1 Nemours Children's Clinic, Jacksonville, Florida 32207; and 2 Genentech, South San Francisco, California 94080
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ABSTRACT |
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We investigated the pharmacokinetics of recombinant human insulin-like growth factor I (rhIGF-I) in growth hormone deficiency (GHD). Nine GHD adults [age 25 ± 3 (SE) yr] received rhIGF-I (60 µg/kg sc) twice, 10 h apart, and blood was sampled over 24 h. IGF-I and free IGF-I concentrations increased, whereas IGF binding protein 3 (IGFBP-3) and acid labile subunit (ALS) were unchanged during treatment. There was no correlation between absorption or terminal half-life of IGF-I and IGFBP-3 or ALS, but negative correlations with IGF-I clearance (CL/F) and volume of distribution (V/F). Positive correlations between both IGFBP-3 and ALS and IGF-I maximal concentration (Cmax) and time of Cmax (Tmax) were observed. Compared with normal individuals studied similarly (using 80 µg/kg), GHD subjects showed a normal absorption half-life, a faster elimination half-life, lower Cmax, yet normal Tmax and V/F. In conclusion, GHD is associated with normal absorption and distribution of IGF-I yet faster elimination kinetics. Additionally, IGFBP-3 and ALS concentrations modulate the peak concentrations of IGF-I achieved and correlate reciprocally with its V/F and CL/F, underscoring the critical importance of binding proteins in modulating the bioavailability of IGF-I in vivo in humans.
growth hormone deficiency; insulin-like growth factor binding protein; acid labile subunit; growth hormone
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INTRODUCTION |
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GROWTH HORMONE (GH) production results in increased concentrations of insulin-like growth factor I (IGF-I), which mediates the protein anabolic and growth-promoting actions of GH, yet not its lipolytic nor its insulin-antagonistic effects (15). IGF-I in the circulation is bound to circulating binding proteins, the most abundant of which, IGF-I binding protein 3 (IGFBP-3), coupled with its acid labile subunit (ALS), form a ternary complex that serves as a reservoir for IGF-I in plasma, modulating its biological effects (10). IGFBP-3 and ALS are GH-dependent moieties, hence their concentrations are typically low in syndromes of GH deficiency (GHD).
The physiological role of this ternary complex has been studied extensively in a variety of experimental situations in vitro and in vivo with sometimes paradoxical results. IGFBP-3 has been shown to have inhibitory actions on IGF-I function, to directly inhibit cell growth, and to also potentiate the actions of IGF-I (10). Patients with GHD typically show marked decreases in circulating IGF-I, IGFBP-3, and ALS, and all significantly increase during GH treatment (2). IGF-I administration to GHD subjects or to subjects with GH insensitivity (GH-receptor deficiency patients) on the other hand, markedly increases IGF-I concentrations but has either minimal or no effect on circulating IGFBP-3 or ALS (19).
GH replacement therapy is now well established for purposes other than growth in the GHD adult (4). Additionally, recombinant human IGF-I (rhIGF-I) has been used extensively in a variety of clinical trials in humans and has been found to have similar protein-anabolic effects as GH, as well as glucose lowering effects (7), suggestive of both GH-like and insulin-like actions in vivo. Because the biological behavior of IGF-I is modulated in part by its binding proteins, we investigated the pharmacokinetics (PK) of rhIGF-I in states of profound GHD and correlated these parameters with changes in specific binding proteins. We hypothesized that because GHD is characterized with marked decreases in some of the pivotal IGF-I binding proteins, the PK of IGF-I would be markedly altered in the GHD state. To accomplish this, a group of adult patients with severe GHD were studied during the subcutaneous administration of rhIGF-I.
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METHODS |
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Study subjects.
These studies were approved by the Nemours Children's Clinic Clinical
Research Review Committee and the Baptist Medical Center Institutional
Review Committee. Nine subjects with physical and biochemical features
of GHD were recruited for these studies (6 males and 3 females) after
informed written consent. Their clinical characteristics are summarized
in Table 1. All but two had childhood onset
GHD. Those who had been treated with GH previously all had discontinued
GH at least 1 yr before the studies. GH production was assessed using
insulin-induced hypoglycemia. Data available from five healthy
GH-sufficient volunteers studied previously [mean age 22.6 ± 1.1 yr, body mass index (BMI) 22.0 ± 0.7 kg/m2, 4 females, 1 male]
were used for comparison (Table 1). All subjects had normal renal
function as measured by serum creatinine and blood urea nitrogen
concentrations and urinalysis.
