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International Bureau 


(51) International Patent Classification 5 
A61K 37/43 


(11) Internationa) Publication Number: 
(43) International Publication Date: 

WO 91/10444 

25 July 1991 (25.07.91) 

(21) International Application Number: PCT/US 9 1/00074 

(22) International Filing Date: 4 January 1991 (04.01.91) 

(30) Priority data: 

8 January 1990 (08.01.90) US 

(71) Applicant: GENENTECH, INC. [US/US]; 460 Point San 

Bruno Boulevard, South San Francisco, CA 94080 (US). 

(72) Inventors: ATTIE, Kenneth, M ; 326 Carl Street, San 

Francisco, CA 94117 (US). MATHER, Jennie, P. ; 269 
La Prenda Drive, Millbrae, CA 94030 (US). 

(74) Agents: HASAK, Janet, E. et al.; Genentech, Inc., 460 
Point San Bruno Boulevard, South San Francisco, CA 
94080 (US). 

(81) Designated States: AT (European patent), AU, BE (Euro 
pean patent), CA, CH (European patent), DE (Euro- 
pean patent), DK (European patent), ES (European pa- 
tent), FR (European patent), GB (European patent), GR 
(European patent), IT (European patent), JP, LU (Euro- 
pean patent), NL (European patent), SE (European pa- 


With international search report. 
Before the expiration of the time limit for amending the 
claims and to be republished in the event of the receipt of 

(57) Abstract 

A method is provided for increasing fertility in a male mammal exhibiting germinal epithelium failure comprising adminis- 
tering to the mammal an effective amount of activin. Preferably, the administration is to the testis of the mammal. 


Codes used to identify States party to the PCT on the front pages of pamphlets publishing international 
applications under the PCT. 


























Burkina Faso 


United Kingdom 




























. Central African Republic 








Democratic People's Republic 





of Korea 




C6le d 'I voire 


Republic of Korea 


Soviet Union 










Sri Lanka 








United States of America 






W0 91/10444 PCT/US91/00074 


Field of the Invention 

This invention relates to a method for increasing fertility in male mammals having a low 
sperm count. 
5 Description of Related Art 

Inhibin is a glycoprotein produced by diverse tissues, including the gonads, pituitary, 
brain, bone marrow, placenta, and adrenal gland. It was initially identified by its ability to 
inhibit the secretion of follicle stimulating hormone (FSH) by the pituitary. De Jong and 
Sharpe, Nature , 2£3_: 71-72 (1 976); Schwartz and Channing. Proc. Natl. Acad. Sci. USA 74: 
5721-5724 (1977). Such preferential regulation of the gonadotropin secretion has generated 
a great deal of interest and prompted many laboratories in the past fifty years to attempt to 
isolate and characterize this substance from extracts of testis, spermatozoa, rete testis fluid, 
seminal plasma, and ovarian follicular fluid using various bioassays. Rivier et a/., Biochem. 
Biophys. Res. Commiin , 133_: 1 20 (1 985); Ling et el., Proc. Natl. Arad. Sci. USA £2: 721 7 
15 (1985); Fukuda et el.. Mol. Cell Endocrjnol,, M : 55 (1985). The structure of inhibin, 
characterized from several species, consists of two disulfide-linked subunits: an a and either 
ajJAora 0B chain. 

After the identification of inhibin. activin was shown to exist in follicular fluid as a 
naturally occurring substance. Activin was found to be capable of stimulating FSH release 
by rat anterior pituitary cells. Vale et a/., Nature. 221: 776-779 (1 986); Ling et ah, Nature. 
321: 779-782 (1 986). Activin consists of a homodimer or heterodimer of inhibin 0 subunits, 
which may be ft or ft subunits. Vale et a/.. Recent Proo. Hnrm Roc M: 1 . 34 (1988) 
There is 95-1 00% amino acid conservation of fi subunits among human, porcine, bovine, and 
rat activins. The 0 A and 0 B subunits within a given species are about 64-70% homologous. 
The activin /? A and ft homodimers ("Activin A" and "Activin B," respectively) have been 
identified in follicular fluid, and both molecules have been cloned and their genes expressed. 
Mason et a/.. Biochem. Biophvs. Res, Cnmm^n j^5 : g 57 (1986); EP Pub. No. 222,491 
published May 20. 1987; Mason eta/., Molecular Enrinrrinnl 3: 1352-1358 (1989). The 
complete sequence of the ft subunit is published in Serono Symposium Publications, entitled 
■Inhibin- Non-Steroidal Regulation of Follicle Stimulating Hormone Secretion", eds. H:G. 
Burger et a/., abstract by A.J. Mason et a/., vol. 42. pp. 77-88 (Raven Press: New York 
1987), entitled "Human Inhibin and Activin: Structure and Recombinant Expression in 
Mammalian Cells." 

