AU649994B2 - Drug for improving the reactions of the ovary and the production of ova and embryos in domestic mammals in connection with biotechnological embryo transfer - Google Patents

Description

D326 COMPOUND FOR IMPROVING THE OVARY REACTIONS AND THE OVA AND EMBRYO PRODUCTION IN DOMESTIC MAMMALS IN COMBINATION WITH BIOTECHNOLOGICAL EMBRYO TRANSFER It is known that with regard to mammals which within a single reproduction cycle as a rule produce in a natural manner only a single fertilized ova (monotocous animal species), to be able to cause an increase in the production of ova and, as a result therefrom, of embryos through the utilization of gonodotropenes; for example, such as HCG, HMG, PMSG, FSH, LH and/or GnRH, which controls the release of gonadotropenes from the hypophysis.

Consequently, it is thereby possible to successively produce a series of ovas which are fertilizable, and to -ecover these as embryos subsequent to their fertilization, and to transfer these to recipient animals. These stimulation methods for the achieving of superovulations has the disadvantage that this can be achieved only through an excess dosing with gonadotropic hormones. Due to the endogenic counterregulation against multiple ovulations, especially with monotocous domestic mammals, unsatisfying ovary reactions result from such known stimulation methods especially with respect to the extense interacting and internal and individual variability, as well as the therefrom resulting number of qualitatively high-grade embryos. A significant multiple usage of oogenises and oozytes which 1is necessitated through an overdosing, especially with gonadotropenes, leads to a reduction in the potential live or vital production yield of a donor animal.

Such an intense intervention into the endogenic and endocrinous regulation of folliculogeneses, follicle ripening and into the ovulation processes, lead to temporary and even to permanent derivative damages to the reproductive yield of donor animals.

These disadvantages which are encountered in the state-of-the-technology are avoided by the compound pursuant to the invention.

To the extent that in accordance with the state-of-the-art there are employed materials which act as inhibin, this utilization is implemented as an active immunization or for the examination of the effects on the physiological reproduction parameters. In accordance with the extent that in the state-of-the-art, there are employed materials which are efffective as inhibinneutralizing antibodies, this utilization is effected for 2diagnostic proof and for the attaining of a profertility effect for individual, female mammals.

Fertility signifies the capability for reproduction of an individual and the capacity of the individual to produce a certain number of live offspring during each reproductive period.

-2- Use of inhibin-neutralizing antibodies to cause superovulation is, in contrast therewith, not a fertilitypropagating measure.

The subject matter of the present invention relates to the use of inhibin-neutralizing antibodies for improvement in the ovary reactions, and in the ova and embryo production for domestic mammals in combination with biotechnological embryo transfer or transplantation.

It is an object of the present invention to provide controlled intervention into the central endogenous endocrine regulation of the ovulation rate-control systems, to initiate superovulation and, as a result thereof, develop a more univer al method for stimulation of superovulation for different species of domestic animals.

Furthermore, it is another object of the invention to achieve a higher proportion of qualitatively high-grade embryos which are capable of being transferred or transplanted, and to thereby improve the economics of embryo transfer for domestic mammals.

According to one aspect of the present invention, there is provided a method of inhibiting the biological activity of inhibin in domestic mammals, comprising the administration of inhibin-neutralizing antibodies directed against naturally occurring or recombinant inhibin or its -3- 940407,p:\oper\jms,70552-9La,3 subunits, or its fragments or its derivatives, wherein said antibodies can neutralize the biological activity of the inhibin formed in gonads, to induce superovulation in combination with embryo transfer in domestic mammals.

In one embodiment of this aspect, the inhibinneutralizing antibodies are administered in combination with at least one superovulation-propagating hormone, such as gonadotropin, gonadotropin-releasing hormone, clomiphene or tamoxifene or another anti-estrogen, or androstenedione, testosterone or dihydroxytestosterone or another antiandrogen.

