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Vol. 281, Issue 1, 522-530, 1997
Department of Pharmacology, Cephalon, Inc., West Chester, Pennsylvania
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Abstract |
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 Top
Abstract
Introduction
Methods
Results
Discussion
References
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Recent studies indicate that a daily s.c. injection of 1 mg/kg of recombinant human insulin-like growth factor-1 (rhIGF-1) for 17 days is efficacious in enhancing the functional recovery of injured sciatic nerves in CD-1 mice. To identify and characterize surrogate marker(s) that are altered in association with the administration of an efficacious dose of rhIGF-1, dose-response curves (0.1, 1 and 10 mg/kg) and time course effects (0, 0.5, 3, 6 and 24 hr) were determined after acute (single) and chronic (once daily for 17 days) injections of rhIGF-1 in CD-1 mice. Plasma glucose levels decreased in a dose-dependent fashion after either acute or chronic injections of rhIGF-1 with maximal effects at 0.5 to 1 hr after administration of rhIGF-1. Among the three insulin-like growth factor binding proteins (IGFBPs) evaluated in the study, only IGFBP2 levels were consistently increased in a dose-dependent fashion with maximal effects 3 hr after the last of a series of injections of rhIGF-1. Furthermore, IGFBP2 levels increased at a dose of rhIGF-1 (1 mg/kg) that enhances the regeneration of injured sciatic nerves in mice. Chronic administration of insulin at doses that cause comparable decreases in plasma glucose to that of rhIGF-1 did not alter IGFBP2 levels or enhance hindlimb function suggesting that the beneficial effects of rhIGF-1 occur via activation of the type-I IGF receptor rather than the insulin receptor. Based on these criteria, IGFBP2 appears to be useful as a surrogate marker for determining the in vivo effects of rhIGF-1.
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Introduction |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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IGF-1 is a 70-amino acid peptide
that is a member of the insulin family of peptides and plays an
essential role in growth and development of many tissues (Liu et
al., 1993
; Baker et al., 1993). In the central nervous
system, IGF-1 is expressed during fetal development of the brain and
peripheral nerves (Werner et al., 1989; de Pablo and de la
Rosa., 1995). Recent studies demonstrate that genetically engineered
mice homozygous for a targeted disruption of the IGF-1 gene exhibit a
phenotype that results in severe growth deficiency including a
reduction in the amount of neuronal fibers and cytoplasm of neuroglial
cells (Liu et al., 1993). In vitro, IGF-1
promotes the survival of astrocytes and neuronal precursor cells from
fetal rat brain and motor neurons (Ang et al., 1992; Komoloy
et al., 1992; Hughes et al., 1993; Gammeltoft
et al., 1994). In vivo, local infusion of IGF-1
to the proximal end of a cut sciatic nerve promotes regeneration of the
peripheral nerve (Nachemson et al., 1990). Furthermore,
recent studies indicate that chronic systemic administration of 1 to 3 mg/kg rhIGF-1 is efficacious in enhancing recovery of hindlimb function
after bilateral crush of sciatic nerves in CD-1 mice (Contreras
et al., 1995). Taken together, these studies have prompted
the hypothesis that IGF-1 would be of potential therapeutic value in
certain pathological conditions involving spinal motor neurons such as
amyotrophic lateral sclerosis (Lewis et al., 1993; 1994).
The cellular effects of insulin and IGF-1 are triggered by the binding
of insulin or IGF-1 to the insulin or type-1 IGF receptors, respectively. The type-I IGF and insulin receptors are highly homologous transmembrane glycoproteins that are composed of two 
-subunits (ligand-binding domains) and two 
-subunits (tyrosine kinase domains) (Yarden and Ullrich, 1988; Czech, 1989; Ullrich and
Schlessinger, 1990). The specificity of ligand binding to the type-I
IGF or insulin receptor is determined by the relative affinities of the
ligands for their receptors. For example, IGF-1 has a 100-fold greater
affinity for the type-1 IGF receptor compared to insulin. After
specific binding of IGF-1 or insulin, the receptor autophosphorylates
its -subunit (Rosen et al., 1983) which in turn leads to
tyrosyl phosphorylation of a 131-kDa cytosolic protein named insulin
receptor substrate-1, (Sun et al., 1991). Signaling to the
cytoplasm and nucleus is attained by association of IRS-1 with second
messenger molecules containing Src homology domains, such as
phosphotidylinositol 3
kinase (Myers et al., 1993) and GRB-2 (Myers et al., 1994).
