Composition for controlled ovarian stimulation
The present invention relates to compositions and pharmaceutical products for the
treatment of infertility.
Assisted reproductive technology (ART) techniques such as in vitro fertilisation (IVF)
are well known. These ART techniques generally require a step of controlled ovarian
stimulation (COS), in which a cohort of follicles is stimulated to full maturity. Standard COS
regimens include administration of gonadotrophins, such as follicle stimulating hormone (FSH)
alone or in combination with luteinising hormone (LH) activity to stimulate follicular
development, normally with administration of a GnRH analogue prior to and/or during
stimulation to prevent premature LH surge. The pharmaceutical compositions generally used
for COS include recombinant follicle stimulating hormone (rFSH), urinary derived FSH,
recombinant FSH + LH preparations, urinary derived menotrophin [human menopausal
gonadotrophin (hMG)] and highly purified human menopausal gonadotrophin (HP-hMG). IVF
can be associated with a risk of ovarian hyperstimulation syndrome (OHSS), which can be life
threatening in severe cases.
The ability to predict the response potential of women to controlled ovarian stimulation
(COS) may allow the development of individualised COS protocols. This could, for example,
reduce the risk of OHSS in women predicted to have an excessive response to stimulation,
and/or improve pregnancy outcomes in women classed as poor responders. The serum
concentration of anti-Mullerian hormone (AMH) is now established as a reliable marker of
ovarian reserve. Decreasing levels of AMH are correlated with reduced ovarian response to
gonadotrophins during COS. Further, high levels of AMH are a good predictor of excessive
ovarian response, and an indicator of risk of OHSS.
In a preliminary study of women under 35 years old undergoing ART, the CONSORT
dosing algorithm (incorporating basal FSH, BMI, age and AFC) was used to predict the optimal
FSH starting dose for COS in women at risk of developing OHSS (Olivennes et. al., 2009).
Individualising the dose did lead to adequate oocyte yield and good pregnancy rate. However,
there were high rates of cancellations in the low dose group (75 IU FSH) due to inadequate
response, and OHSS did occur in a significant proportion of the patients.
There is therefore a need for a composition for use in individualised COS protocols
which provides adequate response to stimulation, and/or decreased risk of OHSS.
As indicated above, standard COS protocols may include administration of FSH. FSH
is naturally secreted by the anterior pituitary gland and functions to support follicular
development and ovulation. FSH comprises a 92 amino acid alpha sub-unit, also common to
the other glycoprotein hormones LH and CG, and a 111 amino acid beta sub-unit unique to
FSH that confers the biological specificity of the hormone (Pierce and Parsons, 1981). Each
sub-unit is post translationally modified by the addition of complex carbohydrate residues. Both
subunits carry 2 sites for N-linked glycan attachment, the alpha sub-unit at amino acids 52 and
78 and the beta sub-unit at amino acid residues 7 and 24 (Rathnam and Saxena, 1975, Saxena
and Rathnam, 1976). FSH is thus glycosylated to about 30% by mass (Dias and Van Roey.
2001 . Fox et al. 2001).
FSH purified from post-menopausal human urine has been used for many years in
infertility treatment; both to promote ovulation in natural reproduction and to provide oocytes for
assisted reproduction technologies. The currently approved recombinant FSH (rFSH) products
for ovarian stimulation, such as follitropin alfa (GONAL-F, Merck Serono / EMD Serono) and
follitropin beta (PUREGON / FOLLISTIM, MSD / Schering-Plough), are derived from a Chinese
Hamster Ovary (CHO) cell line. Currently, no rFSH products from a human cell line are
commercially available.
There is considerable heterogeneity associated with FSH preparations which relates to
differences in the amounts of various isoforms present. Individual FSH isoforms exhibit
identical amino acid sequences but differ in the extent to which they are post-translationally
modified; particular isoforms are characterised by heterogeneity of the carbohydrate branch
structures and differing amounts of sialic acid (a terminal sugar) incorporation, both of which
appear to influence the specific isoform bioactivity.
Glycosylation of natural FSH is highly complex. The glycans in naturally derived
pituitary FSH can contain a wide range of structures that can include combinations of mono-, bi-
, tri- and tetra-antennary glycans (Pierce and Parsons, 1981 . Ryan et al., 1987. Baenziger and
Green, 1988). The glycans can carry further modifications: core fucosylation, bisecting
glucosamine, chains extended with acetyl lactosamine, partial or complete sialylation,
sialylation with a2,3 and a2,6 linkages, and sulphated galactosamine substituted for galactose
(Dalpathado et al., 2006). Furthermore, there are differences between the distributions of
glycan structures at the individual glycosylation sites. A comparable level of glycan complexity
has been found in FSH derived from the serum of individuals and from the urine of post
menopausal women (Wide et al., 2007).
The glycosylation of recombinant FSH products reflects the range of glycosyltransferases
present in the host cell line. The commercially available rFSH products are derived
from engineered Chinese hamster ovary cells (CHO cells). The range of glycan modifications in
CHO cell derived rFSH are more limited than those found on the natural products. Examples of
the reduced glycan heterogeneity found in CHO cell derived rFSH include a lack of bisecting
glucosamine and a reduced content of core fucosylation and acetyl lactosamine extensions
(Hard et al., 1990). In addition, CHO cells are only able to add sialic acid using the a2,3 linkage
(Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990); CHO cell derived rFSH only
includes a2,3-linked sialic acid and does not include a2,6-linked sialic acid.
Thus CHO cell derived FSH is different from naturally produced FSH (e.g. human
Pituitary/ serum/ urinary FSH) which contains glycans with a mixture of a2,3 and a2,6-linked
sialic acid, with a predominance of the former.
Further, it has also been demonstrated that the commercially available recombinant
FSH preparation differs in the amounts of FSH with an isoelectric point (pi) of below 4
(considered the acidic isoforms) when compared to pituitary, serum or post-menopausal urine
FSH (Ulloa-Aguirre et al. 1995). The amount of acidic isoforms in the urinary preparations was
much higher as compared to the CHO cell derived recombinant products, Gonal-f (Merck
Serono) and Puregon (Schering Plough) (Andersen et al. 2004). This must reflect a lower molar
content of sialic acid in the recombinant FSH since the content of negatively-charged glycan
modified with sulphate is low in recombinant FSH. The lower sialic acid content, compared to
natural FSH, is a feature of both commercially available recombinant FSH products and may
reflect a limitation in the manufacturing process.
The circulatory life-time of FSH has been documented for materials from a variety of
sources. Some of these materials have been fractionated on the basis of overall molecular
charge, as characterised by their pi, in which more acid equates to a higher negative charge.
As previously stated the major contributor to overall molecular charge is the total sialic content
of each FSH molecule. For instance, rFSH (Organon) has a sialic acid content of around 8
mol/mol, whereas urine-derived FSH has a higher sialic acid content (de Leeuw et al. 1996).
The corresponding plasma clearance rates in the rat are 0.34 and 0.14 ml/min (Ulloa-Aguirre et
al. 2003). In another example where a sample of recombinant FSH was split into high and low
pi fractions, the in vivo potency of the high pi (lower sialic acid content) fraction was decreased
and it had a shorter plasma half-life (D'Antonio et al. 1999). It has also been reported that the
more basic FSH circulating during the later stages of the ovulation cycle is due to the downregulation
of a2,3 sialyl-transferase in the anterior pituitary which is caused by increasing levels
of estradiol (Damian-Matsumara et al. 1999. Ulloa-Aguirre et al. 2001). Results for the a2,6
sialyl-transferase have not been reported.
Thus, as set out above, recombinant proteins expressed using the CHO system will
differ from their natural counterparts in their type of terminal sialic acid linkages. This is an
important consideration in the production of biologicals for pharmaceutical use since the
carbohydrate moieties may contribute to the pharmacological attributes of the molecule.
The present applicants have developed a human derived recombinant FSH which is the
subject of International Patent Application No. PCT/GB2009/000978, published as
WO2009/127826A. Recombinant FSH with a mixture of both a2,3 and a2,6-linked sialic acid
was made by engineering a human cell line to express both rFSH and a2,3 sialyltransferase.
The expressed product is highly acidic and carries a mix of both a2,3- and a2,6-linked sialic
acids; the latter provided by the endogenous sialyl transferase activity. It was found that the
type of sialic acid linkage, a2,3- or a2,6-, can have a dramatic influence on biological clearance
of FSH. Recombinant FSH with a mixture of both a2,3 and a2,6-linked sialic acid has two
advantages over rFSH expressed in conventional CHO cells: first the material is more highly
sialylated due to the combined activities of the two sialyltransferases; and secondly the material
more closely resembles the natural FSH. This is likely to be more biologically appropriate
compared to CHO cell derived recombinant products that have produce only a2,3 linked sialic
acid (Kagawa et al, 1988, Takeuchi et al, 1988, Svensson et al., 1990) and have decreased
sialic acid content (Ulloa-Aguirre et al. 1995., Andersen et al. 2004).
The rFSH product disclosed in International Patent Application No.
PCT/GB2009/000978 contains branched glycan moieties. FSH comprises glycans (attached to
the FSH glycoproteins) and these glycans may contain a wide variety of structures. As is well
known in the art, branching (of a glycan) can occur with the result that the glycan may have 1,
2 , 3 , 4 or more terminal sugar residues or "antennae"; glycans with 1, 2 , 3 or 4 terminal sugar
residues or "antennae" are referred to respectively as mono-antennary, di-antennary, triantennary
or tetra-antennary structures. Glycans may have sialylation presence on monoantennary
and/or di-antennary and/or tri-antennary and/or tetra-antennary structures. An
example rFSH disclosed in International Patent Application No. PCT/GB2009/000978 included
mono-sialylated, di- sialylated, tri- sialylated and tetra- sialylated glycan structures with relative
amounts as follows: 9-15% mono-sialylated; 27 - 30% di—sialylated; 30 - 36% tri—sialylated
and 25 - 29 % tetra-sialylated. As is well known, a mono-sialylated glycan structure carries
one sialic acid residue; a di-sialylated glycan structure carries two sialic acid residues; a trisialylated
glycan structure carries three sialic acid residues; and a tetra-sialylated glycan
structure carries four sialic acid residues. Herein, terminology such as "X% mono-sialylated",
"X% di-sialylated", "X% tri-sialylated" or "X% tetra-sialylated" refers to the number of glycan
structures on FSH which are mono-, di, tri or tetra sialylated (respectively), expressed as a
percentage (X%) of the total number of glycan structures on the FSH which are sialylated in any
way (carry sialic acid). Thus, the phrase "30 - 36% tri-sialylated glycan structures" means that,
of the total number of glycan structures on the FSH which carry sialic acid residues (that is, are
sialylated), 30 to 36% of these glycan structures are tri sialylated (carry three sialic acid
residues). The applicants have surprisingly found that FSH having a specific amount of tetrasialylated
glycan structures (which is different to that of the example rFSH product disclosed in
PCT/GB2009/000978 mentioned above) is markedly more potent then recombinant FSH
products which are currently on the market. The amino acid sequence of the
applicant's products is the native sequence and is identical to natural human FSH and existing
CHO-derived rFSH products. However, the present applicants have found that human derived
recombinant FSH products (i.e. recombinant FSH produced or expressed in a human cell line
e.g. made by engineering a human cell line) which have a mixture of both a2,3 and a2,6-linked
sialic acid and/or a specific amount of tetra-sialylated glycan structures may be particularly
effective when utilised in (e.g. individualised) COS protocols.
According to the present invention in a first aspect there is provided a product (e.g. a
pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the
treatment of infertility in a patient (e.g. a patient having serum AMH level of 0.05 pmol/L or
above, for example 0.5 pmol/L or above), wherein the product comprises a dose of, or a dose
equivalent to, 1-24 mg , for example 2-24 mg , for example 2 to 15 mg , human derived
recombinant FSH. Preferably the product comprises a dose of, or a dose equivalent to, 4.5 to
12.5 mg , for example 5 to 12.5 mg , for example 6 to 12.5 mg , for example 6.3 to 10.5 mg , human
derived recombinant FSH.
According to the invention there is provided a product (e.g. a pharmaceutical
composition) comprising follicle stimulating hormone (FSH) for use in the treatment of infertility
in a patient having serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L), wherein
the product comprises a (e.g. daily) dose of, or dose equivalent to, 9 to 14 mg , for example 11 to
13 mg , for example 12 mg human derived recombinant FSH. Preferably the FSH is a
recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human cell line derived
recombinant FSH. The dose provides an effective response while minimising risk of OHSS.
Preferably the treatment of infertility comprising a step of determining (e.g. measuring) the
serum AMH level of the patient, and administering the dose to a patient having serum AMH
level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9 pmol/L).
