NANOMEDICINE

Journal of Controlled Release 137 (2009) 160165

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Journal of Controlled Release

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / j c o n r e l

Development of a sustained-release recombinant human growth hormone formulation

H.H. Kwak a,b, W.S. Shim b, M.K. Choi b, M.K. Son a, Y.J. Kim a, H.C. Yang a, T.H. Kim a, G.I. Lee a, B.M. Kim a, S.H. Kang a, C.K. Shim b,
a b

Biopharmaceutical Research Laboratory, Dong-A Pharm. Co. Ltd., Gyeonggi, 446-905, Republic of Korea National Research Laboratory for Transporters Targeted Drug Design, College of Pharmacy, Seoul National University, Seoul 151-742, Republic of Korea

a r t i c l e

i n f o

a b s t r a c t
Recombinant human growth hormone (rhGH) therapy for short stature must be administered as a daily injection because of its poor bioavailability and short half-life. In the present study, a sustained-release formulation of rhGH (SR-rhGH), DA-3003, was prepared using double emulsion solvent evaporation with poly(D,L-lactide-co-glycolide) (PLGA), zinc oxide and hydroxypropyl--cyclodextrin (HPCD) as the release modulator, stabilizer, and aggregationprevention agent, respectively. After a single administration of DA3003, the elevated concentration of rhGH in plasma was sustained for 14 days in rats and 28 days in monkeys. The plasma concentration of insulin-like growth factor-1 (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3), which are pharmacodynamic markers of rhGH administration, increased and remained elevated for approximately 28 days in monkeys. Monkeys administered DA-3003 did not develop antibodies to hGH, indicating safety of the SR-rhGH formulation comparable to that observed with daily rhGH injections (Growtropin II). There were no signicant differences in efcacy between Growtropin II (daily dose of 5 g/animal for 14 days) and DA-3003 (weekly dose of 35 g/animal for 14 days with a dosing interval of a week) in hypophysectomized rats, as assessed by changes in body weight and the width of the tibial growth plate. These results show that a sustained-release rhGH formulation, DA-3003, has the potential to be used safely and efcaciously in a weekly dosing regimen. 2009 Elsevier B.V. All rights reserved.

Article history: Received 2 December 2008 Accepted 21 March 2009 Available online 28 March 2009 Keywords: Protein delivery Sustained-release formulation Recombinant human growth hormone Poly(D,L-lactide-co-glycolide) Solvent evaporation

1. Introduction Human growth hormone (hGH), which is a 191-amino acid, singlechain polypeptide with two disulde bonds, is stored in the anterior pituitary and secreted in a pulsatile fashion mostly during sleep [1]. Since the introduction of recombinant human growth hormone (rhGH) in 1985, it has been used to treat pediatric short stature that results from growth hormone deciency (GHD), Turner's syndrome and chronic renal insufciency, and adult GHD. In general, these patients require daily subcutaneous injections of rhGH over a period of several years. Smith et al. [2] reported that more than 50% of patients treated with growth hormone fail to comply with some aspect of the treatment regimen. The complexity of the rhGH treatment regimen, including the frequency of administration and duration of treatment, negatively affects compliance [3]. Therefore, patient compliance might be signicantly enhanced by reducing the frequency of administration and increasing the convenience of rhGH treatment. Because secretion of endogenous hGH is pulsatile, reducing the frequency of rhGH administration, while maintaining efcacy, is difcult to achieve. However, several clinical studies have shown that
Corresponding author. Tel.: +82 2 880 7873; fax: +82 2 888 5969. E-mail address: shimck@snu.ac.kr (C.K. Shim). 0168-3659/$ see front matter 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.jconrel.2009.03.014

