Two-Year Treatment of Growth Hormone (GH) Receptor Deficiency ...

0021-972X/97/$03.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1997 by The Endocrine Society

Vol. 82, No. 2 Printed in U.S.A.

Two-Year Treatment of Growth Hormone (GH) Receptor Deficiency with Recombinant Insulin-Like Growth Factor I in 22 Children: Comparison of Two Dosage Levels and to GH-Treated GH Deficiency* JAIME GUEVARA-AGUIRRE, ARLAN L. ROSENBLOOM, OSWALDO VASCONEZ, VICTOR MARTINEZ, SHARRON E. GARGOSKY, LINDA ALLEN, AND RON G. ROSENFELD Instituto Endocrinologia, Metabolismo y Reproduccion (J.G.A., O.V., V.M.) Quito, Ecuador; University of Florida College of Medicine (A.L.R., L.A.), Gainesville, Florida 32610; and Oregon Health Sciences University (S.E.G., R.G.R.), Portland, Oregon 97201 ABSTRACT We have reported 1-yr results of a double blind, placebo-controlled trial of recombinant human insulin-like growth factor I (rhIGF-I) replacement in 16 children from the Ecuadorian GH receptor-deficient (GHRD) population. This report extends observations of rhIGF-I efficacy at two dosage levels [120 mg/kg BW twice daily (n 5 15) and 80 mg/kg twice daily (n 5 7)] over 2 yr, compares biochemical responses [serum IGF-I and IGF-binding protein-3 (IGFBP-3)] and their relationship to growth effects, and compares treatment effects of rhIGF-I in GHRD to rhGH in idiopathic GH deficiency (GHD). There were no baseline differences between the low and high dose groups for growth velocity (GV), bone age (BA), SD score for height, or percent mean body weight for height (MBWH). Over 2 yr of rhIGF-I treatment, there were no differences in GV or in changes in height SD score, height age (HA), or BA between the two groups; a subgroup of six subjects at the higher dose followed for a third year continued at the second year GV. The higher dose resulted in a greater change in percent MBWH. GV in yr 1 and 2 for the entire group and in yr 3 for a subgroup were greater for GH-treated GHD (n 5 11). The GHD group showed a greater change in SD score for height and HA, but did not differ from the rhIGF-I-treated GHRD group in the change in BA (DBA) or DHA/

DBA over 2 yr. There was a greater change in percent MBWH in GHRD. There were no differences between dosage groups for serum IGF-I levels at baseline or the near-normal trough levels 12 h after rhIGF-I injection; these individual levels correlated with HA gain in yr 1 and 2. IGFBP-3 levels were markedly low, with no changes of significance with treatment. Comparable growth responses to the two dosage levels and the biochemical changes indicate a plateau effect at or below 80 mg/kg BW twice daily. The growth response and favorable trough levels of IGF-I despite the overall lack of increase in circulating IGFBP-3 levels suggest an alternative mechanism for sustaining IGF-I levels and avoiding rapid clearance of rhIGF-I. The greater increase in MBWH with treatment of GHRD than with treatment of GHD may reflect comparable effects on lean body mass without the lipolytic effects of GH in the GHRD subjects. The difference in growth response between rhIGF-I-treated GHRD and rhGH-treated GHD groups is consistent with the hypothesis that 20% or more of GH-influenced growth is due to the direct effects of GH on bone. Nonetheless, the comparable DHA/DBA suggests similar long term effects of replacement therapy in the two conditions. (J Clin Endocrinol Metab 82: 629 – 633, 1997)