According to the invention in a further aspect there is provided a product (e.g. a
pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the
treatment of infertility in a patient having serum AMH level of ³15 pmol/L, wherein the product
comprises a (e.g. daily) dose of, or dose equivalent to, 5 to 12.5 mg , for example 6 to 10.5 mg
human derived recombinant FSH. Preferably the FSH is a recombinant FSH ("rFSH" or
"recFSH"). Preferably the FSH is a human cell line derived recombinant FSH. The dose
provides an effective response while minimising risk of OHSS. Preferably the treatment of
infertility comprising a step of determining (e.g. measuring) the serum AMH level of the patient,
and administering the dose to a patient having serum AMH level of ³15 pmol/L. In one
embodiment, the product is for use in the treatment of infertility in a patient having serum AMH
level of 15 to 24.9 pmol/L, and the product is for administration at a (e.g. daily) dose of, or dose
equivalent to, 5 to 12 mg , for example 7 to 12 mg , for example 8.7 to 10 mg , human derived
recombinant FSH (preferably 9 to 10 mg human derived recombinant FSH) In this embodiment,
the treatment of infertility may comprise a step of determining (e.g. measuring) the serum AMH
level of the patient, and administering the dose to a patient having serum AMH level of 15 to
24.9 pmol/L. In another embodiment, the product is for use in the treatment of infertility in a
patient having serum AMH level of 25 to 34.9 pmol/L, and the product is for administration at a
(e.g. daily) dose of, or dose equivalent to, 5 to 12 mg , for example 6 to 9 mg , for example 7 to 8
mghuman derived recombinant FSH (preferably 7.3 to 8 mg human derived recombinant FSH).
In this embodiment, the treatment of infertility may comprise a step of determining (e.g.
measuring) the serum AMH level of the patient, and administering the dose to a patient having
serum AMH level of 25 to 34.9 pmol/L. In another embodiment, the product is for use in the
treatment of infertility in a patient having serum AMH level of ³ 35 pmol/L, and the product is for
administration at a (e.g. daily) dose of, or dose equivalent to, 5 to 11 mg , for example 6.3 to 7
mg , human derived recombinant FSH (preferably 6 to 7 mg human derived recombinant FSH).
In this embodiment, the treatment of infertility may comprise a step of determining (e.g.
measuring) the serum AMH level of the patient, and administering the dose to a patient having
serum AMH level of > 35 pmol/L.
The doses above may be for treatment of infertility in the patient's (subject's) first
stimulation protocol. It will be appreciated that for further stimulation cycles, the doses may be
adjusted according to actual ovarian response in the first cycle.
The applicants have found that it is generally necessary to retrieve in the region of nine
oocytes in order to enable selection of two high quality oocytes for transfer.
The applicants have found that for subjects having low AMH (AMH < 15 pmol/L per litre)
a reasonably high dose of recombinant FSH is required (for example 12 mg) to achieve this. At
this dose, 8 to 14 oocytes will be retrieved from 60% of subjects with low AMH. This is an
unexpected and significant improvement over treatment of subjects with low AMH treated with
150 IU Gonal-f, where 8 to 14 oocytes are retrieved from only 33% of subjects. The applicants
have found that there is no need to adjust this dose according to the bodyweight of the patient.
However, 60 % of the population (and 80% of women under 30 treated for infertility)
have high AMH (that is, AMH of ³15 pmol/L). For these subjects it is generally fairly
straightforward to retrieve a mean of 9 to 11 oocytes; the problem with stimulation protocols is
the risk of OHSS. The applicants have found that in patients dosed at low doses of human
recombinant FSH that there is a relationship between oocytes retrieved and body weight of the
subject. This means that there may be a risk associated with treatment with a fixed dose of
FSH (which is usual in the art). The present applicants have established a relationship between
dose of FSH and AMH level and weight of the subject which provides an improved safety profile
(reduced risk of OHSS) with acceptable or improved oocyte retrieval compared to the known
treatment protocols (see example 10).
According to the invention in a further aspect there is provided a product (e.g. a
pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the
treatment of infertility in a patient having serum AMH level of ³15 pmol/L, wherein the product is
for administration at a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 mg (for example
0.09 to 0.17 mg) human derived recombinant FSH per kg bodyweight of the patient. Preferably
the treatment of infertility comprises a step of determining (e.g. measuring) the serum AMH
level of the patient, and administering the dose to a patient having serum AMH level of ³15
pmol/L. In one embodiment, the product is for use in the treatment of infertility in a patient
having serum AMH level of 15 to 24.9 pmol/L, and the product is for administration at a (e.g.
daily) dose of, or dose equivalent to, 0.14 to 0.19 mg human derived recombinant FSH
(preferably 0.15 to 0.16 mg human derived recombinant FSH) per kg bodyweight of the patient.
In this embodiment, the treatment of infertility may comprise a step of determining (e.g.
measuring) the serum AMH level of the patient, and administering the dose to a patient having
serum AMH level of 15 to 24.9 pmol/L. In another embodiment, the product is for use in the
treatment of infertility in a patient having serum AMH level of 25 to 34.9 pmol/L, and the product
is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.1 1 to 0.14 mg human
derived recombinant FSH (preferably 0.12 to 0.13 mg human derived recombinant FSH) per kg
bodyweight of the patient. In this embodiment, the treatment of infertility may comprise a step of
determining (e.g. measuring) the serum AMH level of the patient, and administering the dose to
a patient having serum AMH level of 25 to 34.9 pmol/L. In a still further embodiment, the
product is for use in the treatment of infertility in a patient having serum AMH level of ³ 35
pmol/L, and the product is for administration at a (e.g. daily) dose of, or dose equivalent to, 0.10
to 0.1 1 mg human derived recombinant FSH per kg bodyweight of the patient. In this
embodiment, the treatment of infertility may comprise a step of determining (e.g. measuring) the
serum AMH level of the patient, and administering the dose to a patient having serum AMH
level of > 35 pmol/L. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH").
Preferably the FSH is a human cell line derived recombinant FSH. The doses provide an
effective response while minimising risk of OHSS.
The doses above may be for treatment of infertility in the patient's (subject's) first
stimulation protocol. It will be appreciated that for further stimulation cycles, the doses may be
adjusted according to actual ovarian response in the first cycle.
According to the invention in a still further aspect there is provided a product (e.g. a
pharmaceutical composition) comprising follicle stimulating hormone (FSH) for use in the
treatment of infertility in a patient having serum AMH level of <15 pmol/L, wherein the product is
for administration at a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 mg , (for example
0.19 to 0.21 mg) human derived recombinant FSH per kg bodyweight of the patient. Preferably
the treatment of infertility comprises a step of determining (e.g. measuring) the serum AMH
level of the patient, and administering the dose to a patient having serum AMH level of <15
pmol/L. However, it is not required that patients having serum AMH level of <15 pmol/L are
dosed by body weight. It will be appreciated that these doses may be readily converted
to treat patients with dosing according to their BMI, using conversions well known in the art.
The product (e.g. pharmaceutical compostion) may be for use in the treatment of
infertility in a patient having serum AMH of 5.0-14.9 pmol/L, wherein the product comprises a
dose of, or dose equivalent to, 6 to 18 mg , for example 8 to 11 mg , for example 8.5 to 10.2 mg
human derived recombinant FSH. The product may be for use in the treatment of infertility in a
patient having serum AMH 15.0-29.9 pmol/L, wherein the product comprises a dose of, or a
dose equivalent to, 4.8 to 15 mg , for example 6 to 9 mg , for example 6.8 to 8.5 mg human
derived recombinant FSH. The product may be for use in the treatment of infertility in a patient
having serum AMH 30-44.9 pmol/L, wherein the product comprises a dose of, or a dose
equivalent to, 3.6 to 12 mg , for example 4 to 7 mg , for example 5.1 to 6.8 mg human derived
recombinant FSH. The product may be for use in the treatment of infertility in a patient having
serum AMH 45 pmol/L or greater, wherein the product comprises a dose of, or a dose
equivalent to, 2 to 9 mg , for example 2.4 to 9 mg (for example 3.4 to 5.1 mg) or 2 to 5 mg human
derived recombinant FSH. The product may comprise follicle stimulating hormone (FSH) for use
in the treatment of infertility in a patient having serum AMH of 5 pmol/L or less, wherein the
product comprises a dose of, or a dose equivalent to 7.2 to 24 mg , for example 10 to 15 mg for
example 10.2 to 13.6 mg , human derived recombinant FSH. The product may be for use in the
treatment of infertility in a patient wherein the product comprises a dose of, or dose equivalent
to, 4.8 to 18 mg , for example 6 to 11 mg , for example 6.8 to 10.2 mg human derived recombinant
FSH. Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a
human cell line derived recombinant FSH.
Preferably the rFSH (e.g. human cell line derived recombinant FSH) includes a2,3- and
a2,6- sialylation. The FSH (rFSH) for use according to the invention may have 1% to 99% of
the total sialylation being a2,3-sialylation. The FSH (rFSH) according to the invention may have
1% to 99% of the total sialylation being a2,6-sialylation. Preferably, 50 to 70%, for example 60
to 69%, for example about 65%, of the total sialylation is a2,3-sialylation. Preferably 25 to 50%,
for example 30 to 50 %, for example 3 1 to 38%, for example about 35%, of the total sialylation
is a2,6- sialylation.
Preferably the rFSH (e.g. human cell line derived recombinant FSH) includes mono-, di-
, tri- and tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the
sialylated glycan structures are tetrasialylated glycan structures (e.g. as shown by WAX
analysis of charged glycans, as set out in the Examples below). The FSH comprises glycans
(attached to the FSH glycoproteins). It is well known that glycans in FSH may contain a wide
variety of structures. These may include combinations of mono, bi, tri and tetra-antennary
glycans. Herein, terminology such as "X% of the sialylated glycan structures are tetrasialylated
glycan structures" refers to the number of glycan structures on the FSH which are tetra
sialylated, i.e. carry four sialic acid residues, expressed as a percentage (X%) of the total
number of glycan structures on the FSH which are sialylated in any way (carry sialic acid).
Thus, the phrase "15-24% of the sialylated glycan structures are tetrasialylated glycan
structures" means that, of the total number of glycan structures on FSH which carry sialic acid
residues (that is, are sialylated), 15 to 24% of these glycan structures are tetra sialylated (carry
four sialic acid residues).
The rFSH may be present as a single isoform or as a mixture of isoforms.
The applicants have devised "individualised" COS protocols wherein specific doses of
recombinant FSH having specific characteristics are used to treat patients based on their
specific AMH levels, thereby increasing the likelihood of adequate response to stimulation (e.g.
in patients having a low response potential), and/or decreased risk of OHSS (e.g. in patients
classed as high or excessive responders).
The serum level of AMH may be determined (e.g. measured) by any method known in
the art. Preferably the serum AMH level is measured using the AMH Gen-ll enzyme linked
immunosorbent assay, a kit (Beckman Coulter, Inc., Webster, Texas). This assay can detect
can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of
1. 1 pmol/L. Other assays may be used.
Herein, serum AMH values are generally recited in terms of pmol/L. This may be
converted to ng/mL using the conversion equation 1ng/ml AMH = 7.1 pmol/L AMH.
Herein the terms "patient" and "subject" are used interchangeably.
The product (e.g. pharmaceutical composition) preferably comprises a daily dose of, or
a daily dose equivalent to, the amounts of human derived rFSH defined above, herein, and in
the claims. The (daily) dose may be an initial dose (i.e. it may be reduced, increased, or
maintained during the treatment).
The product (e.g. pharmaceutical composition) may be for (daily) administration of FSH
starting on day one of treatment and continuing for seven to thirteen days, for example nine to
thirteen days, for example 10 to 13 days, for example 10 to 11 days. The product (e.g.
pharmaceutical composition) may be for administration 12 to 16, e.g. 13 to 15, e.g. 14 days
after administration of (e.g. after initiation of administration of, e.g. after initiation of daily
administration of) a GnRH agonist (e.g. Synarel, Lupron, Decapeptyl). The product (e.g.
pharmaceutical composition) may be for administration with a GnRH agonist. The product (e.g.
pharmaceutical composition) may be for administration prior to administration of a GnRH
antagonist (e.g. ganirelix, cetrorelix), for example for administration five or six days prior to
administration of a GnRH antagonist. The product (e.g. pharmaceutical composition) may be
for administration with a GnRH antagonist. Preferably the product (e.g. pharmaceutical
composition) is for administration prior to administration of a high (ovulatory) dose of hCG (for
example 4,000 to 11,000 IU hCG, e.g. 5,000 IU hCG, 10,000 IU hCG etc.; or 150 to 350
microgram recombinant hCG, for example 250 microgram recombinant hCG) to induce final
follicular maturation.
It will be appreciated that the product may be for dosing at frequencies more (or less)
than daily, in which case the relevant doses will be equivalent to the (daily) doses specified
herein.