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Study design. After an overnight fast, subjects were admitted to the clinical studies unit at the Wolfson Children's Hospital in the early morning and an intravenous heparin lock was placed in a forearm vein. At time 0 (0800) blood was withdrawn for measurement of different hormones and growth factors as will be detailed. Immediately thereafter rhIGF-I, kindly provided by Genentech, was administered at 60 µg/kg sc and subjects were allowed to eat. In the subjects who were overweight the dose was calculated based on 120% above ideal body weight (Metropolitan Life Insurance tables). Blood was withdrawn for the determination of total IGF-I and glucose at 0, 30, 60, 120, 180, 240, 300, 360, 480, 600, and 1,440 min. IGF-II, IGFBP-1, IGFBP-2, IGFBP-3, free IGF-I, and the ALS were measured at 0, 120, 240, 600, and 1,440 min. Meals were served again at 240 and 600 min, the latter immediately following the second dose of rhIGF-I. Trough levels were obtained after another overnight fast at 1,440 min the following morning.
Previously studied normal volunteers had received a subcutaneous injection of rhIGF-I at 80 µg/kg, and their blood was sampled similarly as the GHD subjects.Assays. Total IGF-I was measured in all plasma samples by RIA after acid ethanol extraction at Endocrine Sciences Laboratories (Calabassas Hills, CA). The IGF-I assay sensitivity was 1.6 µg/l with an intra- and interassay coefficient of variation (CV) of ~6 and 7%, respectively, in the concentration ranges observed, with a linear range of 1.6-64 µg/l. At 0, 120, 240, 600, and 1,440 min, duplicate samples for IGF-I concentrations were run by similar methods but using pure rhIGF-I standards as determined at Genentech. This assay has a sensitivity of 0.31 µg/l, a linear range of 0.31-40 µg/l, an intra-assay CV of 11.0%, and an interassay CV of 14.3% as previously described (16). The Genentech RIA was standardized against the exact same material used to treat patients, and hence the results from this assay probably reflect absolute mass values.
Most total IGF-I assays use impure, quantitatively incorrect standards. Moreover, most total IGF-I assays are calibrated to the World Health Organization (WHO) international reference reagent for IGF-I immunoassays (87/518), and these assays report concentrations that are in excess of actual values by approximately twofold. Thus much of the plasma total IGF-I data in the literature are of questionable accuracy as absolute values. The Genentech total IGF-I assay uses a highly pure, quantitatively correct standard, and it is not calibrated against the WHO standard (16). IGF-II, IGFBP-1, IGFBP-2, and IGFBP-3 were measured by RIA at Endocrine Sciences Laboratories. The sensitivity, intra- and interassay CV for these assays were IGF-II <80 µg/l, ~7 and 9%, respectively; IGFBP-1 <1.0 µg/l, ~7.4 and 9.0%; IGFBP-2 <178 µg/l, ~9.5 and 12%; IGFBP-3 <0.3 mg/l, ~5.1 and 5.5%. Free IGF-I was measured at Genentech, using a pure IGF-I standard by size exclusion HPLC with an interassay CV of 17% as previously described (12). A two-site ALS ELISA was developed at Genentech using monoclonal antibodies raised against rhALS. Standard rhALS and diluted samples (50 µl of 1:50, 1:150, 1:450, and 1:1,350 dilutions) were added to microtiter plate wells coated with capture antibody and incubated 2 h at 22°C together with enzyme-labeled detection antibody. After washing, chromogen solution was added, color development was stopped after 15 min, and absorbances were read on a plate reader at 450 nm. The intra- and interassay CV were 3.1 and 5.5%, respectively. Glucose was measured by a glucose oxidase method using a Beckman glucose analyzer.PK analysis. The PK parameters for the 24-h plasma IGF-I concentration vs. time profile for each subject were calculated according to a one-compartment model (first order absorption and elimination kinetics) using standard methods (WinNonlin Software; Scientific Consulting, Apex, NC). The plasma concentration vs. time data were fit to a two-exponential model, one exponential for absorption and one for elimination, and the PK parameters were calculated from the slopes and intercept. It was assumed that there was no absorption lag time. Parameters calculated included the apparent absorption and terminal half-life, distribution volume (V/F) and clearance (CL/F). The distribution volume and clearance were not corrected for bioavailability (F). This is due to the fact that bioavailability cannot be calculated without an intravenous PK study and intravenous rhIGF-I cannot be given to human subjects as controlled by the Food and Drug Administration current regulations. Cmax and Tmax represent the maximal concentration and the time when maximal IGF-I concentration were observed, respectively, as fitted by the model. The IGF-I concentration at 0 time was subtracted prior to performing the PK analysis.
