KAZUE TAKANO, KAZUO SHIZUME, ITSURO HIBI and Members of Committee for the Treatment of Pituitary Dwarfism in Japan

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1 Endocrinol. Japon. 1988, 35 (3), A Comparison of Subcutaneous and Intramuscular Administration of Human Growth Hormone (hgh) and Increased Growth Rate by Daily Injection of hgh in GH Deficient Children KAZUE TAKANO, KAZUO SHIZUME, ITSURO HIBI and Members of Committee for the Treatment of Pituitary Dwarfism in Japan Department of Medicine, Institute of Clinical Endocrinology, Tokyo Women's Medical College and the Foundation of Growth Science in Japan Abstract Plasma human growth hormone (hgh) profiles and biological activities of recombinant hgh were compared after im and sc injection in 8 normal volunteers. The time to reach maximal plasma GH and plasma hgh concentrations and the areas under the curve of hgh profiles did not differ significantly after im and sc injections. The biological effect of hgh in increasing nonesterified fatty acid and insulin-like growth factor-i (IGF-I) was the same after both im and sc injections. During 6 months of daily sc administration of recombinant hgh in 20 naive patients, their height increased between 5 and 16.5cm with a mean of 11.0 }3.0cm/year. In 27 patients who switched from hgh injections of 2-4 times/week to daily injections, the height increased between 5.3 and 16.5cm with a mean of 8.3 }2.2 cm/year. These values were greater than those observed in a previous study in which the same amount of hgh was injected in 2-4 doses per week. Plasma IGF-I increased more with daily sc administration than with 2-4 doses per week. The rate of appearance of an antibody to hgh was low (0.5%) and there were no notable changes in blood cell count, urinalysis and/or routine chemistries during the 6 months of daily recombinant hgh treatment. These results show that sc daily administration of hgh is safe, has a greater growth promoting effect, and can be recommended for the treatment of patients with GH deficiency. There is no doubt that human growth hormone (hgh) treatment has growth promoting effects in patients with hgh deficiency. The dose of hgh commonly used has been IU/kg/week, generally administered intramuscularly in 2-4 doses per week. Recently, several investigators (Kastrup Received February 10, 1988 et al., 1983, Wilson et at., 1985, Hermaussen et al., 1985, Albertsson-Wikland et al., 1986) have reported that the daily administration of hgh into such children has a greater growth promoting effect than the 2-4 times/week injection mode, even though the weekly dosage of hgh was the same. In this paper, we report plasma GH profiles and biological effects of hgh after im and

2 TAKANO et al. Endocrinol. June 1988 Japon. sc administration of hgh to normal volunteers and the growth rate during daily sc administration of hgh to patients with Table 1. Clinical data from 47 patients with hgh deficiency growth hormone deficiency. Materials and Methods Study design with normal volunteers Eight healthy male volunteers aged years were studied. All were within }20% of ideal body weight with a mean of 62.4 }7.1kg. They received 8IU of recombinant somatropin at by either im or sc injection at 2 week intervals (cross-over test). Plasma hgh was determined at 0, 1, 2, 3, 4, 10 and 24 hours after injection. Non-esterified fatty acid (NEFA) was measured at 0 and 4 hours after injection and plasma IGF-I was measured before and 24 hours after injection. Study design for patients with hgh de Forty-seven patients aged years with hgh deficiency were investigated. The clinical data are shown in Table 1. Informed consent was obtained from all patients. The diagnosis of hgh deficiency was established by the failure of plasma hgh to respond to general GH stimulation tests. In those cases with additional hormone deficiencies, suitable replacement therapy was given. Twenty out of 47 patients had never been treated with hgh before (Group I), 9 patients had previously been treated with recombinant methionyl hgh (somatrem; Somatonorm(R))(Group II) and 18 patients with recombinant methionine-free-hgh (somatropin; Genotropin(R))(Group III), respectively. Patients in groups II and III had been treated with the hgh preparations by intramuscular administration at a dosage of 0.5IU/kg/week 2-4 times/ week for 7-92 months with a mean of 24 }17.9. The aetiology of the hgh deficiency in 37 patients was idiopathic. The rest of the patients had secondary hgh deficiency; 5 were operated on for craniopharyngioma, 3 were irradiated for germinoma, one had Histiocytosis X, and one had optic glioma. Each patient received somatropin, 0.5IU/kg/ week, by daily sc administration for 6 months. During the treatment, vital signs, height and body weight were checked. Blood cell count, (M }SD) CA: chronological age, BA: bone age, BH: body height BW: body weight, GR : growth rate, *SD of Japanese boys and girls in similar age groups Group I: GH deficient patients who had never been treated before Group II: GH deficient patients who had been treated with methionyl hgh (Somatonorm(R)) Group III: GH deficient patients who had been treated with methionine-free hgh (Genotropin(R)) urinalysis, routine biochemistry and measurement of antibody to hgh were performed before and every month during somatropin treatment. Bone age was checked before and 6 months after the treatment. This study was started in April of Growth hormone preparations Biosynthetic recombinant hgh (somatropin; Genotropin(R)) was obtained from KabiVitrum AB, Stockholm, Sweden. Each vial contained 4 IU of recombinant authentic hgh determined by radioimmunoassay (RIA), 37mg of glycine, and 7mg of sodium phosphate. Assay methods Plasma hgh was determined by double antibody RIA, a gift from the National Pituitary Agency, NIADDK (hgh, AFP-4793 B; anti-

