Method for Transplantation of Fertilized Ova Methode Zur Transplantation Von Befruchteten Eiern Methode De Transplantation D'un Ovule Feconde

~™ llll II II II llll llll III III I II I II (19) J European Patent Office

Office europeen des brevets (11) EP 0 399 423 B1

(12) EUROPEAN PATENT SPECIFICATION

(45) Date of publicationation and mention (51) |nt. CI.6: A61 K 31/40, A61 K 31/46 of the grant of the patent: 19.03.1997 Bulletin 1997/12

(21) Application number: 90109571.1

(22) Date of filing : 1 9.05.1 990

(54) Method for transplantation of fertilized ova Methode zur Transplantation von befruchteten Eiern Methode de transplantation d'un ovule feconde

(84) Designated Contracting States: (56) References cited: AT BE CH DE DK ES FR GB IT LI LU NL SE GB-A- 968 776

(30) Priority: 25.05.1989 JP 133927/89 • PATENT ABSTRACTS OF JAPAN, vol. 13, no. 29.12.1989 JP 343985/89 487, (C-649)[3835], 6th November 1989; &JP-A-1 906 630 (43) Date of publication of application: • ARZNEIM.-FORSCH., vol. 26, no. 7, 1976, pages 28.11.1990 Bulletin 1990/48 1359-1361 ; R. BAUER et al.: "Cholinerge Kontraktion der Harnblase des Hundes durch (73) Proprietor: FUJISAWA PHARMACEUTICAL CO., elektrische Reizung des N.pelvicus und der LTD. Nerven in den Ligamenta lateralia und ihre Osaka-shi Osaka 541 (JP) Beeinflussung durch Atropin und Scopolaminbutylbromid" Inventor: Akira (72) Katsumi, • NIPPON YAKURIGAKU ZASSHI, vol. 69, no. 2, Higashimurayama-gun, Yamagata 990-03 (JP) 1973, pages 257-267; T. WAKABAYASHI et al.: "Pharmacological effects of butoxybenzyl (74) Representative: Turk, Gille, Hrabal, Leifert bromide isolated smooth Brucknerstrasse 20 hyoscyamine on muscle in animals" 40593 Dusseldorf (DE) organs • ARZNEI.-FORSCH., vol. 17, no. 7, 1967, pages 882-885; C. VALLVE: "Die wirkung von Spasmolytica auf die mechanische und elektrische Aktivitat des isolierten Rattenuterus" • ACTA PHYSIOLOGICA SCANDINAVICA, vol. 1 24, no. 3, July 1985, pages 429-436; M. STJERNQUIST et al.: Cholinergic and adrenergic neural control of smooth muscle function in the non-pregnant rat uterine cervix" • ACTA PHYSIOLOGICA SCANDINAVICA, vol. 484, suppl., 1980, pages 1-24, Stockholm, SW; M. HAMMARSTROM: "Uterine secretomotor innervation" • FOLIA MEDICA, vol. 24, no. 1 , 1982, pages 31-36; A. TANEV et al.: "The effect of some CO clinically used spasmolytics on the uterus and other CO smooth muscles" CM

<7> <7> Note: Within nine months from the publication of the mention of the grant of the European patent, give CO any person may notice to the European Patent Office of opposition to the European patent granted. Notice of opposition shall be filed in O a written reasoned statement. It shall not be deemed to have been filed until the opposition fee has been paid. (Art. Q_ 99(1) European Patent Convention). LU Printed by Rank Xerox (UK) Business Services 2.13.16/3.4 EP 0 399 423 B1