In another aspect, the present invention provides a composition for inducing superovulation and increasing embryo production in combination with embryo transfer in domestic mammals comprising an inhibin-neutralizing antibody and at least one gonadotropin, gonadotropinreleasing hormone, anti-estrogen or anti-androgenas4diraa h4 ry Qr% i g re 1 .i r J- 4 The invention further provides a method for inducing superovulation in a female domestic mammal which comprises administering a superovulation-inducing amount of an inhibin-neutralizing antibody to said mammal thereby inducing superovulation in the mammal.

In yet another aspect, the invention provides a method for increasing the number of fertilized embryos in a female domestic mammal which comprises administering an embryo-increasing amount of an inhibin-neutralizing l 940408,p:\oper\jms,70552-91.ch,4 untibody to said mammal thereby increasing the number of fertilized embryos in the mammal.

In a preferred embodiment, the inhibinneutralizing antibodies are specific polyclonal or monoclonal antibodies (antiserum) which neutralize the biological activity of inhibin in living organisms.

These antibodies are directed against naturally occurring or recombinant inhibins or their subunits, or their fragments, or their derivatives, and whose complete molecule represents a glycoprotein which preferably consists as heterodimers from two different subunits (a and bonded through di-sulfide bridges, and whose molecular weight consists of 5 to 120 kDalton (kDa) and which selectively suppress the FSH-secretion.

The antibodies are preferably administered while in a liquid phase, preferably through intravenous and/or subcutaneous and/or through intramuscular application.

Preferably also, the antibodies are administered as multiple injections at specified time intervals, preferably of 12 or 24 hours, over a number of successively following days, preferably on 3.5 to 5 days.

This process is preferably characterized in that the administration is implemented during the middle luteal phase, preferably on the 7th through the llth day of the cycle.

5-940408,p:\oper\j,70552-9.cla,5 (V 940408,p:\oper\jms,70552-91.da,5 Furthermore, the process of the invention is also characterized in that the administration is preferably carried out with equal or reducing dosages, with presently two equal successively following quantities.

The following authors describe the characterization of an inhibin (antigen) for the species of animal designated as cattle. Robertson, D.M. et al. Molecular and Cellular Endocrinology, 44 (1986) pages 271-277.

Forage, R.G. et al. Proc. National Academy of Science; (USA) (1986) pages 3091-3095.

Described is the isolating and characterization through cloning and sequence analysis of DNA, which code for the subunits of inhibin from bovine follicle fluid (bFF).

-6- 940407,p:\oper\jnis,70552-91.cl,6 Produced was the 31 kDa-form of the inhibin through the introduction of a pH- 4.75 precipitation step into the described purifying method for the 58 kDa- inhibin from bFF. Consequently, the former is a cleavage product from 58 kDa-inhibin.

The antibodies used in accordance with this invention neutralize and thereby render ineffective the biological activity of the inhibins which are produced in the gonads of the animal.

Miyamoto et al. in Biochemical and Biophysical Research Communications, Vol.136, No.3 (1986), pages 1103-1109, describe inhibin-antibodies for the species of animals designated as cattle. These are monoclonal antibodies employed against the bFF 32 kDainhibin from bovine follicle fluid.

Monoclonal antibodies against bFF 32 kDa-inhibin are known from the utilization of the hybridizing technique in mice (origin Balb/c).

One particular antibody, Type 256H, recognizes specifically the bovine 20 kDa-inhibin. In comparison with other antibodies this evidences a strict specificity for the bovine 20 kDa-subunit.

94040p\oper\ms705529.a7 94040B8,p:\oper\jms,70552-91.cla,7 The method of the invention is advantageous in the equine species of animals, due to the lacking in the response of this species of animal to gonadotropene preparates.

The method of the invention in which the inhibinneutralizing antibodies are administered in combination with a superovulation-propagating hormone, is exemplified as follows: There is verified the effectiveness of the combination of inhibin-neutralizing antibodies (antiserum) in combination with hormones which propagate superovulations (gonadotropin). Available from an agricultural operation are 15 head of cattle of the type "Deutsche Schwarzbunte" with a cross-breading of approximately 75% "Holstein-Frisian" within the age groups of 17.5 to 25 months and in the weight classes of about 350 to 410 kg.