In addition to liver, which is the main source of IGF-1 in plasma,
there are several local sources of IGF-1 (Schwander et al.,
1983; Gosteli-Peter et al., 1994) and consequently its
actions are relatively widespread. There exists a highly complex family of IGFBPs that are thought to facilitate transport of IGF-1 and modulate the actions of IGF-1 on target cells. Although six different IGFBPs (1-6) have been identified (for review see Drop et
al., 1992), in plasma three different IGFBPs which range between
24 and 55 kDa can be visualized reliably using a Western/ligand blot technique (Fazleabas and Donnelly, 1992). IGFBP3 appears as a doublet
(48-55 kDa) due to different glycosylation states, IGFBP2 is a 34- to
36-kDa protein, and IGFBP4 has an Mr of 24 kDa
(Camacho-Hubner et al., 1991; Clemmons et al.,
1989; Clemmons, 1993). In some studies, IGFBP1 (31 kDa) which is
present in plasma at lower concentrations can be visualized by the
Western/ligand blot technique. In addition to the IGFBPs, an ~ 150 kDa ternary complex exists consisting of IGFBP-3, IGF-1 and an acid
labile subunit. IGF's bioavailibilty is increased after proteolysis of
this complex (Blat et al., 1994). The IGFBPs are thought to
participate in several functions, including control of IGF transport in
blood and out of the vascular compartment, localizing and modulating
IGF's to specific cell types and binding to receptors and regulating
blood glucose levels (Lewitt and Baxter, 1991; Lewitt et
al., 1991; Holly, 1991; for review see Clemmons, 1993).
Recently, rhIGF-1 has been under clinical evaluation for the treatment of amyotrophic lateral sclerosis. To facilitate selection of optimal dosage regimens and to assess the potential for tolerance it would be useful to identify a surrogate marker for rhIGF-1. By definition, a surrogate marker is a measure of some parameter associated with, but not a direct measure of, drug efficacy. It should be easily obtained from tissue or plasma, dose-responsive and, as a result, provide a basis for dosage selection in efficacy trials. The objectives of our study were to characterize biochemical effects of rhIGF-1 administration in an effort to identify a surrogate marker. As a secondary objective, we compared the effects of IGF-1 with those of insulin to determine the specificity of the regenerative effects of IGF-1.
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Methods |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Animals. Male CD-1 mice (Charles River, Raleigh, NC) between 30 to 60 days of age were used in the studies. Before use, mice were housed with five mice per cage and maintained on a 12 hr light/dark cycle. All mice were allowed access to food and water ad libitum.
All studies were carried out under the guidelines of the Cephalon Institutional Animal Care and Use Committee. After either acute or chronic s.c. injections of rhIGF-1 (dosed at the same time of day), mice were anesthetized under CO2 and blood was obtained via intracardiac puncture. Blood samples were immediately introduced into Microtainer heparinized tubes (Becton Dickinson, Rutherford, NJ) and kept on ice for 10 min. Samples were spun at 20,000 × g in a table top Eppendorf centrifuge for 10 min. Plasma (supernatant) was aliquoted and frozen at -80°C until use.Experimental design. For the acute rhIGF-1 injection study, 0.1, 1 or 10 mg/kg of rhIGF-1 were administered s.c. to mice that were killed at 0, 0.5, 1, 3, 6, 12 and 24 hr after the acute injection of rhIGF-1 (n = 5 at each time point/dose).
