Biomedical Engineering Reference
In-Depth Information
FIGURE 15.5
The in vitro biological activity of hGH and
GH-LRv0. The LR-fusions all have similar activities and are
10 times less active than hGH. Source: This research was
originally published in Nature Medicine, Reference [13].
#
FIGURE 15.4
Analytical gel filtration shows that GH-LRv0 is
present in two forms, monomeric and dimeric.
Source: This
research was originally published in Nature Medicine, Refer-
ence [13].
#
Nature Publication Group.
Nature Publication Group.
the LR-fusion was in two distinct forms. Analytical gel
filtration confirmed the presence of two distinct forms of
LR-fusion (Figure 15.4) and this was further corroborated by
analytical ultracentrifugation. These analyses suggested that
the LR-fusion existed in solution in monomeric and dimeric
forms, with the monomer being
orbital plexus under isoflurane anesthesia, the serum sam-
ples were stored at
70
C. The serum concentrations of GH
and GH-LRv0 in these samples were measured using an
ELISA against GH. The pharmacokinetic profiles were
estimated by fitting the values of hormone concentration
versus time to compartmental models using nonlinear least-
square regression analysis. The values were normalized for
animal weight and the clearance rates per animal weight and
terminal half-lives were calculated using coefficient and
exponents obtained from an intravenous bolus model.
GH-LRv0 had delayed clearance when administered
intravenously and subcutaneously, delayed absorption was
also observed for the subcutaneously administered LR-
fusion (Figure 15.6; Table 15.1). When administered by
an intravenous route, GH-LRv0 had a clearance rate of
3.3
75 kDa in size.
15.4.2 Biological Activity
Measurement of biological activity was carried out using a
dual luciferase assay. Briefly, human embryonic kidney
cells, which stably expressed GHR at a high level (HEK
293 Hi), were transfected with an inducible firefly luciferase
gene and a constitutive Renilla luciferase gene. Firefly
luciferase expression was stimulated by GH activity through
the Lactogenic hormone response element (LHRE)
sequence; the Renilla luciferase expression was used to
correct for differences in transfection efficiency and cell
number between the various samples.
GH-LRv0 had agonistic activity; however, the activity
was
2 h; this
is 300 times slower than that of GH [2,18]. After a single
subcutaneous dose, the GH-LRv0 had a delayed peak at
0.9mL/h/kg and a terminal half-life of 21
30 h, compared to 1 h for GH. GH-LRv0 was also still
detectable 8 days after subcutaneous administration,
whereas GH was undetectable after 0.25 days. GH-LRv2
and GH-LRv3 showed similar pharmacokinetic profiles to
GH-LRv0 with clearance rates of 2.8
10-fold lower than the ligand alone (Figure 15.5), GH-
LRv2 and GH-LRv3 showed similar activity to GH-LRv0.
The decreased in vitro bioactivity of the LR-fusions may be
a consequence of the dimer formation of these molecules;
the LR-fusions dimers maybe inactive but in dynamic
equilibrium with the active monomeric form, hence the
reduced in vitro bioactivity.
0.2mL/h/kg and
2.7
0.2mL/h/kg, respectively (Figure 15.6A; Table 15.1).
To investigate if the pharmacokinetic profiles of the LR-
fusions were a consequence of size alone, the pharmaco-
kinetic profiles of two other LR-fusions were also evaluated.
One LR-fusion consisted of GH linked to only domain B of
GHRec (55 kDa) the other consisted of two GH molecules
linked to GHRec, that is, GH-GH-GHRec, (100 kDa); iden-
tical linkers were used between the domains in these LR-
fusions. Both the 55 and the 100 kDa proteins showed
greater clearance rates compared to the 75 kDa GH-LRv0,
the terminal half-life of both were less than 4 h after
15.4.3 Pharmacokinetics
Seven-week-old Sprague-Dawley rats were injected with
vehicle, GH and the LR-fusions. The rats (n
4-6 per
group) were injected subcutaneously or intravenously (pen-
ile vein) with 0.1mg/kg (body weight) of GH or GH-LRv0.
Blood samples were collected periodically from the retro-
ΒΌ
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