Description

This invention relates to a use of a parasympatholytic agent for the manufacture of a veterinary composition as an adjunct to transplantation of fertilized ova. More particularly, the invention relates to use of a parasympatholytic agent 5 such as prif inium bromide or scopolamine butylbromide for the manufacture of a veterinary composition as an adjunct to transplantation of fertilized ova, wherein the agent is used for treating recipient cattle. The invention finds application in the field of animal production. For the purpose of producing improved species of beef cattle or the like and reducing the production cost thereof, the technique of transplanting fertilized ova is spreading. 10 For the transplantation of fertilized ova, there are two approaches, namely surgical transplantation which involves a surgical operation and a non-surgical transplantation using a local anesthetic, e.g. lidocaine. Prif inium bromide, one of the parasympatholytics used in this invention, is known by the chemical name of "1 -1 -die- thyl-3-(diphenylmethylene)-2-methylpyrrolidinium bromide" and has been used as a veterinary drug. Among the known indications of this drug are relief of the increased tone and spasm, increased motor function and pain of the alimentary 15 canal associated with gastrointestinal diseases (forestomach diseases such as ruminal impaction, and gastrointestinal diseases such as gastroenteritis and gastrointestinal catarrh and colic) and relief of the increased tone and spasm and pain of the urinary tract associated with urolithiasis. Scopolamine butylbromide has been used as a drug for humans as well. The known indications include relief of spasm and increased motor function in gastric/duodenal ulcer, esophagospasm, pylorospasm, gastritis, enteritis, intes- 20 tinal colic, spastic constipation, functional diarrhea, cholecystitis, cholangitis, cholelithiasis, biliary dyskinesia, sequelae to gastrectomy/cholecystectomy, urinary stone, cystitis, urethrospasm/cystospasm due to insertion of an instrument, dysmenorrhea and spasm of the lower uterus at delivery and pretreatment for roentgenography and for endoscopy of the digestive tract. Of the methods for transplanting fertilized ova, surgical transplantation achieves a high conception rate but is dis- 25 advantageous in that it is laborious and time-consuming. Non-surgical transplantation does not take as much time but has the disadvantage of low conception rates. As to the non-surgical transplantation method, the procedure under anesthesia with a local anesthetic, such as lidocaine, has been broadly used but since the procedure relaxes the sphincter ani as well as the rectal wall, the air finds its way into the rectum when the hand is inserted for transplantation. This air expands the rectum to interfere with the transplanta- 30 tion procedure and, hence, acts as a factor in reducing the conception rate. Nippon Yakurigaku Zasshi, Vol. 69, no. 2, 1 973, pages 257 - 267 discloses the inhibitory effects of atropine and scopolamine butylbromide on serotonin-induced contraction of isolated estrus rat uterus and the effect of butoxybenzyl hyoscyamine bromide. Arzneim.-Forsch., vol. 17, no. 7, 1967, pages 882 - 885 discloses the effect of spasmolytic agents on the mechanic 35 and electric activity of the isolated rat uterus. Among the various compounds tested, there is also N-n-butyl-scopolamine, whereby this compound showed the strongest effect against the mechanic activity of the rat uterus induced by acetylcholine. Folia Medica, vol. 24, no. 1, 1982, pages 31 - 36 describes the effect of some clinically used spasmolytics on the uterus and other smooth muscles. The tested compounds include papaverine, no-spa, alcozin, spasmalgon, and bus- 40 colysin, wherein the latter compound is identical to n-butyl scopolamine bromide. Arzneim.-Forsch., vol 26, no. 7, 1976, pages 1359-1361 describes the cholinergic contraction of the dog urinary bladder by electrical stimulation of the N. pelvicus and and the Ligamenta lateralia Nerves and its affection by atropine and scopolamine butylbromide. In this document it is described that scopolamine butylbromide possesses antimus- carine-like peripheral effects, and antinicotine-like ganglion-blocking properties. The result of stimulation of the contrac- 45 tion of urinary bladder can be quantitatively abolished by this compound. Acta Physiologica Scandinavica, vol. 484, suppl., 1980, pages 1 - 24, describes the uterine secretomotor innervation and the secretory responses to different types of stimulation including transmural field stimulation, carbachol, meth- oxyamine, isoprenaline and the influences of estrogen and progesterone on this response. Acta Physiologica Scandinavia, vol. 124, no. 3, 1985, pages 429 - 436 describes the cholinergic and adrenergic so neural control of the smooth muscle function in the non-pregnant rat uterine cervix. It also discloses blocking of uterine contractions by atropine, scopolamine and tetrodotoxin and states that contractions were unaffected by propranolol, phenoxybenzamine and yohimbine. GB-A-968776 discloses a feeding composition for animals which comprises an animal feed and a small amount of peristalsis-regulating substance contained therein. This substance is substantially specific as to smooth gastro-intesti- 55 nal muscles and is preferably propantheline bromide, atropine or methantheline bromide. The inventor of this invention conducted an assiduous research for improving the conception rate following fertilized ovum transplantation in an expedient manner and found that the administration of a parasympatholytic agent such as prif inium bromide or scopolamine butylbromide to recipient cattle relaxes not only the rectal wall but also the uterine wall without accompanying a relaxation of the sphincter ani, with the result that the transplanting instrument can be inserted