continuous infusion of rhGH via a pump results in growth velocity and insulin-like growth factor-1 (IGF-I) concentrations that are similar to those achieved with daily rhGH injections [46], demonstrating that pulsatile release is not mandatory for clinical efcacy of rhGH. Thus, to improve patient compliance, development of a sustained-release (SR) rhGH formulation has been actively pursued since the advent of rhGH treatment. The rst SR microsphere formulation of rhGH, Nutropin Depot, which is prepared using poly(D,L-lactide-co-glycolide) (PLGA), was rst launched in 1999 in the United States. Unfortunately, problems with scale-up during manufacturing [79], resulted in the withdrawal of Nutropin Depot from the market in 2004. Since then, several other SR-rhGH formulations, such as hyaluronate microparticles [10], dextran-derivative microspheres [11], PEGylated [12] and crystalline formulations [13], have been developed. However, except for the hyaluronate microparticles, none of these products are commercially available. In addition, the pharmacokinetic and pharmacodynamic proles of rhGH for dextran-derivative microspheres [11] and crystal formulations [13] indicate that the half-life of rhGH in circulation was inadequate. A hyaluronate microparticle formulation of rhGH (HArhGH), which was developed and launched exclusively in Korea by LG Life Science (LB03002, LGLS, Seoul, Korea) in 2007 is the only SR-rhGH product currently available [10]. However, data from cynomolgus

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monkeys showed that the concentration of rhGH in serum remained elevated for only 30 h following administration of HA-rhGH [14]. Therefore, the purpose of the present study was to develop a safe and effective sustained-release formulation of rhGH (SR-rhGH). rhGH was formulated into microspheres using PLGA, zinc oxide and hydroxypropyl--cyclodextrin (HPCD), and the feasibility of clinical use of the SR-rhGH formulation was evaluated. Plasma concentrations of rhGH in rats and monkeys (pharmacokinetic markers), plasma concentrations of IGF-I and insulin-like growth factor binding protein3 (IGFBP-3) in monkeys (pharmacodynamic markers), plasma concentrations of antibodies against rhGH in monkeys (safety markers) as well as weight gain and increase in the width of tibial growth plate in hypophysectomized rats (efcacy markers) of the SRrhGH formulation were compared with that of a commercial daily rhGH preparation (Growtropin II injection solution, Dong-A Pharm. Co., Seoul, Korea).

Fig. 2. Scanning electron micrographs of the surface morphology (left) and crosssection (right) of the DA-3003 microspheres.

2.3. Physicochemical characterization of DA-3003 Please refer to supplementary methods for detailed procedures.

2. Materials and methods 2.1. Materials Please refer to the supplementary documents for more information.

2.4. In vivo study Please refer to supplementary methods for detailed procedures. 3. Results

2.2. Preparation of SR-rhGH microspheres, DA-3003 SR-rhGH microspheres were prepared using water-in-oil-in-water (w/o/w) double emulsion solvent evaporation, as previously described [15], with appropriate modications (Fig. 1). PLGA, zinc oxide and HPCD were used as the release modulator, stabilizer and aggregationprevention agent, respectively. Briey, the rhGH solution (100 mg/mL in water-for-injection, WFI) containing HPCD (2.5% in WFI, w/v) (denoted as W1 in Fig. 1) was emulsied in dichloromethane containing PLGA (32%, w/v), zinc oxide (0.4%, w/v) and Span 40 (0.2%, w/v) (denoted as the Oil phase in Fig. 1) at a W1:Oil volume ratio of 15:25 by homogenization (Lab mixer, Silverson, MA, USA). This rst emulsion was immediately added to the continuous phase (W2: 1% PVA, 500 mM NaCl in WFI), and the solution was further emulsied using a TK mixer (Primix, Tokyo, Japan) to obtain the second emulsion. After emulsication, the dichloromethane was evaporated by stirring at 37 C for 3 h. The resultant microspheres were collected by ltration (pore size: 20106 m), washed three times with WFI, and freeze dried. This microsphere formulation was termed, DA-3003, in the present study.

3.1. Physicochemical characteristics of the SR-rhGH microspheres The mean particle size of the DA-3003 microspheres was approximately 50 m. The SEM images showed that SR-rhGH microspheres were spherical with a smooth surface and internal porous structures (Fig. 2). The content of rhGH in DA-3003 was 13 3%, and its encapsulation efciency was approximately 87%. Size exclusion chromatographic (SEC) proles of DA-3003 for rhGH were identical to those of standard rhGH and starting rhGH (raw material), suggesting that rhGH-derived impurities, such as dimers, aggregates and isoforms, did not form during manufacturing (Fig. 3). Electrophoresis of the rhGH extracted from DA-3003 showed negligible bands for rhGH isoforms (data not shown). This result implies that rhGH retained its monomeric conguration during manufacturing. Overall, DA-3003 was prepared without signicant loss of the physicochemical properties of rhGH in terms of content and purity. The in vitro release of rhGH from both DA-3003 and reference microspheres (i.e., microspheres similarly prepared in the absence of zinc oxide and HPCD) for 14 days was not different between the formulations and remained less than 10% of the loaded amount. The results of sterility and endotoxin tests demonstrated the sterile integrity of DA-3003. The residual solvent (i.e., dichloromethane) in DA-3003 corresponded to concentration limits for a residual solvent in EP.