These cows were presynchronized with injections twice of each respectively of 500 gg chloprosterol, sold under the trademark "Estrumate"; a commercial product of the Coopers Tierarzneimittel GmbH, 3006 Burgwedel, Germany.

The injections were administered at an interval of 11 days. Thereafter, the cows were divided into two groups in accordance with the random principle.

940408p:\oper\js-8- 940408,p:\oper\jms,70552-91.cla,8 At the beginning of the treatment, the cows were at the 9th day of the cycle.

The cattle from Group I, the control group, with n 8 cows, were administered a total dosage of 23.8 mg NIH-FSH unit-equivalent (National Institute of Health-FSH) at 12 hour intervals over 3.5 days in a decreasing dosages with 4.2 4.2 3.5 3.5 2.8 2.8 2.8 mg FSH-equivalent. There were additionally included in each of the 6th and 7th injections respectively 500 ig chloprostenol for the initiation of the luteolysis. With regard to chloprostenol is water there is to be understood as water-soluble sodium salt.

The artificial insemination was carried out 36, 48, 72 hours after the initiation of the luteolysis with deep-frozen sperm from a single bull.

The cattle from Group II, with n 7 cows, in addition to the above-described treatment, and concurrently with the FSH injections, were intravenously injected each with 20 ml of an antiserum pooled from four sheep against inhibin S-antiserum.

For the production of this serum, four sheep rams were actively immunized with a purified preparate of inhibin from bovine follicle fluid, bFF, which was obtained in a slaughterhouse for cattle.

The cattle from whose ovaries there was obtained the follicle fluid, were previously treated with -9of;7S C, t ;TI' O PMSG for increasing the inhibin concentration of inhibin in the bFF.

The rams were initially each basically immunized with respectively 2.5 mg protein of a purified Inhibin-preparate, dissolved in 2.0 ml of an 0.9% NaCl solution and with 2.0 ml complete Freund's adjuvant, The booster administrations were effected on the 20th and 50th days subsequent to the basic immunization with, respectively, 2.0 and 1.8 mg protein, dissolved in 2.0 ml of an 0.9% NaCl solution and with ml of incomplete Freund's adjuvant.

The serum recovery was carried out on the 7th and 10th days subsequent to the second boostering. The antiserum from the four rams were pooled with the same volumetric parts and'fractionated, 150 ml, and stored at -20 0

C.

Achieved were the following results: Treatment: Ova/Embryos Components n Cl total Folltropin 23.8 mg 8 10.5 5.88 7.9 Folltropin 23.8 mg 7 14.1 3 .29 9.6 S-Antibodies against inhibin Treatment: Ova/Embroys .1 Components fertilized transferable Folltropin 23.8 mg 4.76 5.1 3.27 66 3.8 3.06 48 Folltropin 23.8 mg+ 2.64 8.6 2.44 90 8.3 2.21 86 S-Antibodies against inhibin The numerical values are indicated with the media- deviations (on-1) as average values, which result from the arithmetic median of the sum of the individual values of the animals.

Folltropin is a gonadotropin preparate obtained from hyperphasis cells of slaughtered pigs with a low lh constituent. There were employed: Falltropin, a commercial product of Vetrepharm Inc., London, Ontaric, Canada. Selled in Germany by: Praemix GmbH, Mannheim, Germany.

In the foregoing table there signifies: n number of experimental animals, G1 yellow body, determined through rectal palpation, as a feature of the number of implemented ovulations, Total number of all obtained ovas, unfertilized and the embryos, numerical values referring to "total", -11- Superovulation-propagating hormone Folltropin, commercial product.