Both Activin A and Activin AB. but thus far not Activin B, have been isolated from 
natural sources. Activin mRNA (ft and 0 B subunits). bioactivity. and immunoactivity have 
been reported to be produced by testicular Leydig cells from immature rat and pig. Lee-er el., 
Ssienss, 243: 396-398 (1 989); Lee et al., in Serono Symposium Publications, entitled "The 
Molecular and Cellular Endocrinology of the Testis." Cooke and Sharpe, eds.. Vol. SO (Raven 





WO 91/10444 



Press: New York, 1 988), p. 21-27. Activin A has been found recently to have erythropoietic- 
stimuiating activity as well as FSH-releasing activity. See EP Publ. No. 210,461 published 
February 4, 1987 (where the protein is called BUF-3), Eto et aL, Biochem. Biophvs. Res. 
Commun. , 142: 1095-1103 (1987) and Murata et aL, Proc. Natl. Acad, Sci. U.S.A., 85: 
5 2434-2438 (1988) (where the activin is called EDF), and Yu et aL. Nature . 330 : 765-767 
(1987) (where the activin is called FRP). In these systems, inhibin antagonized the actions 
of activin. 

A protein known as follicle or follicular regulatory protein having a molecular weight 
of 1 2,00 to 1 5,000 is found to inhibit aromatase levels, modulate the formation of mature 

10 ova substantially independently of steroidal sex hormones, and reduce fertility in the male rat 
by systemic treatment. It does not directly affect the gonadotropin output of the pituitary. 
See U.S. Pat. No. 4,734,398; Tsutsumi et aL, Fertil. Steril.. 47: 689 (1987); Lew et aL, 
Obstet. and GvnecoL 70: 157-162 (1987); diZerega et aL, Meiotic Inhibition: Molecular 
Control of Meiosis (Alan R. Uss, Inc., 1988), p. 201-226; diZerega et aL, J. Steroid 

15 Biochem.. 27: 375-383 (1987); Montz et aL, Am. J. Obstet. GvnecoL. 436-441 (Feb. 15, 
1984); Ahmad et aL, the Anatomical Record. 224 : 508-513 (1989). This protein, also 
named FRP, has been purified and partially sequenced, and is not related in any way to the 
FSH-releasing protein known as activin, referred to as FRP by the Salk researchers in their 
early work. 

20 Recently, the expression of inhibin subunits, each encoded by a separate gene, was 

demonstrated in several tissues in addition to ovary and testis. Inhibin a, 0 A , and 0 B mRNAs 
were detected in placental, pituitary, adrenal, bone marrow, kidney, spinal cord, and brain 
tissues. Meunier et aL, Proc. Natl. Acad. Sci. USA. £5: 247 (1988). The expression of the 
inhibin subunit mRNAs varied by several-fold in a tissue-specific manner, suggesting different 

25 functions for these proteins depending on their pattern of association and their site of 

Inhibin and activin are members of a family of growth and differentiation factors. The 
prototype of this family is transforming growth factor-beta (TGF-/?) (Derynck et aL, Nature, 
316: 701-705 (1985)), which, according to one source, possesses FSH-releasing activity. 

30 Ying et aL, Biochem. Biophvs. Res. Commun.. 135: 950-956 (1 986). Other members of the 
TGF-A family include the Mullerian inhibitory substance, the fly decapentaplegic gene 
complex, and the product of Xenoous Vg-1 mRNA. 

In the human, growing preovulatory follicles and corpus luteum secrete inhibin into the 
circulation in response to FSH stimulation. Lee and Gibson, Aust. J. Biol. Sci.. 3g : 1 1 5-120 

35 (1 985); McLachlan et aL, Fertil. Steril.. 48: 1 001 (1 987). Thus, inhibin-related peptides play 
important roles in the modulation of gonadal functions via a pituitary feedback loop. In rat 
primary cultures of testis cells and ovarian thecal-interstitial cells, inhibin has been reported 
to enhance androgen biosynthesis stimulated by leutinizing hormone (LH), whereas activin 

WO 91/10444 PCT/US91/00074 


suppresses androgen production. Hsueh et al.. Proc. Natl. Acad. Sci. USA. £4j 5082-5086 
(1987). Other workers have been unable to repeat these observations. deKretser and 
Robertson, Biology of Renmrii.rtinn 4Q: 33.47 (1 989), particularly p. 41 . Inhibitory effects 
of TGF-0 on Leydig cell steroidogenesis have also been described. Lin et al., Biochem. 
5 Bjppfrys. pes. Commun., Jifi: 387 (1987); Fauser and Hsueh, Life Sci.. 4^: 1363 (1988); 
Avallet et a/., Biochem. Biophvs. Res. Cnmmim. 14§: 575 (1 987). In granulosa cells, activin 
has been reported to inhibit (and TGf-0 to enhance) progesterone production. Ignotz and 
Massague, J. Biol, Chem.., 261: 4337 (1986). In primary cultures of granulosa cells, activin 
and inhibin as well as TGF-0 were found to affect hormone synthesis and secretion, each in 
10 a different fashion. Adashi and Resnick, Endocrinology. 119; 1879 (1986); Ying et a/., 
Biochem. Bipphvs. Res. Common. , JL3£: 969 (1 986); Hutchinson et al., Biochem. Bionhvs. 
Res. Commun. , 14£: 1405 (1987); Mondschein er at., Endocrinology. 123 : 1970 (1988); 
Feng et el., J. Biol, Chem., 26J.: 14167 (1986). These molecules have both positive and 
negative effects on FSH-dependent granulosa cell function. Carson et al.. J. Reprod. Fert.. 
1 5 8J5: 735-746 (1 989). Also suggested is that individual members of the TGF-0/inhibin gene 
family regulate ovarian function, not only by direct action on follicle cells, but also indirectly 
by influencing the production rate of other members of that family. Zhiwen et al., Molecular 
and Cellular Endocrinology £g : 161-166 (1988). 

