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Original Studies |
Department of Endocrinology (C.C.-H., F.M.-M., A.J.C.), Pediatric Endocrinology Section (C.C.-H., K.A.W., M.O.S.), and Department of Clinical Pharmacology (A.J.), St. Bartholomew’s Hospital, London, United Kingdom EC1A 7BE; Cobbold Laboratories (P.C.H.), University College, London, United Kingdom; Pharmacia & Upjohn, Inc. (Y.H.), Stockholm, Sweden; and the Kolling Institute of Medical Research (R.B.), Sydney, Australia
Address all correspondence and requests for reprints to: Cecilia Camacho-Hübner, M.D., Department of Endocrinology and Chemical Endocrinology, 51–53 Bartholomew Close, London, United Kingdom EC1A 7BE.
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Abstract |
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Introduction |
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Prolonged therapy with recombinant human IGF-I (rhIGF-I) in children with GHIS and those with GH gene deletion has proved to be safe and effective, with side-effects presenting mainly when high doses of rhIGF-I are used (7, 8, 9, 10). These studies have demonstrated good growth responses to rhIGF-I therapy in these patients using doses in the range of 80–120 µg/kg, twice daily, which maintained appropriate circulating IGF-I levels. Pharmacokinetic studies after single sc injections independently of the dose used have shown a significantly reduced IGF-I half-life (11, 12), possibly due to the marked reduction in serum IGFBP-3 and ALS found in these patients. The aim of this study was to investigate the effect of rhIGF-I therapy on the GH-IGF axis given to a patient with partial deletion of the IGF-I gene. This report describes the changes in the circulating levels of the most pertinent serum IGFBPs (IGFBP-3, -2, and -1), ALS, IGF-II, and insulin after rhIGF-I administration. The pharmacokinetics of rhIGF-I and its effect on GH secretory capacity assessed by overnight GH profiles were further evaluated. The present data support the importance of rhIGF-I in restoring circulating IGF-I levels and thus, by normalizing GH levels, improving insulin sensitivity as well as other biochemical markers of the GH-IGF axis.
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Subjects and Methods |
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Blood samples were obtained at the start of treatment and after 1, 3, 6, and 12 months for analysis of serum concentrations of IGF-I, IGF-II, ALS, IGFBP-3, IGFBP-2, IGFBP-1, and insulin. The samples were obtained usually at noon, nearly 2–4 h after the last sc rhIGF-I injection. In addition, fasting levels of insulin and IGFBP-1 were measured in the last sample obtained from the overnight GH profile described below.
Overnight GH profiles were performed on the night before the first study day (day 0). Blood samples were obtained from an indwelling peripheral venous catheter at 20-min intervals from 2000–0800 h. Four studies were performed at 0, 1, 6, and 12 months.
Pharmacokinetic studies were performed after an overnight fast following the first GH profile; rhIGF-I at a dose of 40 µg/kg was given by sc injection followed by a standard breakfast. Serial blood samples were obtained at 60-min intervals from 0900–1600 h after the injection. The patient was then treated with rhIGF-I (40 µg/kg·day), and blood samples were obtained at 0900 h for 6 continuous days. The same protocol was repeated at 3 months, when the dose of rhIGF-I was increased to 80 µg/kg·day given in a single sc injection. All serum samples were kept at -20 C until analysis. The study protocol was approved by the local ethics committee, and informed written consent was obtained from the patient’s parents.
Immunoassays
GH. Serum GH concentrations were determined in duplicate with an immunoradiometric assay (IRMA), using materials developed by the North Thames Regional Assay. Standards have been calibrated against International Reference Preparation 85/05. The results are expressed in milliunits per L (conversion factor: 1 ng/mL = 2.6 mU/L). The lower limit of sensitivity was 0.5 mU/L, with intra- and interassay coefficients of variation less than 5%. The volume requirement of this assay was 200 µL, permitting blood sampling every 20 min without exceeding a total blood volume of 7 mL/kg.
Total IGF-I and IGF-II. IGF-I was measured by RIA (15), using rhIGF-I as the radioligand. All serum samples were extracted with formic acid-acetone before analysis to remove interfering IGFBP. Intra- and interassay CVs were less than 10%. The lower limit of sensitivity of this assay is 20 ng/mL. In addition, all pretreatment samples and all samples obtained for the pharmacokinetic studies were measured in a commercial IGF-I assay (Diagnostic Systems Laboratories, Inc., Webster, TX) with a lower limit of sensitivity of less than 1 ng/mL. Serum was extracted by acid-ethanol and then subjected to the IRMA as recommended by the manufacturer. The interassay coefficient of variation was 8.9%.