3 Vol. 35, No. 3 DAILY INJECTION OF hgh INCREASES GROWTH body, AFP ). The intra-and interassay coefficients of variation for hgh were 6.6% and 8.0%, respectively. EDTA plasma was used for the determination of IGF-I by RIA (Nicholas Institute, San Juan Capistrano, California, USA). The intraassay coefficient of variation was 4.1%. Anti-hGH and-body was determined by the polyethylene glycol methods described previously (Takano et al., 1983). Bone age was estimated by the method of Tanner et al. (1975). The analysis of variance, Student's t-test, and paired t-test were used for statistical analysis. Results Study in normal volunteers The changes in plasma hgh concen-

4 Table 2. Serum non-esterified fatty acid (NEFA) levels (meq/l) in normal volunteers before and 4 hours after im and sc injections of 8 IU recombinant somatropin Fig.2. Mean plasma GH profiles after im ( --- ) and sc( œ- œ)injections of 8 IU recombinant hgh (Mean }SD, N=8). tration after im or sc injection of 8 IU of somatropin are shown in Figs.1 and 2. After im injection, plasma hgh reached a peak at 3 hours after injection with a mean value of 42.9 }15.2ng/ml (mean } SD) and declined gradually and reached the initial value after 24 hour injection. After sc injection, plasma hgh reached a peak at 4 hours after injection with a mean value of 39.5 } 10.1ng/ml and declined gradually to the intial value at 24 hours. The time taken to reach the maximal hgh level (T max), the maximal plasma hgh concentration (C max), and the areas under the curve of plasm hgh (AUC) after im and sc administration did not differ significantly (T max; 3.3 } 0.7 vs 3.6 } 0.7 hour, C max; 45.1 } 13.2 vs 40.8 } 11.1ng/ ml, AUC; } 81.1 vs } 74.1ng/ml. Table 3. Plasma IGF-I (U/ml) in normal volunteers before and 24 hours after im and sc injections of 8 IU recombinant somatropin W. Serum NEFA increased significantly from 0.70 } 0.25 to 1.02 } 0.26mEq/liter at 4h after im injection and from to 1.04 }0.30mEq/liter at 4 h after sc injection (Table 2). These two increased values did not differ significantly. Plasma IGF-I was 0.75 }0.61 and 0.91 } 0.37 U/ml before injection and incrto 1.37 } 0.29

5 and 1.41 }0.22U/ml at 24 hours after im and sc injections, respectively (Table 3). These latter two values also did not differ significantly. Table 5,Plasma IGF-I (U/ml) before and during daily administration of recombinant somatropin to naive (Group I) and switched (Group (III+III patients Daily somatropin injection in pituitary dwarfism The growth rate, the changes in bone age, and the appearance of anti-hgh antibody during the treatment are shown in Table 4. During the 6 months of daily hgh treatment in naive patients (G-1), their height increased between 2.5 and 8.25cm, corresponding to 5.0 and 16.5cm with a mean of 11.0 }3.0 (mean } SD) cm/ year. In the switched patients in groups II and III, their height increased between cm and cm, with a mean of 9.3 }3.2 and 7.8 }1.5 cm/year, respectively. In group II, the growth rate was greater than that observed in the previous year (7.5 }2.5cm, p>0.05). During the 6 month treatment, the bone age advanced between , and years with a mean of 0.6 }0.4, 0.6 }0.5 Table 4.Growth rate, change in bone age, and the appearance of anti-hgh antibody during 6 months of daily sc administration of recombinant somatropin to naive (Group I) and switched (Group II, Group III) patients and 0.8 }0.6 in groups I, II and III, respectively. Antibody to hgh was observed in two of 20 naive patients at the end of 6 months of the treatment with titers of 102 and 10, respectively. Antibody was already present in 6 of 9 Somatonorm(R) treated patients (G-II). During the daily sc administration of somatropin for 6 months, the antibody disappeared in two patients and the titer of antibody decreased in three others. The antibody did not appear in 3 patients who did not have the antibody at the beginning of this study. The antibody was already present in one of the 18 patients previously treated with somatropin (G-III) with a titer of 10. The antibody titer in this patient did not change during the daily sc administration of somatropin. The antibody did not appear in the other 17 patients during this study. Thus, in switched patients, the antibody did not appear de novo in any patient during daily sc administration of somatropin. During the treatment, no notable changes in blood cell count, urinalysis and/or routine biochemistry were seen. During somatropin treatment, plasma IGF-I increased significantly to the same