The treatment with Folltropin in Group I or the control group, displays an average obtention of 7.9 embroys and ova for each individual animal. The proportion of fertilized embryos consisted of 66% (5.1) and the proportion of implantable or transferable embryos of 48% The compound pursuant to the invention with the combination of antibodies and gonadotropenes leads to an unexpected and significant increase in the obtention of ova and embryos to 9.6 and the proportion of fertilized embryos up to 90% and the transferable embryos up to 86% When an average rate of pregnancies of 60% was employed as the basis resulting from the embryo transfer, then from 3.8 transferable embryos, Group I, in practice there could be expected an average of 2,3 pregnancies for the animals.

The 8.3 embryos which were capable of transfer by means of the compound of the invention, in contrast therewith led unexpectedly to an increase to 5.0 expected pregnancies.

The compound pursuant to the invention thus leads in a surprising manner to a doubling in the number of offspring of a donor animal in contrast with the state-of-the-art.

-12r 9 A further practical experiment was carried out under similar conditions in another opera in with cattle.

Available ware 7 cattle of the same species in the age classes of about 16 to 19 months and in the weight classes of about 320 to 360 kg. The synchronization, as well as the further treatment of the cattle was carried out as in the first experiment.

The cattle in Group I, control group, with n 3, in a deviation from Experiment 1 were treated with a total dosage of 16.9 mg NIH-FSH standard-equivalent, Folltropin. The individual dosages consisted of 3.15 3.15 2.45 2.45 1.9 1.9 mg for the respective injections.

The cattle in the Group II, Experimental group with n 4 cows, in addition to the treatment of the Control Group I, were concurrently intravenously injected against inhibin with the FSH injections each of 15 ml of a.i antiserum pooled from 6 rabbits.

The production of the rabbit antiserum, K-antiserum, against inhibin was carried out analogously with the method employed for the rams.

-13- S I Achieved were the following results: Treatm~ent: Ova/Embryos Components n Cl +-total Folltropin 16.9 mg 3 14.7 4.9 5.3 Foiltropin 16.9 mg+ 4 17.0 1.4 11.8 K-Antiserum against inhibin -14- 1 Treatment: Ova/Embryos Components fertilized transferable Folltropin 16.9 mg 4.2 4.0 3.6 75 2.0 2.0 38 Folltropin 16.9 mg+ 2.2 10.3 1.3 87 6.5 1.9 63 K-Antiserum against inhibin The compound pursuant to the invention with the combination of antibodies and gonadotropenes leads also in this experiment to a significant increase of the average obtained ova and embryos from 5.3 to 11.8, as well as to a significant increase in the proportion of fertilized embryos from 75% to 87% (10.3) and in the transferable embryos from 38% to 63% At an achievable pregnancy rate of 60% from the embryo transfers, from 2.0 transferable embryos there could be expected an average of 1.2 pregnancies.

The utilization of the compound of the invention, in contrast therewith, with 6.5 transferable embryos for each donor animal led to an increase of up to 3.9 expected pregnancies.

Thereby, the result of the first experiment was independently verified. This result is surprising.

The experiments were carried out by the inventor.

There could also be determined the following surprising result from the experiments: In both experiments, the median deviation of the individual values from a median value (average value) at higher absolute values of all investigated parameters in the groups treated with the compound pursuant to the invention were significantly lower than in the control groups. An improvement in the stimulation is especially effected also through the more constant ovary reactions from the compound of the invention.

The utilization of antibodies which neutralize inhibin is carried out pursuant to the state-of-the-art through single injections on the second or on the eighth day of the cycle.

This is verified by the following publications: J. H. M. Wrathall, et al., Journal of Endocrinology (1990) 124, 167 176. Refer to page 169, right-hand column under "Experiment lines 7 through 9: "On day 2".

G. E. Mann, et al., first column (1989) 123, pages 383 391. See page 385, left-hand column, under "Immunization experiment", line 15: "On day 8 of the luteal phase".

In contrast therewith, the compound pursuant to the invention is applied as follows: -16- There are administered a plurality of injections at defined time intervals, preferably in 12 or 24 hours over a number of successively following days, preferably between 3.5 and 4 days.