Activin A and inhibin were reported to modulate growth of two gonadal cell lines, 
20 suggesting that these proteins may regulate proliferation as well as functions of gonadal cells. 
Gonzalez-Manchon and Vale, Endocrinology. 1666-1672 (1989). The secretion of 
inhibin by the corpus luteum has been proposed to suppress the concentration of FSH in the 
luteal phase of the cycle and hence the inhibition of follicular development. Baird et al., Ann. 
N. Y. Acad. Sri.. E41- 153-161 (1988). 
25 a review article postulates that inhibin is at least one of the factors that determines 

the number of follicles destined to ovulate, and that interference with the action of inhibin 
might contribute to the regulation of fertility. De Jong, Phvsiol. Rpv. 68: 555 (1 988). Many 
investigators have speculated that due to its FSH-inhibiting effect, inhibin may be useful in 
male and female contraception. Sheth and Moodbidri, Adv. Contracept . 2: 131-139 (1986); 
30 Hndlay. Fertil. Steril., 46: 770 (1 986); van Dissel-Emiliani er al. . Endocrinology. 125 : 1 898- 
1903 (1989); Bhasin eta/., Endocrinology, 124: 987991 (1989). However, another author 
doubts that inhibin can inhibit spermatogenesis (citing Bremner et al., J. Clin. Invest.. gg : 
1044 (1981)). and states that inhibin might also have some direct stimulatory effects on 
spermatogenesis. Baker et al.. Clin. Rcnrnri. ^ F fn , g : 161-174 (1983). 
35 The distributions of the 0, 0„ and 0 B subunits of inhibin/activin polypeptides were 

studied in the testis of rats. It was found that in the rat testis, both Sertoli and interstitial 
cells produce inhibin/activin subunits, and the o and 0 subunits are produced by different 
types of interstitial cells in immature rats. Roberts et al., Endocrinology . J2&: 2350 (1989). 

WO 91/10444 



Also it was found that immunoreactive inhibin subunits are present in multiple cells in the 

testis and that the amounts of immunostainable subunits in the seminiferous epithelium are 

differentially regulated. Shaha et aL. Endocrinology , 125: 1941 (1989). 

Activin bioactivity has been reported to be secreted by interstitial cells in in vitro , 
5 while Sertoli cells secrete inhibin or a mixture of inhibin and activin. Lee et aL, in Serono 

Symposium Publications, supra; Lee et aL, Science, supra. 

Many substances produced in the testes have been shown to regulate testicular 

function locally. Mather, in Mammalian Cell Culture, ed. J. Mather (Plenum Publishing Corp. 

1984), p. 167-193. While inhibin and activin have primarily been considered as feedback 
0 regulators of pituitary function, in light of recent data on multiple sites of production and 

action in the testis, it seems likely that they may also play a role as local regulators of 

testicular function. 

Failure to conceive is a complaint that leads as many as one in six married couples to 
seek medical attention. Of these couples, at least 40% will be discovered to have a male 

5 factor deficiency. Approximately 61% of infertile men have hypospermatogenesis on 
testicular biopsy. These patients have partial germinal epithelium failure and present with 
oligospermia and normal testosterone levels. The etiology is often idiopathic, but may be 
associated with antineoplastic agents, cryptorchidism, or varicoceles. 

In the male, the maturation of immature germ cells into spermatozoa (spermatogenesis) 

0 is thought to be regulated by the gonadotropins (LH and especially FSH) and androgens 
(testosterone). The current treatments for male infertility due to oligospermia are based on 
this assumption. These include induction of rebound from testosterone or anabolic androgen- 
induced azospermia; administration of exogenous gonadotropins or gonadotropin releasing 
hormone; use of clomiphene citrate or tamoxifen to stimulate endogenous gonadotropin 

5 secretion; administration of low doses of mesterolone, an oral synthetic androgen; and use 
of an aromatase inhibitor such as testolactone. 

No study has conclusively established the benefit of these treatments, although one 
report suggests that a statistically significant effect is exerted only by clomiphene citrate. 
However, clomiphene has been shown to be problematic in the high doses used by women. 