The time course of the chronic effects of rhIGF-1 treatment was evaluated by comparing the effects at 0, 0.5, 3, 6 and 24 hr after an acute challenge injection of 1 mg/kg of rhIGF-1 in two chronic treatment groups (n = 6 at each time point): 1) mice receiving injections with vehicle (0.1 M acetic acid) for 17 days and 2) mice receiving injections 1 mg/kg of rhIGF-1 for 17 days. The dose-response effects of chronic administration of rhIGF-1 was evaluated in mice injected daily with either 1) vehicle or 2) rhIGF-1 (0.1, 1 and 10 mg/kg) for 17 days (n = 6 at each dose). On the last day, both treatment groups were challenged with either 0.1, 1 or 10 mg/kg rhIGF-1. These mice were killed at 3 hr after the last challenge injection. Insulin (from bovine pancreas) was obtained from Sigma Chemical Co. (St. Louis, MO) and was prepared in phosphate buffered saline and administered via s.c. route (1U/kg = 0.036 mg/kg; 10 U/kg = 0.36 mg/kg).Sciatic crush. Mice were anesthetized on day 0 with a s.c. injection of ketamine (50 mg/kg), acetapromazine (0.75 mg/kg) and xylazine (5 mg/kg). Both sciatic nerves were exposed near the hip and crushed for 10 sec using hemostats whose tips were covered with plastic tubing to produce a fairly uniform amount of damage. The incision was closed with autoclips and the mice allowed to recover from the anesthetic before being randomly assigned to different treatment groups.
Both sciatic nerves were crushed and vehicle, insulin (1 or 10 U/kg) or rhIGF-I (1 mg/kg) was administered s.c. on the day of surgery and for the next 17 days. At various times thereafter, the mice were tested for return of function in both hind limbs by measuring the ability to grip an inverted wire screen.Grip assay. Mice were placed individually on a wire screen mesh, which was turned over to assess the ability of the mice to correctly grip the screen with their hind paws. Each mouse was tested 10 times and the number of failed trials recorded. Each failed trial was scored as a "1" with a maximum score of "10" for the test. A correct response was when a mouse gripped the screen with its hind toes. If a mouse held onto the screen by hooking its ankles or legs through the screen, the trial was still recorded as a failure.
IGF-1 immunoradiometric assay. After s.c. injections of rhIGF-1, the levels of rhIGF-1 attained in the plasma compartment were evaluated using the IGF-1 immunoradiometric assay kit from Diagnostic Scientific Laboratories Inc. (Webster, TX). The IGF-1 monoclonal antibody used in this assay detects human IGF-1 and does not cross-react with rat or mouse IGF-1. Hence the detection of IGF-1 in plasma reflects that of the rhIGF-1 injection only. Briefly, samples (100 µl, in duplicate) were acidified, to dissociate any complex bound to IGF-1, neutralized and assayed. Typically, 50 µl of the plasma sample and 200 µl of [125I]IGF-1 were added to IGF-1 antibody coated plastic tubes and incubated at room temperature for 3 hr on an orbital shaker set at 140 r.p.m. Tubes were then overturned to drain off excess liquid and washed three times with 3 ml of distilled deionized water. Radioactivity in tubes were determined in a Wizzard gamma-counter (Wallac, Inc., Gaithersburg, MD). Pharmacokinetic parameters were analyzed by non-compartmental pharmacokinetic analysis using a computer program written for PC-SAS (PC-SAS for Windows, Version 6.08, SAS institute, Cary, NC).
Plasma glucose determinations. Glucose levels were assessed in plasma samples of mice injected with rhIGF-1 or insulin using a glucose assay kit (Sigma Diagnostics, St. Louis, MO). Briefly, 10 µl of plasma samples (in duplicate) or glucose standards were incubated with 1 ml of glucose assay reagent for 10 min at room temperature. The reaction was terminated by the addition of 10 ml of 0.1 N hydrochloric acid. Samples were transferred to cuvettes and the absorbance was determined in a spectrophotometer at 520 nm.
Protein determination. Protein concentrations were determined in plasma after a dilution of 1:20 in distilled deionized water. Bovine plasma albumin (0.0625-2 mg/ml) was used to create a standard curve. Five µl of sample in triplicate was incubated with 300 µl of a 1:5 diluted stock solution of BioRad protein reagent (BioRad, Richmond, CA). Absorbance at 520 nm was detected in a spectrophotometer. Linear regression analysis was used to generate the standard curve and protein values were calculated from the linear regression equation.
Western/ligand blotting.