The treatment is carried out, in general, most optionally during the luteal phase, preferably from the 7th to the 11th cycle. During this interval, there appears on the ovaries a new wave of growing follicles.

A similar treatment scheme is applied during the embryo transfer in the treatment with gonadotropin.

The compound pursuant to the invention affords the technical advantage of an increase production of qualitatively high-grade, transferable embryos for each scavenging of a donor animal at considerably more constant ovary reactions.

The resultingly increased production of valuable offspring produces a significant increase in the economics of embryo transfer programs, (ET-Programs), especially with cattle.

The compound pursuant to the invention affords the further advantage that its employment is also possible in connection with such species of animals which could heretofore not be stimulated or only with a slight degree of success; especially, such as with mares.

-17w L

Claims (32)

1. A method of inhibiting the biological activity of inhibin in domestic mammals, comprising the administration of inhibin-neutralizing antibodies directed against naturally occurring or recombinant inhibin or its subunits, or its fragments or its derivatives, wherein said antibodies can neutralize the biological activity of the inhibin formed in gonads, to induce superovulation in combination with embryo transfer in domestic mammals.

2. The method of claim 1 wherein said domestic mammals are horses or cattle.

3. The method of claim 1 wherein said inhibin-neutralizing antibodies are administered in combination with at least one superovulation-propagating hormone.

4. The method of claim 3 wherein said hormone is selected from gonadotropin, gonadotropin-releasing hormone, clomiphene or tamoxifene or another anti-estrogen, and androstenedione, testosterone or dihydroxytestosterone or another anti-androgen.

A composition for inducing superovulation and increasing embryo production in combination with embryo transfer in domestic mammals comprising an inhibin- eA 4\-V eAo) neutralizing antibody and jat least one gonadotropin, gonadotropin-releasing hormone, anti-estrogen or anti-androgen aid.-cornm sition being a nnn-nturally occurring composition.

6. The composition of claim 5 wherein said inhibin-neutralizing antibody is an anti-bovine inhibin antibody.

7. The composition of claim 5 wherein said inhibin-neutralizing antibody is a polyclonal antibody.

8. The composition of claim 5 wherein said inhibin-neutralizing antibody is a monoclonal antibody. S -18- 940408,p:\oper\jms,70552-91.cla8

9. The composition of claim 5 wherein said anti-estrogen is clomiphene or tamoxifene.

The composition of claim 5 wherein said anti-androgen is androstenedione, testosterone or dihydroxytestosterone.

11. A method for inducing superovulation in a female domestic mammal which comprises administering a superovulation-inducing amount of an inhibin-neutralizing antibody to said mammal thereby inducing superovulation in the mammal.

12. The method of claim 11 which further comprises administering to said mammal at least one gonadotropin, gonadotropin-releasing hormone, anti-estrogen or anti-androgen.

13. The method of claim 11 wherein said inhibin-neutralizing antibody is administered to said mammal for 3 to 5 days.

14. The method of claim 13 wherein said inhibin-neutralizing antibody is administered to said mammal in multiple injections at about 12-24 hour intervals.

The method of claim 11 wherein said inhibin-neutralizing antibody is administered to said mammal during 'te middle luteal phase.

16. The method of claim 15 wherein said inhibin-neutralizing antibody is administered to said mammal when said mammal is growing follicles.

17. The method of claim 11 wherein said inhibin-neutralizing antibody is an anti- bovine inhibin antibody.

18. The method of claim 11 wherein said inhibin-neutralizing antibody is a polyclonal antibody. 19 940407,p:\oper\jms,70552-91.cla,19

19. The method of claim 11 wherein said inhibin-neutralizing antibody is a monoclonal antibody.

The method of claim 12 wherein said anti-estrogen is clomiphene or tamoxifene.

21. The method of claim 12 wherein said anti-androgen is androstenedione, testosterone or dihydroxytestosterone.

22. A method for increasing the number of fertilized embryos in a female domestic mammal which comprises administering an embryo-increasing amount of an inhibin-neutralizing antibody to said mammal thereby increasing the number of fertilized embryos in the mammal.