3 Furthermore, the gonadotropins and androgens may act primarily in an indirect manner, via 
stimulation of Sertoli and/or Leydig cell factors that affect the germinal epithelium directly. 

Accordingly, it is an object of the present invention to provide a method for increasing 
fertility of men with oligospermia using a fertility agent that causes direct stimulation of 
sperm production by local administration. 

5 It is another object to provide a fertility agent to treat hypospermatogenesis that is 

both safe and efficacious. 

This object and other objects will be apparent to one of ordinary skill in the art. 

WO 91/10444 



Summar v of the Invention 
The present invention provides a method of increasing fertility in a male mammal 
exhibiting germinal epithelium failure comprising administering to the mammal an effective 
amount of activin. 

In another aspect, the invention provides a pharmaceutical composition for increasing 
fertility in male mammals exhibiting germinal epithelium failure comprising an effective 
amount of activin in a pharmaceutical^ acceptable carrier. 

Brief Desc ription of the Drawings 

Figure 1 shows graphs of the level of incorporation of 3H-thymidine after 24 hours, 48 
hours and 72 hours of treatment of rat Sertoli and germ cell cocultures with added activin A 
(A, 100 ng/mi), added inhibin (I, 100 ng/ml), or control (C). All conditions contained medium 
plus 5F (which is insulin, 5 //g/ml; transferrin, 5 //g/ml; c-tocopherol, 5 //g/ml; EGF, 5 ng/ml; 
and aproteinin, 25 //g/ml). 

Figure 2A shows a graph of DNA flow cytometric quantification of inhibin-treated rat 
Sertoli and germ cell cocultures at 48 hours compared with a control (5F). Figure 2B shows 
a similar graph comparing activin treatment with a control (5F). 

Description of the Preferred Embodiment^ 

As used herein, the term "activin" refers to homo- or heterodimers of 0 chains of 
inhibin, prepro forms, and pro forms, together with glycosylation and/or amino acid sequence 
variants thereof. After cleavage from the mature protein, the precursor portion may be non- 
covalently associated with the mature protein. Activin A refers to activin with the two chains 
of 0 A . Activin AB refers to activin with the chains 0 A and ft. Activin B refers to activin with 
the two chains of 0 B . 

The intact isolated prepro or prodomain or mature 0 A and 0 B sequences are suitably 
synthesized by any means, including synthetic and/or recombinant means, but are preferably 
synthesized in recombinant cell culture, for example, as described in U.S. Pat. No. 4,798,885 
issued January 17, 1989. 

It is within the scope hereof to employ activin from animals other than humans, for 
example, porcine or bovine sources, to treat humans. For example, the nucleotide and 
deduced amino acid sequences of the porcine activin 0 chain are found in Figures 2A and 2B 
of U.S. Pat. No. 4,798,885, supra. Likewise, if it is desirable to treat other mammalian 
species such as domestic and farm animals and sports, zoo, or pet animals, human activin, 
as well as activin from other species, is suitably employed. 

Generally, amino acid sequence variants will be substantially homologous with the 
relevant portion of the mammalian 0 chain sequences set forth in, e.g., U.S. Pat. No. 
4,798,885, supra. Substantially homologous means that greater than about 60% of the 
primary amino acid sequence of the homologous polypeptide corresponds to the sequence 
of the activin chain when aligned to maximize the number of amino acid residue matches 

WO 91/10444 



between the two proteins. Alignment to maximize matches of residues includes shifting the 
amino and/or carboxyl terminus, introducing gaps as required, and/or deleting residues present 
as inserts in the candidate. Typically, amino acid sequence variants will be greater than 
about 70% homologous with the corresponding native sequences. 

While the site for introducing a sequence variation is predetermined, it is unnecessary 
that the mutation per se be predetermined. For example, in order to optimize the 
performance of mutation at a given site, random mutagenesis may be conducted at the target 
codon or region and the expressed activin mutants screened for the optimal combination of 
desired activity. Techniques for making substitution mutations at predetermined sites in DNA 
having a known sequence are well known, for example, M13 primer mutagenesis. 

Mutagenesis is conducted by making amino acid insertions, usually on the order of 
about from 1 to 10 amino acid residues, or deletions of about from 1 to 30 residues. 
Substitutions, deletions, insertions, or any subcombination may be combined to arrive at a 
final construct. Preferably, however, substitution mutagenesis is conducted. Obviously, the 
mutations in the encoding DNA must not place the sequence out of reading frame and 
preferably will not create complementary regions that could produce secondary mRNA 

Covalent modifications of activin are included within the scope of the invention, and 
include covalent or aggregative conjugates with other chemical moieties. Covalent 
derivatives are prepared by linkage of functionalities to groups that are found in the activin 
amino acid side chains or at the N- or C-termini, by means known in the art. For example, 
these derivatives will include: aliphatic esters or amides of the carboxyl terminus or residues 
containing carboxyl side chains, e.g., aspartyl residues; O-acyl derivatives of hydroxyl group- 
containing residues such as aryl or alanyl; and N-acyl derivatives of the amino terminal amino 
acid or amino-group containing residues, e.g., lysine or arginine. The acyl group is selected 
from the group of alkyl moieties (including C3 to CIO normal alkyl), thereby forming alkanoyl 
species, and carbocyclic or heterocyclic compounds, thereby forming aroyl species. The 
reactive groups preferably are difunctional compounds known per se for use in crosslinking 
proteins to insoluble matrices through reactive side groups, e.g., m-maleimidobenzoyl-N- 
hydroxy succinimide ester. Preferred derivatization sites are at histidine residues. 