IGFBPs were analyzed from plasma
samples using a modified Western/ligand blot method (Hossenlopp
et al., 1986; Fazleabas and Donnelly, 1992). Ten µl of 3x
Laemmlli sample buffer (Laemelli, 1970), [2% sodium dodecylsulfate,
125 mM Tris-HCl, 10% (w/v) glycerol] was added to each 20-µl sample
of plasma and 2 µl of bromophenol blue. Samples were then diluted 1:4
with 1× sample buffer and boiled for 5 min. Seven µg of total
protein were electrophoresed on a 12% sodium
dodecylsulfate-polyacrylamide gel at 75V for 3 hr and then transferred
to nitrocellulose membranes overnight in Tris-glycine buffer containing
0.125 mM ethanolamine (pH 9.5). Blots were washed in 125 mM TBS, pH
7.4, for 30 min at room temperature followed by a 2-hr incubation with
TBS containing 1% bovine serum albumin. Blots were then incubated with
50 pM (3-[125I]iodotyrosyl) IGF-1; [specific activity
2000 Ci/mmol (Amersham, Cleveland, OH
)] in TBS containing 1% bovine serum albumin and 0.1% Tween-20 for 2 nights. Blots were washed three times (15 min for each wash) with TBS
containing 0.1% Tween-20 followed by two washes in TBS. Blots were
air-dried, placed in a Phosphor-Imager cassette and exposed for 1 day.
Data analysis. Data were analyzed using a two-way analysis of variance. When significant effects of treatment were observed data were further analyzed by Duncan's multiple range test. For the behavioral and biochemical studies, results are presented as mean ± S.E.M.. To assess whether any treatment resulted in responses that were different from control values (sham/vehicle) on any given day, a Dunnett's t test was carried out comparing all values to that of the control group. To determine whether any treatment was better than another treatment, the results of all groups were compared to one another using the Newman-Keuls test. Any comparisons with a P < .05 were considered to be significant.
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Results |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Determination of plasma rhIGF-1 levels after acute and chronic
injections of rhIGF-1.
The levels of rhIGF-1 in the plasma were
determined in plasma samples after acute injections and were assayed
using an IRMA assay. As shown in figure 1a, plasma
concentrations of rhIGF-1 increased in a dose-dependent manner with the
Cmax achieved 0.5 hr after administration. The levels of
rhIGF-1 in plasma at 0.5 hr were 104.8 ± 5.8, 1197 ± 43.4 and 11580 ± 570 ng/ml after an acute injection of 0.1, 1 and 10 mg/kg of rhIGF-1, respectively.
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Determination of plasma glucose levels after acute and chronic
injections of rhIGF-1.
Because IGF-1 has been reported to result
in hypoglycemia at high doses, plasma glucose levels were determined in
samples from mice treated with an acute injection of rhIGF-1. Plasma
glucose levels were significantly reduced between 0.5 and 1 hr, the
time of peak plasma concentrations of rhIGF-1, after either the acute injection of 1 or 10 mg/kg of rhIGF-1 (fig. 2a). In
order to determine the effects of chronic treatment with 1 mg/kg of
rhIGF-1, the time course for the decrease in plasma glucose levels was
determined in mice after the last of a series of repeated injections.
As shown in figure 2b, neither potentiation nor tolerance to the peak
effect of rhIGF-1 was observed after the acute challenge injection of 1 mg/kg in both treatment groups. However, although plasma glucose levels
returned to baseline levels by 3 hr after the acute rhIGF-1 challenge
injection in chronic vehicle-treated mice, they were still
significantly lower in chronic rhIGF-1-treated mice. By 6 hr, plasma
glucose levels in both groups returned to control levels. These results
suggest that the decrease in plasma glucose levels is slightly
prolonged after chronic treatment with rhIGF-1. This prolonged effect
on plasma glucose at 3 hr after chronic treatment with rhIGF-1 was
observed at all challenge doses of rhIGF-1 when compared to the chronic
vehicle treated group (fig. 2c).