Fig. 1. Schematic illustration of the solvent evaporation method used to prepare the DA3003 microspheres.

Fig. 3. Size exclusion chromatography of rhGH in-house standard (A), starting rhGH (raw material, B) and rhGH extracted from DA-3003 (C).

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H.H. Kwak et al. / Journal of Controlled Release 137 (2009) 160165 Table 1 Pharmacokinetic parameters of Growtropin II, DA-3003 and HA-rhGH in Sprague Dawley rats. Daily rhGH (Growtropin II, 0.1 mg/kg) Cmax (ng/mL) Tmax (h) AUC01d (ngd/mL) AUC014d (ngd/mL) Initial burst (%) 77.2 6.43 0.65 0.15 5.22 0.144 (DA-3003, 6 mg/kg) 16.8 2.56 65.4 3.69 25.8 3.91 SR-rhGH (HA-rhGH, 6 mg/kg) 655 15.2 3.2 0.49 264 8.94 270 9.28 97.8 0.313

3.2. Plasma levels of rhGH in SD rats from DA-3003 The concentration of rhGH in the plasma of SD rats following a single subcutaneous injection of the daily preparation (Growtropin II) at an rhGH dose of 0.1 mg/mL/kg rapidly reached Cmax of 77.2 ng/mL at a Tmax of 0.65 h, and declined to baseline values within 5 h of administration (Fig. 4). The plasma concentration of rhGH after treatment with HArhGH at an rhGH dose of 6 mg/mL/kg was much higher for 24 h with a Cmax of 655 ng/ml at a Tmax of 3.2 h, but declined rapidly to baseline values within 48 h of administration (Fig. 4, Table 1). However, the plasma concentration of rhGH after treatment with DA-3003 at an rhGH dose of 6 mg/mL/kg remained signicantly elevated for at least 14 days, with the highest plasma level at the 1st sampling period of 2 h (Fig. 4). The initial burst of rhGH from HA-rhGH was 98%, while it was less than 26% for DA-3003 (Table 1). The burst was determined from the ratio, AUC01d/AUC014d [16]. These results indicated that the release of rhGH from DA-3003 was substantially sustained compared to Growtropin II and HA-rhGH. 3.3. Pharmacokinetic and pharmacodynamic study of SR-rhGH in rhesus monkeys DA-3003 was subcutaneously injected into juvenile rhesus monkeys at an rhGH dose of either 3 or 7.5 mg/mL/kg, and the plasma concentrations of rhGH were compared with those observed following the rst subcutaneous injection of Growtropin II at an rhGH dose of 0.5 mg/mL/kg. The baseline plasma concentration of rhGH in the Growtropin II group was 1.56 0.68 ng/mL, compared with 1.90 0.68 ng/mL and 2.83 1.40 ng/mL for the 3 and 7.5 mg/mL/kg doses of DA-3003, respectively. The plasma concentration of rhGH from Growtropin II rapidly declined to baseline values within 24 h of administration. However, the plasma concentrations from DA-3003 remained signicantly elevated for at least 28 days for both doses (3 and 7.5 mg/mL/kg) (Fig. 5, Table 2). The initial bursts of rhGH released from DA-3003, which were determined from the ratio of AUC01d/AUC028d [16], were approximately 29 and 18%, respectively, for the 3 and 7.5 mg/mL/kg doses. The results presented in Fig. 5 and Table 2 conrmed that the release of rhGH from DA-3003 was substantially sustained in monkeys as well as in rats (Fig. 4). It is well known that growth hormone increases the concentration of IGF-I in plasma [17]. Thus, the concentration of IGF-1 in plasma was monitored after the subcutaneous administration of either Growtro-

d: day, : p b 0.05, vs. Growtropin II, : p b 0.05, vs. DA-3003.