Statistics. Unpaired Student's t-test was used to estimate the differences in specific parameters between the two study groups. Regression analysis using the least-squares method was used to assess the correlation between the observed IGF-I and binding protein concentrations, as well as between IGF-I PK parameters and IGFBP-3 and ALS concentrations in the GHD patients. Repeated-measures ANOVA was used to estimate the differences in plasma concentrations for the different growth factors and binding proteins in the GHD group. Significance was established at P < 0.05.
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RESULTS |
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Total IGF-I assays.
A comparison between Endocrine Sciences plasma IGF-I concentrations and
the limited IGF-I concentration profile measured in Genentech's assay
in duplicate samples in the same GHD patients revealed that the IGF-I
concentrations measured by the Genentech's assay were about one-half
the magnitude of those measured at the Endocrine Sciences Laboratories
(Fig. 1). This difference is probably related to differences in IGF-I assay calibration as described previously.
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IGF-I binding proteins, IGF-II, free IGF-I, and ALS.
Table 2 summarizes the mean changes in the
aforementioned factors in the GHD patients during these studies. ANOVA
showed significant increases in IGF-I and free IGF-I concentrations
during rhIGF-I treatment and significant decreases in IGF-II and
IGFBP-1. IGFBP-2 increased slightly and IGFBP-3 and ALS
concentrations remained invariant during treatment.
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PK parameters.
PK parameters for IGF-I in the GHD patients are summarized in Table
3. As a result of the differences in IGF-I
assays and because the Genentech IGF-I assay was run on a limited time
series, the V/F and CL/F were adjusted to reflect PK parameters that
would be obtained if all the samples were run by Genentech's assay so these parameters could be compared with those obtained in normal individuals studied previously using the Genentech assay. The V/F and
CL/F parameters were divided by 0.452, the slope of a plot Genentech
vs. Endocrine Science IGF-I assay results (intercept ~0). According
to the model, the half-lives and
Tmax (time when that maximal concentration was achieved) do not depend on concentration and need not be adjusted. Cmax
values (maximal IGF-I concentration) were adjusted by multiplying the
Endocrine Science value by 0.452. For comparison, PK parameters of
normal, GH sufficient controls are shown. The GHD subjects had lower
Cmax but similar
Tmax as the
control subjects; absorption half-lives were similar between the two
groups but the terminal (elimination) half-life was shorter in the GHD
subjects; V/F was not different between the two groups and there was a
trend toward higher clearance in the GHD group even though the latter
did not achieve statistical significance.
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DISCUSSION |
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The study of the PK of IGF-I was performed because of the potential use of this peptide as a protein-anabolic, and even bone-anabolic agent in humans (14, 15, 18). GHD patients have been shown to have comparable effects to rhIGF-I administration as to GH therapy (9), yet rhIGF-I has the advantage of having insulin-like effects (7), making it potentially appealing to patients with GHD who also have carbohydrate intolerance. This study of subcutaneous rhIGF-I administration in severe GHD young adults illustrates the marked influence of IGFBP-3 and ALS in the PK of IGF-I in humans. The binding proteins were low in GHD subjects, yet their concentrations showed a significant reciprocal correlation with the volume of distribution and clearance of IGF-I. Both at baseline and 4 h after the administration of rhIGF-I, the higher the IGFBP-3 concentration, the lower the volume of distribution and the lower the clearance of the peptide. This reciprocal relationship was also observed with the ALS. The maximal IGF-I concentration achieved in plasma (Cmax), and the time of achievement of Cmax (Tmax) were positively correlated with the available amounts of circulating IGFBP-3 and ALS, clearly indicative of the pivotal regulatory role of these binding proteins in the distribution and clearance of IGF-I in humans and the potential modulator role of these compounds on the biological behavior of this peptide. The observed decrease in IGFBP-1 concentrations may well be secondary to the nutritional dependency of this binding protein as it is reciprocally related to insulin. IGF-II concentrations decreased after rhIGF-I treatment and IGFBP-2 increased as typically observed (15). IGFBP-3 concentrations did not increase after IGF-I, as expected, as IGFBP-3 is a GH-dependent protein.
