Clinical assessment of human gonadotrophins
produced by recombinant DNA technology
Ernest Loumaye l ,4, Isabelle Martineau l , Angela Piazzil ,
Louis O'Dea2, Susan Inee l , Colin Howles3, Genevieve
Deeosterd l , K.Van Loonl and A.Galazka l
lCorporate Medical Affairs Department, Ares-Serono, CH-1211 Geneva 20,
Switzerland, 2Serono Inc., Norwell, Massachusetts, USA, 3Europe Region,
Ares-Serono, CH 1211 Geneva, Switzerland
4To whom correspondence should be addressed
Human gonadotrophins are follicle stimulating hormone (FSH), luteinizing
hormone (LH) and human chorionic gonadotrophin (HCG). All three gonadotrophins are now produced in vitro by recombinant DNA technology. Being complex
heterodimeric proteins, an eukaryotic cell line, has been selected for expression
and large scale production (Chinese hamster ovary cells). The advantages of
producing the gonadotrophins in vitro rather than extracting them from human
fluids are: (i) a fully controlled production process from bulk to finished product;
(ii) high purity and specific activity; (iii) batch-to-batch consistency; and (iv)
complete absence of contamination by the other gonadotrophins. Pharmacokinetic
characterization of recombinant human gonadotrophins has shown that this first
generation have pharmacokinetic characteristics very similar to the extractive
gonadotrophins with an apparent terminal half-life after s.c. administration of
~37 h, 12 hand 32 h for recombinant FSH (rhFSH), recombinant LH (rhLH)
and recombinant HCG (rhHCG) respectively. Clinical efficacy and safety have
been demonstrated in several randomized, well-controlled studies, comparing
rhFSH administered s.c. with uhFSH administered i.m. for stimulating follicular
development prior to assisted reproductive technology and in WHO Group II
anovulation. To date, ~ 1300 patients have been treated with rhFSH. Moreover,
rhLH has recently been successfully used in association with rhFSH for inducing
ovulation and pregnancy in WHO I anovulatory patients and rhCG has been
successfully used for triggering final follicular maturation prior to assisted
reproductive technology.
Key words: anovulationlgonadotrophins/IVFlhypogonadotrophic hypogonadism!
PCOD
Introduction
Human gonadotrophins include follicle stimulating hormone (FSH), luteinizing
hormone (LH) and human chorionic gonadotrophin (HCG). They are members
of a family of complex heterodimeric glycoproteins secreted by the pituitary
gland. They share a common a-subunit. The ~-subunit amino acid sequence is
Human Reproduction Volume II Supplement I 1996
© European Society for Human Reproduction and Embryology
95
E.Loumaye et al.
different for each of these gonadotrophins. LH and HCG display a high homology
in their ~-subunits leading to an essentially similar biological function, although
they are different in terms of pharmacokinetic characteristics. In contrast, the ~­
subunit of hFSH is different from hLH and HCG ~-subunits, leading to distinct
biological actions. Gonadotrophins are responsible for controlling reproductive
functions both in males and females, i.e. FSH primarily for the processes
concerned with germ cell development in the gonads and LHIHCG for promoting
steroidogenesis in the gonads (Catt and Dufau, 1991).
Pharmaceutical preparations of human FSH play an important role in the
treatment of human infertility. FSH is widely used to stimulate follicular
development for inducing ovulation in anovulatory women wishing to conceive
(Speroff et aZ., 1989). It is also routine treatment for stimulating multiple follicular
development in infertile ovulatory women undergoing assisted reproductive
techniques (ART) such as in-vitro fertilization (IVF) and embryo transfer
treatment (Hughes et aZ., 1992; Bustillo, 1995; FIVNAT, 1995). In men, FSH is
used in combination with HCG to initiate and maintain spermatogenesis in
hypo gonadotrophic hypogonadism (Whitcomb and Crowley, 1990).
Human LH is used in association with FSH to promote adequate steroidogenesis
during stimulation of follicular development in hypo gonadotrophic hypogonadal
women pertaining to World Health Organization (WHO) group I anovulation
(Couzinet et aZ., 1988; Shoham et aZ., 1991; Hull et ai., 1994). HCG is used as
a surrogate drug to mimic the mid-cycle LH surge and induce final follicular
maturation, early luteinization and ovulation after FSH-induced follicular development. In men, HCG is used for promoting testosterone secretion (Whitcomb and
Crow~ey, 1990).