The expression "administering to the testis" means not only injection into the testis, 
but also techniques that result in flooding the area surrounding the testis with activin such 
that the activin is absorbed into the testis. In addition, the activin can be injected into a 
vessel that feeds the testis, preferably using a microscopic procedure. Furthermore, the 
activin can be put into an implant that is placed near the testis and through which the activin 
is absorbed into the testis. Examples include an intratesticular long-acting depot (e.g., 
microsphere) or slow-release implant. Other techniques may be employed, provided that the 

WO 91/10444 



result is that activin is applied locally to the testis and is effective for the purposes stated 

The expression "germinal epithelium failure" refers to disorders of male mammals that 
may be characterized as complete or partial germinal epithelium failure, provided that some 
5 spermatogonal stem cells are present, as determined, e.g., by a testis biopsy analysis. 
Examples of such disorders include those characterized as partial germinal epithelium failure 
as well as azoospermia presenting in patients who have some spermatogonal stem cells. 
Complete failure is associated with high basal FSH levels. 

The expression "partial germinal epithelium failure" refers to a disorder of mammals 
1 0 that present with oligospermia and intact Leydig cell steroidogenic capacity and pituitary cells. 
Such males have normal testosterone levels but low sperm counts. Most clinicians consider 
a sperm density of less than 20 million/ml with adequate volume, motility and morphology 
to indicate low sperm count. Sperm morphology is another indication, with one suggestion 
that low sperm count is evidence when the percentage of abnormal spermatozoa is above 40. 
15 The disorders characterized as partial germinal epithelium failure may be caused by 

chemicals or drugs such as chemotherapeutic drugs and sulfa antibiotics, as well as alcohol 
and illicit drugs. Other possible causes include genetic disorders, genital tract infections, and 
varicoceles. The largest group of infertile men falls into the category of idiopathic 
oligospermia, without an evident etiology. The need for an increase in fertility is generally 
20 due to a primary testicular disorder, i.e., not at the hypothalamic or pituitary level. 

The present invention concerns itself with using activin to increase fertility in male 
mammals in the patient population identified above, including sports, zoo, pet, and farm 
animals such as dogs, cats, cattle, pigs, horses, monkeys, and sheep, as well as humans. 
Preferably the disorder is partial germinal epithelium failure. 
25 The activin is administered to the mammal by any suitable technique, including 

parenteral, sublingual, intratesticular, intrapulmonary, and intranasal administration. The 
specific route of administration will depend, e.g., on the medical history of the patient. 
Examples of parenteral administration include intramuscular, subcutaneous, intravenous, 
intraarterial, and intraperitoneal administration. Preferably, the activin is administered via the 
30 testis, as discussed above. 

The activin compositions to be used in the therapy will be formulated and dosed in a 
fashion consistent with good medical practice, taking into account the clinical condition of 
the individual patient, the site of delivery of the activin composition, the method of 
administration, the scheduling of administration, and other factors known to practitioners. 
35 The "effective amount" for purposes herein is thus determined by such considerations. 

As a general proposition, the total pharmaceutical^ effective amount of the activin 
administered per dose will be in the range of about 1 //g/kg/day to 10 mg/kg/day of patient 
body weight, although, as noted above, this will be subject to a great deal of therapeutic 

WO 91/10444 PCI7US91/00074 


discretion. Preferably, this dose is no more than about 10 //g/kg/day. The key factor in 
selecting an appropriate dose is the result obtained, as measured by increases in sperm 
density by serum analysis or the number of spermatocytes, or by other criteria as deemed 
appropriate by the practitioner, e.g., biopsy. 
5 For administration, the activin is formulated generally by mixing it at the desired degree 

of purity, in a unit dosage injectable form (solution, suspension, or emulsion), with a 
pharmaceutical^ acceptable carrier, i.e., one that is non-toxic to recipients at the dosages 
and concentrations employed and is compatible with other ingredients of the formulation. 
For example, the formulation preferably does not include oxidizing agents and other 
1 0 compounds that are known to be deleterious to polypeptides. 

Generally, the formulations are prepared by contacting the activin uniformly and 
intimately with liquid carriers or finely divided solid carriers or both. Then, if necessary, the 
product is shaped into the desired formulation. Preferably the carrier is a parenteral carrier, 
more preferably a solution that is isotonic with the blood of the recipient. Examples of such 
1 5 carrier vehicles include water, saline, Ringer's solution, and dextrose solution. Non-aqueous 
vehicles such as fixed oils and ethyl oleate are also useful herein, as well as liposomes. 