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The placebo-controlled trial also demonstrated that the persistent low levels of IGFBP-3 did not result in increased risk for hypoglycemia from the expected surge in free IGF-I after injection. The growth responses we reported, as well as those from other reports, in the absence of an increase in IGFBP-3 levels, suggested an alternative mechanism for sustaining therapeutic levels of IGF-I in the circulation or at the tissue level. The growth response to rhIGF-I injection was also thought to argue against the importance of the direct effects of GH on prechondrocyte maturation and local (autocrine/paracrine) IGF-I elaboration, a mechanism postulated to account for 20% of GH-influenced growth (4). Initial enthusiasm for the response to rhIGF-I in GHRD was tempered by a comparison of first year growth velocities (GV) to those of children with isolated GH deficiency (GHD) treated with rhGH (5). This report extends our observations of growth response to rhIGF-I in the Ecuadorian population with GHRD to 2 yr for 22 patients at 2 dosage levels and to 3 yr for 6 of them receiving the higher dose. Their growth

F THE APPROXIMATELY 200 reported instances of Laron syndrome due to GH receptor deficiency (GHRD), 1 of 3 (n 5 70) come from an area 120 km in diameter in southern Ecuador (1, 2). Children from this unique population, the only large, genetically homogeneous group of persons with GHRD, participated in a double blind, placebocontrolled trial of safety and efficacy of recombinant human insulin-like growth factor I (rhIGF-I). We confirmed a therapeutic response over 1 yr of treatment, but without an increase in circulating levels of IGF-binding protein-3 (IGFBP-3), the principal carrier for IGF-I in the circulation (3).

Received June 28, 1996. Revision received October 3, 1996. Accepted October 21, 1996. Address all correspondence and requests for reprints to: Dr. Jaime Guevara, Iemir, Casilla 6337-CCI, Quito, Ecuador. * This work was supported by Pharmacia Peptide Hormone Division, NIH Grant R01-DK-45830 (to A.L.R., J.G.-A., R.G.R.), and FDA Orphan Drug Grant FDA-R-000860 (to J.G.-A., R.G.R., and A.L.R.). Presented at the Annual Meetings of the Society for Pediatric Research and American Pediatric Society, Washington, D.C., May 7, 1996.

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responses are also compared to those of 11 patients with isolated GHD in the same setting who were being treated with rhGH. Subjects and Methods Patients Subjects with GHRD were aged 3.1–17.1 yr, with height sd scores of 25.8 to 211.5. Fifteen of the 22 subjects were female, consistent with the disparate sex ratio in 1 of the 2 provinces that are the origin of the Ecuadorian population (1). All had typical phenotypic features of GHRD, very low or unmeasurable levels of GH-binding protein in the circulation, random GH levels above 10 mg/L, and homozygosity for the alternative splice mutation site of codon 180 in exon 6 of the GH receptor gene (6). All were prepubertal, and bone age estimations were 1.5–9.3 yr (7). GH-deficient patients without organic etiology were from the same Andean population. All had serum GH responses to 2 or more stimuli of less than 4.5 mg/L by RIA using a polyclonal antibody. They were treated with 3–7 injections per week of rhGH in doses of 0.16 – 0.30 mg/kg BWzwk. Five had associated TSH deficiency that was treated with T4 replacement, and 1 also had ACTH deficiency treated with cortisol replacement. These 11 subjects were predominantly male (n 5 8), which corresponds precisely to the sex distribution for idiopathic GHD in the large U.S. postmarketing survey (8). Ages were 3–15.2 yr, and bone ages ranged from 4 months to 8.8 yr.

Study design Studies in the GHRD population were carried out under protocols approved by the ethics committee of the Institute of Endocrinology, Metabolism, and Reproduction (Quito, Ecuador) and the human subjects review boards of Stanford University (Stanford, CA) and the University of Florida (Gainesville, Florida) in compliance with the laws and regulations of the United States and Ecuador. All parents, and children over 7 yr of age, signed and received copies of informed consent forms written in Spanish. Initiation of treatment was as previously described (3). The first 15 subjects received 120 mg/kg every 12 h throughout the study, and the next 7 patients received 80 mg/kg twice daily by sc injection.