Initially, for clinical use, hFSH was extracted from pituitary glands and the
first pregnancies were reported in 1960 (Gemzell et aZ., 1960). Soon afterwards,
gonadotrophins extracted from post-menopausal urine [menotrophin, human
menopausal gonadotrophin (HMG)] also proved to be effective (Lunenfeld et aZ.,
1962) and became the standard gonadotrophin preparation for three decades.
HMG contains a mixture of FSH and LH and is of low specific activity (see
Table I). Since then, more and more purified urine-derived FSH preparations
have been developed (see Table I).
Initially, some very limited amounts of LH were produced by extraction from
pituitary glands (Van de Wiele et aZ., 1970). Since then, hLH has been extracted
from the urine of post-menopausal women, and has only been available in
combination with FSH, i.e. HMG. This fixed LHIFSH ratio does not allow
independent adaptation of the doses of LH and FSH. Up to now, HCG has
always been extracted from urine of pregnant women.
Through the application of recombinant DNA technology, it is now possible
to produce hFSH, hLH and HCG for therapeutic use without the need for
extraction from human fluids. Recombinant human FSH (rhFSH), recombinant
human LH (rhLH); and recombinant HCG (rHCG) are produced in vitro by
genetically engineered mammalian cells [Chinese hamster ovary (CHO) cells]
(Keene et aZ., 1989; Mannaerts et aZ., 1991; Chappel et ai., 1992, Loumaye
96
Human gonadotrophins and recombinant DNA technology
Table I. Pharmaceutical preparations of human follicle stimulating hormone (FSH)
Preparation
Source of
FSH
FSH activity
(lU/vialampoule)
LH activity
(lU/vialampoule)
FSH specific
Co-purified
activity (FSH lUI non-FSH
mg proteins)
human proteins
HMG
Urine
75
75
75-150
>95%
uhFSH
Urine
75
<0.7
100-150
>95%
uhFSH-HP
Urine
75-150
<0.001
-10 000
<1%
rhFSH
CHO cells
75-150
none
-10000
none
HMG = human menopausal gonadotrophin; u = urinary; h = human; r = recombinant; HP =
highly purified.
et aI., 1995). The production of human gonadotrophins by recombinant technology
has been coupled with advanced protein purification processes, e.g. immunoaffinity purification and high purification liquid chromatography (HPLC) , resulting
in the formulation of high specific activity, pharmaceutical preparations suitable
for clinical use.
The reasons for producing gonadotrophins for pharmaceutical use in vitro
rather than extracting them from human fluids can be summarized.
Full control of the source of bulk materials
The production of rhFSH is independent of urine collection. Currently, annual
collection of urine for worldwide gonadotrophin supply represents an excess of
60 X 106 I and hundreds of thousands of donors are participating in these
programmes. Being independent of this source of material avoids shortages of
material for clinical use due to lack of raw materials. This is particularly
relevant considering the constantly increasing demand for gonadotrophins due to
development of new ART, e.g. the recently developed intracytoplasmic sperm
injection (ICSI) procedure. Further, it allays concerns over the possibility that
infectious agents, drugs or drugs metabolites derived from the human-recovered
raw materials, could (despite precautions) find their way into the finished
products. Finally, since cells derived from a single transfected cell will be used
to supply FSH for the decade to come, recombinant technology should offer
unprecedented consistency in product characteristics.
Purity and specific activity
This is related to the up-to-date purification processes which were developed in
parallel with the in-vitro production process. High purity is clinically relevant
since it results in products suitable for s.c. administration. It also allows quality
control of the preparations (see below).
97
E.Loumaye et al.
Batch-to-batch consistency
Recombinant human gonadotrophin preparations are expected to present fewer
batch-to-batch variations compared with extractive preparations, e.g. rhFSH
displays a lower level of amino acid chain truncation and less deaminated
degradation forms than the urine-derived material. The carbohydrate structures
of rhFSH are a subset of those found in urinary preparations; moreover, the high
purity allows the use of physico-chemical characterization of the product for
quality control and product specification purpose in addition to the currently
used in-vivo bioassay.