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Determination of plasma IGFBP levels after chronic injections of rhIGF-1. In initial experiments, commercially available mouse plasma was used to analyze the three IGFBPs by Western/ligand blots. Incubation of a second nitrocellulose blot with a 1000-fold excess of unlabeled rhIGF-1 prevented specific binding of [125I]IGF-1 to the IGFBPs (data not shown). Addition of high concentrations (100 ng/ml) of rhIGF-1 to mouse plasma samples did not alter the levels of the IGFBPs suggesting that endogenous amounts of IGF-1 are not carried along with IGFBPs during gel electrophoresis (data not shown).
After Western/ligand blot analysis, three major bands can be visualized as shown in lane 1 (fig. 3a). The 48- to 55-kDa doublet reflects IGFBP3 and the 34- to 36-kDa singlet reflects IGFBP2. The estimated Mr sizes of each of the forms was determined by comparison with defined Mr size standards and are consistent with previous published reports (Camacho-Hubner et al., 1991; Clemmons, 1993). The identity of the two IGFBPs was
further confirmed by comparison of the bands with those of purified
rhIGFBP2 and rhIGFBP3 (lanes 2 and 3, fig. 3a). The 24-kDa band has
been previously reported to be IGFBP4 (Camacho-Hubner et
al., 1991; Clemmons et al., 1993). Due to the
unavailability of purified recombinant IGFBP4, direct identity
comparisons could not be made, hence we can only assume that this band
is IGFBP4. In some of the experiments, a faint band at ~ 31 kDa
(presumably IGFBP1) was observed. Densitometric analyses using the
Phosphor Imager revealed that the value of this band were near
background levels and could not be measured reliably. Therefore,
further characterization and quantitation of only IGFBP2, IGFBP3 and
24-kDa protein (IGFBP4) were carried out.
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Comparison of the effects of insulin with rhIGF-1 after sciatic nerve crush. To examine whether a peptide similar to rhIGF-1 that also has the ability to decrease plasma glucose could be efficacious in 1) enhancing regeneration of the sciatic nerve and 2) increasing IGFBP2 levels, we determined the effect of insulin on grip and IGFBPs after sciatic nerve injury.
In initial experiments the time course and dose-response effects of insulin on plasma glucose levels were determined (data not shown). From these studies, we determined that 1 and 10 U/kg of insulin caused a similar decrease in plasma glucose to that of 1 and 10 mg/kg of rhIGF-1. To compare the effects of multiple injections of insulin and rhIGF-1 on behavioral parameters associated with sciatic nerve function and IGFBP2 levels, CD-1 mice with bilateral crushes of the sciatic nerves were injected daily for 17 days with vehicle for insulin (phosphate-buffered saline), insulin (1, 10 U/kg), vehicle for rhIGF-1 or 1 mg/kg of rhIGF-1. Mice were tested for their ability to grip an inverted screen in 10 trials. As shown in figure 5a, the rate of recovery of grip ability was significantly enhanced after daily injections with rhIGF-1 but not after insulin. The effects of multiple injections of insulin and rhIGF-1 on plasma glucose at 0.5 hr (significant decrease in plasma glucose) after the last injection is shown in figures 5b. Both agents significantly decreased plasma glucose 0.5 hr after the injection. The effects of 1 U/kg insulin on plasma glucose was not significantly different from that after 1 mg/kg of rhIGF-1.
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Discussion |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Several lines of evidence suggest that IGF-1 plays a key
role in nerve regeneration and sprouting (Henderson et al.,
1983; Near et al., 1992; Gehrmann et al., 1994;
Caroni et al., 1994; Contreras et al., 1995).
Furthermore, recent studies suggest that systemic administration of
rhIGF-1 for 17 days enhances the rate of functional recovery of injured
sciatic nerve of adult mice (Contreras et al., 1995). The
results of the latter study suggest that a bell-shaped dose-response
curve exists for rhIGF-1, and although 1 mg/kg of rhIGF-1 was the most
efficacious dose for enhancing functional recovery in injured sciatic
nerves of mice, 10 mg/kg of rhIGF-1 was modestly effective in recovery
of hindlimb function. Our purpose was to identify a biochemical
surrogate marker in plasma that is altered at a dose that causes a
therapeutic effect and does not result in biochemical tolerance after
chronic treatment. Based on the results of our study, changes in IGFBP2 fit the criteria for a surrogate marker for chronic rhIGF-1 treatment.