Herein the term "treatment of infertility" includes treatment of infertility by controlled
ovarian stimulation (COS) or methods which include a step or stage of controlled ovarian
stimulation (COS), for example Intra Uterine Insemination (IUI), in vitro fertilisation (IVF), or
intracytoplasmic sperm injection (ICSI). The term "treatment of infertility" includes treatment of
infertility by ovulation induction (Ol) or by methods which include a step or stage of ovulation
induction (01). The term "treatment of infertility" includes treatment of infertility in a subject
having tubal or unexplained infertility, including treatment of infertility in a subject having
endometriosis, for example stage I or stage I I endometriosis, and/or in a subject having
anovulatory infertility, for example WHO type I I anovulatory infertility, and/or in a subject with a
partner with male factor infertility. The product (or composition) may be for (use in) the
treatment of infertility (and/or for controlled ovarian stimulation) in a subject having
endometriosis, for example in a subject having stage I or stage I I endometriosis, as defined by
The American Society for Reproductive Medicine (ASRM) classification system for the various
stages of endometriosis, (stage IV most severe; stage I least severe) [American Society for
Reproductive Medicine. Revised American Society for Reproductive Medicine classification of
endometriosis: 1996. Fertil Steril 1997; 67,817 821 .].
The product (composition) may be for (use in) the treatment of infertility (and/or for
controlled ovarian stimulation) in a subject having normal serum FSH level of 1 to 16 IU/L, for
example 1 to 12 IU/L, in the early follicular phase.
The product (composition) may be for (use in) the treatment of infertility (and/or for
controlled ovarian stimulation) in a subject aged 18 to 42 years, for example 25 to 37 years.
The product may be for (use in) the treatment of infertility (and/or for controlled ovarian
stimulation) in a subject having BMI > 1 and BMI < 35 kg/m2, for example a subject having BMI
>18 and BMI < 25 kg/m2, for example a subject having BMI >20 and BMI < 25 kg/m2.
The rFSH may preferably include 27 - 33%, for example 30 - 32%, tri-sialylated glycan
structures. The rFSH may preferably include 24 - 33%, for example 26 - 30%, di-sialylated
glycan structures. The rFSH may preferably include 12 - 2 1%, for example 15 - 17%, monosialylated
glycan structures. The rFSH preferably includes mono-sialylated, di- sialylated, trisialylated
and tetra- sialylated glycan structures with relative amounts as follows: 15 to 17%
mono-sialylated; 26 - 30% di-sialylated; 27 - 33% (e.g. 29 to 32%, e.g 30-32%, e.g 30 to 3 1%)
tri-sialylated and 17 - 23 % tetra-sialylated (e.g. as shown by WAX analysis of charged
glycans, as set out in the Examples). The rFSH may include from 0 to 7%, for example 0.1 to
7%, for example 3 to 6%, for example 5 to 6%, neutral sialylated structures. The FSH
comprises glycans (attached to the FSH glycoproteins). Herein, terminology such as "X%
mono-sialylated", "X% di-sialylated", "X% tri-sialylated" or "X% tetra-sialylated" refers to the
number of glycan structures on FSH which are mono-, di, tri or tetra sialylated (respectively),
expressed as a percentage (X%) of the total number of glycan structures on the FSH which are
sialylated in any way (carry sialic acid). Thus, the phrase "27 - 33% tri-sialylated glycan
structures" means that, of the total number of glycan structures on FSH which carry sialic acid
residues (that is, are sialylated), 27 to 33% of these glycan structures are tri sialylated (carry
three sialic acid residues).
The rFSH may have a sialic acid content [expressed in terms of a ratio of moles of sialic
acid to moles of protein] of 6 mol/mol or greater, for example between 6 mol/mol and 15
mol/mol, e.g between 8 mol/mol and 14 mol/mol, for example between 10 mol/mol and 14
mol/mol, e.g between 11 mol/mol and 14 mol/mol, e.g between 12 mol/mol and 14 mol/mol,
e.g. between 12 mol/mol and 13 mol/mol. The rFSH may be produced or expressed in a
human cell line.
The FSH (rFSH) for use according to the invention may have 1% to 99% of the total
sialylation being a2,3-sialylation. The rFSH may have 10% or more of the total sialylation being
a2,3-sialylation. For example, 20, 30, 40, 50, 60, 70, 80 or 90% or more of the total sialylation
may be a2,3-sialylation. The rFSH may preferably include a2,3-sialylation in an amount which is
from 50 to 70% of the total sialylation, for example from 60 to 69% of the total sialylation, for
example from 63 to 67%, for example around 65% of the total sialylation. The FSH (rFSH) for
use according to the invention may have 1% to 99% of the total sialylation being a2,6-
sialylation. The rFSH (or rFSH preparation) of the invention may have 5% or more, for
example 5% to 99%, of the total sialylation being a2,6-sialylation. The rFSH may have 50% or
less of the total sialylation being a2,6-sialylation. The rFSH may preferably include a2,6-
sialylation in an amount which is from 25 to 50% of the total sialylation, for example from 30 to
50% of the total sialylation, for example from 3 1 to 38%, for example around 35% of the total
sialylation. By sialylation it is meant the amount of sialic residues present on the FSH
carbohydrate structures. a2,3-sialylation means sialylation at the 2,3 position (as is well known
in the art) and a2,6 sialylation at the 2,6 position (also well known in the art). Thus "% of the
total sialylation may be a 2,3 sialylation" refers to the % of the total number of sialic acid
residues present in the FSH which are sialylated in the 2,3 position. The term "% of the total
sialylation being a2,6-sialylation" refers to the % of the total number of sialic acid residues
present in the FSH which are sialylated in the 2,6 position.
The rFSH may have a sialic acid content (amount of sialylation per FSH molecule) of
(based on the mass of protein, rather than the mass of protein plus carbohydrate) of 6% or
greater (e.g. between 6% and 15%, e.g. between 7% and 13%, e.g. between 8% and 12%, e.g.
between 11% and 15%, e.g. between 12% and 14%) by mass.
The rFSH may be rFSH or a rFSH preparation in which 16 % or fewer (e.g. 0.1 to 16%)
of the glycans comprise (e.g. carry) bisecting N-acetylglucosamine (bisecting GlcNAc or
bisGlcNAc). Preferably the rFSH (or rFSH preparation) is an rFSH or rFSH preparation in
which 8 to 14.5% of the glycans comprise (e.g. carry) a bisecting N-acetylglucosamine
(bisecting GlcNAc or bisGlcNAc).
It will be understood that FSH comprises glycans attached to the FSH glycoproteins. It
will also be understood that 100% of the glycans refers to or means all of the glycans attached
to the FSH glycoproteins. Thus, herein, the terminology "8 to 14.5% of the glycans comprise
(carry) bisecting N-acetylglucosamine" means that 8 to 14.5% of the total number of glycans
attached to the FSH glycoproteins include/carry bisecting N-acetylglucosamine; "16% or fewer
of the glycans comprise (carry) bisecting N-acetylglucosamine" means that 16 % or fewer of the
total number of glycans attached to the FSH glycoproteins include/carry bisecting Nacetylglucosamine,
and so on.
The applicants have found that recombinant FSH (rFSH preparations; rFSH
compositions) in which 16% or fewer (e.g. 8 to 14.5%) of the glycans comprised in the FSH
glycoproteins carry bisecting GlcNac may have advantageous pharmacokinetic properties. It is
believed the advantageous properties may arise because the amount of glycans which carry
bisecting GlcNac is similar to that in the human urinary derived product Bravelle, which is rather
less than that of other recombinant FSH preparations such as those disclosed in
WO20 12/0 17058.
The rFSH (or rFSH preparation) may be an rFSH or rFSH preparation in which 20% or
more of the glycans comprise (e.g. carry) N-Acetylgalactosamine (GalNAc), for example in
which 20% or more of the glycans comprise (e.g. carry) a terminal GalNAc. Preferably the rFSH
(or rFSH preparation) is an FSH or FSH preparation in which the 40 to 55%, for example 42%
to 52%, of the glycans comprise (e.g. carry) GalNAc. Preferably the rFSH (or rFSH
preparation) is an FSH or FSH preparation in which the 40 to 55%, for example 42% to 52%, of
the glycans comprise (e.g. carry) terminal GalNAc.
It will be understood that FSH comprises glycans attached to the FSH glycoproteins. It
will also be understood that 100% of the glycans refers to or means all of the glycans attached
to the FSH glycoproteins. Thus, herein, the terminology "wherein 20% or more of the glycans
comprise (e.g. carry) GalNAc" means that 20% or more of the total number of glycans attached
to the FSH glycoproteins include/carry N-Acetylgalactosamine (GalNAc); "40 to 55%, for
example 42% to 52%, of the glycans comprise (e.g. carry) terminal GalNAc" means that 40 to
55 %, for example 42% to 52%, of the total number of glycans attached to the FSH
glycoproteins include/carry terminal GalNAc, and so on.
It appears that the availability of the a2,6- linkage increases the number of tetra
sialylated structures, compared to CHO cell derived products which have only the a2,3- linkage
available. The applicants have also found that their rFSH is distinguished over other approved
products because of the sugar composition: it includes, or may include, a specific amount of
GalNac. This may be linked to tetrasialylation and potency because the 2,6- sialylation is
associated with GalNac. In other words, the present applicants have developed an rFSH
product which includes specific characteristics (2,6- linker sites, GalNac) which provide rFSH
with high degree of sialylation, which appears to lead to improved potency in vivo.
The rFSH (or rFSH preparation) may have 16 to 24% of the glycans comprising (e.g.
terminal) 1 fucose-lewis, for example 16.5 to 18% of the glycans comprising (e.g. terminal) 1
fucose-lewis. The rFSH (or rFSH preparation) may have 1.5 to 4.5%, for example 2 to 4%, for
example 3.7%, of the glycans comprising (e.g. terminal) 2 fucose -lewis. The content of
fucose-lewis may have an effect on potency.
The rFSH may be produced or expressed in a human cell line, for example a Per.C6
cell line, a HEK293 cell line, a HT1080 cell line etc.. This may simplify (and render more
efficient) the production method because manipulation and control of e.g. the cell growth
medium to retain sialylation may be less critical than with known processes. The method may
also be more efficient because there is little basic rFSH produced compared to production of
known rFSH products; more acidic rFSH is produced and separation/removal of basic FSH is
less problematic. The rFSH may be produced or expressed in a PER.C6® cell line, a PER.C6®
derived cell line or a modified PER.C6® cell line. rFSH which is produced or expressed in a
human cell line (e.g. PER.C6® cell line, HEK293 cell line, HT1080 cell line etc.) will include
some a2,6-linked sialic acids (a2,6 sialylation) provided by endogenous sialyl transferase
activity [of the cell line] and will include some a2,3-linked sialic acids (a2,3 sialylation) provided
by endogenous sialyl transferase activity. The cell line may be modified using a2,3-
sialyltransferase. The cell line may be modified using a2,6-sialyltransferase. Alternatively or
additionally, the rFSH may include a2,6-linked sialic acids (a2,6 sialylation) provided by
endogenous sialyl transferase activity [of the cell line]. Herein, the term "human derived
recombinant FSH" means recombinant FSH which is produced or expressed in a human cell
line (e.g. recombinant FSH made by engineering a human cell line).
The rFSH may be produced using a2,3- and/or a2,6-sialyltransferase. In an example,
rFSH is produced using a2,3- sialyltransferase. The rFSH may include a2,6-linked sialic acids
(a2,6 sialylation) provided by endogenous sialyl transferase activity.
The product may be a pharmaceutical composition. The pharmaceutical composition is
for the treatment of infertility. The treatment of infertility may comprise assisted reproductive
technologies (ART), ovulation induction or intrauterine insemination (IUI). The pharmaceutical
composition may be used, for example, in medical indications where known FSH preparations
are used.
The product or composition can be formulated into well-known compositions for any
route of drug administration, e.g. oral, rectal, parenteral, transdermal (e.g. patch technology),
intravenous, intramuscular, subcutaneous, intrasusternal, intravaginal, intraperitoneal, local
(powders, ointments or drops) or as a buccal or nasal spray. A typical composition comprises a
pharmaceutically acceptable carrier, such as aqueous solution, non toxic excipients, including
salts and preservatives, buffers and the like, as described in Remington's Pharmaceutical
Sciences fifteenth edition (Matt Publishing Company, 1975), at pages 1405 to 1412 and 1461 -
87, and the national formulary XIV fourteenth edition (American Pharmaceutical Association,
1975), among others.
Examples of suitable aqueous and non-aqueous pharmaceutical carriers, diluents,
solvents or vehicles include water, ethanol, polyols (such as glycerol, propylene glycol,
polyethylene glycol, and the like), carboxymethylcellulose and suitable mixtures thereof,
vegetable oils (such as olive oil), and injectible organic esters such as ethyl oleate. The
compositions of the present invention also can contain additives such as but not limited to
preservatives, wetting agents, emulsifying agents, surfactants and dispersing agents.
Antibacterial and antifungal agents can be included to prevent growth of microbes and includes,
for example, m-cresol, benzyl alcohol, paraben, chlorobutanol, phenol, sorbic acid, and the like.