pin II (seven daily administrations at a dose of 0.5 mg/mL/kg) or DA3003 (single injections at doses of either 3 or 7.5 mg/mL/kg) to juvenile rhesus monkeys (Fig. 6a). The mean baseline concentration of IGF-I in the monkeys was 662 38 ng/mL for the Growtropin II group and 789 126 and 896 105 ng/mL for the low- and high-dose DA-3003 groups, respectively. The IGF-I concentration increased markedly in all groups 4 ~ 8 h after administration, regardless of the type of rhGH formulation (Fig. 6a). However, in the Growtropin II group, the concentration of IGF-I decreased rapidly after administration of the nal (7th) dose, returning to the baseline concentration by day 11. On the other hand, the concentration of IGF-I in plasma remained elevated for 28 days after administration of DA-3003. This result is consistent with the sustained elevation in plasma rhGH concentration observed for the DA-3003 groups (Fig. 5). The Cmax of IGF-I for both doses of DA-3003 was 35 ~ 43% greater than that observed after treatment with Growtropin II. Moreover, the AUC028d for both doses of DA-3003 was approximately 50% greater than that observed for Growtropin II (Table 2). Interestingly, the level of IGF-I from DA-3003 declined on the 17th day and thereafter (Fig. 6a) despite the stabilized level of rhGH for the period (Fig. 5). The mechanism of this discrepancy needs further investigation, although a similar phenomenon was previously reported [18]. The plasma concentration of IGFBP-3, which is also up-regulated by elevated rhGH concentrations [19], was monitored in the monkeys (Fig. 6b). The concentration of IGFBP-3 in all groups demonstrated a prole similar to that of IGF-I (Fig. 6a). The Cmax and AUC028d of IGFBP-3 from DA-3003 were 28 ~ 49% and 25 ~ 59% greater than the values observed for Growtropin II, respectively (Table 2).

Fig. 4. Average plasma rhGH concentrations in SD rats treated with Growtropin II (0.1 mg rhGH/mL/kg, ), DA-3003 (6 mg rhGH/mL/kg, ) and HA-rhGH (6 mg rhGH/mL/kg, ). Data are means SE (n = 5).

Fig. 5. Average plasma rhGH concentrations in rhesus monkeys treated with Growtropin II for 7 days (0.5 mg rhGH/mL/kg, ) and DA-3003 (3 mg rhGH/mL/kg, ; 7.5 mg rhGH/ mL/kg, ). Data are mean SE (n = 4).

H.H. Kwak et al. / Journal of Controlled Release 137 (2009) 160165 Table 2 Pharmacokinetic and pharmacodynamic parameters of Growtropin II and DA-3003 in rhesus monkeys. Daily injection of rhGH (Growtropin II, 0.5 mg/kg for 7d) rhGH Cmax (ng/mL) Tmax (h) AUC01d (ngd/mL) AUC028d (ngd/mL) Initial burst (%) IGF-I Cmax (ng/mL) AUC028d (ngd/mL) IGFBP-3 Cmax (ng/mL) AUC028d (ngd/mL) 340 33.7 1.8 0.25 66.7 5.08 1310 60.0 23700 1280 3430 245 71200 3330 Single injection of SR-rhGH (DA-3003) 3 mg/kg 7.5 mg/kg Body weight gain (g) Width of the tibial growth plate (m) 57.9 8.94 46.1 6.69 197 7.25 295 48.7 29.3 4.10 18.2 5.52 1870 220 1770 111 34600 3940 36300 1650 4400 359 5120 408 89100 8280 113000 10200

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Table 3 The efcacy of DA-3003, HA-rhGH and Growtropin II in hypophysectomized rats, assessed based on cumulative body weight gain and increase in the width of the tibial growth plate on day 14. SR-rhGH DA-3003 17 0.87 436 14.8 HA-rhGH 12 0.59, 390 4.97, Daily rhGH Growtropin II 20 1.5 462 10.2 6.6 0.64 339 18.3 Non-treatment

The efcacy of DA-3003, HA-rhGH and Growtropin II were compared at equivalent doses of rhGH = 70 g/animal. DA-3003 and HA-rhGH was administered twice as a weekly subcutaneous injection at an rhGH dose of 35 g/animal. Growtropin II was administered for 14 days as a daily subcutaneous injection at an rhGH dose of 5 g/animal. : p b 0.05, vs. Non-treatment, : p b 0.05, vs. DA-3003.

d: day, : p b 0.05, vs. Growtropin II.