6 extent in naive and switched patients (Table 5). The mean values at 2, 4 and 6 months of the treatment ranged between 1.2 and 1.6U/ml. Discussion We have studied the plasma GH profiles and biological activities after im and sc injection of recombinant hgh (somatropin). We did not observe any difference in the time to reach the maximal plasma hgh (T max) or plasma hgh concentration (C max), or the area under the curve of hgh profiles (AUC). Furthermore, the biological effects of hgh, such as increasing NEFA and IGF-I, were the same with either im or sc injections. Thus, the route of hgh administration, im or sc, did not affect the plasma GH profile or biological activity. Christiansen et al. (1983) reported that sc injecction resulted in a more delayed and lower-sustained plasma GH concentration than did im injection. They injected 4 IU/m2 hgh into GH deficient children. A median peak value of 160ng/ml was observed 2h after im injection; but sc injection resulted in a more sustained increase, reaching 41ng/ml at 6h and subsiding slowly with a median concentration of 15ng/ml persisting after 14 h. On the other hand, Russo and Moore (1982) reported that the peak and integrated concentrations of hgh were similar after both im and sc injections. They injected 0.1 U/kg of pituitary extracted hgh into children with GH deficiency and observed that the peak and integrated concentrations of plasma hgh were 43.1 } 8.8 and 93.1 } 16.6ng/ml, respectively, for the im group, and 43.1 }11.1 and }36.1ng/ml, respectively, for the sc group. These results were similar to ours. The differences in the GH profiles between Christiansens et al. (1983) and our data can be attribute to the hgh preparation, injection volume, the needle used, the injection site and/or the subjects used. J. S. Christiansen stated that they injected 2 IU of pituitary extracted hgh in 0.5ml with 21G ~1.5 (0.8 ~40) needle into the gluteal region for im, and with 27G ~3/4 (0.4 ~20) into the anterior lateral femoral region for sc into GH deficient children (personal communication). We injected 8 IU of recombinant hgh in 2 ml with 23G ~25 needle into the lateral brachial region of normal adults for both im and sc. During the six months of daily injection of somatropin into naive patients, their height increased cm/year. This value was significantly greater than that observed in a previous study of 2-4 times/ week injections of the same total dosage of somatropin (8.1 }1.7cm/year, p<0.05) (Takano et al., 1987), p-hgh (6.5 }0.6 cm/year Shizume 1984), or m-hgh (Somatonorm(R), 8.2 }1.7 cm/year) (Takano et al., 1986). In switched patients (Group II and III), the mean growth rate was 8.3 } 2.2cm/year with daily sc injection, which was greater than that observed in a previous study, where the same amount of hgh was injected in 2-4 divided doses/week (6. }1.2 cm/year) (Takano et al., 1987). These increased growth rates with daily administration are similar to those reported by others (Kastrup et al., 1983, Wilson et al., 1985, Hermanussen et al., Albertsson-Wikland et al., 1986, Albertsson-Wikland 1987). For many years, we have used im injection of hgh for the treatment of GH deficient children. The major objection to sc administration of hgh was the potential for increased immunogenicity and lipoatrophy at the injection site (Underwood et al., 1974, Moore and Leppert 1980). Because of the high purity of the somatropin, antibody to hgh was observed in very few patients and the titers were very low. It is well accepted that methionyl-hgh has more antigenicity than p-hgh; six out of 9 patients previously treated with m-hgh had the antibody at

