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Jun 2, 2012 - BioTherapeutics R&D, Pfizer Inc., Chesterfield, Missouri 63017, United States. ABSTRACT: An effective and safe formulation of sustained-...
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A Novel Sustained-Release Formulation of Recombinant Human Growth Hormone and Its Pharmacokinetic, Pharmacodynamic and Safety Profiles Yi Wei,†,‡ Yuxia Wang,*,† Aijun Kang,§ Wei Wang,∥ Sa V. Ho,∥ Junfeng Gao,§ Guanghui Ma,*,† and Zhiguo Su† †

National Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China ‡ Graduate School of the Chinese Academy of Sciences, Beijing, 100190, People’s Republic of China § Department of Laboratory Animal Science, Peking University Health Science Center, Beijing, 100191, People’s Republic of China ∥ BioTherapeutics R&D, Pfizer Inc., Chesterfield, Missouri 63017, United States ABSTRACT: An effective and safe formulation of sustainedrelease rhGH for two months using poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEGPLA, PELA) microspheres was developed to reduce the frequency of medication. The rhGH-loaded PELA microspheres with a narrow size distribution were successfully prepared by a double emulsion method combined with a premix membrane emulsification technique without any exogenous stabilizing excipients. The narrow size distribution of the microspheres would guarantee repeatable productivity and release behavior. Moreover, the amphiphilic PELA improved the bioactivity retention of protein drugs since it prevented protein contact with the oil/water interface and the hydrophobic network, and modulated diffusion of acidic degradation products from the carrier system. These PELA microspheres were compared in vivo with commercial rhGH solution, conventional poly(D,L-lactic acid) (PLA) and poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres. Administration of rhGH-PELA could extend the duration of rhGH release (for up to 56 days) and increase area under the curve (AUC) compared to rhGH solution, PLA or PLGA microspheres in Sprague−Dawley (SD) rats. In addition, rhGH-PELA microspheres induced a greater response in total insulin-like growth factor-1 (IGF-1) and insulin-like growth factor binding protein-3 (IGFBP-3) than other rhGH formulations. With a hypophysectomized SD rat model, the pharmacological efficacy of rhGH-PELA microspheres was shown to be better than that from daily administration of rhGH solutions over 6 days based on body weight gain and width of the tibial growth plate. Histological examination of the injection sites indicated a significantly milder inflammatory response than that observed after injection of PLA and PLGA microspheres. Neither anti-rhGH antibodies nor the toxic effects on heart, liver and kidney were detectable after administration of rhGH-PELA microspheres in SD rats. These results suggest that rhGHPELA microspheres have the potential to be clinically effective and safe when administered only once every two months, a dose regimen for better patient acceptance and compliance. KEYWORDS: PELA microspheres, narrow size distribution, growth hormone, controlled release, high bioactivity, safety

1. INTRODUCTION Human growth hormone (hGH) is a protein of 191 amino acids with a molecular weight of approximately 22 kDa. It has a unique role in promoting longitudinal bone growth, and in the regulation of protein, lipid, and carbohydrate metabolism.1−3 Since its first introduction in 1985, recombinant human growth hormone (rhGH) has been used for treatment of pediatric short stature caused by growth hormone deficiency, Turner’s syndrome, and chronic renal failure.4−7 Due to its short halflife, the current rhGH product requires frequent subcutaneous injection daily or three times a week, which leads to poor patient compliance.8 Thus, development of a sustained-release © 2012 American Chemical Society

rhGH formulation could ameliorate patient quality of life and alleviate the burden of expense attendant on frequent injections. A variety of release strategies for encapsulation, or modifications to rhGH such as polymeric microspheres,9−14 biodegradable implants,15 injectable hydrogels,16−20 and PEGylation,21,22 have been proposed and researched extensively. Among them, entrapment of proteins in biodegradable Received: Revised: Accepted: Published: 2039

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proper pore size,36 because there exists a linear relationship between the microcapsules' size and the pore size of the membrane. Herein, we used a double emulsion combining with premix membrane emulsification technique to prepare PELA microspheres with narrow size distribution, which can realize process reproducibility and repeatable release behavior.36−40 To support potential clinical use of the rhGH-PELA microspheres, preclinical studies need to be conducted to demonstrate efficacy and safety of rhGH-PELA microspheres. In this study, the efficacy and safety of sustained-release rhGH-PELA uniform microspheres were evaluated in vivo in comparison with rhGH-loaded PLA and PLGA microspheres. rhGH serum concentrations were assessed after a single subcutaneous injection with rhGH-loaded microspheres and daily rhGH solution. The serum levels of two pharmacodynamic biomarkers for rhGH, IGF-1 and IGFBP-3, were also monitored. Body weight gain and tibial growth in the hypophysectomized (Hpx) rat model were used as indicators for rhGH pharmacological activity. Finally, plasma concentrations of antibodies against rhGH in rats and histological examination of the injection sites were monitored, along with potential toxic effects of rhGH-PELA microspheres on cardiac, renal, and hepatic functions of the experimental animals to probe the safety and tolerability of rhGH-PELA microspheres.