Monotherapeutic agent
The rhFSH preparation is free of LH activity providing a monotherapeutic agent
for clinical use and rhLH is the first ever therapeutic preparation containing only
LH, available in significant quantities.
The objective of this review is to provide updated information on results of
clinical evaluation of a preparation of rhFSH (Gonal-F®; Serono, Switzerland),
a preparation of rhLH (LHadi®; Serono, Switzerland) and a preparation of rHCG
(Ovidrel®; Serono, Switzerland). One other therapeutic preparation of rhFSH
resulting from a similar recombinant DNA production process is in development
(Puregon®; Organon, The Netherlands) (Devroey et ai., 1994; Out et aZ., 1995).
Since rhFSH is the most advanced in terms of clinical assessment, a large part
of this paper will be related to it.
Clinical assessment of rhFSH
Before entering the clinical phase of rhFSH assessment, several studies were
conducted in non-human primates and human volunteers to determine the
pharmacokinetic characteristics of rhFSH and to compare them with those of
urinary (u)hFSH. The data indicate that the pharmacokinetic characteristics of
rhFSH are very similar to those of uhFSH preparations (Porchet et ai., 1993,
1994; Le Cotonnec et ai., 1994a,b). The bioavailability is -60% and comparable
after s.c. and i.m. administration. After s.c. administration the apparent terminal
half-life of FSH is -37 h. For clinicians, this indicates that rhFSH can be
administered using the same doses and schedules that had been used for uhFSH.
Clinical indications for FSH
Since no other gonadotrophin preparations were initially available, HMG had
been used for stimulating follicular development in all of the main FSH indications
in female patients, i.e. induction of follicular development in WHO group I
anovulation, in WHO group II anovulation [mainly patients with a polycystic
ovarian disease (PCOD)], and for stimulating multiple follicular development in
98
Human gonadotrophins and recombinant DNA technology
ovulatory patients, pre-treated or not with a GnRH agonist, prior to ART. The
experience accumulated since then with urinary preparations of FSH practically
devoid of LH activity supports the concept that FSH is effective alone and
actually leads to significantly higher pregnancy rates than HMG in IVF (FIVNAT,
1995; Wikland et al., 1994; Daya et al., 1995).
WHO group II anovulation is characterized by asynchronous gonadotrophin
and oestrogen production and normal concentrations of prolactin (PRL) (WHO,
1973). These patients present with a variety of menstrual disorders ranging from
regular but anovulatory cycles to oligomenorrhoea and amenorrhoea. The
persistence of oestrogen production is demonstrated by the presence of spontaneous bleeding or progesterone-induced withdrawal bleeding. When ovarian
morphology is assessed by ultrasound in these patients, a high proportion of
them (>90%) present a polycystic aspect of the ovary (Franks, 1989; Fox
et al., 1991).
Since the majority of these patients are already over-exposed to excessive
endogenous LH secretions, it was not a surprise to show that treatment with
uhFSH is at least as effective as HMG for stimulating follicular development
and achieving ovulation and pregnancy after HCG administration in this population (Seibel et al., 1985; Venturoli et al., 1987; Butt et al., 1988; Homburg et al.,
1990; Fulghesu et at., 1990; Gadir et al., 1990; Larsen et al., 1990; Sagle et al.,
1991). Moreover, recently, it has been shown that the FSH threshold concentration
required to initiate follicular growth is not influenced by co-administration of
LH, confirming that addition of LH to FSH is unnecessary in this indication (van
Weissenbruch et at., 1993).
In male patients, FSH is indicated in association with HCG to initiate
and maintain the spermatogenesis in patients deficient in gonadotrophins, i.e.
hypo gonadotrophic hypogonadism.
rhFSH assessment in patients undergoing superovulation for ART
The first clinical indication which was investigated with rhFSH was the stimulation
of multiple follicular development in women undergoing ART.
An early case report first indicated that rhFSH alone stimulates multiple
ovarian follicular growth and oestradiol secretion in patients undergoing IVF
with embryo transfer who were pre-treated with a GnRH agonist (Germond
et al., 1992). Retrieved oocytes were fertilized and a viable pregnancy obtained.