In response to either acute or chronic administration of rhIGF-1,
plasma glucose levels decrease to similar levels with a maximal effect
observed at 0.5 hr after injection. This suggests that neither
potentiation nor tolerance to the peak hypoglycemic effects of rhIGF-1
were observed. A decrease in plasma glucose in response to IGF-1
administration is consistent with previous reports which show that
IGF-1 has insulin-like activity on blood glucose levels (Zapf et
al., 1986; Snyder and Clemmons, 1990; for review see Froesch
et al., 1985). The decrease in plasma glucose levels was
found to recover to control levels more slowly after chronic rhIGF-1
treatment when compared to an acute rhIGF-1 injection. Although the
mechanism of action and physiological role of this effect is unclear,
it is possible that this effect could be due to altered IGFBP levels.
For example, IGFBP1 has been shown to regulate blood glucose levels by
counteracting the insulin-like activity of IGF-1 in vitro
(Drop et al., 1979; Ritvos et al., 1988) and
in vivo (Lewitt et al., 1991). In our assay we
cannot reliably measure IGFBP1, however, it is tempting to speculate that changes in IGFBP1 levels or function after chronic treatment with
rhIGF-1 could retard the return of plasma glucose to normal levels.
Another possibility for observing this prolonged decrease in plasma glucose would be if the levels of rhIGF-1 accumulate in plasma after repeated rhIGF-1 injections, i.e., there is an alteration in the pharmacokinetic parameters of rhIGF-1 after chronic treatment. However, this does not appear to be the case since changes in the clearance rate or t1/2 of rhIGF-1 after chronic treatment were not observed in our studies. Because both acute and chronic injections of rhIGF-1 alter plasma glucose levels at a dose that is efficacious at enhancing the functional recovery of injured sciatic nerves (1 mg/kg), it fits the criteria for a surrogate marker. However, it is important to note that circadian rhythms, nutrition and stress can also affect plasma glucose levels and, therefore, plasma glucose cannot be used as a surrogate marker.
In response to 1 mg/kg of acute or repeated rhIGF-1 injections, plasma
IGFBP2 levels showed a dramatic increase (208%) with peak effects
attained at 3 hr after the challenge injection. This effect is
consistent with a previous study showing that in human subjects, a
daily injection of IGF-1 for 7 days resulted in an increase in IGFBP2
levels (Baxter et al., 1993). Furthermore, in our study, a
prolonged effect of rhIGF-1 on IGFBP2 is observed after chronic
treatment. This effect could be due to alterations in either the rate
of translation or the rate of degradation of IGFBP2. A recent study
showed that after sciatic nerve axotomy in neonates, IGFBP2 mRNA
increases with a concomitant increase in IGF-1 (Gehrmann et
al., 1994). Whatever the case may be, it is clear that biochemical
tolerance to the increase in IGFBP2 was not observed after chronic
rhIGF-1 treatment.
Chronic administration of 10 mg/kg of rhIGF-1 led to greater increases
in IGFBP2 levels in plasma. Although efficacy has been observed at this
dose of rhIGF-1, it appears to be less effective than 1 mg/kg of
rhIGF-1 at promoting recovery of hindlimb function (Contreras et
al., 1995). However, after administration of 10 mg/kg rhIGF-1, we
find that plasma glucose levels decrease to about 50% of control
levels. Therefore, it is possible that complications such as decreases
in plasma glucose at high doses could negate the positive effect of
neuronal regeneration.
Previous studies have shown that plasma levels of IGFBP2 are not
regulated by glucose infusion, insulin or circadian rhythms (Clemmons
et al., 1991). We have also demonstrated that the response of IGFBP2 is specific for rhIGF-1 because chronic administration of
insulin, failed to significantly alter IGFBP2 levels. Furthermore, we
have shown that low doses of insulin, which decreases plasma glucose
comparable to that after rhIGF-1 does not enhance hindlimb function.