IGF-II concentrations were measured after formic acid-acetone extraction in a procedure similar to that used for IGF-I (16). The assay sensitivity was 125 ng/mL. The mean intra- and interassay coefficients of variation were 7.9% and 10.4%, respectively. rhIGF-I and -II peptides used for standard and tracer were provided by Pharmacia & Upjohn, Inc. (Stockholm, Sweden).
ALS. ALS was measured in serum using a RIA described previously (17). The intra- and interassay coefficients of variation were 5.4% at 24 mg/L and 3.3% at 20.8 mg/L, respectively. The normal range of ALS in healthy subjects was 15–34 mg/L. ALS levels were also examined by immunoblot as described by Liu et al (18), using a rabbit polyclonal antiserum generated against a synthetic N-terminal 1–34 amino acid fragment of ALS provided by P. D. K. Lee (Diagnostics System Laboratories, Inc.).
IGFBP-3, IGFBP-2, and IGFBP-1. Serum IGFBP-3, IGFBP-2, and IGFBP-1 were determined using commercially available immunoassays (Diagnostic System Laboratories, Inc.). Serum IGFBP-3 levels were measured using an enzyme-linked immunosorbent assay kit with a detection limit of 0.04 ng/mL. The intra- and interassay coefficients of variation were 7.2% and 8.3%, respectively. The normal reference range for 13- to 17-yr-old males is 2.03–5.96 mg/L. IGFBP-2 was measured using a RIA kit with an assay sensitivity of 0.5 ng/mL. The intra- and interassay coefficients of variation were 6.2% and 4.9%, respectively. The median serum IGFBP-2 level in normal healthy subjects was 453 ng/mL (range, 198–769). IGFBP-1 was measured using an IRMA kit with a detection limit of 0.11 ng/mL. The intra- and interassay coefficients of variation were 7.2% and 5.3%, respectively. The serum IGFBP-1 concentration (mean ± SEM) in normal healthy subjects was 35.6 ± 8.8 in the fasting state (19).
Plasma insulin. Immunoreactive insulin was determined using a double antibody RIA as previously described (20). Briefly, 125I-labeled insulin (Amersham International, Aylesbury, UK) was used as tracer, with human insulin as the standard (Novo Biolabs, Bagsvaerd, Denmark). The first antibody was a guinea pig antiporcine insulin antibody (Immunodiagnostic Systems Ltd., Washington, UK), and sheep antiguinea pig Fc (International Laboratory Services, London, UK) was used as the second antibody.
Detection of antibodies against IGF-I by time-resolved fluoroimmunoassay (trFIA)
IGF-I antibodies were determined using 96-well microtiter plates coated with rhIGF-I, followed by incubation with serum test sample and protein G labeled with europium, used for detection in the DELFIA technology (Wallac OY, Turku, Finland). The patients’ serum samples were obtained before and after 1 month of treatment and every 6 months thereafter. Antibody activity was detected by the fluorescence generated at serum dilutions of 1:900 and 1:1800. Positive controls were tested using rabbit serum, and negative controls were tested using human serum obtained from a pool of normal donors.
Calculations
Pharmacokinetic parameters were derived from the IGF-I levels and were calculated using a two-compartment model (21). The following pharmacokinetic variables were estimated: Cmin, observed trough period (predose) IGF-I concentration; Cmax, observed maximum IGF-I concentration; V, volume of distribution; tmax, observed time of maximum IGF-I concentration; and t1/2, half-life of terminal exponential phase. Baseline IGF-I concentrations were undetectable, and a cut-off level of 2 ng/mL was used. Data analysis was performed with the computer program PCModfit version 1.25 (21).
To evaluate the effects of rhIGF-I therapy on baseline GH levels as well as peak values, the distribution method for analysis of GH profiles was used (22). In brief, for each 24-h serum GH concentration profile, a cumulative frequency distribution was calculated. The results were then plotted on probability paper to yield a linear plot, and from this, discrete probabilities were derived. The threshold concentration at or below which the profile spreads 5% (trough) or 95% (peak) of the time was then estimated from the regression equation. The values in this subject were compared with values in midpubertal healthy male subjects (22).