Growth evaluation Standing heights were determined as the means of three measurements made with a Harpenden stadiometer. The sd score for height was calculated using U.S. National Center for Health Statistics data (9). X-Ray films of the left hand and wrist were read for determination of bone age (BA) using the method of Greulich and Pyle (7). National Center for Health Statistics data for the U.S. population (9) was also used to estimate height age (HA) and percent mean body weight for height (% MBWH).

Measurements of IGF-I and IGFBP-3 Serum specimens were obtained before treatment and at regular intervals during treatment 12 h after the evening rhIGF-I injection; serum concentrations of IGF-I at these times are defined as trough levels. IGF-I assays were performed by RIA after removal of binding proteins by acid gel chromatography, as previously described (3). IGFBP-3 concentrations were estimated by immunoradiometric assay (Diagnostic Systems Laboratories, Webster, TX). This IGFBP-3 immunoradiometric assay has been validated in our laboratory and shown to correlate with RIA results employing antibody a-IGFBP-3-gl (10) with r . 0.95 (data not shown).

Statistical analysis Auxologic data are expressed as the mean 6 sd, and means are compared using Student’s t test, with P , 0.05 considered significant. Biochemical data, age adjustment, and correlations were performed using the Statistical Analysis System (t test, general linear models).

Results Dose comparison

Growth. As shown in Table 1, there were no significant differences in age, baseline GV, BA, sd score for height, or % MBWH between the subjects receiving 120 mg/kg and those receiving 80 mg/kg twice daily, although the latter were, on the average, 2 yr younger. There were no significant differences in GV in yr 1 or 2, or in changes of height sd score, HA, or BA over these time periods when the 120 and 80 mg/kg groups were compared. The higher dose, however, resulted in a greater change in % MBWH (Table 2). The mean baseline GV for the six subjects followed in yr 3 was not different from that of the entire group (2.9 6 1.5 vs. 3.3 6 1.5 cm/yr). The mean increment in GV over baseline in yr 3 (2.9 6 1.2 cm/yr) was similar to that in yr 2 for those six subjects (3.6 6 1.9). IGF-I. Baseline IGF-I concentrations in serum ranged from less than 2 to 26 mg/L, all well below 22 sd from normal (Fig. 1). There were no significant differences in serum IGF-I concentrations between the dosage groups (low dose, 4.4 6 5.5 mg/L; high dose, 9.5 6 8.5; P 5 0.16). Mean trough levels of IGF-I rose comparably after 12 months of rhIGF-I treatment in both dosage groups (low dose, 68.8 6 88.3 mg/L; high dose, 102.2 6 123.7; P 5 0.52). All but one of the high dose and one of the lower dose subjects showed a substantial elevation of the IGF-I level. Three of the high dose and two lower dose patients failed to show a persistent elevation of trough levels of IGF-I at 24 months, but mean values were not significantly changed (Fig. 1). There was a significant correlation between the increases over baseline in serum IGF-I trough levels at 1 and 2 yr and annual increments in HA for the entire group (Fig. 2). IGFBP-3. The mean baseline IGFBP-3 level in the 120 mg/kg twice daily group (328 6 118 mg/L) was greater than that in the lower dose group (195 6 86; P 5 0.016), and this difference persisted with age adjustment (P 5 0.017). Baseline IGFBP-3 levels in serum correlated with sd score for height for the entire population (r 5 0.54; P 5 0.009), as previously described (1). There were no changes in IGFBP-3 concentration between baseline, 1 yr (284 6 158 mg/L), and 2 yr (336 6 145) of treatment in the higher dose group. The increase from baseline to 1 yr (278 6 65 mg/L) in the lower dose group was not significant (P 5 0.07), but eliminated the difference from the higher dose group (Fig. 3). Annual growth responses for the entire group, measured as HA increase, did not correlate significantly with the change in (D) IGFBP-3 levels at yr 1 (r 5 0.18; P 5 0.41) or yr 2 (r 5 0.30; P 5 0.17). TABLE 1. Comparison of baseline data for patients with GHRD treated with rhIGF-I [120 mg/kg BW (n 5 15) or 80 mg/kg (n 5 7)] twice daily by sc injection