7 the beginning of this study. During this study, the antibody titers decreased. In naive patients, 2 out of 20 had the antibody at a low titer after 6 months of treatment. Thus, daily sc injection of somatropin did not induce much antibody formation. There are some studies which report that subcutaneous and intramuscular growth hormore therapy are equal in promoting linear growth (Russo and Moore 1982, Wilson et al., 1985). Therefore, the increased growth promoting effect of daily sc injection is due to the increased frequency of GH administration, not to the route of administration. Since the subcutaneous method is safer, less painful, and easier than im injection, sc administration can be recommended for the daily injection of hgh. Human GH is secreted in pulses; therefore, daily injection seems to be more physiologically sound than 2-4 times/week injection. Furthermore, plasma IGF-I increased more with daily injections. There remains the question whether more frequent injections of hgh might produce an even greater height increase. Further investigation will be required to answer this question. Acknowledgements The authors are very grateful to Sumitomo Pharmaceutical Co., Ltd., (Osaka, Japan) for supplying methionine-free hgh preparations. This work was supported by Grants in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan (No ) and by a research grant from the Foundation for Growth Science in Japan. References Albertsson-Wiklanp, (1987). The effect of human growth hormone injection frequency on linear growth rate. Acta Peodiatr. Scand. (suppl.) 337, Albertsson-Wikland, K., O. Westphal and U. U. Westgren (1986). Daily subcutaneous administration of human growth hormone in growth hormone deficient children. Acta Paediatr. Scand. 75, Christiansen, J. S., H. Orskov, C. Binder and K. W. Kastrup (1983). Imitation of normal plasma growth hormone profile by subcutaneous administration of human growth hormone to growth hormone deficient children. Acta. Endocrinol. (Copenh.) 103, Hermanussen, M., K. Feiger-Benoit and W. P. Sippell (1985). Catch-up growth following transfer from three times weekly i. m. to daily. s. c. administration of hgh in GH deficient patients, monitored by knemometry. Acta. Endocrinol. 109, Kastrup, K. W., J. S. Christiansen, J. K. Andersen and H. Orskov (1983). Increased growth rate following transfer to daily sc administration from three weekly im injection of hgh in growth hormone deficient children. Acta Endocrinol. (Copenh) 104, Russo L. and W. V. Moore (1982). A comparison of subcutaneous and intramuscular administration of human growth hormone in the the therapy of growth hormone deficiency. J. Clin. Endocrinol. Metab. 55, Shizume, K. (1984). Long term effects of human growth hormone on 1959 patients with pituitary dwarfism throughout Japan. Endocrinol. Japon. 31, Takano, K., K. Shizume, N. Hizuka, I. Hibi, K. Kato, H. Kohno, T. Kondo, M. Ogawa, Y. Okada, M. Sudo, S. Suwa, and J. Takahara (1983). Treatment of idiopathic pituitary dwarfism with methionyl human growth hormone. Endocrinol. Japon. 30, Takano, K., K. Shizume, H. Hizuka, A. Okuno, T. Umino, Y. Kobayashi, S' Kusano, H. Nakajima, M. Irie, I. Hibi, K. Kato, S. Suwa, T. Koshimizu, M. Ogawa, M. Sudo, H. Imura, Y. Okada, T. Kondo, K. Hashimoto, M. Miyao, H. Kohno, N. Iwatani and S. Ono. (1986). Treatment of pituitary dwarfism with methionyl Lhuman growth hormone in Japan. Endocrinol. Japon. 33, Takano, K., K. Shizume, I. Hibi, A. Okuno, K. Hanyu, S. Suwa, H. Nakajima, T. Kondo, K. Kato, N. Iwatani, T. Momoi, K. Chihara, E. Shirakawa and H. Kohno (1987). Treatment of pituitary dwarfism with authentic recombinant human growth hormone (SM- 9500). Endocrinol. Japon. 34,

8 Tanner, J. M., R. H. Whitehouse, W. A. Marchall, M. J. R. Healy and H. Goldstein (1983). Assessment of Skeletal Maturity and Prediction of Adult Height (TW2 Method). Academic Press INC., London. (Second edition). Moore, W. (1978). The role of aggregated hgh in the therapy of hgh deficient children. J. Clin. Endocrinol. Metab. 46, Moore, W. and P. Leppert (1980). Role of aggregated human growth hormone (hgh) in development of antibodies to hgh. J. Clin. Endocrinol. Metab. 51, Underwood, L., S. Viona, J. Van Wyk (1974). Restoration of growth by human growth hormone (Roos) in hypopituitary dwarfs immunized by other human growth hormone preparations; clinical and immunological studies. J. Clin. Endocrinol. Metab. 38, Wilson, D. M., B. Baker, R. L. Hintz and R. G. Rosenfeld (1985). Subcutaneous versus intramuscular growth hormone therapy: Growth and acute somatomedin response. Pedirtrics 76,