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Results |
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Between 16.2–16.5 yr of age the patient was treated with rhIGF-I at 40
µg/kg in a single sc injection, and from 16.5–17.2 yr of age his
dose was increased to 80 µg/kg daily. During the first 6 months of
treatment, his growth rate was 7 cm/yr, but it declined slightly during
the following 6 months to 6.5 cm/yr. During this time he has progressed
normally through puberty, and abdominal magnetic resonance imaging
revealed no organomegaly of liver, spleen, or kidneys. rhIGF-I pharmacokinetics
The variables derived from the two-compartment model are shown in
Table 2, and the IGF-I profiles are shown
in Fig. 1. After the first injection of
40 µg/kg·day, the maximum increase in IGF-I levels to 340 ng/mL was
reached around 7–8 h postinjection and slowly declined thereafter.
Elimination profiles remained more or less unchanged from days 1–6,
indicating no significant accumulation of IGF-I between doses. The
elimination profiles obtained with the low dose and the high dose (40
and 80 µg/kg·dose, respectively) were similar, demonstrating the
expected dose-dependent increase in circulating IGF-I levels at the
higher dose. The time course of serum IGFBP-3 coincided with the IGF-I
peak in both studies (data not shown).
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Figure 2 compares the four overnight
GH profiles performed in this patient. The first study (panel A) was
performed before treatment. Very large peaks (>300 mU/L) and little
return to baseline was observed. After 1 month on IGF-I at a dose of 40
µg/kg·day (Fig. 2B), the GH peak
decreased to 75 mU/L. After 3 months on the 80 µg/kg dose (Fig. 2C),
there was a further reduction of GH peaks as well as more defined
baseline levels. After 1 yr of rhIGF-I treatment (Fig. 2D), an
accumulative effect of IGF-I was observed, with a further decline in
baseline and peak levels. Table 3 shows
the results of occupancy analysis, which confirm these findings and
demonstrate characteristics similar to data obtained from healthy
normal subjects in midpuberty (n = 5).
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Table 4 show changes in other
members of the IGF system. Serum ALS concentrations were initially high
and normalized with the high IGF dose. IGFBP-3 was only slightly
increased at the beginning of the study, returning to baseline values
later in the study. IGFBP-2 levels before treatment were low compared
to reference ranges, and serum levels increased to normal levels as GH
levels declined.
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As GH levels decreased during the course of the study, fasting insulin levels also decreased, and IGFBP-1 levels rose. Insulin and IGFBP-1 concentrations were also measured in the overnight samples, and there was an inverse relationship between the two (data not shown). Blood glucose levels were carefully monitored in this patient, and his fasting glucose levels remained normal throughout the study period (data not shown).
IGF-I antibodies
IGF-I antibodies were detected after 6 months of therapy, and
antibody titers have continued to increase over time, as shown in Fig. 3.
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Discussion |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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In the present study we describe the short term concurrent changes in the serum IGF system in response to rhIGF-I in this patient as well as the effect of treatment on his linear growth. We used two different doses of rhIGF-I, and examination of the kinetics of the IGF system indicated that this patient’s IGF-I pharmacokinetic parameters were similar to those reported in healthy subjects (11). This is in marked contrast to the 5- to 7-h IGF-I half-lives reported in GH-insensitive children (11, 12). Guler et al. (24), using bolus injections of [125I]IGF-I and -II, demonstrated that the 150-kDa complex is responsible for the relatively long half-life of IGFs and that the 50-kDa and free IGF pool has a rapid turnover. Our data support the concept that the pharmacokinetic pattern of exogenous IGF-I is determined by normal serum IGFBP-3 and ALS concentrations.
Our findings also support several previous studies examining the effects of sc injections of rhIGF-I resulting in decreased overnight secretion of GH (25, 26). The 12-h GH levels were significantly lower after 1 month of rhIGF-I treatment compared to baseline values, reflecting the negative feedback exerted by IGF-I levels on GH release. However, when the data were further analyzed using the GH occupancy model (22), it was clear that GH levels were still elevated. The higher rhIGF-I dosing schedule normalized these parameters. Chronic rhIGF-I treatment also caused a decline in total IGF-II levels; this decline could result from displacement of IGF-II from IGFBP-3.