3.5. Efcacy of DA-3003 in hypophysectomized rats 3.4. rhGH antibody formation The immunogenicity of Growtropin II and DA-3003 was evaluated by measurement of anti-hGH antibodies. Non-native proteins, such as aggregated rhGH, produced during manufacturing or release of rhGH from the microparticles, are immunogenic. The absorbances at 450/620 nm for the plasma samples from all groups (Growtropin II and DA-3003 groups) were below the cut-off value (i.e., twice the average of the negative control), indicating that antibody formation in response to administration of either Growtropin II or DA-3003 was negligible. The efcacy of DA-3003, HA-rhGH and Growtropin II was compared in hypophysectomized (Hpx) rats based on weight gain and increase in the width of the tibial growth plate. Growtropin II was administered daily via subcutaneous injection for 14 days at an rhGH dose of 5 g/animal. DA-3003 and HA-rhGH were administered weekly via subcutaneous injection at an rhGH dose of 35 g/animal. Body weight increased over the course of the study, regardless of the type of formulation (Table 3). Weight gain in both rhGH-treated groups was signicantly greater than that observed in the control group, but there was no difference between the DA-3003 and Growtropin II groups. However, the weight gain for the HA-rhGH group was signicantly lower than that of the DA-3003 group (Table 3). In addition, representative stained tibial sections are shown in Fig. 7. The width of the tibial growth plate was signicantly increased in both rhGH-treated groups compared with the control group (Fig. 7, Table 3). No signicant differences in the width of the tibial growth plate were found between the DA-3003 and Growtropin II groups, but the width for the HA-rhGH group was smaller compared to the DA-3003 group. These results indicate that, at a dose of 70 g/animal, administration of DA-3003, as two injections separated by one week, is comparable to 14 daily injections of Growtropin II and is superior to HA-rhGH.

Fig. 6. The average plasma IGF-I (a) and IGFBP-3 (b) concentrations in rhesus monkeys treated with Growtropin II for 7 days (0.5 mg rhGH/mL/kg, ) and DA-3003 (3 mg rhGH/ mL/kg, ; 7.5 mg rhGH/mL/kg, ). Data are means SE (n = 4).

Fig. 7. Representative sections of the proximal tibial growth plate from hypophysectomized rats administered two weekly subcutaneous injections of DA-3003 (a) and HArhGH (b) at an rhGH dose of 35 g/animal, or 14 daily subcutaneous injections of Growtropin II (c) at an rhGH dose of 5 g/animal. The control group had no treatment (d). The tissue sections were stained with the hematoxylin and eosin.

