecc of eposcanc on blood plnsma progesterono
concentration in ~namrnals tlO significant overall ofE~ct of do50 or
route Oe fldrnlnl~r~tion Oe ~po~n~ on thls vnrl~bl~ w~s ob~rvod ln
the curk6y h~n. A 91gnLEie~tle ~p ~ 0.0001) docr~Q ~hQrO1n WA9
corr~ L ci~ n~ a ~1 ~ 6 nr~ r ~ llnll le~ n, b~
ehLs wa~ ov~rcoln~3 hy r~bound rl~ 2l~ ~In~ 8 ho-lr~ ~ose~n~dleaeioR ~o
thnt no sL~nlELc~nt ov~rall e~ ct o~ sa~npllll~ tilll~ was shown.
Significant r~duc~Lon of estradiol concen~ration based overall on
doso Oe Qpostntle (p e 0.0001) and sampling timo (p ~ 0.02) WA9
observed, bue ~he effect of rouee oE adrnlnlscr~tLon w~s rloC signl-
ficant. SLgnlican~ reduction of corticoseerone conc~ntraeion based
overall on dose (p < 0.0001) and rou~e of administration (p <0.02) of
epostane and sampling time (p < 0.03) was observed. No significant
overall effect of dose or route of administration of epostane or
; sampling time on prolactin concentration was observed.
Egg production W2S measured as percent hen-day egg production,
which is defined for a given group of hens and a given number of days
as:
percent hen-day eg8 production ~ the number of eg~s laid x 100
the number of hen9 x the number oE
days
Egg produccion results o~ ~h~ ~lrs~ cesc ~rc prescn~d in T~bl~ 1.
~va~u~
~0 90/~560X ' ' PCT/US90/02933
-5-
Table I
Flrst Test - E~P Production
Epos~ane Percent Hen-Dav E~g Produc~ion
Dose Premedicatlon Postmedicaeion
5 (mg./k~.~ Qral Parenteral Oral Parenteral
0 47.0 34.5 24.0 51.7
1.25 37~3 35,5a 26,7 6o~oa
2.50 27.1 30.9 12.0 ~6.7
;.00 '~9.8 ~0~9b 21.0 70~ob
10.~0 57.3 39.1 66.7 56.7
a~D Significantly diferent (p < 0.03)
As shown by Table I the 1.25 and 5.00 mg./kig. parenteral dose groups
showed slgnLf'icanc increases in egg production. This was consldered
l; encourflglng bocause onlv .~ singl~ do5~ W~S gLvon to e~ch han nnd only
threc days' egg pro~luction pos~n~ecllcaticln w~s. mo~aurocl,
Ll~ CO~ ti o~ lv~nel~n ~ih~ 0,
0,625, 1.25 ~nd 2.50 l~lg,/k~. ornl do~;~9 n~ flpo~ ln~ dnLly ~r ~Lv~
W~ ;3 ~tl hlo~ 6~ ,0R~ eL~ r~s~it~ r~ L,
corcLcos~rona, prol~cein, c~lelwn nncl protaln, ~gg production and
egg charac~rlscLcs ln the broocly-pronc t~lrke~ hen were decermined.
Two hundred ~200) Elrsc cycle turkev hens prone to broodlnoss were
divid~d r~ndomly inco 5 ~roups oE 40 hens each ancl lnduced to molt
and ~herebv to be~in the'ir second laying cycles synchronously. The
daily oral doses of epostane were given in gelatin capsules beginning
with the third week of the egg laying cycle. Blood samples were
taken daily from 10 hens in each dose group. Egg production and
nesting behavior were determined six times daily. The hens were
sacrificed two weeks after termination of medication and at necropsy
the o~aries and uterus were examined. Analysis of progesterone,
estradiol, corticosterone and prolactin concentrations were done by
radiommunoassay. Analysis of calcium and protein concentrations was
done colorimetrically. Data were analy~ed by analysis oE variance
except egg charac~eris~ics and necropsy data, which were analy~ied by
chi square analysis,
A~i necropsy ~he ovary o~ ~ch hcn whlch cornplo~ecl ~ho ~esc WA~
examined. A normal ovary was considered indicatLve of egg produc~
.. , .. , . .. ~ ~ .