These experiments suggest that the effects of rhIGF-1 on IGFBP2 levels
and regeneration of hindlimb function are modulated via the type-1 IGF
receptor and not via the insulin receptor. Because both acute and
chronic injections of rhIGF-1 alter plasma IGFBP2 levels at a dose that
is efficacious at enhancing regeneration of the sciatic nerve function,
and do not result in biochemical tolerance, IGFBP2 appears to be useful
as surrogate marker for the chronic effects of rhIGF-1.
Previous studies indicate that changes in IGFBP3 are more variable and
can be influenced by the particular conditions of treatment. For
example, twice daily s.c. injections of 0.04 mg/kg of IGF-1 for 7 days
in growth hormone receptor-deficient patients did not alter IGFBP3
(Gargosky et al., 1993). However, low doses of IGF-1 (0.1-0.125 mg/kg) in fasted human subjects for 7 days slightly decreased IGFBP3 levels (Baxter et al., 1993). These studies
have not used high enough doses of IGF-1 to observe any increases in IGFBP3. In contrast to injections, infusion studies in humans show that
IGFBP3 increases in response to IGF-1 (Clemmons et al., 1989; Quin et al., 1994). Our results suggest that the
effects of rhIGF-1 on IGFBP3 are dose-dependent. Although low doses of rhIGF-1 (0.1 mg/kg) decrease IGFBP3 levels, a dose of 10 mg/kg of
rhIGF-1 was found to modestly increase IGFBP3 levels. However, because
IGFBP3 did not increase in association with a functionally efficacious
dose of rhIGF-1, IGFBP3 is not a useful surrogate marker for the
chronic in vivo effects of rhIGF-1.
IGFBP4 (24-kDa protein) did not change in response to any of the
chronic rhIGF-1 treatments. Consistent with previous reports, this
binding protein is not altered after rhIGF-1 administration (Clemmons
et al., 1989) and therefore does not fit the criteria for a
surrogate marker for the effects of rhIGF-1.
In conclusion, we have identified significant increases in the levels of IGFBP2 after repeated injections of an efficacious dose (1 mg/kg) of rhIGF-1. Furthermore, we have determined that this repeated administration did not result in tolerance to IGFBP2 increases. Taken together, we suggest that IGFBP2 would be useful as a biochemical marker for determining the chronic in vivo effects of rhIGF-1. The observation that rhIGF-1, but not insulin, increases both IGFBP2 levels as well as recovery of hindlimb function is consistent with the hypothesis that the effects of rhIGF-1 are mediated via the type-1 IGF but not the insulin receptor.
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Acknowledgments |
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The authors thank Dr. David R. Clemmons (University of North Carolina) for helpful suggestions and Dr. Peter King and Susan Neidlinger for assistance with the pharmacokinetic data analysis.
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Footnotes |
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Accepted for publication December 4, 1996.
Received for publication July 25, 1996.
Send reprint requests to: Dr. Ratan Bhat, Cephalon Inc., 145 Brandywine Parkway, West Chester, PA 19380.
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Abbreviations |
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IGF-1, insulin-like growth factor-1; rhIGF-1, recombinant human insulin-like growth factor-1; IGFBP, insulin-like growth factor binding protein; TBS, Tris-buffered saline.
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References |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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borg, M.,
Hansen, L. B. H.,
Holm, N. R.,
Drakenberg, K.,
Sara, V. R.,
Hultberg, B. and
Nilsson, C.:
Insulin-like growth factors in the nervous system: evolution, fetal development, maintenance and tumor formation. In The Insulin-Like Growth Factors and their Regulatory Proteins, ed. by R. C. Baxter,
P. D. Gluckman and
R. G. Rosenfeld, pp. 295-305, Elsevier Science B. V, Amsterdam, The Netherlands, 1994.-kinase.
Endocrinology
132: 1421-1430, 1993[Abstract].This article has been cited by other articles:
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 H. Zhang, D. Chung, Y.-C. Yang, L. Neely, S. Tsurumoto, J. Fan, L. Zhang, M. Biamonte, J. Brekken, K. Lundgren, et al. Identification of new biomarkers for clinical trials of Hsp90 inhibitors Mol. Cancer Ther., May 1, 2006; 5(5): 1256 - 1264. [Abstract] [Full Text] [PDF]
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