2 EP 0 399 423 B1 deeper into the uterus to help achieve a remarkable improvement in conception rate. This invention is predicated on the above finding. The veterinary composition which is used according to present invention is applicable to cattle, e. g. beef cattle and dairy cattle, swine, horses and so on. Examples of the parasympatholytics to be used in this invention include prifinium bromide, scopolamine butylbromide, atropine and so on. The administration and dosage of the parasympatholytic agent are not critical but can be chosen in accordance with the particular agent to be employed. Generally, however, satisfactory results are obtained when prifinium bromide is used in an intravenous dose of 30 to 50 mg/aminal or scopolamine butylbromide is used in an intravenous dose of 80 to 140 mg/animal to recipient cattle. When prifinium bromide is used at ovum collection as well, satisfactory results are assured by intravenous injection of 50- 100 mg/animal to donor cattle. The technique for transplantation of fertilized ova is the same as that used conventionally but it is preferable to go through the transplantation procedure while the rectal and uterine walls remain relaxed after drug administration.

[Examples]

Example 1

Prifinium bromide (7.5 g) was dissolved in distilled water for injection (1 ,000 ml) and the resultant aqueous solution was filled, in 5 ml portions, into ampoules, which were then sealed. The injection thus prepared was used in Example 3.

Example 2

Scopolamine butylbromide (2.0 g) was dissolved in distilled water for injection (100 ml) and the resultant aqueous solution was filled, in 5 ml portions, into ampoules, which were then sealed. The injection thus prepared was used in Example 4 as below.

Example 3

In donor cattle at collection of ova, comparison was made between a lidocaine caudal epidural anesthesia group consisting of 5 Holstein and 10 Japanese Black cattle (a total of 15 animals) and a prifinium bromide intravenous injection group consisting of 1 Holstein and 6 Japanese Black cattle (a total of 7 animals). A similar comparison was made in recipient cattle at transplantation using 60 animals for lidocaine caudal epidural anesthesia and 23 animals for prifinium bromide intraveous injection. The dose for donor cattle at ovum collection was 75 mg/animal (10 ml) intravenously and the dose for recipient cattle at transplantation was 37.5 mg/animal (5 ml) intravenously. Immediately after intravenous injection of prifinium bromide, arrest of rectal peristalsis, relaxation of the rectal wall and relaxation of the wall of the uterus were found in remarkable degrees in both donor and recipient cattle. The duration of action in donor cattle was 59.6 minutes on the average. The average duration of action in recipient cattle was 28.4 minutes. The results are shown in the following table. EP 0 399 423 B1

Number of trans- Number of impreg- Conception rate planted cattle nated cattle Epidural anesthesia One fresh ovum 4 1 25.0% with lidocaine TwoT fresh.. ova 8_ 4. 50.0%c. „. One frozen ovum 39 10 25.6% Two frozen ova 9 4 44.4% Total 60 19 31.7% Intravenous injection One fresh ovum 2 2 100.0% of prifinium bromide ^ TTwo fresh, .. ova 1 0n 0.0%n no. One frozen ovum 14 8 57.1% Two frozen ova 6 6 100.0% Total 23 16 69.6%

Example 4

25 Scopolamine butylbromide 100 mg/animal (5 ml), instead of prifinium bromide 37.5 mg/animal (5 ml) in Example 3, was injected intravenously to recipient cattle at transplantation and fertilized ova were transplanted in the same manner as in Example 3. The fertilized ova were obtained in the same manner as in Example 3. The results are shown in the following table.