There was no correlation between free IGF-I and IGFBP-3 or ALS concentrations at any time point. Contrary to the sustained increase in IGF-I concentrations observed even 24 h after the first rhIGF-I injection, free IGF-I concentrations quickly increase after rhIGF-I administration with peak concentrations at 120 min yet markedly decline reaching nearly the same baseline levels at 24 h after first injection. These data indicate that in severe GHD states free IGF-I is rapidly cleared from plasma.
Direct comparison data of the GHD subjects studied here with those of normal, GH-sufficient individuals given rhIGF-I and studied similarly showed interesting results. Whereas the apparent absorption half-lives were comparable between the two groups, the elimination half-life was shorter in the GHD state. Cmax was much greater in the normal volunteers compared with the GHD patients, and the volume of distribution was similar in both groups. Even though the doses used in the normal controls studied previously were 33% greater than in the GHD subjects, the data strongly suggest that the rate of subcutaneous absorption and distribution of IGF-I is not altered in GHD states. Even though the GHD group had a higher range of BMI than the normal controls, separate analysis of the differences in PK parameters between the two groups, excluding four subjects in the GHD group with BMI more than 30 kg/m2, revealed the same relative differences in PK as including all subjects (data not shown). This strongly suggests that greater adiposity in the GHD group did not significantly contribute to the findings reported here. Taken in aggregate, the data reported here suggest that in states of GHD, doses of 60 µg/kg of rhIGF-I are absorbed and distributed normally yet cleared faster from plasma. IGF-I has been shown in both experimental animals and in humans to increase renal plasma flow and glomerular filtration rate (5); however, it is unlikely that differences in kidney function between the GHD and control groups could account for the faster elimination kinetics because renal function measures were normal in both groups. This suggests that IGF-I administration may require either higher or more frequent dosing to achieve the biological effects desired in states of GHD, and this would necessitate further studies.
The PK of IGF-I have been previously studied in a few clinical situations in experimental animal models and in humans. Both in syndromes of GH insensitivity (Laron's syndrome) and in GHD children, studies after one single injection of IGF-I showed that the PK profile was strongly dependent on the IGFBP-3 concentrations (1). In hypophysectomized rats the volume of distribution of intravenous rhIGF-I decreased and the total body clearance increased (11). This increased elimination of IGF-I was also observed in younger (4 wk) compared with older rats (7 wk), presumably because of decreased levels of IGFBP-3 in growing rats (8). In adult patients with chronic renal failure in either hemodialysis or chronic peritoneal dialysis, the half-life and volume of distribution of IGF-I (50 or 100 µg/kg sc) were significantly decreased compared with normals, yet the Tmax and clearance were normal (6). These results compare with those found by Rabkin et al. (17) in a similar group of subjects in renal failure. Taken in aggregate, the data gathered in the present studies suggest that normal kidney function and IGFBP-3 are two key modulators of the PK of IGF-I both in GH-deficient and -sufficient states.
The effect of binding proteins on the kinetics and biological effects of circulating peptides has been observed in several other hormone systems. Corticosteroid binding globulin concentrations, for example, have been shown to be reciprocally related to the disappearance of cortisol in plasma (3). Similarly, GH-binding protein reciprocally correlates with GH production rates as measured by deconvolution modeling in children (13). In all these systems it is becoming increasingly clear that the binding proteins can affect the distribution and bioavailability of the given hormones; hence, the concomitant assessment of binding protein levels with those of the given hormone may be critical in specific disease states.
In conclusion, GHD in humans is associated with normal absorption and distribution of IGF-I yet faster elimination kinetics. IGFBP-3 and ALS modulate the peak concentrations of IGF-I achieved and correlate reciprocally with the volume of distribution and clearance of the peptide. These PK data should be considered when administering rhIGF-I in GHD-like states.
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ACKNOWLEDGEMENTS |
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We are deeply grateful to Annie Rini and Susan Welch, Nemours Children's Clinic, for coordinating these studies; Burnese Rutledge and the nursing staff, Wolfson Children's Hospital, for the care of the patients; Gregory Bennett and Tauri Senn, Genentech, for statistical support and assay development, respectively; and the pharmacological assay services at Genentech for assay support.
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FOOTNOTES |
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This work was supported by the National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-51360 (N. Mauras) and Nemours Research Programs (Jacksonville, FL).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: N. Mauras, Div. of Endocrinology, Nemours Children's Clinic, 807 Nira St., Jacksonville, FL 32207 (E-mail: nmauras{at}nemours.org).
Received 28 December 1998; accepted in final form 4 June 1999.
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