If a preservative is included, benzyl alcohol, phenol and/or m-cresol are preferred; however, the
preservative is by no means limited to these examples. Furthermore, it may be desirable to
include isotonic agents such as sugars, sodium chloride, and the like. The product or
composition may further comprise a salt comprising a pharmaceutically acceptable alkali metal
cation selected from the group consisting of Na+- or K+- salts, or a combination thereof.
Preferably the salt is a Na+- salt, for example NaCI or Na2S0 4.
Preferably the product or composition comprises recombinant FSH and one or more of
Polysorbate 20, L-methionine, phenol, disodium sulphate and sodium phosphate buffer.
In some cases, to effect prolonged action it is desirable to slow the absorption of FSH
(and other active ingredients, if present) from subcutaneous or intramuscular injection. This can
be accomplished by the use of a liquid suspension of crystalline or amorphous material with
poor water solubility. The rate of absorption of FSH then depends upon its rate of dissolution
which, in turn, can depend upon crystal size and crystalline form. Alternatively, delayed
absorption of a parenterally administered FSH combination form is accomplished by dissolving
or suspending the FSH combination in an oil vehicle. Injectable depot forms can be made by
forming microencapsule matrices of the FSH (and other agents, if present) in biodegradable
polymers such as polylactide-polyglycolide. Depending upon the ratio of FSH to polymer and
the nature of the particular polymer employed, the rate of FSH release can be controlled.
Examples of other biodegradable polymers include polyvinylpyrrolidone, poly(orthoesters),
poly(anhydrides) etc. Depot injectable formulations are also prepared by entrapping the FSH in
liposomes or microemulsions which are compatible with body tissues.
Injectable formulations can be sterilized, for example, by filtration through a bacterialretaining
filter, or by incorporating sterilizing agents in the form of sterile solid compositions
which can be dissolved or dispersed in sterile water or other sterile injectable medium just prior
to use. Injectable formulations can be supplied in any suitable container, e.g. vial, pre-filled
syringe, injection cartridges, and the like.
The product or composition may be formulated for single use or for multiple use
(multiple dose). If the product or composition is formulated for multiple use, it is preferred that a
preservative is included. If a preservative is included, benzyl alcohol, phenol and/or m-cresol
are preferred; however, the preservative is by no means limited to these examples. The single
use or multiple use formulated product or composition may further comprise a salt comprising a
pharmaceutically acceptable alkali metal cation selected from the group consisting of Na+- or
K+- salts, or a combination thereof. Preferably the salt is a Na+- salt, for example NaCI or
Na2S0 4.
The product or composition may be included in a container such as a vial, prefilled
cartridge (e.g. for single administration or multiple use) or an injection device such as a "pen"
for e.g. administration of multiple doses .
The product or composition may be a formulation (e.g. injectable formulation) including
FSH (optionally with hCG, LH, LH activity etc.) The LH activity, if present, may originate from
LH or human chorionic gonadotropin, hCG. If there is more than one active ingredient (i.e.
FSH and e.g. hCG or LH) these may be suitable for administration separately or together. If
administered separately, administration can be sequential. The product can be supplied in any
appropriate package. For example, a product can include a number of containers (e.g. prefilled
syringes or vials) containing either FSH or hCG, or a combination (or combination) of both
FSH and hCG. The hCG may be recombinant hCG or urinary hCG. If the product includes a
number of containers (e.g. pre-filled syringes or vials) containing FSH, e.g. recombinant FSH,
each container may include the same amount of FSH. One or more containers may include
different amounts of FSH. The syringes or vials may be packaged in a blister package or other
means to maintain sterility. Any product can optionally contain instructions for using the FSH
(and e.g. hCG if present) formulations. The pH and exact concentration of the various
components of the pharmaceutical composition are adjusted in accordance with routine practice
in this field. See GOODMAN and GILMAN's THE PHARMACOLOGICAL BASIS FOR
THERAPEUTICES, 7th ed. In a preferred embodiment, the compositions of the invention are
supplied as compositions for parenteral administration. General methods for the preparation of
the parenteral formulations are known in the art and are described in REMINGTON; THE
SCIENCE AND PRACTICE OF PHARMACY, supra, at pages 780-820. The parenteral
compositions can be supplied in liquid formulation or as a solid which will be mixed with a sterile
injectable medium just prior to administration. In an especially preferred embodiment, the
parenteral compositions are supplied in dosage unit form for ease of administration and
uniformity of dosage.
According to the present invention in a further aspect there is provided a method of
treatment of infertility comprising: (a) measuring the serum AMH level of a subject; and (b)
administration to the subject a dose of, or a dose equivalent to, 1-24 mg , for example 2-24 mg ,
for example 2 to 15 mg , human derived recombinant FSH. Preferably the dose is, or is
equivalent to, 4.5 to 12.5 mg , for example 5 to 12.5 mg , for example 6 to 12.5 mg , for example
6.3 to 12 mg , human derived recombinant FSH.
According to the present invention in a further aspect there is provided a method of
treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a
subject; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 9 to 14 mg , for
example 11 to 13 mg , for example 12 mg human derived recombinant follicle stimulating
hormone (FSH) to a (the) subject having serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to
14.9 pmol/L). Preferably the FSH is a recombinant FSH ("rFSH" or "recFSH"). Preferably the
FSH is a human cell line derived recombinant FSH. The dose provides an effective response
while minimising risk of OHSS.
According to the present invention in a further aspect there is provided a method of
treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a
subject; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 5 to 12.5 mg human
derived recombinant follicle stimulating hormone (FSH) to a (the) subject having serum AMH
level of ³15 pmol/L. The (e.g. daily) dose may be, or be equivalent to, 6 to 10 mg human
derived recombinant follicle stimulating hormone (FSH). Preferably the FSH is a recombinant
FSH ("rFSH" or "recFSH"). Preferably the FSH is a human cell line derived recombinant FSH.
The dose provides an effective response while minimising risk of OHSS.
In one embodiment, the method includes a step of administering a (e.g. daily) dose of,
or dose equivalent to, 5 to 12 mg , for example 7 to 12 mg , for example 8.7 to 10 mg , human
derived recombinant FSH (preferably 9 to 10 mg human derived recombinant FSH) to a (the)
subject having serum AMH level of 15 to 24.9 pmol/L In another embodiment, the method
includes a step of administering a (e.g. daily) dose of, or dose equivalent to, 5 to 12 mg human
derived recombinant FSH (for example 7 to 12 mg , for example 6 to 9 mg , for example 7 to 8 mg ,
for example 7.3 to 8 mg human derived recombinant FSH) to a (the) subject having serum AMH
level of 25 to 34.9 pmol/L. In another embodiment, the method includes a step of administering
a (e.g. daily) dose of, or dose equivalent to, 5 to 11 mg human derived recombinant FSH (for
example 6 to 7 mg , for example 6.3 to 7 mg , human derived recombinant FSH) to a (the) subject
having serum AMH level of ³ 35 pmol/L.
According to the present invention in a further aspect there is provided a method of
treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a
subject; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 mg (for
example 0.09 to 0.17 mg) human derived recombinant FSH per kg bodyweight of the subject,
wherein the subject has serum AMH level of ³15 pmol/L. Preferably the FSH is a recombinant
FSH ("rFSH" or "recFSH"). Preferably the FSH is a human cell line derived recombinant FSH.
The dose provides an effective response while minimising risk of OHSS.
In one embodiment, the method includes a step of administering a (e.g. daily) dose of,
or dose equivalent to, 0.14 to 0.19 mg human derived recombinant FSH (preferably 0.15 to 0.16
mg human derived recombinant FSH) per kg bodyweight of the subject, the subject having
serum AMH level of 15 to 24.9 pmol/L . In another embodiment, the method includes a step of
administering a (e.g. daily) dose of, or dose equivalent to, 0.1 1 to 0.14 mg human derived
recombinant FSH (preferably 0.12 to 0.13 mg human derived recombinant FSH) per kg
bodyweight of the subject, the subject having serum AMH level of 25 to 34.9 pmol/L. In another
embodiment, the method includes a step of administering a (e.g. daily) dose of, or dose
equivalent to, 0.10 to 0.1 1 mg human derived recombinant FSH per kg bodyweight of the
subject, the subject having serum AMH level of ³35 pmol/L. Preferably the FSH is a
recombinant FSH ("rFSH" or "recFSH"). Preferably the FSH is a human cell line derived
recombinant FSH. These doses provide an effective response while minimising risk of OHSS.
According to the present invention in a further aspect there is provided a method of
treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a
subject; and (b) administering a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 mg (for
example 0.19 to 0.21 mg) human derived recombinant FSH per kg bodyweight of the subject,
wherein the subject has serum AMH level of <15 pmol/L.
The administration preferably comprises a daily dose of, or a daily dose equivalent to,
the amount of FSH defined above and in the claims. The (daily) dose may be an initial dose (it
may be reduced, increased, or maintained during the treatment).
The method may be a method of treatment of infertility in the patient's (subject's) first
stimulation protocol. It will be appreciated that for further stimulation cycles, the doses may be
adjusted according to actual ovarian response in the first cycle.
According to the present invention in a further aspect there is provided a method of
treatment of infertility comprising: (a) determining (e.g. measuring) the serum AMH level of a
subject;
and (b) if the subject has serum AMH level of <15 pmol/L (e.g. 0.05 pmol/L to 14.9
pmol/L), administering to the subject a dose of, or dose equivalent to, 10 to 14 mg , for example
11 to 13 mg , for example 12 mg , human derived recombinant follicle stimulating hormone (FSH);
or
if the subject has serum AMH level of 15 to 24.9 pmol/L, administering to the subject a
dose of, or dose equivalent to, 0.14 to 0.19 mg human derived recombinant FSH (preferably
0.15 to 0.16 mg human derived recombinant FSH) per kg bodyweight of the subject; or
if the subject has serum AMH level of 25 to 34.9 pmol/L pmol/L, administering to the
subject a dose of, or dose equivalent to, 0.1 1 to 0.14 mg human derived recombinant FSH
(preferably 0.12 to 0.13 mg human derived recombinant FSH) per kg bodyweight of the subject;
or
if the subject has serum AMH level of ³35 pmol/L pmol/L, administering to the subject a
dose of, or dose equivalent to, 0.10 to 0.1 1 mg human derived recombinant FSH per kg
bodyweight of the subject.
For a patient (subject) having serum AMH of 5.0-14.9 pmol/L, a dose of, or dose
equivalent to, 6 to 18 mg , for example 8 to 11 mg , for example 8.5 to 10.2 mg human derived
recombinant FSH may be administered. For a patient (subject) having serum AMH 15.0-29.9
pmol/L, a dose of, or a dose equivalent to, 4.8 to 15 mg , for example 6 to 9 mg , for example 6.8
to 8.5 mg human derived recombinant FSH may be administered. For a patient (subject) having
serum AMH 30-44.9 pmol/L, a dose of, or a dose equivalent to, 3.6 to 12 mg , for example 4 to 7
mg , for example 5.1 to 6.8 mg human derived recombinant FSH may be administered. For a
patient (subject) having serum AMH 45 pmol/L or greater, a dose of, or a dose equivalent to, 2
to 9 mg , for example 2.4 to 9 mg (for example 3.4 to 5.1 mg) or 2 to 5 mg human derived
recombinant FSH may be administered. For a patient (subject) having serum AMH of 5 pmol/L
or less, a dose of, or a dose equivalent to 7.2 to 24 mg , for example 10 to 15 mg for example
10.2 to 13.6 mg , human derived recombinant FSH may be administered. In some examples, a
dose of, or dose equivalent to, 4.8 to 18 mg , for example 6 to 11 mg , for example 6.8 to 10.2 mg
human derived recombinant FSH is administered. Preferably the FSH is a recombinant FSH
("rFSH" or "recFSH"). Preferably the FSH is a human cell line derived recombinant FSH. The
administration preferably comprises a daily dose of, or a daily dose equivalent to, the amount of
FSH defined above and in the claims. The (daily) dose may be an initial dose (it may be
reduced, increased, or maintained during the treatment.
Detailed description of the invention
The present invention will now be described in more detail with reference to the attached
drawings in which:
Figure 1 shows a plasmid map of the pFSHalpha/beta expression vector;
Figure 2 shows the a2,3-sialyltransferase (ST3GAL4) expression vector;
Figure 3 shows the a2,6-sialyltransferase (ST6GAL1 ) expression vector;
Figure 4 shows % abundance sialic acid distribution of examples of recombinant FSH produced
by PER.C6® cells stably expressing FSH after engineering with a2,3- sialyltransferase;
Figure 5 shows % abundance of glycan charge distribution of examples of recombinant FSH
produced by PER.C6® cells stably expressing FSH after engineering with a2,3-
sialyltransferase;
Figure 6 shows a comparison of concentration of inhibin-B following administration of 225IU
Gonal f (bottom line, dotted line) and 225 IU of the Example (top line, full line) of Invention;
Fig 7 shows the effect of body weight on oocytes retrieved in the low AMH treatment group
(Example 10, 10A); and
Fig 8 shows the effect of Body weight on oocytes retrieved in the high AMH treatment group
Sequence Selection
Human FSH
The coding region of the gene for the FSH alpha polypeptide was used to according to
Fiddes and Goodman. (1981). The sequence is banked as AH007338 and at the time of
construction there were no other variants of this protein sequence. The sequence is referred
herein as SEQ ID NO:1 .