Number of trans- Number of impreg- Conception rate planted cattle nated cattle Scopolamine butylbromide One fresh ovum 2 1 50.0% Two fresh ova One frozen ovum 4 4 100.0% Two frozen ova 1 0 0.0% Total 7 5 71.4%

The conception rate after transplantation of fertilized ova in accordance with this invention is higher than 50%, which is said to be "a parameter of fertilized ovum transplantation technology which is expected to be achieved in the 45 near future by the non-surgical method" and even higher than 60% which is said to be "a parameter of fertilized ovum transplantation technology which is desirably achieved" (Chikusan-no-kenkyu 42(1 1), 9-14) and, therefore, this invention is believed to provide a far-reaching benefit to the animal industry.

Claims 50 Claims for the following Contracting States : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE

1 . Use of a parasympatholytic agent for the manufacture of a veterinary composition as an adjunct to transplantation of fertilized ova. 55 2. Use according to claim 1 , wherein the parasympatholytic agent is prifinium bromide.

3. Use according to claim 1 , wherein the parasympatholytic agent is scopolamine butylbromide.

4 EP 0 399 423 B1

Claims for the following Contracting State : ES

1. Process for preparing a veterinary composition for use as an adjunct to transplantion of fertilized ova, wherein a parasympatholytic agent is brought into a form which can be used as intravenous injection. 5 2. Process according to claim 1 , wherein a parasympatholytic agent is dissolved in distilled water for injection in a concentration of 0,75 to 2,0 g/100 ml, filled into ampoules and then sealed.

3. Process according to claim 1 or 2, wherein the parasympatholytic agent is scopolamine butylbromide or prifinium 10 bromide.

Patentanspruche

Patentanspruche fur folgende Vertragsstaaten : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE 15 1. Verwendung eines parasympatholytischen Mittels fur die Herstellung einer tiermedizinischen Zusammensetzung als Hilfsstoff zur Transplantation befruchteter Eizellen.

2. Verwendung nach Anspruch 1 , wobei das parasympatholytische Mittel Prif iniumbromid ist. 20 3. Verwendung nach Anspruch 1 , wobei das parasympatholytische Mittel Scopolaminbutylbromid ist.

Patentanspruche fur folgenden Vertragsstaat : ES

25 1. Verfahren zur Herstellung einer tiermedizinischen Zusammensetzung fur die Verwendung als Hilfsmittel fur die Transplantation befruchteter Eizellen, wobei ein parasympatholytisches Mittel in eine Form gebracht wird, die als intravenose Injektion verwendet werden kann.

2. Verfahren nach Anspruch 1 , wobei ein parasympatholytisches Mittel in destilliertem Wasser zur Injektion in einer 30 Konzentration von 0,75 bis 2,0 g/ml gelost, in Ampullen gefullt und dann versiegelt wird.

3. Verfahren nach Anspruch 1 oder 2, wobei das parasympatholytische Mittel Scopolaminbutylbromid Oder Prifinium- bromid ist.

35 Revendications

Revendications pour les Etats contractants suivants : AT, BE, CH, DE, DK, FR, GB, IT, LI, LU, NL, SE

1 . Utilisation d'un agent parasympatholytique pour la preparation d'une composition a usage veterinaire sous la forme 40 d'un additif a la transplantation d'ovules fertilises.

2. Utilisation selon la revendication 1 , dans laquelle I'agent parasympatholytique est le bromure de prifinium.

3. Utilisation selon la revendication 1 , dans laquelle I'agent parasympatholytique est le bromure de butyle de la sco- 45 polamine.

Revendications pour I'Etat contractant suivant : ES

1 . Procede de preparation d'une composition a usage veterinaire destinee a etre utilisee sous la forme d'un additif a so la transplantation d'ovules fertilises, dans lequel on prepare un agent parasympatholytique sous une forme qui peut etre utilisee en injection intraveineuse.

2. Procede selon la revendication 1 , dans lequel on dissout un agent parasympatholytique dans de I'eau distillee, a des fins d'injection, a une concentration de 0,75 a 2,0 g/100 ml, on verse la preparation dans des ampoules que 55 Ton scelle.

3. Procede selon la revendication 1 ou 2, dans lequel I'agent parasympatholytique est le bromure de butyle de la scopolamine ou le bromure de prifinium.