Subsequently, several phase III clinical studies have been completed. The first
completed study was a prospective, randomized, parallel group, open, multicentre
study performed to compare rhFSH (Gonal-F®) administered s.c. with uhFSH
(Metrodin®) administered i.m. in women undergoing IVF with embryo transfer
(Recombinant Human FSH study Group, 1995). In this study all patients were
pre-treated with buserelin (200 Ilg/day s.c.) for inducing pituitary desensitization
prior to stimulation with FSH. No significant difference was observed in the
mean number of growing follicles, of retrieved oocytes and of fertilized oocytes.
The duration of FSH treatment to achieve full follicular and the average dose
99
E.Loumaye et al.
Table II. Safety summary for subjects treated with recombinant human follicle stimulating
hormone (rhFSH, Gonal-F®)
Indication
No. patients (0/0)
Exposure (months)
Serious adverse event (0/0)
11.3
0
202 (37.9)
55.2
9 (4)
Clinical pharmacology 68 (12.8)
ART
WHO II
227 (42.6)
227.4
6 (3)
Male
36 (6.8)
119.1
2 (5.5)
Total
533 (100)
413.0
17 (3)
ART = assisted reproductive techniques; WHO II = WHO group 11 anovulation.
of FSH to reach this stage were not significantly different for rhFSH and uhFSH.
In all, 83 and 82% of the patients achieved embryo transfer in the rhFSH and
uhFSH groups respectively. The embryo implantation rate was 13% in both
treatment groups. The 'take home baby' rate was 18 and 15% per embryo transfer
in the rhFSH and uhFSH groups respectively.
Results from a second independent, prospective, randomized, parallel group,
study conducted in the USA, comparing rhFSH (Gonal-F®) with uhFSH (Metrodin®) support the conclusion of the first study (O'Dea et ai., 1993). It is
noteworthy that no anti-FSH antibodies were found in any of the patients treated
with rhFSH, nor in any of those treated with uhFSH. Local tolerance was good
with both treatments.
Clinical assessment in WHO group II anovulation
The second indication for rhFSH which was evaluated is the stimulation of
(single) follicular development in WHO group II anovulatory patients. In this
population, the clinical challenge is to achieve the maturation and ovulation of
a minimal number of follicles (and preferably only one) to obtain a singleton
pregnancy and no ovarian hyperstimulation syndrome (OHSS). Therefore, in the
multicentre studies which were conducted to compare rhFSH with uhFSH, a
'chronic low dose' regimen was used for administering FSH (Buvat et at., 1989;
Hamilton-Fairley et at., 1991; Shoham et at., 1991c).
The first case report published indicated that rhFSH alone is effective in
stimulating follicular development and in promoting oestradiol and inhibin
secretion. Successful ovulation and pregnancy was achieved following HeG
administration (Hornnes et at., 1993).
More recently, a multinational, prospective, open, randomized, parallel group
study comparing the efficacy of rhFSH (Gonal-F®) (n = 110 patients) with
uhFSH (Metrodin®) (n = 112 patients) for inducing ovulation in WHO group II
anovulatory patients has been completed. Using a chronic low dose protocol, the
ovulation rate was 64 and 59% (P = 0.36) in the first treatment cycle and the
cumulative ovulation rate over three treatment cycles was 84 and 91 % (P =
0.09) for rhFSH and uhFSH respectively. Monofollicular development, defined
100
Human gonadotrophins and recombinant DNA technology
Table III. List of serious adverse events from subjects
treated with recombinant human follicle stimulating
hormone (rhFSH, Gonal-F®)
Female
Mild-moderate OHSS
4
Abortions
3
Ectopic pregnancy
Congenital malformation
Severe intra-uterine growth retardation
Severe abdominal pain and fever
Pelvic inflammatory disease
Severe headache
Drop-outs for ovarian cysts
2
Male
Viral myositis
Submandibular abscess
as one follicle ;:::::16 mm on the day of HCG, was achieved in 60 and 51 % of the
first treatment cycle (cycle A), treated with rhFSH and uhFSH respectively.