23. The method of claim 22 which further comprises administering to said mammal at least one gonadotropin, gonadotropin-releasing hormone, anti-estrogen or anti-androgen.

24. The method of claim 22 wherein said inhibin-neutralizing antibody is administered to said mammal for 3 to 5 days.

The method of claim 24 wherein said inhibin-neutralizing antibody is administered to said mammal in multiple injections at 12-24 hour intervals.

26. The method of claim 22 wherein said inhibin-neutralizing antibody is administered to said mammal during the middle luteal phase.

27. The method of claim 22 wherein said inhibin-neutralizing antibody is administered to said mammal when said mammal is growing follicles.

28. The method of claim 22 wherein said inhibin-neutralizing antibody is an anti- bovine inhibin antibody. 940407,p:\oper\jms,70552-91.cla,20

29. The method of claim 22 wherein said inhibin-neutralizing antibody is a polyclonal antibody.

The method of claim 22 wherein said inhibin-neutralizing antibody is a monoclonal antibody.

31. The method of claim 23 wherein said anti-estrogen is clomiphene or tamoxNifene.

32. The method of claim 23 wherein said anti-andirogen is ancirostenedione, testosterone or dihydroxytestosterone. Dated this 7th day of April, 1994 Stefan Heiden By his Patent Attorneys Davies Collison Cave 21 940407,p:\oper\jms70552-9l.cla,21 '1r INTERNATIONAL SEARCH REPORT International Application No PCT/EP 91/00004 I. CLASSIFICATION OF SUBJECT MATTER (if several classification symbols apply, Indicate all) According to International Patent Classification (IPC) or to both National Classification and IPC Int.C. 5 A 61 K 39/395, A 61 K 45/06, (A61 K 39/395, 37:38), (A 61 K 39/395, 31:135) II. FIEL' a SEARCHED Minimum Documentation Searched 7 Classification System j Classification Symbols Int.Cl. 5 A 61 K Documentation Searched other than Minimum Documentation to the Extent that such Documents are Included In the Fields Searched III. DOCUMENTS CONSIDERED TO BE RELEVANT I Category I Citation of Document, with indication, where appropriate, of the relevant passages 12 Relevant to Claim No. 13 X WO, A, 8600078 (MONASH UNIVERSITY et al.) 3 January 1,3-5 1986, see claims 1-4,8,9,38,39,59,60; page 19, line 13 page 20, line 9 X Chemical Abstracts, volume 112, No.l, 1 January 1,3-5 1990, (Columbus, Ohio, US), G.E. Mann et al.: Passively immunizing ewes against inhibin du- ring the luteal phase of the estrous cycle raises the plasma concentration of FSH", see page 90, abstract 915h, J. Endocrinol. 1989, 123(3), 383-91 (cited in the application) A EP, A, 0276166 DONALDSON) 27 July 1988, 2 see claims A US, A, 4864019 VALE et al.) 5 September 1989, 1,3-5 see claims 1-9; column 15, lines 3-32 A Biological Abstracts, volume 82, No.10, 1986, 1,3-5 (Philadelphia, PA, US), L.J. Cummins et al.: "Increase in ovulation rate after immunization of Merino cuves with a fraction of bovine Special categories of cited documents: 10 later document published after the International filing date document defining the general state of the art which is not or pority date and not In conlict with the application but considered to be of particular relevance invted to understton and the principle or theory underlying the invention earlier document but published on or after the international document of particular relevance; the claimed Invention Scannot be considered novel or cannot be considered to document which may throw doubts on priority claim(s) or involve an Inventive step which s cited to establish the publication date of another document of particular relevance;' the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docu- other means ments, such combination being obvious to a person skilled document published prior to the International filing date but in the art. later than the priority date claimed document member of the same patent family IV. CERTIFICATION Date of the Actual Completion of the International Search Date of Mailing of this International Search Report 12 April 1991 (12.04.91) 24 May 1991 (24.05.91) International Searching Authority Signature of Authorized Officer European Patent Office Form PCT/ISA/210 (second sheet) (January 1985). -2- Intmatloal Appication No. PCT/EP 91/00004 III. DOCUMENTS CONSIDERED TO BE RELEVANT (CONTINUED FROM THE SECOND SHET) Category* C ato of Documnmt, wtth dIt-dton, whrm pprop rt, of tm nalemvt pemages Relevant to Claim No follicular fluid containing inhibin activity", see page AB-1026, abstract 97579, J. Reprod. Fertil., 77(2): 365-372, 1986 P,X Chemical Abstracts, volume 112, No. 15, 9 April 1,3-5 1990, (Columbus, Ohio, US) M. Mizumachi et al.:"Superovulation of ewes immunized against the human recombinant inhibin alpha-subunit associated with increased pre- and postovulatory follicle- stimulating hormone levels", see page 89, abstract 132566e, Endocrinology (Baltimore) 1990, 126