WC~90~60X 2 0 ~ 3 9 ~ ~ P~T/US90/02933
-6- ~
tivicy during ~he test period. A tumorous, juvenile or regressed
ovary or one having a~resic follicles was considered indicative o~
lack of egg productivity during the tese period. The number of hens
having normal ovaries (11/37, 10/38, 19/27, 13/40) was significantlv
; Breater (p < 0.27) in the groups medicated with epostane tO.313,
O.625, 1.25, 2.50 mg./kg. respectively) than in ~he unmeclicated group
(7/40). Each ovary was also examined for los5 of hierarchy oE
follicles ~ncl numbcr of prlmnry folllcles. The numbcr of hens
showLrlg los~ o~ hl~rarchy oE Colllcl~3 ~5~35, 9/38, 7~36, 8/40~ was
sLgnlfic~ ly gr~ater ~p ~ 0.03~-) ln the groups medicated with
epostane (0.313, 0.62S, 1.25 ~g./kg. respectively) ehan in the
unmedicated group (none). The number of primary follicles was
significantly greater (p < 0.028) in the groups medicaeed with
epost~ne than in the u~ncdicntccl gro-lp. Eposean~ thor~forc slgnl
icantly revors~d ~he t~ncl~ncy cow~lrd n~re~.ic ov~rLfs ~nd tll~
con~cqucn~ inabiLl~y CO procluc~ og~ ncl slgni~lcnnel~ Incr~n~3Q
rccrulemenc o~ Polllcle3 fcr ov~llnelon ~In~l ehQ eon~l~aq~ne ~Ibllley eo
prod-lc~ Inorc~ Ln ellrkoy hal~ prono e~ LoodillQ!~
Ov~arall e~3ge.q o~ a~o~ednQ ro~a~llen~3.0Fl orl bloocl pln9raa ~nrn~nuC-
ers are shown in Tabl~
T~bl~ I~
Sec.ond Tes~ e~all alood Pl~s!na ~ara~ee~. E ~ç c es
: Parameter Concentration
25 (Concentration Units) Unmedicated Hens EPostane Medicated Hens
Progesterone 561a 825a
(pg./ml.)
Estradiol 57.66b 61.14b
(pg./ml.) -
Corticosterone 3 93C 5 07c
(ng./ml.)
Prolactin 1049 1095
(ng./ml.)
Calci~m 15.8 16~1
tm~./dl.)
Pro~ein /~ . 98 5 . 02
(g./dl. )
W O 90/1560~ 2 0 5 3 9 0 8 PCT/US90/02933
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. . ~
a Si~nificanely different (p < 0.006)
b Significantly different (p < 0.01)
c Significantly differen~ (p < 0.005)
Increases in progescerone, estradiol and corticosterone concentra-
tions were even more significantly (p < 0.0001) increased in produc-
tive hens than in nonproductive hens. Plasma protein concencracions
were also significancly (p < 0.0001) increased in productive hens.
Pla~ma prolacc~n concen~ra~lons were slgniflcanclv ~p ~ 0.0001)
described in prcducelve hans.
Egg productlon resulcs of tho second test are shown in Table
III.
Table III
~ w~ iQo
Wock _ Q ~_Q~ Q~ 5
l ~0 ~ 3 2 5 l~
20 2 31 25 ~9 ~8 28
3 22 12 19 20 20
4 19 ~ 11 16 18
14 7 8 22 23
6 12 lS 11 32 25
25 7 12 9 15 30 21
8 4 ; 12 19 11
9 4 4 11 7 9
Egg production declined steadily in the unmedicated group due to
increased broodiness during the test period. Epostane appeared to
reverse this tendency partially in the medicated groups. During week
5 (the third week of ~edication) egg production was significantly
greater (p 5 0.04) in the 1.25 and 2.50 mg./kg.dose groups than in
35 the 0.313 and 0.625 mg./kg. dose groups. During we~ks 6 and 7 ~the
fourth and PlPth w~ks of medicfltion) egg productl~n w~Y slgnlEl~
cantly great~r ~p 5 0.04 and 0.01, respectively) in the 1.25 and 2.50
W O 90/1SfiO8 PCT/US90/02933
205~90~ -8- ~ I
Gsg./kg. dose groups ehan in the 0, 0.313 and 0.625 mg./~g. dose
groups. During ~eek 8 (~he firs~ week postmedication) e~g production
was significan~ly greater (p < 0.008) in the 1.25 mg,/kg, dose group
than in the O and O.313 mg./kg. dose groups. This pattern of '
significance is not greatlY affeoted by correcting che results for
hens which were unproduc~ive or which died or by normalizing the
medicated groups against the un~edicated group,
E8g weLgh~s and eggshell weights were sllghely decr~sasod in the
~ucllcat~sd do~s zroups, bue ~hes specL~ic g-avle~y of ch~ ~sggs WAS not
a~feceed by ns~sdlcatlon. Th~so efEtscts are no~ consLder~scl important.
COMPARATTVE TEST I~ T~IE CHICKEN
A test was conducted to determine the effects of epostane on
blood plasma hormone concentratic)ns and egg laying efficiency in
chickens nearing the ~erminaeion of theLr a,ctive egg produc~lon, Two
hundred ~200) hens were clivlded lneo 10 groups o~ 20 hens ~ach for
the tu~t. ~n dupllcato groups opostancS was glven Ln the Eo~cl ~ally
ior four weeks ae 0, 1.25, 2.5, 5,0 and 10 mg.~kg. IlloQtl plas~mrS.
concentra~lon~ o~ prog~raeQrono, oseracl:Lol, coreLcc,~e~,ronso ~nd
~rol~c~ln w~sro d~srlnLllt~sd l~s ~ t~ fl~Q ~ ~
Egg produceion rlnd ogg qualiey wesrQ tloeormln~d ln ~ll groups.
Preliminary evaluaeion Oe ehe data showJ thae eposeflne dLd not have a
maJor eect on blood plasma hormone concenerations or egg laying
eficlency in eh~ chicken.
: `~
