Biomedical Engineering Reference
In-Depth Information
were carried out to stabilize hGH and extend its half-life. It
was shown that complexation of hGH with heparin did not
cause a major distribution in the tertiary structure of hGH but
decreased the hydrophilic environment and stabilize the
hormone. Other study demonstrated that crystals of hGH
coated with positively charged poly (arginine) allowed
delivery of hGH over a period of several days. Other studies
designed a long-acting hGH by fusing the CTP of hCG
b
-subunit that contains four O-linked oligosaccharide
recognition sites to the coding sequence of the hormone.
Crystallographic studies indicated that N-terminal and
C-terminal of hGH are not important for binding of the
hormone to its receptor. Therefore, CTP was ligated to
the N-terminal and C-terminal of hGH. The results indicate
that ligation of CTP to the coding sequence of GH did not
affect secretion of the chimeric protein into the medium.
In vivo studies in hypophysectomized rats indicated that,
bioactivity and pharmacokinetic parameters, area under the
curve (AUC) and mean residence time (MRT), T
max
,
C
max
, and half-life, of hGH bearing the CTP were dramati-
cally enhanced.
Efficacy of hGH-CTPs was assessed by weight gain in a
hypophysectomized rat model. Weight gain was measured in
all animals before treatment, 24 h after first injection and
then twice weekly until the end of the study on day 13.
Subcutaneous injections of GH-WT, or CTP-GH-CTP-CTP
(50
m
g/rat) once a week for 2 weeks or with Biotropin
(10
m
g/rat) daily for 2 weeks, resulted a dramatic increase
(P
CTP-hGH-CTP-CTP chimera were injected IV into hypophy-
sectomized male mice. At selected intervals after injection
of 50
m
g/kg, blood samples were collected and the concen-
trations of hGH were determined. The results indicated
that a higher level of the chimera is still detectable in serum
after 50 h where the level of Biotropin after 24 h was
undetectable. The estimated half-life of CTP-hGH-CTP-
CTP is increased by fourfold tofivefold comparing to
Biotropin. These data suggest that the mechanism of hGH
metabolic clearance is affected by the presence of CTP [32].
Preliminary studies of hGH-CTPs clinical trials indicated
that this peptide is safe for use and clinical trials Phase II is in
the way.
13.4 CONCLUSIONS
In this chapter, we have described the role of O-linked
oligosaccharide chains on glycoprotein function. Moreover,
the design of long-acting glycoprotein hormone (FSH, TSH,
EPO, and hGH) agonists was explained. This was achieved
by ligation of the sequence of hCG
b
-carboxyl-terminal
peptide, containing the recognition signal of four O-linked
oligosaccharide chains, to the coding sequence of the hor-
mone. Ligation of the CTP to different proteins indicated
that the O-linked glycosylation recognition sites of the CTP
are preserved. Moreover, this ligation is not involved in
assembly of the subunits, secretion of the dimer, receptor-
binding affinity and in vitro bioactivity. CTP can readily be
attached to a wide array of existing therapeutic proteins,
stabilizing the therapeutic protein in the bloodstream and
greatly extending its life span without additional toxicity or
loss of desired biological activity. Both the in vivo bio-
activity and half-life in circulation of hormones bearing the
CTP were significantly enhanced. CTP-modified proteins
can be manufactured using established recombinant DNA
techniques in widely used mammalian protein expression
systems. Clinical trials of proteins containing the CTP
indicated that these chimeras are not immunogenic and
safe for use. This strategy may have wide applications for
enhancing the in vivo bioactivity and half-life of diverse
proteins.
0.001) in weight gain. These results indicated that
accumulation of CTP-hGH-CTP-CTP variant induces an
incremental weight gain of 16.5 and 16.8 g, respectively.
Similar results were obtained when Biotropin was injected
daily.
Injection of GH-CTPs (0.4, 0.8, or 4 mg/kg) once every 4
days significantly (P
<
0.001) increased the weight gain
compared to controls. A similar effect was achieved by
daily administration of 0.1mg/kg Biotropin. Injection of
higher amounts (1.05mg/kg) of CTP-hGH-CTP-CTP once
every 4 days dramatically increased the weight gain. These
results indicated the importance of sustained blood levels,
rather than total dose of hGH for higher bioactivity in vivo.
These findings are consistent with the hypothesis that the
ability of a single injection of hGH-CTPs to increase weight
gain, results from its increased stability in the circulation.
This may support the strategy of hGH administration of at
least once a week.
Pharmacokinetic studies indicated the importance of
sustained blood levels, rather than total dose of hGH. These
findings are consistent with the hypothesis that the ability of
a single injection of hGH-CTP to increase weight gain,
results from its increased stability in the circulation. The
increased biopotency of the chimera may reflect a change in
their metabolic clearance in vivo. Therefore, the circulatory
half-lives of the hormones were determined. hGH-WT or
<
REFERENCES
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GlcNAcylation and phosphorylation: roles in insulin resistance
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2. Wopereis S, Lefeber DJ, Morava E, Wevers RA. (2006)
Mechanisms in protein O-glycan biosynthesis and clinical
and molecular aspects of protein O-glycan biosynthesis
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