Pregnancy rate was 23 and 25% (P = 0.75) in the first treatment cycle and the
cumulative pregnancy rate over three treatment cycles was 42 and 48% (P =
0.33) for rhFSH and uhFSH respectively. Multiple birth rates were 6 and 14%
for rhFSH and uhFSH respectively. One case of moderate OHSS was recorded
in each treatment group (0.4% per cycle). No patients were found to be positive
for anti-FSH antibody (Serono, unpublished data). Data on local tolerance was
available for 9327 injections of the 9494 injections performed in this study. The
local tolerance was good. The fact that rhFSH can be injected s.c. led to voluntary
self-administration in -50% of cases, contrasting with <10% in the uhFSH group
in the context of this study.
Clinical assessment in male hypogonadism
Recombinant hFSH is also currently evaluated in association with HCG in
hypo gonadotrophic hypogonadal male patients for restoring spermatogenesis and
fertility. Successful induction of fertility by Gonal-F® in a patient with complete,
primary hypogonadism (testicular volume of 1.0 ml bilaterally) has been reported
(Quinton et ai., 1994).
Overall safety experience with rhFSH
Experience with Gonal-F® is quickly growing and to date> 1300 patients have
been treated worldwide. All clinical studies performed worldwide with
101
E.Loumaye et aL
Gonal-F® are monitored through Serono's Drug Event Report procedure. Table II
lists the number of serious adverse events reported for the first 533 subjects!
patients exposed to rhFSH in the respective clinical indications. Table III
indicates that some observed serious adverse events (SAE) are related to the
pharmacological property of the compound, e.g. OHSS and abdominal pain, but
the majority are expected complications of pregnancy in general such as e.g.
miscarriage, ectopic pregnancy and congenital malformations, not related to the
drug itself. In addition, to date, no sera have been found positive for anti-FSH
antibody.
Clinical assessment of rhLH
Before entering the clinical phase of rhLH assessment, several studies have been
conducted in non-human primates and human volunteers to determine the
pharmacokinetic characteristics of rhLH and compare these with pituitary LH
and uhLH (Porchet et aI., 1995; Serono, unpublished data). The data indicate
that the pharmacokinetic characteristics of rhLH preparations are very similar to
those of pituitary and urine-derived hLH preparations. After s.c. administration
the apparent terminal half-life is -10-12 h for all three preparations. This came
as some surprise since the apparent terminal half-life of hLH in serum has
usually been reported to be -3-4 h (Yen et aI., 1968; Diczfalusy et aI., 1988).
Clinical assessment of rhLH in WHO group I anovulation
A rare cause of anovulation is that described as WHO group I (WHO, 1973). It
is also named as central failure or hypo gonadotrophic hypogonadism and is
characterized by reduced hypothalamic or pituitary activity. As a consequence,
serum gonadotrophins are abnormally low and ovarian oestrogen secretion is
negligible.
In WHO group I anovulation, co-administration of LH is required during FSH
therapy to obtain optimal follicular development. This is supported by the
following clinical evidence: (i) in a crossover design, uhFSH was compared with
HMG in 10 hypogonadotrophic hypogonadal patients. Ovulation was assessed
by luteal phase progesterone concentrations and ultrasonography. During HMG
treatment, two cycles were cancelled because of risk of OHSS, and ovulation
occurred in the other eight cycles. During FSH treatment, one cycle was cancelled
because of a risk of OHSS, and ovulation occurred in six out of the nine
remaining cycles. Pre-ovulatory oestradiol concentrations were, on average, three
times lower with FSH than with HMG (Couzinet et al., 1988); (ii) in a similar
crossover study, FSH was compared to HMG in nine hypo gonadotrophic
hypogonadal patients, ovulation was assessed by measuring luteal phase progesterone and assessing the ultrasonographic appearance of the corpus luteum (CL).
All HMG-treated cycles led to ovulation and only three out of nine FSH-treated
cycles led to ovulation. Moreover, in FSH-treated cycles mean pre-ovulatory
102
Human gonadotrophins and recombinant DNA technology
9000
o
8000
-
7000
..oJ
:::: 6000
o
0
-&.
5000
o
N
W
E
...
4000
::l
CI)
tn
oT
3000
2000
1000
0
T
o -
0""-
001
0
75
225
rQ.,
01
8 0-.......... db··············
............... ~ ....... 'o'ai,
.Oc?
o
25
r-hLH dose (IU/day)
Figure 1. Individual serum oestradiol (E l ) concentrations on the day of human chorionic gonadotrophin
(HCG) administration or the last day of recombinant human follicle stimulating hormone (rhFSH)
administration (if no HCG was injected) in WHO group I anovulatory women treated with 150 rhFSH/day
and a fixed dose of recombinant human luteinizing hormone (rhLH) i.e. 0, 25, 75, or 225 IU LH/day.
oestradiol concentrations were three times lower and endometrial thickness was
reduced by almost 30% when compared with HMG-treated cycles (Shoham
et oZ., 1991d).