Age (yr) Growth velocity (cm/yr) Bone age (yr) SD score for ht MBWH (%)

120 mg/kg

80 mg/kg

9.8 6 3.4 3.4 6 1.4 4.9 6 2.0 28.5 6 1.3 97.3 6 10.2

7.6 6 4.7 3.0 6 1.8 4.0 6 3.5 28.0 6 1.8 97.9 6 22.6

MBWH, Mean body weight for height.

rhIGF-I IN GHRD AT TWO DOSES FOR 2 YR

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TABLE 2. Comparison of growth responses of children with GHRD to 120 and 80 mg/kg rhIGF-I twice daily

Growth velocity (cm/yr) Yr 1 Yr 2 Yr 3 (n 5 6) Change in SD score for ht Yr 1 Yr 2 Change in ht age (yr) over 2 yr Change in bone age (yr) over 2 yr Change in MBWH (%) over 2 yr

120 mg/kg

80 mg/kg

8.8 6 1.1 6.4 6 1.1 5.7 6 1.4

9.1 6 2.2 5.6 6 2.1

1.0 6 0.4 0.5 6 0.3 1.8 6 0.6 2.2 6 1.2 16.5 6 11.0a

0.8 6 0.2 0.5 6 0.3 1.6 6 1.1 1.7 6 0.7 7.9 6 8.7a

MBWH, Mean body weight for height. a P , 0.05.

FIG. 2. Correlation of annual changes in height age and differences between baseline and trough levels of serum IGF-I with rhIGF-I treatment in 22 children with GHRD. The dashed line represents the 1 yr correlation (r 5 0.54; P 5 0.009), and the solid line represents the 2 yr correlation (r 5 0.58; P 5 0.005).

FIG. 1. Fasting IGF-I serum levels at baseline (pretreatment) and at 1 and 2 yr of treatment of 15 children with GHRD receiving rhIGF-I injections of 120 mg/kg BW every 12 h and 7 children receiving 80 mg/kg every 12 h. Treatment specimens were obtained 12 h after rhIGF-I injection. Means are represented by solid squares, boxes encompass the 25th to 75th percentile, and vertical bars indicate the 10th to 90th percentile. Medians are horizontal bars within the boxes.

Comparison to rhGH-treated GHD. The comparable baseline characteristics between the 22 GHRD subjects and the 11 GHD patients are shown in Table 3. Although the GHD subjects were growing at a significantly slower pretreatment velocity, the GHRD subjects had a significantly more deviant sd score for height. The GVs in yr 1 and 2 for the entire group and in a third year subgroup were significantly greater for the GHD patients treated with rhGH than for the rhIGF-Itreated GHRD subjects (Table 4). The GHD subjects also had a greater D height sd score and HA over the 2 yr of treatment, although there was no difference in D BA or the ratio of D HA to D BA. The D % MBWH was significantly greater in the GHRD group (Table 4). Discussion

We have previously demonstrated that Ecuadorian patients with GHRD are significantly shorter than others with GHRD reported in the literature (11). This is reflected in both their lower sd score for height and their lower GV. For example, a recent report from Israel (12) notes a mean baseline GV in 8 subjects over 3 yr of age, who would be comparable to our population, of 4.6 6 1.3 vs. 3.3 6 1.5 cm/yr in

FIG. 3. IGFBP-3 levels at baseline and after 1 and 2 yr of treatment of 15 children with GHRD receiving 120 mg/kg BW rhIGF-I every 12 h and at 1 yr of treatment of 7 children with 80 mg/kg every 12 h. See Fig. 1 for explanation of box plot.