The carrier suitably contains minor amounts of additives such as substances that 
enhance isotonicity and chemical stability. Such materials are non-toxic to recipients at the 
dosages and concentrations employed, and include buffers such as phosphate, nitrate, and 
20 other organic acid salts; antioxidants such as ascorbic acid; low molecular weight (less than 
about ten residues) polypeptides, e.g., polyarginine or tripeptides; proteins, such as serum 
albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; 
amino acids, such as glycine, glutamic acid, aspartic acid, or arginine,; monosaccharides, 
disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, 
25 mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or 
sorbitol; counterions such as sodium, and/or nonionic surfactants such as Tween, Pluronics, 
or PEG. 

The activin is typically formulated in such vehicles at a concentration of about 0.1 
mg/ml to 100 mg/ml at physiological pH. It will be understood that use of certain of the 
30 foregoing excipients, carriers, or stabilizers will result in the formation of activin salts. 

Activin to be used for therapeutic administration must be sterile. Sterility is readily 
accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes). 

Therapeutic activin compositions generally are placed into a container having a sterile 
access port, for example, a vial having a stopper pierceable by a hypodermic injection needle. 
35 Activin ordinarily will be stored in unit or multi-dose containers, for example, sealed 

ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitute. 
As an example of a lyophilized formulation, 10-ml vials are filled with 5 ml of sterile-filtered 
1% (w/v) aqueous activin solution, and the resulting mixture is lyophilized. The infusion 

WO 91/10444 


solution is prepared by reconstituting the lyophilized activin using 5 ml of sterile water or 
Ringer's solution. 

The activin is also suitably administered by sustained release systems. Suitable 
examples of sustained release compositions include semi-permeable polymer matrices in the 
5 form of shaped articles, e.g., films, or microcapsules. Sustained release matrices include 
polyiactides (U.S. Pat. No. 3.773.919. EP 58.481), copolymers of L-glutamic acid and 
gamma-ethyl-L-glutamate (U. Sidman et al., Biopolvmers. 2j>, 547-556 (1983H, poly(2- 
hydroxyethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res. . i§ : 167-277 (1981). 
and R. Langer, Chem. Tech., J2: 98-1 05 (1 982)). ethylene vinyl acetate (R. Langer et ah. IdJ 

1 0 or poly-D-(-)-3-hydroxybutyric acid (EP 1 33,988). Sustained release activin compositions also 
include liposomally entrapped activin. Liposomes containing activin are prepared by methods 
known per se: DE 3,21 8, 1 2 1 ; Epstein et al., Proc. Natl. Arari. Sri U.S.A., fi2j 3688-3692 
(1985); Hwang et al.. Proc. Natl. AraH s ff |, 1 1 g a yj. 4030-4034 (1980); EP 52,322; EP 
36,676; EP 88.046; EP 143,949; EP 142,641; Japanese Pat. Appln. 83-118008; U.S. Pat. 

1 5 Nos. 4,485.045 and 4.544,545; and EP 1 02.324. Ordinarily, the liposomes are of the small 
(about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 
30 mol. percent cholesterol, the selected proportion being adjusted for the optimal activin 

Activin therapy is suitably combined with other proposed or conventional fertility 
20 increasing therapies. For example, activin can be administered with other fertility agents used 
to stimulate proliferation and differentiation of germ cells. 

Examples of other therapies or agents include induction of rebound from testosterone 
or anabolic androgen-induced azospermia; administration of exogenous gonadotropins or 
gonadotropin releasing factors such as human chorionic gonadotropin (hCG), human 
25 menopausal gonadotropin (hMG), purified FSH, or gonadotropin releasing hormone (GnRH). 
Alteratively, ciomiphene citrate or tamoxifen may be used in conjunction with activin to 
stimulate endogenous gonadotropin secretion. In addition, low doses of mesterolone, an oral 
synthetic androgen, or an aromatase inhibitor such as testolactone may be administered. 

The inhibin and fertility agents are suitably delivered by separate or the same means, 
by separate or the same administration route, and at the same or at different times, 
depending, e.g.. on dosing, the clinical condition of the patient, etc. It is not necessary that 
such fertility agents be included in the activin compositions per se, although this will be 
convenient where such drugs are delivered by the same administration route. 

When employed together with the activin, such agents typically are employed in lesser 
35 dosages than when used alone. If hCG is used, preferably the effective amount is 1 500 to 
2000 I.U. twice weekly until testosterone levels are in the adult male range. At that point. 
hMG in a dose of about one ampule every other day is also administered. |f ciomiphene 
citrate therapy is employed, treatment is typically 25 mg of ciomiphene citrate daily for 21 


WO 91/10444 



to 25 days, followed by a 5- to 7-day rest period. This cycle is generally repeated for at least 
24 weeks. 