Together, these observations indicated that in the hypogonadotrophic hypogonadal female population, a significant proportion of patients do not have the
threshold concentration of endogenous LH required to achieve optimal follicular
development and steroidogenesis during therapy with FSH alone. The availability
of rhFSH devoid of any residual LH activity has prompted researchers to readdress this question. A first case of hypo gonadotrophic hypogonadism treated
with rhFSH was reported by Schoot et aZ. (1992). Multiple follicular development
(defined as fluid cavities in the ovaries, visualized with ultrasound) was achieved
while the oestradiol concentration remained very low (about four times lower
than the expected average value). Follicular fluid oestradiol concentrations were
1500 times lower than expected. Serum progesterone showed no elevation
following HeG administration. This first observation has been recently confirmed
in a larger number of patients. Some follicular development was recorded in
all patients, but interestingly, although oestradiol secretion was very low,
103
E.Loumaye et al.
immunoreactive inhibin secretion did not appear to be as significantly impaired
(Schoot et aI., 1994).
Recombinant hLH thus appears as an ideal adjunct therapy to rhFSH in
hypogonadotrophic hypogonadal women, and early case reports clearly showed
that rhLH (LHadi®) is bioactive in humans and efficiently supports FSH-iRduced
follicular development leading to pregnancies in this indication (Hull etaZ., 1994;
Kousta et aI., 1996).
A randomized, parallel group, multicentre, dose-finding clinical study has been
completed for the evaluation of rhLH in this indication. A total of 38 patients
completed the study. Fixed doses of either 25 IV or 75 IV or 225 IV/day of
rhLH in combination with a fixed dose of 150 IV rhFSH were compared with
rhFSH alone (150 IV/day). Doses ranging from 75-225 IV/day of rhLH,
administered simultaneously with rhFSH were able to promote optimal ovarian
oestradiol secretion (Figure 1). In addition, these doses enhanced the ability of
the follicle to luteinize when exposed to a surrogate pre-ovulatory LH surge, i.e.
HCG 5000 IV (Shoham et aZ., 1995).
Recombinant LH administered s.c. was well-tolerated and no pre-treatment
and post-treatment sera were found positive for anti-LH antibodies in any of the
patients (Serono, unpublished data).
Clinical assessment of rHCG
The clinical assessment of rHCG was started after completion of preclinical and
human pharmacology studies during which the pharrnacokinetic characteristics
of rHCG have been shown to be very similar to those of urine-derived HCG. In
humans, the apparent terminal half-life after s.c. administration is -32 h.
Clinical evaluation of rHCG is ongoing in both indications, i.e. ART and
WHO group II anovulation. More than 10 pregnancies have been recorded after
rHCG administration to trigger final follicular maturation and luteinization prior
to ART (Serono, unpublished data).
Conclusion
All three human gonadotrophins are now produced in vitro by recombinant DNA
technology and have been purified and formulated to lead to preparations suitable
for therapeutic use in humans using s.c. administration. All three preparations
are progressing well through clinical evaluation programmes designed to permit
the registration of these agents by Health Authorities.
Recombinant hFSH appears to be safe and effective for stimulating follicular
development prior to ART or in WHO group II anovulation without any coadministration of exogenous LH. An effective regimen of administration is a
single daily, s.c., self-administered injection into the anterior wall of the abdomen.
Recombinant FSH is also effective in stimulating follicular development in
WHO group I anovulation, but co-administration of rhLH is mandatory in order
104
Human gonadotrophins and recombinant DNA technology
to obtain adequate follicular steroidogenesis as demonstrated by oestradiol
secretion, endometrial growth and luteal phase progesterone secretion.
In addition to the important clinical uses of the recombinant gonadotrophins,
their safety, specificity and the consistency of the preparations, makes them
useful tools for further pre-clinical and clinical research to optimize gonadotrophin
therapeutic regimens and protocols.
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