the 22 Ecuadorian children (P , 0.025). Thus, the first year GV increment of 3.7 6 1.6 cm in the Israeli children is significantly less than the first year GV increment of 5.5 6 1.4 cm/yr in the Ecuadorian group (P , 0.05). The Ecuadorian patients’ mean GV in the first year of rhIGF-I treatment (8.9 6 1.5 cm) is similar to the first year mean GV for 24 GHRD and 2 GH gene deletion patients in the European multicenter study treated with 40 –120 mg/kg twice daily (8.5 6 2.1 cm) (13). Eighteen patients in the European study treated for 2 yr also had second year GV (6.4 6 2.2 cm) comparable to that of the Ecuadorian children (6.1 6 1.5 cm). The 5 Israeli subjects over 3 yr of age who had data recorded for the second and third years had comparable second year incremental growth and overall GV to those of the Ecuadorian patients, but had a slowing to less than pretreatment velocities in the third year in 3 of 5 subjects and velocities of 5.5 and 6.5 cm/yr in the other 2. The 6 Ecuadorian patients with third year treatment data maintained second year velocities, with only

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TABLE 3. Comparison of baseline data for 22 patients with GHRD and 11 patients with idiopathic GHD

Age (yr) Growth velocity (cm/yr) Bone age (yr) SD score for ht MBWH (%)

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TABLE 4. Comparison of growth responses to replacement therapy with rhIGF-I of 22 children with GHRD to those of 11 children with idiopathic GHD treated with rhGH

GHRD

GHD

P

9.1 6 4.9 3.3 6 1.5 4.8 6 2.5 28.4 6 1.4 97.5 6 14.7

8.9 6 4.7 2.2 6 1.4 3.7 6 2.6 26.6 6 1.9 103.3 6 9.2

NS ,0.05 NS ,0.01 NS

MBWH, Mean body weight for height.

one growing less than 2 cm/yr above baseline, providing encouragement that further decreases might not occur. The major difference in treatment between the Ecuadorian and Israeli studies is that the Israeli patients were treated with 150 –200 mg/kg BW rhIGF-I as a single daily injection. This is a comparable or greater total dose than our more recent patients are receiving, but with possibly less satisfactory pharmacodynamics than a twice daily regimen provides. Improvement in mean height sd score over 2 yr was 1.2 in the European study (13), 1.3 for 5 patients with GHRD receiving 80 –120 mg twice daily in the U.S. (14), and 1.4 for the 22 patients in this study. The European and Ecuadorian patients achieved two thirds of their improvement in the initial year; 1 yr data were not reported in the U.S. study. The Israeli study reported mean sd score improvement of only 0.4 at 1 yr, remaining at this level of improvement for the 6 completing 2 yr of treatment. That the twice daily regimen is salutary is suggested by the trough levels of IGF-I noted 1 and 2 yr after the start of IGF-I therapy. These long term effects are consistent with our earlier findings of trough levels of IGF-I comparable to serum IGF-I concentrations in normal control subjects 12 h after injections of rhIGF-I (40 mg/kg twice daily) in six young adults with GHRD (15). The need for and efficacy of every 12 h administration is further supported by the response of six adults with GHD given sc injection of 40 mg/kg BW rhIGF-I. Serum IGF-I levels rose from markedly subnormal into the normal range over 1–3 h and were sustained for 10 –16 h despite no increase in IGFBP-3 levels (16). We have reported that adults with GHRD had significant correlation between both IGF-I and IGFBP-3 levels in serum and statural deviation (height sd score) and found comparable correlation for children between IGFBP-3 levels and sd score (1). Children in the European study of rhIGF-I treatment of GHRD had significant correlations between baseline sd score for height and sd score for serum IGF-I, IGF-II, and IGFBP-3 levels (17). In this study we demonstrated an important correlation between growth response to rhIGF-I injections in GHRD and trough levels of IGF-I 12 h after injection. Maintenance of therapeutic levels of IGF-I in the absence of an increase in IGFBP-3 suggests an alternative mechanism for sustaining the circulating level of IGF-I and avoiding the clearance of free hormone too rapidly for therapeutic effect at the cellular level. A rise in the already elevated IGFBP-2 levels may be substituting for the absence of an IGFBP-3 response (15). The potential advantage of IGFBP-2 binding would be that this IGFBP diffuses more readily into the interstitial space than does IGFBP-3 and could improve the bioavailability of IGF-I (18).