IGFBP-3 and ALS are known to be stimulated by GH in GH-deficient subjects (27). Lee et al. (28) reported that in a group of untreated GHD subjects, a single sc dose of rhGH led to an increase in serum IGF-I 3 h posttreatment coincident with peak GH concentrations. Furthermore, the rise in IGF-I preceded that in IGFBP-3, the major carrier for IGFs. ALS, a glycoprotein, is almost exclusively of hepatic origin and may therefore reflect hepatic GH responsiveness better than IGF-I and/or IGFBP-3, which are produced in many tissues. Our patient had considerably increased ALS levels, indicative of elevated GH levels. In normal subjects, ALS levels decreased after rhIGF-I administration (29), but were maintained within normal reference ranges after simultaneous administration of rhGH. Our data demonstrate that ALS levels normalized to midpubertal values during treatment with the higher dose of rhIGF-I which normalized GH levels, and these normal levels were maintained during the first year of therapy.
Serum IGFBP-2 may be suppressed by GH and insulin under certain conditions, although the published data are not always in agreement (30, 31, 32). In the current study, fasting IGFBP-2 and IGFBP-1 levels did change after 1 month and particularly after 3, 6, and 12 months of higher dose rhIGF-I treatment coinciding with the significant changes in GH and insulin concentrations.
A previous study in which GH, insulin, and glucose levels were separately controlled showed that insulin acutely suppresses serum IGFBP-1 concentrations and that GH does not have a significant acute independent action in vivo (33). This has been supported by various studies in which IGFBP-1 varies inversely with insulin overnight (34). A recent study by Lee et al. (28) showed that the increase in IGFBP-1 levels also followed the posttreatment decrease in GH during the overnight sampling. Multiple regression analysis revealed a stronger inverse relationship of IGFBP-1 with insulin than with GH. These data are consistent with the current knowledge that insulin is one of the most important regulators of IGFBP-1 synthesis (35).
The hypersecretion of GH in this patient was associated with marked hyperinsulinemia and euglycemia. Presumably to maintain normoglycemia, a significant increase in insulin release was required to compensate for a marked reduction in insulin sensitivity. The degree of insulin insensitivity seen in this patient was more severe than the reduction in insulin sensitivity associated with puberty (36). Fasting insulin levels decreased concomitantly with a reduction of GH levels. Hofman et al. (37) have shown that in short subjects with intrauterine growth retardation, reduced insulin sensitivity occurred in childhood. Their study formally documented that impaired insulin sensitivity is associated with intrauterine growth retarded subjects and may be an early metabolic marker of the development of syndrome X later in life (38).
During the first year of treatment the patient had an almost doubling of growth velocity compared to his pretreatment velocity. During treatment with rhIGF-I his skeletal maturation progressed and reached a bone age of 15 yr; therefore, it was beyond that associated with peak growth velocity. During this time he also developed IGF-I antibodies; however, it appears that the increasing antibody titers have not diminished the bioactivity of rhIGF-I, as assessed using the effect of the negative feedback of IGF-I on GH release.
The growth response observed in this patient is not as marked as that observed in naive GH-deficient patients receiving GH replacement therapy and is possibly less marked than that of GHIS patients after receiving rhIGF-I. Contributing factors for this clinical response may be related to his history of severe intrauterine growth retardation, his severe short stature associated with dysmorphic features, and the timing of rhIGF-I treatment, which was initiated when the patient was already progressing through puberty. Treatment with rhIGF-I given systemically may not completely replace the local response of target tissues to locally produced IGF-I.
In summary, we have presented studies characterizing the acute and chronic responses to exogenous rhIGF-I on several parameters of the IGF system in a patient with a partial deletion of the IGF-I gene. Our results showed that in response to rhIGF-I serum GH and insulin showed the most dramatic acute responses. This, in turn, caused significant changes in serum ALS, IGFBP-1, and IGFBP-2 levels. IGFBP-3 showed smaller changes, possibly reflecting changes in rates of proteolysis and clearance. In conclusion, this study has demonstrated that the absence of a functional IGF-I gene has marked effects on linear growth as well as important metabolic consequences for insulin sensitivity.
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Acknowledgments |
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Received June 1, 1998.
Revised December 23, 1998.
Accepted January 25, 1999.
