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stability in blood and improved in vivo bioactivity compared to rhFSH
Follistim
s
in rats.
148
6.1.2 Addition of the Carboxy terminal peptide (CTP). This
approach consists in constructing chimeric genes containing the
sequence encoding the CTP of the hCGb-subunit fused to the human FSH
b-subunit. The bFSH-CTP chimera was co-transfected with the common
glycoprotein a-subunit and expressed in CHO cells.
149
The presence of
the CTP sequence did not affect assembly of FSH a- and b-subunits nor
the secretion of the dimer. In vitro receptor binding and steroidogenic
activity of chimerical rhFSH were the same as wild-type rhFSH. CTP is a
31 amino acids sequence including four additional O-linked glycans,
resulting in a 10-fold prolonged half-life of hCG compared to other
gonadotropins.
150
Both the in vivo potency and half-life in circulation of
rhFSH bearing CTP unit(s) were enhanced.
149,151
Further studies showed
a longer absorption and elimination half-lives compared to rhFSH
Puregon
s
152
or Gonal-F
s
,
153
10-fold increase in biopotency
compared with pituitary hormone.
154
In rat that a single injection of this
fusion molecule stimulated follicular maturation suciently to facilitate
ovulation induction. In comparison, a single injection of the same dose
of pituitary FSH was ineffective in increasing ovarian ovulatory poten-
tial.
154
Interestingly, no rhFSH-CTP antibody formation was observed.
152
These studies on rhFSH-CTP molecule led to the development of cor-
ifollitropin alfa (Elonva
s
, Merck formerly Organon), the first long-acting
hybrid molecule with sustained follicle-stimulating activity marketed in
2010. Corifollitropin alfa demonstrated a 2- to 4-fold increase in bioac-
tivity (ovarian weight, serum estradiol and progesterone, ovulated ova)
over rhFSH across all in vivo parameters assessed. These data demon-
strate that corifollitropin alfa is a specific ligand with high anity for
FSH receptor lacking intrinsic activity for LH and TSH receptors.
151
At the
production level, CTP is an interesting fusion approach because it
increases FSH secretion by a factor 3.
155
The use of a medium dose of this
long-acting FSH is a safe treatment option and equally effective com-
pared to daily FSH.
156
and a
B
6.1.3 Addition of N-glycosylation sites. Long-acting FSH molecules
have also been engineered by introducing additional N-glycosylation
sites. The half-life in vivo and bioactivity of rhFSH including additional
N-glycans was definitely longer than those of rhFSH
153,157-159
and roughly
similar to that corifollitropin alfa.
153,158
It is therefore demonstrated that
carbohydrate-targeted engineering is successful and worth being con-
sidered to develop long acting analogs.
6.2 Glycosylation based engineering
Engineering glycans aims at both reducing clearance mediated by the
receptor of asialoglycoproteins,
95
GalNAc-sulfate,
96
mannose-rich
160
and
fucosylated proteins.
161
Increasing the sialic acid helps masking this
carbohydrate signals and thus prevents lectin-mediated uptake. Indeed,
controlling sialylation of protein drugs is still a major challenge in the
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