GHRD

Growth velocity (GV) Yr 1 Yr 2 Yr 3 (6 GHRD; 8 GHD) Increment of GV (above baseline) Yr 1 Yr 2 Yr 3 Change in ht SD scorea Change in ht age (HA)a Change in bone age (BA)a Change in HA/change in BAa Change in MBWH (%)a

GHD

P

8.9 6 1.5 6.1 6 1.5 5.7 6 1.4

10.9 6 1.6 8.1 6 2.2 8.3 6 1.9

,0.0001 ,0.02 ,0.001

5.5 6 1.4 2.9 6 1.6 2.9 6 1.2 1.4 6 0.6 1.7 6 0.8 2.0 6 1.1 0.97 6 0.41 13.8 6 10.4

8.8 6 2.5 6.1 6 3.1 6.5 6 2.4 2.2 6 1.0 2.5 6 0.7 2.6 6 1.3 1.06 6 0.37 4.8 6 7.3

,0.0001 ,0.01 ,0.005 ,0.01 ,0.01 NS NS ,0.01

MBWH, Mean body weight for height. a Over 2 yr of treatment.

Several studies have confirmed that rhIGF-I treatment of GHRD does not increase IGFBP-3 levels, in contrast to what occurs with rhGH treatment of GHD, indicating that the GH induction of IGFBP-3 is a direct effect (3, 13, 15). We also found that the acid-labile subunit of the IGFBP-3 is not increased by rhIGF-I therapy in these patients (data not shown). IGF-I administration also fails to raise serum IGFBP-3 levels in adults with GHD (16). The recent report of the first instance of GH insensitivity due to a defect in the IGF-I gene further confirms the direct role of GH in IGFBP-3 generation; despite absent IGF-I, the affected patient had normal levels of IGFBP-3 (19). That IGF-I concentrations were not significantly different at the two dose levels suggests a plateau effect beyond 80 mg/kg twice daily, as does the similar clinical responses to the twice doses. The only noted difference in response to the dosages of IGF-I used was in MBWH, with the higher dose resulting in a greater weight for height increase. Similarly, the GHRD subjects as a group had a greater increase in MBWH than did the rhGH-treated GHD group. It would be expected that rhGH treatment in GHD would have a lipolytic effect that is absent in GHRD subjects treated with rhIGF-I, but that both forms of treatment would affect lean mass accretion comparably. Thus, total fat mass in GHRD would diminish relatively little, while lean mass increases, perhaps in a dose-dependent fashion, to explain these observations. Daughaday and Rotwein (4) estimated that 20% of GHinfluenced growth is the result of direct effects of GH on bone. The difference between average growth of GH-treated GHD patients and rhIGF-I-treated GHRD patients is consistent with this estimate. It is not possible to attribute this difference to direct GH effects on bone without the ultimate experiment of treating naive GHD patients with IGF-I. Comparable D HA/D BA in the two groups over 2 yr of treatment suggests that long term growth in rhIGF-I-treated GHRD may be comparable to that in GH-treated GHD. Acknowledgments The authors thank Dr. Ana Lucia Martinez and Nancy Davila (Quito) for assistance with data and specimen collection; Diane Simpson and Dan Wanek (Portland, OR) for laboratory assistance; Rolf Gunnarsson,

rhIGF-I IN GHRD AT TWO DOSES FOR 2 YR M.D., Ph.D., Cecilia Frostegard, Cheryl Vitow, and co-workers of Pharmacia Peptide Hormones; and Margaret Stanley (Gainesville, FL) for manuscript preparation.

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