A typical combined composition will contain the above-noted amount of activin and 
about 25 mg of clomiphene citrate in a suitable intraperitoneal fluid such as lactated Ringer's 
5 solution. 

The invention will be more fully understood by reference to the following examples. 
They should not however, be construed as limiting the scope of the invention. 


Sertoli cells and germ cells from 20-day-old male Sprague-Dawley rats (Charles River 
10 Laboratories, Inc., Wilmington, MA) were co-cultured in serum-free media. Cultures were 
prepared according to the glycine/collagenase method described by Mather and Phillips in 
Methods for Serum-Free Culture of C ells of the Endocrine System. Barnes and Sato eds. (Alan 
R. Liss, inc.: New York, 1984), p. 29-45, and Rich et aL, Endocrinol. . 113 : 2284-2293 

15 Briefly, testis were removed and decapsulated and the tubules teased apart in a 

hypertonic glycine solution. The return of the tubules to isoosmotic medium results in lysis 
of the interstitial tissue without harming the tubules. The tubules were then minced into 
smaller segments and enzymatically treated with collagenase/dispase to remove the basement 
membranes and peritubular cells. The peritubular cells were discarded and the tubular pieces 

20 of 1-5 mm in length, which contain Sertoli cells and spermatogonia and spermatocytes, were 
plated in serum-free Ham's F1 2/DME medium supplemented with HEPES and insulin, 5 //g/ml; 
transferrin, 5 //g/ml; a-tocopherol, 5 ^g/ml; epidermal growth factor (EGF), 5ng/ml; and 
aproteinin, 25 //g/ml (5F). After 20-24 hours the Sertoli cells had attached to the substrate 
and spread to form a monolayer. Spermatocytes could be seen adhering to the monolayer 

25 as single cells or groups of two cells or floating unattached in the medium. 

At 24 hours after plating the medium was changed and unattached ceils were 
discarded. Fresh 5F medium was added to all cultures, and additionally 100 ng/ml human 
recombinant inhibin A or activin A (prepared and purified as described in U.S. Pat. No. 
4,798,885 issued January 17, 1989) was added to the experimental conditions. All 

30 conditions were assayed in triplicate and the entire experiment was repeated multiple (> 1 0) 

Between 24 and 48 hours of treatment, clusters of spermatogonia and increased 
numbers of primary spermatocytes appeared in the activin-treated wells. These cells appear 
as connected clusters of 8-32 cells attached to the Sertoli cell monolayer and large cells in 
35 suspension. No such effect was seen with inhibin. 

Each well contained 2 million cells. A total of 1 //Ci of 3H-thymidine was added to 
each well after 24, 48, or 72 hours of treatment with activin or inhibin. 

WO 91/10444 PCT/US91/00074 


Label incorporation into cells was measured after 20 hours of incubation with 3H- 
thymidine. Cells were detached from the substrate by vigorous pipetting with a 1 ml 
Pipettman™ pipettor. and the entire contents of the well was transferred to a 1 0-ml filter well 
containing two glass fiber filters and 5 ml of cold 20% trichloroacetic acid. The precipitated 
5 cells were caught on the filter and washed two times with cold 5% trichloroacetic acid to 
remove unincorporated 3H-thymidine. Filters were washed once with cold methanol and 
counted in a scintillation fluid appropriate for aqueous samples. 

The results are shown in Figure 1 . Incorporation was higher in the activin-treated wells 
compared with untreated control or inhibin-treated wells in all cases. At 48 hours of curturing 
1 0 the activin was at its highest incorporation (1 1 ,040 cpm ±_ 1 572 SEM) relative to the control 
(4515 .+ 597) and inhibin-treated cultures (5355 ± 466). Thus, activin increases the 
proliferation of the spermatocytes. 

The effect of inhibin and activin on germ cell differentiation was quantified by flow 
cytometric analysis. Sertoli cells were stained with Nile red (a selective fluorescent stain for 
intracellular lipid droplets) and non-staining germ cells were electronically gated. A DNA- 
specific fluorochrome (Hoechst 33342) was used to determine the percentage of germ cells 
with N, 2N or 4N DNA content, with 4N = primary spermatocytes. As seen from Figures 2A 
and 2B, activin-treated cultures had a significant increase in the percentage of 4N germ cells 
as compared with control or inhibin-treated cultures at 48 hours. 

In conclusion, activin stimulates the proliferation and differentiation of 20-day old rat 
testicular germ cells in vitro, indicating that it will increase fertility in the male. The data also 
indicate that local administration of activin, being mitogenic for germ cells via, e.g., an 
intratesticular depot method, would cause direct gonadal stimulation of sperm production, 
independent of (or possibly in concert with) changes in gonadotropin secretion. 



WO 91/10444 




1 . A method of increasing fertility in a male mammal exhibiting germinal epithelium 
failure comprising administering to the mammal an effective amount of activin. 