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References |
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TopAbstractIntroductionSubjects and MethodsResultsDiscussionReferences |
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) subunit of the high molecular
weight insulin-like growth factor binding protein complex. J Clin
Endocrinol Metab. 70:1347–1353.This article has been cited by other articles:
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  E. C. Walvoord, A. de la Pena, S. Park, B. Silverman, L. Cuttler, S. R. Rose, G. Cutler, S. Drop, and J. J. Chipman Inhaled Growth Hormone (GH) Compared with Subcutaneous GH in Children with GH Deficiency: Pharmacokinetics, Pharmacodynamics, and Safety J. Clin. Endocrinol. Metab., June 1, 2009; 94(6): 2052 - 2059. [Abstract] [Full Text] [PDF]
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 M J E Walenkamp and J M Wit Genetic disorders in the GH IGF-I axis in mouse and man Eur. J. Endocrinol., August 1, 2007; 157(suppl_1): S15 - S26. [Abstract] [Full Text] [PDF]
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M. J. E. Walenkamp, H. J. van der Kamp, A. M. Pereira, S. G. Kant, H. A. van Duyvenvoorde, M. F. Kruithof, M. H. Breuning, J. A. Romijn, M. Karperien, and J. M. Wit A Variable Degree of Intrauterine and Postnatal Growth Retardation in a Family with a Missense Mutation in the Insulin-Like Growth Factor I Receptor J. Clin. Endocrinol. Metab., August 1, 2006; 91(8): 3062 - 3070. [Abstract] [Full Text] [PDF]
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M. J. E. Walenkamp, M. Karperien, A. M. Pereira, Y. Hilhorst-Hofstee, J. van Doorn, J. W. Chen, S. Mohan, A. Denley, B. Forbes, H. A. van Duyvenvoorde, et al. Homozygous and Heterozygous Expression of a Novel Insulin-Like Growth Factor-I Mutation J. Clin. Endocrinol. Metab., May 1, 2005; 90(5): 2855 - 2864. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, J. Y. Weltman, A. L. Weltman, A. Iranmanesh, E. E. Muller, and C. Y. Bowers Age and Secretagogue Type Jointly Determine Dynamic Growth Hormone Responses to Exogenous Insulin-Like Growth Factor-Negative Feedback in Healthy Men J. Clin. Endocrinol. Metab., November 1, 2004; 89(11): 5542 - 5548. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, S. M. Anderson, P. Kok, A. Iranmanesh, J. Frystyk, H. Orskov, and D. M. Keenan Estradiol Supplementation Modulates Growth Hormone (GH) Secretory-Burst Waveform and Recombinant Human Insulin-Like Growth Factor-I-Enforced Suppression of Endogenously Driven GH Release in Postmenopausal Women J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1312 - 1318. [Abstract] [Full Text] [PDF]
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J. D. Veldhuis, S. M. Anderson, J. T. Patrie, and C. Y. Bowers Estradiol Supplementation in Postmenopausal Women Doubles Rebound-Like Release of Growth Hormone (GH) Triggered by Sequential Infusion and Withdrawal of Somatostatin: Evidence that Estrogen Facilitates Endogenous GH-Releasing Hormone Drive J. Clin. Endocrinol. Metab., January 1, 2004; 89(1): 121 - 127. [Abstract] [Full Text] [PDF]
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J. Rubin, C. L. Ackert-Bicknell, L. Zhu, X. Fan, T. C. Murphy, M. S. Nanes, R. Marcus, L. Holloway, W. G. Beamer, and C. J. Rosen IGF-I Regulates Osteoprotegerin (OPG) and Receptor Activator of Nuclear Factor-{kappa}B Ligand in Vitro and OPG in Vivo J. Clin. Endocrinol. Metab., September 1, 2002; 87(9): 4273 - 4279. [Abstract] [Full Text] [PDF]
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 K. Wallenius, K. Sjogren, X.-D. Peng, S. Park, V. Wallenius, J.-L. Liu, M. Umaerus, H. Wennbo, O. Isaksson, L. Frohman, et al. Liver-Derived IGF-I Regulates GH Secretion at the Pituitary Level in Mice Endocrinology, November 1, 2001; 142(11): 4762 - 4770. [Abstract] [Full Text] [PDF]
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K. A. Woods, C. Camacho-Hübner, R. N. Bergman, D. Barter, A. J. L. Clark, and M. O. Savage Effects of Insulin-Like Growth Factor I (IGF-I) Therapy on Body Composition and Insulin Resistance in IGF-I Gene Deletion J. Clin. Endocrinol. Metab., April 1, 2000; 85(4): 1407 - 1411. [Abstract] [Full Text]
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