The coding region of the gene for FSH beta polypeptide was used according to Keene
et al ( 1989). The sequence is banked as NM_00051 0 and at the time of construction there were
no other variants of this protein sequence. The sequence is referred herein as SEQ ID NO: 2
Sialyltransferase
a2,3-Sialyltransferase - The coding region of the gene for beta-galactoside alpha-2,3-
sialyltransferase 4 (a2,3-sialyltransferase, ST3GAL4) was used according to Kitagawa and
Paulson (1994). The sequence is banked as L23767 and referred herein as SEQ ID NO: 3 .
a2,6-Sialyltransferase - The coding region of the gene for beta-galactosamide alpha-
2,6-sialyltransferase 1 (a2,6-sialyltransferase, ST6GAL1 ) was used according to Grundmann et
al. ( 1990). The sequence is banked as NM_003032 and referred herein as SEQ ID NO: 4 .
EXAMPLES
Example 1 Construction of the FSH expression vector
The coding sequence of FSH alpha polypeptide (AH007338, SEQ ID NO: 1) and FSH
beta polypeptide (NM_003032, SEQ ID NO: 2) were amplified by PCR using the primer
combinations FSHa-fw and FSHa-rev and FSHb-fw and FSHb-rec respectively.
FSHa-fw 5'-CCAGGATCCGCCACCATGGATTACTACAGAAAAATATGC-3' (SEQ ID NO:9)
FSHa-rev 5'-GGATGGCTAGCTTAAGATTTGTGATAATAAC-3' (SEQ ID NO:10)
FSHb-fw 5'-CCAGGCGCGCCACCATGAAGACACTCCAGTTTTTC-3' (SEQ ID NO: 11)
FSHb-rev 5'-CCGGGTTAACTTATTATTCTTTCATTTCACCAAAGG-3' (SEQ ID NO: 12)
The resulting amplified FSH beta DNA was digested with the restriction enzymes Ascand Hpa\ and inserted into the Asc\ and Hpa\ sites on the CMV driven mammalian expression
vector carrying a neomycin selection marker. Similarly the FSH alpha DNA was digested with
SamHI and Nhe\ and inserted into the sites SamHI and Nhe\ on the expression vector already
containing the FSH beta polypeptide DNA.
The vector DNA was used to transform the DH5a strain of E.coli. Colonies were picked
for amplification. Colonies containing the vector containing both FSH alpha and beta were
selected for sequencing and all contained the correct sequences according to SEQ ID NO: 1
and SEQ ID NO: 2 . Plasmid pFSH A+B#17 was selected for transfection (Figure 1).
Example 2 Construction of the ST3 expression vector
The coding sequence of beta-galactoside alpha-2,3-sialyltransferase 4 (ST3, L23767,
SEQ ID NO: 3) was amplified by PCR using the primer combination 2,3STfw and 2,3STrev.
2,3STfw 5'-CCAGGATCCGCCACCATGTGTCCTGCAGGCTGGAAGC-3' (SEQ ID NO: 13)
2,3STrev 5'-TTTTTTTCTTAAGTCAGAAGGACGTGAGGTTCTTG-3' (SEQ ID NO: 14)
The resulting amplified ST3 DNA was digested with the restriction enzymes BamH \ and
AflW and inserted into the BamH \ and AflW sites on the CMV driven mammalian expression
vector carrying a hygromycin resistance marker. The vector was amplified as previously
described and sequenced . Clone pST3#1 (Figure 2) contained the correct sequence according
SEQ ID NO: 3 and was selected for transfection.
Example 3 Construction of the ST6 expression vector
The coding sequence of beta-galactosamide alpha-2,6-sialyltransferase 1 (ST6,
NM_003032, SEQ ID NO: 4) was amplified by PCR using the primer combination 2,6STfw and
2,6STrev.
2,6STfw 5'-CCAGGATCCGCCACCATGATTCACACCAACCTGAAG-3' (SEQ ID NO: 15)
2,6STrev 5'-TTTTTTTCTTAAGTTAGCAGTGAATGGTCCGG-3' (SEQ ID NO: 16)
The resulting amplified ST6 DNA was digested with the restriction enzymes BamH \ and
AflW and inserted into the BamH \ and AflW sites on the CMV driven mammalian expression
vector carrying a hygromycin resistance marker. The vector was amplified as previously
described and sequenced. Clone pST6#1 1 (Figure 3) contained the correct sequence
according SEQ ID NO: 4 and was selected for transfection.
Example 4 Stable expression of pFSH a+b in PER.C6® cells. Transfection isolation and
screening of clones.
PER.C6®clones producing FSH were generated by expressing both polypeptide chains
of FSH from a single plasmid (see Example 1) .
To obtain stable clones a liposome based transfection agent with the pFSH
a+b construct. Stable clones were selected in VPRO supplemented with 10% FCS and
containing G41 8 . Three weeks after transfection G41 8 resistant clones grew out. Clones were
selected for isolation. The isolated clones were cultured in selection medium until 70-80%
confluent. Supernatants were assayed for FSH protein content using an FSH selective ELISA
and pharmacological activity at the FSH receptor in cloned cell line, using a cAMP accumulation
assay. Clones expressing functional protein were progressed for culture expansion to 24 well, 6
well and T80 flasks.
Studies to determine productivity and quality of the material from seven clones were
initiated in T80 flasks to generate sufficient material. Cells were cultured in supplemented
media as previously described for 7 days and the supernatant harvested . Productivity was
determined using the FSH selective ELISA. The isoelectric profile of the material was
determined by Isoelectric focusing (IEF), by methods known in the art. Clones with sufficient
productivity and quality were selected for sialyltransferase engineering.
Example 5 Level of sialylation is increased in cells that over express a2,3-
sialyltransferase. Stable expression of pST3 in FSH expressing PER.C6® cells;
Transfection isolation and screening of clones.
PER.C6® clones producing highly sialylated FSH were generated by expressing a2,3
sialyltransferase from separate plasmids (Example 2) in PER.C6® cells already expressing
both polypeptide chains of FSH (from Example 4). Clones produced from PER.C6® cells as set
out in Example 4 were selected for their characteristics including productivity, good growth
profile, production of functional protein, and produced FSH which included some sialylation.
Stable clones were generated as previously described in Example 4 . Clones were isolated,
expanded and assayed. The a2,3-sialyltransferase clones were adapted to serum free media
and suspension conditions.
As before, clones were assayed using a FSH selective ELISA, functional response in an
FSH receptor cell line, IEF, metabolic clearance rate and Steelman Pohley analysis. Results
were compared to a commercially available recombinant FSH (Gonal-f, Serono) and the
parental FSH PER.C6®cell lines. FSH produced by most of the clones has significantly
improved sialylation (i.e. on average more FSH isoforms with high numbers of sialic acids)
compared to FSH expressed without a2,3- sialyltransferase. In conclusion expression of FSH
together with sialyltransferase in PER.C6®cells resulted in increased levels of sialylated FSH
compared to cells expressing FSH only.
Example 6 Production and purification overview
A procedure was developed to produce FSH in PER.C6®cells that were cultured in
suspension in serum free medium. The procedure is described below and was applied to
several FSH-producing PER.C6®cell lines.
FSH from a2,3- clone (Example 5) was prepared using a using a modification of the
method described by Lowry et al. (1976).
For the production of PER.C6®-FSH, the cell lines were adapted to a serum- free
medium, i.e., Excell 525 (JRH Biosciences). The cells were first cultured to form a 70%-90%
confluent monolayer in a T80 culture flask. On passage the cells were re-suspended in the
serum free medium, Excell 525 + 4 rtiM L-Glutamine, to a cell density of 0.3x1 06 cells/ml. A 25
ml cell suspension was put in a 250 ml shaker flask and shaken at 100 rpm at 37°C at 5% CO2.
After reaching a cell density of > IxlO6 cells/ml, the cells were sub-cultured to a cell density of
0.2 or 0.3x1 06 cells/ml and further cultured in shaker flasks at 37°C, 5% C0 2 and 100 rpm.
For the production of FSH, the cells were transferred to a serum- free production
medium, i.e., VPRO (JRH Biosciences), which supports the growth of PER.C6®cells to very
high cell densities (usually > 107 cells/ml in a batch culture). The cells were first cultured to >
1xlO6 cells/ml in Excell 525, then spun down for 5 min at 1000 rpm and subsequently
suspended in VPRO medium + 6 mM L-glutamine to a density of 1x106 cells/ml. The cells were
then cultured in a shaker flask for 7-10 days at 37°C, 5% CO2 and 100 rpm. During this period,
the cells grew to a density of > 107 cells/ml. The culture medium was harvested after the cell
viability started to decline. The cells were spun down for 5 min at 1000 rpm and the supernatant
was used for the quantification and purification of FSH. The concentration of FSH was
determined using ELISA (DRG EIA 1288).
Thereafter, purification of FSH was carried out using a modification of the method
described by Lowry et al. (1976). Purification using charge selective chromatography was
carried out to enrich the highly sialylated forms by methods well known in the art.
During all chromatographic procedures, enrichment of the sialylated forms of FSH as
claimed herein was confirmed by RIA (DRG EIA 1288) and/or IEF.
Example 7 Quantification of relative amounts of a2,3 and a2,6 sialic acid
The relative percentage amounts of a2,3 and a2,6 sialic acid on purified rFSH (Example
6) were measured using known techniques.
N-Glycans were released from the samples using PNGase F under denaturative
conditions and then labelled with 2-aminobenzamide. Released glycan forms were then
separated and analysed by Weak Anion Exchange (WAX) column for determination of charge
distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid
and 2,3 sialidase for determination of 2,3 sialic acid, were further analyzed by wax column.
The relative percentages of the charged glycans were calculated from structures
present in the undigested and digested glycan pools and are shown in Figure 4 (for 8 samples).
These were found to be in the ranges 50% - 70% (e.g. about 60% or 65%) for a2,3 sialylation
and 28 to 50%, generally 30 to 35% (e.g. about 31% or 35%), for a2,6 sialylation.
Example 8 Quantification of relative amounts mono, di, tri and tetra sialylated glycan
structures
The relative percentage amounts of mono, di, tri and tetra sialylated structures on
glycans extracted from purified rFSH (Example 6) were measured using known techniques.
N Glycans were released from the samples using PNGase F under denaturative
conditions and then were labeled with 2-aminobenzamide. Glycans were released from the
samples using PNGase F under denaturative conditions and then labeled with 2-
aminobenzamide. Released glycan forms were then separated and analysed by Weak Anion
Exchange (WAX) column for determination of sialylation distribution. The relative amounts of
neutral, mono-sialylated, di-sialylated, tri-sialylated and tetra-sialylated structures are shown in
Figure 5 (for the 8 samples shown in Fig 4).
The rFSH includes neutral , mono-sialylated, di- sialylated, tri- sialylated and tetrasialylated
glycan structures with relative amounts as follows: neutral 5-6 %; 15-1 7% monosialylated
; 26-30% di-sialylated ; 30-32% tri-sialylated and 17-23 % tetra-sialylated.
Example 8a
The relative percentage amounts of a2,6 sialic acid on purified rFSH extracted from
nine samples of purified rFSH (produced by the methods of Example 6) were measured using
known techniques.
N-Glycans were released from the samples using PNGase F under denaturative
conditions and then labelled with 2-aminobenzamide. Released glycan forms were then
separated and analysed by Weak Anion Exchange (WAX) column for determination of charge
distribution. Labelled glycans treated with 2,3,6,8 sialidase for determination of total sialic acid
and 2,3 sialidase for determination of 2,3 sialic acid , were further analyzed by wax column (see
Example 8). The analysis allows calculation of a2,6 sialic acid .
The relative percentages of the charged glycans were calculated from structures
present in the undigested and digested glycan pools and are shown in the following Table.
These were found to be in the ranges 25 to 50%, generally 30 to 35% for a2,6 sialylation.
The relative percentage amounts of bisecting GlcNac, GalNac and 1-Fucose Lewis on
glycans extracted from the nine samples of purified rFSH (produced by the methods of Example
6) were measured using known techniques. N-Glycans were released from the glycoprotrein
using PNGase F and labeled with 2-aminobenzamide (2AB). The analysis was done by two
dimensional (2D) HPLC analysis in combination with enzymatic degradation of the glycans. For
verification, the glycans were analyzed by MALDI-MS The relative amounts of alpha 2,6-sialic
acid and the terminal residues are shown in the following table, together with those for Gonal F
(CHO cell derived recombinant FSH) and Bravelle (human urinary FSH).