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4. Discussion The present study developed a sustained-release rhGH formulation comprised of rhGH-containing PLGA microspheres that were prepared using water-in-oil-in-water (w/o/w) double emulsion. Preservation of protein integrity during both manufacturing (e.g. emulsication, spray drying and cryogenic spraying) and drug release is a signicant challenge in the development of sustained-release formulations [20]. The primary cause of protein denaturation during emulsication is not shear stress, but the presence of a large interface between the aqueous and organic phases [21]. In addition to protein/ organic solvent contact, hydrophobic interactions between the protein and carriers, such as polymers, are a problem associated with the emulsion method that can lead to protein unfolding and aggregation [22,23]. Various excipients were investigated for their ability to preserve protein integrity in the DA-3003 microspheres. Among them, zinc oxide and HPCD effectively stabilized the rhGH, as evidenced by the absence of dimers, aggregates and isoforms of rhGH in the sizeexclusion chromatographic (Fig. 3) and gel electrophoretic analyses of DA-3003 (data not shown). It is well-known that zinc both facilitates the formation of hGH-zinc dimers, which are more stable than the monomeric form of hGH [24], and controls the release of hGH from microspheres [7,9]. HPCD has been used to reduce covalent aggregate formation during microsphere preparation. HPCD competes with the water/organic solvent interface, thereby preventing both emulsication-induced denaturation and aggregation of proteins [25]. Moreover, protein solubility is enhanced by retention of the hydrophobic moieties in the cavity of HPCD [26], which results in both a greater initial protein concentration and minimization of the lag phase during release of the protein from the microspheres. Zinc oxide and HPCD have been clinically used as diluents in Humalog (zinc oxide) and Sporanox (HPCD) injections without evoking any signicant side effects. Indeed, local irritation of the injected site was not observed for DA-3003 in preliminary clinical studies (data not shown). To assess the potential clinical use of the sustained-release DA3003 formulation, preclinical studies were conducted in rats and monkeys. As shown in Figs. 4 and 5, the plasma prole of rhGH released from the DA-3003 formulation was dramatically sustained compared to that of the rhGH administered as daily injections of Growtropin II in both SD rats and rhesus monkeys. The plasma concentration of rhGH from DA-3003 at the 1st sampling point (i.e., 2 h) was signicantly lower than Cmax observed for Growtropin II, despite administration of a signicantly greater dose of rhGH with DA3003. Moreover, the initial burst of rhGH released from DA-3003 was less than that of Growtropin II (Tables 1 and 2). In addition, much longer sustaining of plasma rhGH was observed for DA-3003 compared to HA-rhGH. These results indicate that DA-3003 serves as a storage depot for rhGH that allows for sustained release of the hormone in the body. However, DA-3003 demonstrated a low bioavailability of rhGH (21% in rats and 4930% in monkeys) when calculated based on the AUC of daily rhGH. A similar phenomenon was reported for solution dosage forms of rhGH following subcutaneous administration via an osmotic pump [18]. Therefore, incomplete release of rhGH from DA-3003 does not appear to be associated with the bioavailability issue. Instead, possible difference in the systemic clearance between DA-3003 (larger clearance due to low and sustained plasma rhGH) and daily rhGH (saturated and lower clearance due to relatively high plasma rhGH) might explain the low bioavailability of DA-3003. Fig. 6 shows that plasma concentrations of IGF-I and IGFBP-3, which are well-known biomarkers of growth hormone, remained elevated for 28 days after a single injection of DA-3003. Moreover, administration of the DA-3003 to hypophysectomized rats resulted in increases in both body weight and the width of the tibial growth plates that were equivalent to those produced by daily injections with

Growtropin II and superior to those of HA-rHGH (Fig. 7, Table 3). These results provide convincing evidence that the bioactivity of rhGH in DA-3003 is preserved during both preparation of the microspheres and release of the hormone from DA-3003 in vivo. The immunogenicity of peptide-based drugs is an important safety issue that must be considered during development of these therapeutic agents. In the present study, anti-hGH antibody formation in response to administration of either DA-3003 or Growtropin II was negligible, suggesting that the immunogenicity of DA-3003 is comparable to that of Growtropin II. Taken together, the results of the present study suggest that the delivery of rhGH via DA-3003 might have advantages over administration of the daily injection formulations, such as Growtropin II. First, DA3003 may improve patient compliance because the required frequency of administration is signicantly reduced. In addition, because the repetitive burst of plasma rhGH from daily Growtropin II can be avoided with DA-3003, possible side effects associated with the burst would be minimized. Second, the prolonged elevation in plasma concentrations of pharmacodynamic markers, such as IGF-I and IGFBP-3, with DA-3003 treatment suggests that a lower dose of rhGH delivered in a sustained-release formulation may have therapeutic efcacy comparable to that achieved with serial daily injections of rhGH. A similar phenomenon has been documented in monthly LHRH agonists for the treatment of prostate cancer [27]. In conclusion, an rhGH-containing PLGA microsphere formulation, DA-3003, was successfully produced using a double emulsion method. In animal studies, DA-3003 treatment prolonged the increase in plasma rhGH concentration, continuously induced IGF-I and IGFBP-3, promoted body weight gain, and increased the width of the tibial growth plate. The results of these preclinical studies demonstrate the excellent pharmacokinetic prole, pharmacodynamic responses and efcacy of DA-3003. In summary, DA-3003 has the potential to be clinically effective when administered only once either weekly or biweekly, which promises to signicantly improved patient compliance. Acknowledgments This work was supported by the Ministry of Commerce, Industry and Energy, Republic of Korea. (Project #: 10007723) and by a grant from the Korean Ministry of Science and Technology through the National Research Laboratory Program (ROA-2006-000-10290-0). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.jconrel.2009.03.014. References
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