2. The method of claim 1 wherein the activin is porcine or human activin A, activin 
AB, or activin B. 

3. The method of claim 2 wherein the activin is human activin A. 

4. The method of claim 1 wherein the administration is to the testis. 

5. The method of claim 4 wherein the administration is by injection into the testis. 

6. The method of claim 1 wherein the mammal is human. 

7. The method of claim 1 wherein the effective amount is a daily dose of about 1 
pg/kQ to 10 mg/kg. 

8. The method of claim 1 wherein the germinal epithelium failure is partial germinal 
epithelium failure. 

9. A pharmaceutical composition for increasing fertility in male mammals exhibiting 
germinal epithelium failure comprising an effective amount of activin in a pharmaceutical^ 
acceptable carrier. 

10. The composition of claim 9 wherein the activin is porcine or human activin A f 
activin AB, or activin B. 

1 1 . The composition of claim 10 that contains human activin A. 

12. The composition of claim 9 wherein the carrier is sterile water, a buffer, or 
Ringer's solution. 

WO 91/10444 


1 / 1 



FIG. 1 ,5fi0 * 







24 HR 



48 HR 




72 HR 

FIG. 2A 

2N 4N 


FIG. 2B 






InUmatlonal Aepllcatlon No PCT/US 91/00074 

1. CLASSIFICATION OF SUBJECT MATTER (If intra! da ittfi cation symbols apply. Indicate alt) • 

According to International Patsnt Classification (IPC) or to both National Classification and IPC 

IPC 5 : A 61 K 37/43 


Minimum Documentation Ssarchtd » 

Classification System 

Classification Symbols 

IPC 5 

A 61 K, C 07 K' 

Documentation Ssarchtd othar than Minimum Documentation 
to the Extant that such Oocumants art Included In the Fltlds Sesrched • 


Category a 

Citation ot Document, " with Indication, where appropriate, of the rtlevant passages '* 

Relevant to Claim No. " 

US, A, 4864019 (WYLIE W. VALE et al. ) 
5 September 1989 

• Special categories of cited documents: *e 

"A" document defining the general state of the art which la not 
considered to be of particular relevance 

"E" earlier document but published on or after the International 
filing date 

*L" document which may throw doubts on priority clalm(a) or 
which Is cited to establish ths publication date of another 
citation or ether special reason (ss specified) 

*0" document referring to an oral disclosure, use, exhibition or 
othar means 

-P" document published prior to the international filing date but 
later than the priority date claimed 

"T" later document p utile bed attar the International filing date 
or priority date and not In conflict with the application but 
cited to understand the principle or theory underlying the 

"X" document of particular relevance; the claimed Invention 
cannot be considsrad novel or cannot be conaldered to 
involve an Inventive atep 

•Y" document of particular relevance;' the claimed Invention 
cannot be conaldered to Invohre an Inventive sttp when the 
document la combined with one or more other such docu- 
ments, such combination being obvious to a peraon skilled 
in the art 

document member of the same patent family 


Oil* o< th. Actual Completion of tho InUrnatloiul Soarch 

16th April 1991 

Data of Mailing of this International Search Report 

International Searching Authority 


^jgnatn ol Authoriiad Ofllcir 

(jjtJlCU13 Danielle van der Haas 

Form PCT /ISA/210 1 second sheet) (January 1615) 

International Application No. PCT/US 91/00074 



This Internationa ( search report haa not be*n established in respect of certain dalma under Article 17(2) (a) for the following reaaons: 
Claim numbers . !./„.??., because they relate to subject matter not required to be searched by this Authority, namely: 

Please see Rule 39.1 (iv): methods for treatment of the 
human or animal body by surgery or therapy, as well as 
diagnostic methodes 

2.n Claim numbers , becauae they relate to parts of the International application that do not comply with the prescribed require* 

ments to such an extent that no meaningful international search can be carried out. spectneally : 

3 J | Claim numbers because they are dependent claims and are not drafted hi accordance with the aecond and third sentences of 

PCT Ru!e6.4<a). 


This International Searching Authority found multiple Inventions In thla International application as follows: 

As all required additional search fees were timely paid by the applicant, this International search report covers all searchable dalma 
of the International application, 

2Q As only some of the required additional search fees were timely paid by the applicant, this International 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 la restricted to 
the Invention first mentioned In the claims; It Is covered by claim numbers: 

4.| | As all •earchable claims could be searched without effort justifying an additional fee* the International Searching Authority did not 
— invite payment of any additional fee. 

Remark on Protest 

Q The additional search fees were accompanied by applicant's protest 

n No protest accompanied the payment of eddtttonal aearch teea. 

Form PCT /IS A/210 (supplements! sheet (2)) (January 1985) 




SA 44281 

This annex lists the patent family members relatmg to the patent documents cited in the above-mentioned internatioaal search report. 
The members are as contained in the European Patent Office EDP file on 27/05/91 

The European Patent Office is in no way liable for these particulars which are merely given for the purpose of information. 

Patent document 
cited in search report 


Patent family 


US-A- 4864019 






w For more details about this annex : see Official Jearaal of the European Patent Office, No. 12/82 

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