IGalNA 0 11.3
5 1 44.6 50.7 44.7 49 47.6 45.3 46.4 44.9 47.1
c
Bisectin
g 10
GlcNAc 12.4 10.2 8.9 8.7 11.8 11.4 10.6 13.9 10.9 55 14
1
Fucose 2 1. 1 16.7 23.3 16.1 20.3 18.1 17.9 18.7 19.0 19.0 3.1 1 2.2
Lewis
2
Fucose 4 4.1 4.3 1.9 3.1 4.2 3.8 3.9 4.4 3.7 - n.d.2
Lewis
Value of 3.1 is total ½ Fucose Lewis. Not determined.
It can be seen that the amount of GalNac in the FSH of the invention varies between
about 44.9 and 51%, averaging about 47.1 %.
It can be seen that the amount of bisecting GlcNac in the FSH of the invention varies
between 8.7 and 13.9%, averaging approximately at 10.9%.
It can be seen that the amount of 1 Fucose Lewis in the FSH of the invention varies
between 16.1 and 23.3%, averaging approximately at 19%.
It can be seen that the amount of 2 Fucose Lewis in the FSH of the invention varies
between 1.9 and 4.4%, averaging approximately at 3.7%.
Example 9 - A multiple dose study investigating the safety, tolerability,
pharmacokinetics, pharmacodynamics, and immunogenicity of FE 999049 in comparison
to GONAL-F.
Study population
A total of 48 (24 on each drug) healthy women received daily doses of 14.6 mg of FE 999049 (a
composition according to the invention, produced according to Example 6) or 16.5 mg of Gonal-
F for seven days.
Safety results
Multiple dose administration of FE 999049 and GONAL-F was safe and generally well tolerated
as assessed by Adverse Events (AEs), vital signs, ECG, clinical laboratory measurements, and
physical examination. No serious adverse event or death occurred during the study.
Pharmacokinetic results
Following the administration of FE 999049 and GONAL-F over 7 days, the FSH concentration
values as assessed immediately prior to the next injection increased and seemed to reach a
steady state level after 6-7 days. However the exposure (AUC and Cmax) of FE 999049 was
60% higher in comparison to Gonal-F.
Pharmacodynamic results
The concentrations of inhibin-B (see figure 6), oestradiol, and progesterone all increased
subsequent to administration of FE 999049 and GONAL-F, however to a greater extent
following administration of FE 999049 compared to GONAL-F. Both number and size
distribution of follicles showed a greater response to FE 999049 compared to GONAL-F.
Example 9 demonstrates that FSH having a specific amount ( 17-23%) of tetra-sialylated glycan
structures and e.g. specific amounts of a2,3 sialylation and a2,6 sialylation is markedly more
potent then recombinant FSH products which are currently on the market.
Example 10 - A multiple dose study investigating FE 999049 in comparison to GONAL-F.
The following describes a randomised, controlled, assessor-blind, parallel groups,
multinational, multicentre trial assessing the dose-response relationship of FE 999049 in
patients undergoing controlled ovarian stimulation for in vitro fertilisation (IVF) / intracytoplasmic
sperm injection (ICSI). The patient population was 265 IVF patients aged between 18 to 37
years, with BMI 18.5 to 32.0 kg/m2.
The trial was designed as a dose-response trial with number of oocytes retrieved as the primary
endpoint. Secondary endpoints will explore the qualitative and quantitative impact of different
doses of FE 999049 with regard to endocrine profile, follicular development, oocyte fertilisation,
embryo quality and treatment efficiency (i.e. total gonadotropin consumption and duration of
stimulation). The trial is designed to evaluate the efficacy of FE 999049 to establish pregnancy
when used in controlled ovarian stimulation for IVF/ICSI cycles.
Subjects were assessed within 3 months prior to randomisation for compliance with the
inclusion and exclusion criteria, including an anti-Mijllerian hormone (AMH) assessment to
increase homogeneity of the trial population in relation to ovarian response and minimise the
number of potential poor and hyper-responders to the FE 999049 doses and GONAL-F dose
used in the trial. The AMH assessment was measured using the AMH Gen-ll enzyme linked
immunosorbent assay kit (Beckman Coulter, Inc., Webster, Texas). This assay can detect
AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1. 1 pmol/L.
On day 2-3 of their menstrual cycle, subjects were randomised in a 1: 1 : 1 : 1 : 1 : 1 fashion
to treatment with either 90 IU, 120 IU, 150 IU, 180 IU or 210 IU FE 999049 or 150 IU GONAL-F,
and ovarian stimulation initiated. Randomisation was stratified according to AMH level at
screening [5.0-14.9 pmol/L (low AMH) and 15.0 to 44.9 pmol/L (high AMH)).
Gonal-F is filled by mass (FbM) at FDA request; referring to mg dose is therefore
appropriate. The Gonal - F label indicates 600 IU/44 mg , which indicates that 150 IU is 11 mg .
However, there is some variation and the batch certificate for this trial indicated that 11.3 g
Gonal-F was equivalent to 150 IU. The FE999049 doses are presented by protein content (pg)
rather than biological activity. Thus the doses of FE999049 were 5.2pg (90 IU), 6.9pg ( 120 IU),
8.6pg ( 150 IU), 10.3pg ( 180 IU) or 12.1pg (21 0 IU).
The subject and dose distribution is set out as follows (data are number of subjects):
Table 1
FE 999049 GONAL-F
5.2pg 6.9pg 8.6pg 10.3pg 12.1m 11.3 ( 1 1) g Total
Screened 334
Randomised
42 45 44 45 46 43 265
and exposed
High AMH
strata
23 26 24 24 26 25 148 (56%)
( 15.0 - 44.9
pmol/L)
Low AMH
strata
19 19 20 20 2 1 18 117 (44%)
(5.0 - 14.9
pmol/L)
Per-protocol 40 42 42 44 44 43 255
The daily dose level of FE 999049 or GONAL-F is fixed throughout the entire
stimulation period. During stimulation, subjects are monitored on stimulation day 1, 4 and 6 and
hereafter at least every second day. When 3 follicles of > 5 mm are observed, visits are
performed daily. Subjects are treated with FE 999049 or GONAL-F for a maximum of 16 days.
To prevent a premature LH surge, a GnRH antagonist (ganirelix acetate,
ORGALUTRAN, MSD / Schering-Plough) may be initiated on stimulation day 6 at a daily dose
of 0.25 mg and continued throughout the stimulation period. Triggering of final follicular
maturation is done on the day when >3 follicles with a diameter > 17 mm are observed. If there
are <25 follicles with a diameter >12 mm, 250 pg recombinant hCG (choriogonadotropin alfa,
OVITRELLE, Merck Serono / EMD Serono) is administered. If there are 25-35 follicles with a
diameter ³12 mm, 0.2 mg GnRH agonist (triptorelin acetate, DECAPEPTYL / GONAPEPTYL,
Ferring Pharmaceuticals) is administered. In case of excessive ovarian response, defined as
>35 follicles with a diameter >12 mm, the treatment is cancelled. In case of poor ovarian
response, defined as <3 follicles with a diameter >10 mm observed on stimulation day 10, the
cycle could be cancelled.
Oocyte retrieval takes place 36h (± 2h) after triggering of final follicular maturation and
the oocytes inseminated by IVF and/or ICSI. Fertilisation and embryo development are
assessed from oocyte retrieval to the day of transfer. For subjects who underwent triggering of
final follicular maturation with hCG, one blastocyst of the best quality available is transferred on
day 5 after oocyte retrieval while remaining blastocysts are frozen. For subjects who undergo
triggering of final follicular maturation with GnRH agonist, no embryo transfer takes place in the
fresh cycle and blastocysts are instead frozen on day 5 . Vaginal progesterone tablets
(LUTINUS, Ferring Pharmaceuticals) 100 mg 3 times daily are provided for luteal phase support
from the day after oocyte retrieval until the day of the clinical pregnancy visit. A i Q test is
performed 13-15 days after embryo transfer and clinical pregnancy will be confirmed by
transvaginal ultrasound (TVU) 5-6 weeks after embryo transfer.
Results
The number of oocytes retrieved (primary endpoint) is shown in the following Table.
Table 2
FE 999049 GONAL-F
5.2 g 6.9 g 8.6 pg 10.3 pg 12.1 g 11.3 ( 11)ug
Oocytes
retrieved
All 5.2 (3.3) 7.9 (5.9) 9.2 (4.6) 10.6 (7.0) 12.2 (5.9) 10.4 (5.2)
High AMH 5.9 (3.9) 9.1 (6.4) 10.6 (4.8) 13.6 (7.8) 14.4 (5.8) 12.4 (5.4)
Low AMH 4.5 (2.2) 6.3 (4.9) 7.4 (3.8) 6.9 (3.6) 9.4 (4.9) 7.8 (3.4)
Data are mean (SD)
The primary objective was met: a significant dose-response relationship was established for FE
999049 with respect to number of oocytes retrieved. This finding was observed not only for the
overall trial population, but also for each of the two AMH strata used at randomisation.
A significant dose-response for FE 999049 was demonstrated for all key objective
pharmacodynamic parameters, e.g. estradiol, inhibin B and inhibin A. At a similar microgram
dose level, the pharmacodynamic responses with FE 999049 were larger than with GONAL-F
(these results not shown).
The serum FSH concentrations after exposure to FE 999049 were significantly higher than for
GONAL-F. The results confirm that the PK profile of FE 999049 differs from that of GONAL-F.
Fertilisation rates, blastocyst development and pregnancy rates in IVF/ICSI patients treated with
FE 999049 were within expectations.
There were no safety concerns with the use of FE 999049. A good local tolerability was
documented.
Further Analysis
The applicants have further analysed the data to identify the FE 999049 dose(s) that fulfil the
following criteria with respect to number of oocytes retrieved :
· Oocytes retrieved in the range 8-1 4
Minimise proportion of patients with <8 oocytes
Minimise proportion of patients with ³20 oocytes
The applicants also investigated the impact of body weight . If relevant, the dose is converted
into mg g for an average subject. This value of pg/kg and ±0.01 g kg are evaluated in a model
with respect to distribution of oocytes retrieved as well as safety profile, and the optimal dose is
identified.
Low A H strata
As seen in Table 2 , the dose of FE999049 which fulfilled the first criterion (Oocytes retrieved in
the range 8-14) was 12.1 g (mean 9.4 oocytes retrieved). The distribution of oocytes is shown
in Table 3 below.
Table 3
FE 999049 GONAL-F
5.2pg 6 8.6pg 10 .3 g 12.1 g 11.3 ( 11) g
Oocytes
retrieved
<4 32% 24% 15% 10% 10% 6%
4-7 63% 42% 45% 60% 20% 56%
8-14 5% 24% 35% 30% 60% 33%
15-19 0% 5% 5% 0% 5% 6%
>20 0% 5% 0% 0% 5% 0%
Data are % of subjects
As shown by the box and arrow, a dose of 12.1 pg FE999049 provides retrieval of the
most desirable number of oocytes in 60% of subjects in the low AMH group. This is a marked
improvement on Gonal-F (most desirable number of oocytes in only 33% of subjects).
Table 4 below shows the analysis of signs of excessive response in the low AMH strata
(data are number of subjects). It can be seen that there were no indications of early OHSS of a
moderate or severe nature and there were no incidences of preventative action being required;
there are no concerns associated with the dose of 12.1 pg FE999049 in a patient having low
AMH.
Table 4
FE 999049 GONAL-F
5.2 g 6.9pg 8.6pg 10.3pg 12.1pg 11.3(1 1)pg
All subjects 19 19 20 20 2 1 18
Early OHSS, mod/sev
GnRH agonist
triggering
Preventive action*
³15 oocytes
Any of the above
Fig 7 shows the effect of body weight on oocytes retrieved (for the low AMH strata), for the
various doses. The arrows indicate the number of oocytes retrieved from subjects of
bodyweight between 45kg and 90kg treated at the 12.1 mg dose. As can be seen (text box) the
variation between patients of bodyweight 45kg and those of 90kg is less than around 0.5
oocytes; in other words dosing by body weight is not required in patients with low AMH when
dose of FE999049 is at least 12 mg , because there is not a significant variation in oocytes
retrieved with body weight, at this dose.
Thus the applicants have found that a dose of, or dose equivalent to, 6 to 18 mg , for example 9
to 14 mg , for example 12 mg , human derived recombinant FSH is suitable for use in the
treatment of infertility in a patient having serum AMH <15 pmol/L, for example 0.05-14.9 pmol/L
for example 5.0-14.9 pmol/L. The dose provides an effective response while minimising risk of
OHSS.
High AMH strata
As seen in Table 2 , three doses of FE999049 fulfilled the first criterion (oocytes retrieved in the
range 8-14): 6.9 mg (mean 9.1 oocytes retrieved), 8.6 mg (mean 10.6 oocytes retrieved), and
10.3 mg (mean 13.6 oocytes retrieved).