1058-63 P,X Chemical Abstracts, volume 112, No. 9, 1,3-5 26 February 1990, (Columbus, Ohio, US), J.H.M. Wrathall et al.:"Inhibin immunoneutrali zation by antibodies raised against synthetic peptide sequences of inhibin alpha subunit: effects on gonadotropin concentrations and ovulation rate in sheep", see page 106, abstract 70202x, J. Endocrinol. 1990, 124(1), 167-76 (cited in the application) Form PC!ISAEIO (eira ahet) (Juiary 1345) nternatlonal Application No. PCT/EP 91/00004 1 FURTHER INFORMATION CONTINUED FROM THE SECOND SHEET SIincompletely searched V. OBSERVATIONS WHERE CERTAIN CLAIMS WERE FOUND jC This International search report has not been established In respect of certain claims under Article 17(2) ftr the following rasons: 1.3 Claim numbers...6.-., because they relate to subject matter not required to be seerched by this Authority, namely: See PCT rule 39.1(iv) Methods for treatment of the human or animal body by surgery or therapy, as well as diagnostic methods. Claim because they relate to parts of the International application that do not comply wih the prescribed require- ments to such an extent that no meaningful International search can be carried out. apacificay: 3 Ctam because they are dependent claims nd are not drafted in accordan with the econd and thi sentences of PCT Rule 6.4(a). VI.Q OBSERVATIONS WHERE UNITY OF INVENTION IS LACKING 2 This International Searching Authority found multiple inventions In this international application as follows: As alt required additional search fees wore timely paid by the applicant, this International search report covers all searchable claims of the International application. 2. As only some of the required additional search fees were timely paid by the applicant, this intrnatlonal search report covers only those claims of the International application for which fees were paid, specifically claims: No required additional search fees were timely paid by the applicant. Consequently, this International search report is retrictii to the invention first mentioned In the claims; It Is covered by claim numbers: As all seerchable claims could be searched without effort Justifying an additional fee, the International Searching Authority did not invite payment of any additional tee. Remark on Protest The additional earch tees were accompanied by appllcant's protest SNo protest accompanied the payment of additional March fees. Form PCT/ISA210O (supplemental sheet (January 1985) ANNEX TO THE INTERNATIONAL SEARCH REPORT ON INTERNATIONAL PATENT APPLICATION NO. EP 9100004 SA 43482 This annex lists the patent family members relating to the patent documents cited in the above-mentioned international search report. The members are as contained in the European Patent Office EDP file on 07/05/91 The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. Patent document Publication Patent family Publication cited in search report date member(s) date WO-A- 8600078 03-01-86 EP-A- 0185034 25-06-86 AU-A- 4437485 10-01-86 JP-T- 61502399 23-10-86 EP-A- 0276166 27-07-88 US-A- 4780451 25-10-88 AU-A- 1025988 28-07-88 US-A- 4864019 05-09-89 None o 0 M For more details about this annex see Official Journal of the European Patent Office, No. 12/82