Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
2001-08-29
2003-12-09
Kemmerer, Elizabeth (Department: 1646)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C424S085100, C530S350000, C530S351000, C530S397000, C530S399000
Reexamination Certificate
active
06660717
ABSTRACT:
The present invention relates to the use of Growth Differentiation Factor-9 (GDF-9) in assisted reproduction and to kits comprising GDF-9.
Reproduction involves the growth and selection of ovarian follicles, leading to the ovulation of one healthy (high quality) oocyte that can be fertilised. Subsequently the fertilised oocyte develops to an embryo, which implants in a receptive endometrium and grows to a foetus and finally a healthy baby. Couples with fertility problems, however, need to undergo treatment in order to obtain children. At the moment two major treatment programs are available: Ovulation Induction (OI), for couples with female infertility due to anovulatory cycles, and In Vitro Fertilisation (IVF) for couples with either female or male fertility problems. In the case of OI protocols, low doses of gonadotropins are administered in order to induce mono-follicular growth and the ovulation of one oocyte that can be fertilised naturally. In the case of IVF relatively high levels of gonadotropins are administered in order to obtain multi-follicular development. After the induction of multi-follicular growth, oocyte maturation is induced with Luteinizing Hormone (LH)/human Chorionic Gonadotropin (hCG) and mature oocytes are retrieved by follicular puncture. After in vitro fertilisation and in vitro early embryo cleavage, 2 to 3 embryos are transferred into the uterus.
In both treatments, the gonadotropin dosing has to be adapted to individual patients in order to obtain mono- or multi-follicular development. This is extremely difficult, and success is not guaranteed. Moreover, final pregnancy outcome rates are relatively low (~20%) due to many factors, among which oocyte quality and endometrium receptivity. The cause of the relatively low success rates may lay in the way folliculogenesis and oogenesis are induced.
During the natural menstrual cycle, the gonadotropins FSH and LH induce follicle and oocyte development. Due to selection, which most likely is also controlled by FSH, only one follicle from the growing follicle pool becomes dominant and finally releases a healthy, fertilisable oocyte at ovulation. The other follicles become atretic and never will ovulate but degenerate.
It is well known that not only gonadotropins are needed for optimal follicle and oocyte development, but also growth factors. GDF-9 is such a growth factor. Its nucleotide and amino acid sequence has been described by Incerti et al (Biochim.Biophys.Acta (1994), 1222, 125-128) and McGrath et al (Mol. Endo. (1995), 9, 131-136). In GDF-9 knockout mice it has been demonstrated that follicular development ceases at the primary stage due to GDF-9 deficiency (Dong et al., 1996). Moreover it has been demonstrated that GDF-9 is present in follicles (in the oocytes to be more specific) from the primary stage up to the fully grown preovulatory stage follicles (McGrath et al., 1995). These results indicate that GDF-9 is involved in normal follicle and oocyte development.
Due to selection mechanisms only one follicle from the group of follicles that left the primordial pool, reaches the preovulatory stage, i.e. the dominant follicle, and provides a healthy, fertilizable oocyte. The others become atretic and degenerate. The mechanisms controlling the selection of a dominant follicle are not fully understood, but is the hypothesis is that the follicle that is most sensitive to FSH, is the one that becomes dominant.
Follicular growth thus is controlled by growth factors such as for example IGF-1, GDF-9, and later on by the gonadotropins FSH and LH, and by estrogens. During growth several stages can be identified: the primary stage, the preantral and antral stage, and finally the preovulatory stage.
A major difference between infertility treatment protocols and natural conception is the number of follicles and oocytes that are involved.
During Assisted Reproduction Techniques (ART) treatment, the selection mechanism that leads to the ovulation of one healthy oocyte that will be fertilised and will develop to an implanting embryo, is overruled by administration of relatively high gonadotropin (mainly FSH) doses. This protocol causes many follicles and oocytes to grow and finally many oocytes are obtained from these follicles. However, although oocyte maturation is induced with LH/CG, not all oocytes actually have matured at the moment of retrieval, or oocytes have matured but do not develop properly. In some cases, oocyte maturation can subsequently be induced in vitro, but this in vitro maturation procedure has limited success. As a result only a relatively small proportion of all oocytes obtained from the follicles will eventually develop to an embryo that implants in the endometrium. This leads to relatively low ART success.
The present invention provides for an improvement of stimulating follicle and oocyte development and maturation by administration of GDF-9 in combination with gonadotropins. This will lead to the retrieval of higher quality oocytes with higher fertilisation, cleavage and implantation capacities and thus finally to higher ART success rates. Thus, according to the present invention GDF-9 might be used in therapy. Especially, it might be used for assisted reproduction.
Thus, due to administration of GDF-9, smaller follicles, which are not destined to become apoptotic, will be selected into the growing pool, and will grow to healthy preovulatory follicles. in addition, growing follicles might be rescued by GDF-9 and will develop to a fully grown follicle with a high quality oocyte, leading to an increase in final success rates, i.e. pregnancy outcome.
The induction of follicular growth by a combination of the growth factor GDF-9 and gonadotropins might lead to a more natural follicular development, and herewith might improve oocyte quality and thus final success rate.
According to the present invention GDF-9 can be administered to female patients in order to improve follicle and oocyte development and maturation. The administration can be included in existing treatment protocols for ovarian stimulation and in vitro fertilisation. Alternatively, GDF-9 can also be used for developing isolated follicles and/or oocytes by incubation in vitro in a suitable culture medium in the presence of GDF-9, preferably in addition to FSH.
GDF-9 preferably is prepared by recombinant DNA technology in eukaryotic host cells. DNA encoding GDF-9 has been isolated and characterized and can be used for the preparation of suitable vector system. GDF-9 preferably is encoded by DNA of mammalian origin, more preferably by human DNA.
The nucleotide acid sequence of GDF-9 of one species can be used as a probe or as a source to prepare synthetic oligonucleotides to be used as primers in DNA amplification reactions allotting the isolation and identification of complete gene of other species. Using that sequence information, the complete gene can be derived from cDNA or genomic DNA from other sources or synthesized using known methods.
Under gonadotropins is to be understood FSH as isolated from human sources, such as menopausal urine. Alternatively, recombinant FSH might be prepared by production in eukaryotic host cell. Furthermore, as in current assisted reproduction protocol treatments, maturation can be induced by the administration of LH or hCG. However, also variations thereupon including GnRH antagonists and/or agonists can be used.
The proteins to be used according to the invention are those proteins which have the amino acid sequences as isolated from the relevant vertebrate tissue, and have these known sequences per se, or their allelic variants thereof.
The variations that can occur in a sequence may be demonstrated by (an) amino acid difference(s) in the overall sequence or by deletions, substitutions, insertions, inversions or additions of (an) amino acid(s) in said sequence. Amino acid substitutions that are expected not to essentially alter biological and immunological activities, have been described. Amino acid replacements between related amino acids or replacements which have occurred frequently in evolu
Rose Ursula Maria
van Duin Marcel
Akzo Nobel N.V.
Kemmerer Elizabeth
Milstead Mark W.
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