Fig 8 shows the effect of body weight on oocytes retrieved (for the high AMH strata), for the
various doses. The arrows indicate the number of oocytes retrieved from subjects of body
weight between 45kg and 90kg treated at the 6.9 mg , 8.6 mg and 10.3 mg doses. As can be
seen (text boxes) the variation is significant: for the 6.9 mg dose 6 additional oocytes will be
retrieved from a 45 kg patient compared to a 90 kg patient; for the 8.6 mg dose 4 additional
oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient; and for the 10.1 mg
dose 2.5 additional oocytes will be retrieved from a 45 kg patient compared to a 90 kg patient.
In other words dosing by body weight has an impact in patients with high AMH when the dose
of FE999049 is less than 12 mg , because there is a significant variation in oocytes retrieved with
body weight, at these doses.
Table 5a below shows a further breakdown of oocytes retrieved (from Table 2) by AMH. This
shows the doses which fulfilled the first criterion (oocytes retrieved in the range 8-14) for each
sub strata of AMH (boxes).
5.2 g 6.9 pg 8.6 pg 10.3 pg 12.1 pg
Oocytes retrieved
15-24 pmol/L 4.9 (3. 8) 7.3 (3. 6) 10.6 (5. 1) 11.5 (6.7) 12.3 (5.9)
25-34 pmol/L 7.0 (1.8) 9.1 (6.8) 9.7 (6.7) 15.5 (6.4) 16.7 (4.9)
35-45 pmol/L 8.5 (9.2) \ 21.0 (1.4) \1 1.3 (2.6) \ 18.0 15.7 (6.5)
(12.2)
Table 5 b below shows the analysis of patients where treatment was cancelled due to
either excessive response or agonist triggering, for these subgroups. For example, one patient
in the 25-34 pmol/L AMH strata cancelled due to excessive response following the dose of 10.3
pg and one patient in the 25-34 pmol/L AMH strata cancelled due to excessive response
following the dose of 12.1 pg; one patient in the 35-45 pmol/L AMH strata cancelled following
agonist triggering following dose of 10.3 pg; and one patient in the 35-45 pmol/L AMH strata
cancelled following agonist triggering following dose of 6.9 g.
Table 5b
FE 999049
5.2pg 6.9pg 8.6pg 10.3pg 12.1pg
OHSS*** , cancellation
due to excessive
response or agonist
triggering*** *
15-24 pmol/L 0
25-34 pmol/L 0
35-45 pmol/L 0
It can be seen therefore that tailoring of dose by bodyweight (Fig 8) and AMH level
would be useful in the high AMH strata, to minimise cancellations and maximise oocyte
retrieval.
The applicants have found that the following doses provide an effective response while
minimising risk of OHSS (kg is kg body weight of patient).
The following are appropriate if dosing by bodyweight is not desired.
The following are appropriate if fewer categories of AMH are required.
AMH categories 3 AMH categories 2 AMH categories One dose
AMH Dose AMH Dose AMH Dose AMH Dose
<15 12 pg <15 12 pg <15 12 pg - 0.16
pg/kg
15-24 0.15-0.16 pg/kg 15-24 0.15-0.16 ³15 0.14 pg/kg
pg/kg
25-34 0.12-0.13 pg/kg >25 0.12 pg/kg
³35 0.10-0.11 pg/kg
The following are appropriate if dosing by bodyweight is not desired. .
4 AMH categories 3 AMH categories 2 AMH categories One dose
AMH Dose AMH Dose AMH Dose AMH Dose
<15 12 pg <15 12 pg <15 12 pg - 9.3 pg or 10 pg
15-24 9.3-10 pg 15-24 9.3-10 pg ³15 8.7 pg
25-34 7.3-8 pg >25 7.3 pg
>35 6.3-7 pg
Thus the applicants have found that a dose of, or dose equivalent to, 9 to 14 pg, for example 2
pg, human derived recombinant FSH is suitable for use in the treatment of infertility in a patient
having serum AMH <15 pmol/L, for example 0.05-14.9 pmol/L for example 5.0-14.9 pmol/L.
The dose provides an effective response while minimising risk of OHSS.
The applicants have found that a dose of, or dose equivalent to, 5 to 12.5 pg, for example 6 to
10.5 pg, human derived recombinant FSH is suitable for use in the treatment of infertility in a
patient having serum AMH ³ 5 pmol/L. The dose provides an effective response while
minimising risk of OHSS.
The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.09 to 0.19 pg
human derived recombinant FSH per kg bodyweight of the patient is suitable for use in the
treatment of infertility in a patient having serum AMH level of ³15 pmol/L. The applicants have
found that a (e.g. daily) dose of, or dose equivalent to, 0.14 to 0.19 pg human derived
recombinant FSH (preferably 0.15 to 0.16 pg human derived recombinant FSH) per kg
bodyweight of the patient is suitable for use in the treatment of infertility in a patient having
serum AMH level of 15 to 24.9 pmol/L. The applicants have found that a (e.g. daily) dose of, or
dose equivalent to, 0.1 1 to 0.14 pg human derived recombinant FSH (preferably 0.12 to 0.13 pg
human derived recombinant FSH) per kg bodyweight of the patient is suitable for use in the
treatment of infertility in a patient having serum AMH level of 25 to 34.9 pmol/L. The applicants
have found that a (e.g. daily) dose of, or dose equivalent to, 0.10 to 0.1 1 mg human derived
recombinant FSH per kg bodyweight of the patient is suitable for use in the treatment of
infertility in a patient having serum AMH level of ³ 35 pmol/L. These doses provide an effective
response while minimising risk of OHSS.
The applicants have found that a (e.g. daily) dose of, or dose equivalent to, 0.15 to 0.21 mg (e.g.
0.16 mg) human derived recombinant FSH per kg bodyweight of the patient is suitable for use in
the treatment of infertility in a patient having serum AMH level of <15 pmol/L, for example for
the first stimulation cycle with human derived recombinant FSH. . However, it is not required
that patients are dosed by body weight at this level of AMH.
Example 10 A - Individualised COS protocol (low AMH)
The selected patients are about to undergo COS for in vitro fertilisation (IVF) /
intracytoplasmic sperm injection (ICSI) by methods known in the art. The pre-treatment
protocol includes assessment/screening of the patient's serum AMH using the AMH Gen-ll
enzyme linked immunosorbent assay kit (Beckman Coulter, Inc., Webster, Texas). This assay
can detect AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of
1. 1 pmol/L. AMH may be measured using other Assay kits (e.g. available from Roche).
The COS protocol proceeds in the usual manner apart from administration of the initial
dose of FE 999049 according to AMH level at screening. A patient with an AMH level of <14.9
pmol/L would be administered an initial daily dose of approximately 12 mg FE 999049, a human
derived recombinant FSH product manufactured according to the method of Example 6 . A
patient with an AMH level of 15 to 24.9 pmol/L would receive an initial daily dose of 0.15 to 0.19
mg of the human derived recombinant FSH per kg bodyweight of the patient. A patient with an
AMH level of 25 to 34.9 pmol/L would receive an initial daily dose of 0.1 1 to 0.13 mg of the
human derived recombinant FSH per kg bodyweight of the patient. A patient with an AMH level
of ³ 35 pmol/L would receive an initial daily dose of 0.10 to 0.1 1 mg of the human derived
recombinant FSH per kg bodyweight of the patient.
Example 11 - Individualised COS protocols.
The doses in this protocol are less preferred that Example 10A.
The selected patients are about to undergo COS for in vitro fertilisation (IVF) / intracytoplasmic
sperm injection (ICSI) by methods known in the art. The pre-treatment protocol includes
assessment/screening of the patient's serum AMH using the AMH Gen-ll enzyme linked
immunosorbent assay kit (Beckman Coulter, Inc., Webster, Texas). This assay can detect
AMH concentrations greater than 0.57 pmol/L with a minimum limit of quantitation of 1. 1 pmol/L.
The COS protocol proceeds in the usual manner apart from administration of the initial
dose of FE 999049 according to AMH level at screening in line with the following table. Thus a
patient with an AMH level of 5-14.8 pmol/L would be administered 180 IU FSH in the form of
approximately 8-1 1 mg FE 999049, a human derived recombinant FSH product manufactured
according to the method of Example 6 . A patient with an AMH level of 30-44.9 pmol/L would
be administered 120 IU FSH in the form of approximately 4-7 mg FE 999049, a human derived
recombinant FSH product manufactured according to the method of Example 6 . If the AMH
level is not available, the patient recombinant would be administered 120-180 IU FSH in the
form of approximately 6-1 1 mg FE 999049, a human derived recombinant FSH product
manufactured according to the method of Example 6 .
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Figs 1, 2 and 3: Plasmid maps of the pFSHalpha/beta ,pST3 and pST6 expression vectors.
C V = Cytomegalovirus promoter, BGHp(A) = Bovine Growth Hormone poly-adenylation
sequence, fl ori = f l origin of replication, SV40 = Simian Virus 40 promoter, Neo =
Neomycin resistance marker, Hyg = Hygromycin resistance marker, SV40 p(A) = Simian
Virus 40 poly-adenylation sequence, FSH A = Follicle stimulating hormone alpha
polypeptide, FSH B = Follicle stimulating hormone beta polypeptide, ST3GAL4 = a2,3-
sialyltransferase, ST6GAL1 = a2,6-sialyltransferase, ColEI = ColEI origin of replication,
Amp = ampicillin resistance marker.
SEQ ID NO: 1
Follicle stimulating hormone alpha polypeptide
Accession number AH007338
Nucleotide sequence of FSH alpha
1 ATGGATTACT ACAGAAAATA TGCAGCTATC TTTCTGGTCA CATTGTCGGT GTTTCTGCAT
61 GTTCTCCATT CCGCTCCTGA TGTGCAGGAT TGCCCAGAAT GCACGCTACA GGAAAACCCA
121 TTCTTCTCCC AGCCGGGTGC CCCAATACTT CAGTGCATGG GCTGCTGCTT CTCTAGAGCA
181 TATCCCACTC CACTAAGGTC CAAGAAGACG ATGTTGGTCC AAAAGAACGT CACCTCAGAG
241 TCCACTTGCT GTGTAGCTAA ATCATATAAC AGGGTCACAG TAATGGGGGG TTTCAAAGTG
301 GAGAACCACA CGGCGTGCCA CTGCAGTACT TGTTATTATC ACAAATCTTA A
Protein sequence of FSH alpha (SEQ ID NO: 5)
1 MDYYRKYAAI FLVTLSVFLH VLHSAPDVQD CPECTLQENP FFSQPGAPIL QCMGCCFSRA
61 YPTPLRSKKT MLVQKNVTSE STCCVAKSYN RVTVMGGFKV ENHTACHCST CYYHKS
SEQ ID NO: 2
Follicle stimulating hormone beta polypeptide
Accession number NM_000510
Nucleotide sequence of FSH beta
1 ATGAAGACAC TCCAGTTTTT CTTCCTTTTC TGTTGCTGGA AAGCAATCTG CTGCAATAGC
61 TGTGAGCTGA CCAACATCAC CATTGCAATA GAGAAAGAAG AATGTCGTTT CTGCATAAGC
121 ATCAACACCA CTTGGTGTGC TGGCTACTGC TACACCAGGG ATCTGGTGTA TAAGGACCCA
181 GCCAGGCCCA AAATCCAGAA AACATGTACC TTCAAGGAAC TGGTATATGA AACAGTGAGA
241 GTGCCCGGCT GTGCTCACCA TGCAGATTCC TTGTATACAT ACCCAGTGGC CACCCAGTGT
301 CACTGTGGCA AGTGTGACAG CGACAGCACT GATTGTACTG TGCGAGGCCT GGGGCCCAGC
361 TACTGCTCCT TTGGTGAAAT GAAAGAATAA
Protein sequence of FSH beta (SEQ ID NO: 6)
1 MKTLQFFFLF CCWKAICCNS CELTNITIAI EKEECRFCIS INTTWCAGYC YTRDLVYKDP
61 ARPKIQKTCT FKELVYETVR VPGCAHHADS LYTYPVATQC HCGKCDSDST DCTVRGLGPS
121 YCSFGEMKE
SEQ ID NO: 3
Beta-galactoside alpha-2,3-sialyltransferase 4
Accession Number L23767
Nucleotide sequence of ST3GAL4
1 ATGTGTCCTG CAGGCTGGAA GCTCCTGGCC ATGTTGGCTC TGGTCCTGGT CGTCATGGTG
61 TGGTATTCCA TCTCCCGGGA AGACAGGTAC ATCGAGCTTT TTTATTTTCC CATCCCAGAG
121 AAGAAGGAGC CGTGCCTCCA GGGTGAGGCA GAGAGCAAGG CCTCTAAGCT CTTTGGCAAC
181 TACTCCCGGG ATCAGCCCAT CTTCCTGCGG CTTGAGGATT ATTTCTGGGT CAAGACGCCA
241 TCTGCTTACG AGCTGCCCTA TGGGACCAAG GGGAGTGAGG ATCTGCTCCT CCGGGTGCTA
301 GCCATCACCA GCTCCTCCAT CCCCAAGAAC ATCCAGAGCC TCAGGTGCCG CCGCTGTGTG
361 GTCGTGGGGA ACGGGCACCG GCTGCGGAAC AGCTCACTGG GAGATGCCAT CAACAAGTAC
421 GATGTGGTCA TCAGATTGAA CAATGCCCCA GTGGCTGGCT ATGAGGGTGA CGTGGGCTCC
481 AAGACCACCA TGCGTCTCTT CTACCCTGAA TCTGCCCACT TCGACCCCAA AGTAGAAAAC
541 AACCCAGACA CACTCCTCGT CCTGGTAGCT TTCAAGGCAA TGGACTTCCA CTGGATTGAG
601 ACCATCCTGA GTGATAAGAA GCGGGTGCGA AAGGGTTTCT GGAAACAGCC TCCCCTCATC
661 TGGGATGTCA ATCCTAAACA GATTCGGATT CTCAACCCCT TCTTCATGGA GATTGCAGCT
721 GACAAACTGC TGAGCCTGCC AATGCAACAG CCACGGAAGA TTAAGCAGAA GCCCACCACG
781 GGCCTGTTGG CCATCACGCT GGCCCTCCAC CTCTGTGACT TGGTGCACAT TGCCGGCTTT
841 GGCTACCCAG ACGCCTACAA CAAGAAGCAG ACCATTCACT ACTATGAGCA GATCACGCTC
901 AAGTCCATGG CGGGGTCAGG CCATAATGTC TCCCAAGAGG CCCTGGCCAT TAAGCGGATG
961 CTGGAGATGG GAGC A CAA GAACCTCACG TCCTTCTGA
Protein Sequence of ST3GAL4 (SEQ ID NO: 7)
1 MCPAGWKLLA MLALVLWMV WYSISREDRY IELFYFPIPE KKEPCLQGEA ESKASKLFGN
61 YSRDQPIFLR LEDYFWVKTP SAYELPYGTK GSEDLLLRVL AITSSSIPKN IQSLRCRRCV
121 WGNGHRLRN SSLGDAINKY DWIRLNNAP VAGYEGDVGS KTTMRLFYPE SAHFDPKVEN
181 NPDTLLVLVA FKAMDFHWIE TILSDKKRVR KGFWKQPPLI WDVNPKQIRI LNPFFMEIAA
241 DKLLSLPMQQ PRKIKQKPTT GLLAITLALH LCDLVHIAGF GYPDAYNKKQ TIHYYEQITL
301 KSMAGSGHNV SQEALAIKRM LEMGAIKNLT SF
SEQ ID NO: 4
Beta-galactosamide alpha-2,6-sialyltransferase 1
Accession number NM 003032
Nucleotide sequence of ST6GAL1
1 ATGATTC C CCAACCTGAA GAAAAAGTTC AGCTGCTGCG TCCTGGTCTT TCTTCTGTTT
61 GCAGTCATCT GTGTGTGGAA GGAAAAGAAG AAAGGGAGTT ACTATGATTC CTTTAAATTG
121 CAAACCAAGG AATTCCAGGT GTTAAAGAGT CTGGGGAAAT TGGCCATGGG GTCTGATTCC
181 CAGTCTGTAT CCTCAAGCAG CACCCAGGAC CCCCACAGGG GCCGCCAGAC CCTCGGCAGT
241 CTCAGAGGCC TAGCCAAGGC CAAACCAGAG GCCTCCTTCC AGGTGTGGAA CAAGGACAGC
301 TCTTCCAAAA ACCTTATCCC TAGGCTGCAA AAGATCTGGA AGAATTACCT AAGCATGAAC
361 AAGTACAAAG TGTCCTACAA GGGGCCAGGA CCAGGCATCA AGTTCAGTGC AGAGGCCCTG
421 CGCTGCCACC TCCGGGACCA TGTGAATGTA TCCATGGTAG AGGTCACAGA TTTTCCCTTC
481 AATACCTCTG AATGGGAGGG TTATCTGCCC AAGGAGAGCA TTAGGACCAA GGCTGGGCCT
541 TGGGGCAGGT GTGCTGTTGT GTCGTCAGCG GGATCTCTGA AGTCCTCCCA ACTAGGCAGA
601 GAAATCGATG ATCATGACGC AGTCCTGAGG TTTAATGGGG CACCCACAGC CAACTTCCAA
661 CAAGATGTGG GCACAAAAAC TACCATTCGC CTGATGAACT CTCAGTTGGT TACCACAGAG
721 AAGCGCTTCC TCAAAGACAG TTTGTACAAT GAAGGAATCC TAATTGTATG GGACCCATCT
781 GTATACCACT CAGATATCCC AAAGTGGTAC CAGAATCCGG ATTATAATTT CTTTAACAAC
841 TACAAGACTT ATCGTAAGCT GCACCCCAAT CAGCCCTTTT ACATCCTCAA GCCCCAGATG
901 CCTTGGGAGC TATGGGACAT TCTTCAAGAA ATCTCCCCAG AAGAGATTCA GCCAAACCCC
961 CCATCCTCTG GGATGCTTGG TATCATCATC ATGATGACGC TGTGTGACCA GGTGGATATT
1021 TATGAGTTCC TCCCATCCAA GCGCAAGACT GACGTGTGCT ACTACTACCA GAAGTTCTTC
1081 GATAGTGCCT GCACGATGGG TGCCTACCAC CCGCTGCTCT ATGAGAAGAA TTTGGTGAAG
1141 CATCTCAACC AGGGCACAGA TGAGGACATC TACCTGCTTG GAAAAGCCAC ACTGCCTGGC
1201 TTCCGGACCA TTCACTGCTA A
Op-
Protein Sequence of ST6GAL1 (SEQ ID NO: 8)
1 MIHTNLKKKF SCCVLVFLLF AVICVWKEKK KGSYYDSFKL QTKEFQVLKS LGKLAMGSDS
61 QSVSSSSTQD PHRGRQTLGS LRGLAKAKPE ASFQVWNKDS SSKNLIPRLQ KIWKNYLSMN
121 KYKVSYKGPG PGIKFSAEAL RCHLRDHVNV SMVEVTDFPF NTSEWEGYLP KESIRTKAGP
181 WGRCAWSSA GSLKSSQLGR EIDDHDAVLR FNGAPTANFQ QDVGTKTTIR LMNSQLVTTE
241 KRFLKDSLYN EGILIVWDPS VYHSDIPKWY QNPDYNFFNN YKTYRKLHPN QPFYILKPQM
301 PWELWDILQE ISPEEIQPNP PSSGMLGIII MMTLCDQVDI YEFLPSKRKT DVCYYYQKFF
361 DSACTMGAYH PLLYEKNLVK HLNQGTDEDI YLLGKATLPG FRTIHC
CLAIMS
1. A product comprising follicle stimulating hormone (FSH) for use in the treatment of
infertility in a patient (e.g. a patient having serum AMH level of 0.05 pmol/L or above), wherein
the product comprises a dose of, or a dose equivalent to, 2-24 mg , for example 2 to 15 mg ,
human derived recombinant FSH.
2 . A product for use according to claim 1 comprising follicle stimulating hormone (FSH) for
use in the treatment of infertility in a patient having serum AMH level of <15 pmol/L, wherein the
product comprises a dose of, or dose equivalent to, 9 to 14 mg human derived recombinant
FSH.
3 . A product for use according to claim 2 wherein the product comprises a dose of, or
dose equivalent to 12 mg human derived recombinant FSH.
4 . A product for use according to claim 2 or claim 3 wherein the treatment of infertility
comprises a step of determining the serum AMH level of the patient, and a step of
administering the dose to a patient having serum AMH level of <15 pmol/L.
5 . A product for use according to claim 1 comprising follicle stimulating hormone (FSH) for
use in the treatment of infertility in a patient having serum AMH level of ³15 pmol/L, wherein the
product comprises a dose of, or dose equivalent to, 5 to 12.5 mg human derived recombinant
FSH.
6 . A product for use according to claim 5 wherein the treatment of infertility comprises a
step of determining the serum AMH level of the patient, and a step of administering the dose to
a patient having serum AMH level of ³15 pmol/L.
7 . A product for use according to claim 1 comprising follicle stimulating hormone (FSH) for
use in the treatment of infertility in a patient having serum AMH level of ³15 pmol/L, wherein the
product is for administration at a dose of, or dose equivalent to, 0.09 to 0.19 mg human derived
recombinant FSH per kg bodyweight of the patient.
8 . A product for use according to claim 7 in the treatment of a patient having serum AMH
level of 15 to 24.9 pmol/L, wherein the product is for administration at a dose of, or dose
equivalent to, 0.14 to 0.19 mg human derived recombinant FSH per kg bodyweight of the
patient.
9 . A product for use according to claim 7 in the treatment of a patient having serum AMH
level of 25 to 34.9 pmol/L, wherein the product is for administration at a dose of, or dose
equivalent to, 0.1 1 to 0.14 human derived recombinant FSH per kg bodyweight of the
patient.
10. A product for use according to claim 7 in the treatment of a patient having serum AMH
level of ³ 35 pmol/L, wherein the product is for administration at a dose of, or dose equivalent
to, 0.10 to 0.1 1 mg human derived recombinant FSH per kg bodyweight of the patient.
11. A product for use according to any of claims 7 to 10 wherein the treatment of infertility
includes a step of determining the serum AMH level of the patient, and a step of administering
the dose to a patient having the defined serum AMH level.
12. A product according to any preceding claim wherein the follicle stimulating hormone
(FSH) is a human derived recombinant FSH.
13. A product according to any preceding claim wherein the FSH includes a2, 3- and a2, 6-
sialylation.
14. A product according to claim 13 wherein 25 to 50%, for example 30 to 50%, of the total
sialylation is a2, 6- sialyation.
15. A product according to claim 13 or claim 14 wherein 50 to 70% of the total sialylation is
a 2,3- sialyation.
16. A product according to any preceding claim wherein the FSH includes mono-, di-, triand
tetra-sialylated glycan structures, wherein 15-24%, for example 17-23% of the sialylated
glycan structures are tetrasialylated glycan structures.
17. A product comprising follicle stimulating hormone (FSH) for use in the treatment of
infertility according to any preceding claim, wherein the product further comprises a salt
comprising a pharmaceutically acceptable alkali metal cation selected from the group consisting
of Na+- or K+- salts, or a combination thereof.
18. A method of treatment of infertility comprising:
(a) measuring the serum AMH level of a subject; and
(b) administration to the subject a dose of, or a dose equivalent to, 2-24 mg , for example 2 to 15
mg , human derived recombinant FSH.
19. A method of treatment of infertility comprising: (a) determining the serum AMH level of a
subject; and (b) administering a dose of, or dose equivalent to, 9 to 14 mg human derived
recombinant follicle stimulating hormone (FSH) to a subject having serum AMH level of <15
pmol/L.
20. A method of treatment of infertility comprising: (a) determining the serum AMH level of a
subject; and (b) administering a dose of, or dose equivalent to, 5 to 12.5 mg human derived
recombinant follicle stimulating hormone (FSH) to a (the) subject having serum AMH level of
>15 pmol/L.
2 1. A method of treatment of infertility comprising: (a) determining the serum AMH level of a
subject; and (b) administering a dose of, or dose equivalent to, 0.09 to 0.19 mg human derived
recombinant FSH per kg bodyweight of the subject, wherein the subject has serum AMH level
of >15 pmol/L.
22. A method according to claim 2 1 comprising a step of administering a dose of, or dose
equivalent to, 0.14 to 0.19 mg human derived recombinant FSH per kg bodyweight of the
subject, the subject having serum AMH level of 15 to 24.9 pmol/L.
23. A method according to claim 2 1 comprising a step of administering a dose of, or dose
equivalent to, 0.1 1 to 0.14 mg human derived recombinant FSH per kg bodyweight of the
subject, the subject having serum AMH level of 25 to 34.9 pmol/L.
24. A method according to claim 2 1 comprising a step of administering a dose of, or dose
equivalent to, 0.10 to 0.1 1 mg human derived recombinant FSH per kg bodyweight of the
subject, the subject having serum AMH level of ³ 35 pmol/L.
25. A method of treatment of infertility comprising: (a) determining the serum AMH level of a
subject; and (b) administering a dose of, or dose equivalent to, 0.15 to 0.17 mg human derived
recombinant FSH per kg bodyweight of the subject, wherein the subject has serum AMH level
of <15 pmol/L.
26. A product comprising follicle stimulating hormone (FSH) for use in the manufacture of a
medicament for use in the treatment of infertility in a patient (e.g. a patient having serum AMH
level of 0.05 pmol/L or above), wherein the product comprises a dose of, or a dose equivalent
to, 2-24 mg , for example 2 to 15 mg , human derived recombinant FSH.