poly(lactide-co-glycolide) (PLGA) microspheres has been widely investigated as a technique to produce sustained release formulations for rhGH administration. The best known rhGH sustained delivery system, Nutropin Depot, consisting of polylactide-co-glycolide acid (PLGA), was approved by US FDA in 1999 as a monthly product. However, it was withdrawn from the market in 2004 due to several drawbacks including a high burst release, protein denaturation after administration, and adverse reactions, such as inflammation.23 Protein denaturation is caused by the harsh acidic microenvironment associated with the degradation of PLGA when no stabilizers are added.24,25 High burst release can lead to a significant loss of active drug in a short period of time, promote concentrationdependent toxicities, and reduce the duration of efficacy of the formulation. More importantly, protein aggregation can also take place in the unfavorable environment, and the release of protein aggregates may potentially result in an enhanced immunogenic response. Currently, there are no commercial rhGH sustained delivery systems that last for longer than a month. It is still a challenge to develop a suitable rhGH controlled release system with preservation of rhGH integrity both in vitro and in vivo because rhGH is not stable when exposed to an organic solvent/water interface during preparation of the system and matrix polymer affects on rhGH stability.26−28 It is generally known that amphiphilic polymers bearing both hydrophilic and hydrophobic structures in their molecules behave like surfactants.29 Compared to the widely utilized PLGA, protein delivery systems based on hydrophilic− hydrophobic block copolymers may have clear advantages.30 Incorporation of hydrophilic blocks in a hydrophobic polymer can be utilized to modify the degradation rate as well as the permeability of the matrix, leading to copolymer compositiondependent release kinetics.31 The improved diffusion of acidic degradation products within the microsphere system would minimize formation of an extreme, e.g., acidic, environment and minimize degradation of encapsulated protein during encapsulation and release.32−34 In addition, the amphiphilic property can minimize protein contact with the oil/water interface and the hydrophobic network, improving the stability of protein drugs.32 In our previous study, we successfully prepared amphiphilic poly(monomethoxypolyethylene glycol-co-D,L-lactide) (mPEG-PLA, PELA) microspheres with a narrow size distribution for sustained release of rhGH without any additional stabilizing excipients.35 The in vitro experiments confirmed several advantages of PELA microspheres, higher encapsulation efficiency, lower initial burst, more releasable amount, and better stability of rhGH than PLA and PLGA microspheres. We found that fewer aggregates were formed and less hydrolysis of rhGH took place in PELA microspheres due to the stable microenvironment. This is because the hydration of the hydrophilic mPEG sequences in the releasing medium promoted the swelling structure of the microspheres and then improved diffusion of water-soluble acids generated by the polyester hydrolysis, which minimized the acidic microenvironment. Furthermore, conventional processes for preparing microspheres usually involved mechanical stirring, homogenization, or spray techniques. In these cases, the size distributions of microspheres were very broad and the particle sizes were difficult to control, which decreased the reproducibility and compromised the experiment results. By using the membrane emulsification technique, the size of microcapsules can be controlled accurately by the choice of a membrane with

2. MATERIALS AND METHODS 2.1. Materials. Recombinant human growth hormone (rhGH) was kindly supplied by Pfizer (USA). PLA and PLGA (lactide:glycolide molar ratio of 50:50) with a molecular weight Mw ∼20000 Da were purchased from the Institute of Medical instrument (Shandong, China). PELA with a molecular weight Mw ∼20000 Da was supplied by the Dai Gang Company (Shandong, China), in which the mPEG block has a molecular weight of 2000 Da. Poly(vinyl alcohol) (PVA-217, degree of polymerization 1700, degree of hydrolysis 88.5%) was provided by Kuraray (Japan). Shirasu porous glass (SPG) membrane (pore size of the membrane was 5.2 μm) was kindly provided by SPG Technology Co. Ltd. (Japan). Fast membrane emulsification equipment (FMEM-500M) was provided by National Engineering Research Center for Biotechnology (Beijing). ELISA kits were purchased from Cusabio Biotech Co., Ltd. (USA). All other reagents were of analytical grade. 2.2. Preparation of Microspheres. Microspheres loaded with rhGH were prepared by a two-step procedure.38 Briefly, the coarse double emulsions were prepared at first; 0.4 mL of rhGH aqueous solution (32 mg/mL) was mixed with 4 mL of ethyl acetate containing PELA (200 mg) by homogenization (IKA T18) at 10000 rpm for 30 s in an ice bath to form a primary emulsion. The W/O emulsion was further emulsified into external aqueous phase containing 1% w/v PVA and 0.9% w/v NaCl by magnetic stirring for 60 s at 300 rpm to prepare coarse double emulsions. The coarse double emulsions were further refined by extruding the emulsion through the SPG membrane under a high pressure of 110 kPa. The obtained uniform double emulsions were poured quickly into 800 mL of solution containing 0.9% w/v NaCl (solidification solution) under magnetic stirring at 500 rpm for 5 min to solidify the microspheres. The obtained microspheres were collected by centrifugation (Sigma 3K30, German, rotor 19776H) at 2600g for 10 min and washed with distilled water three times. The resulting microspheres were filtered (Durapore Membrane Filter, 0.45 μm, Fisher Scientific, Pittsburgh, PA), washed three times with double distilled water, and vacuum freeze-dried 2040

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the control and daily rhGH groups, blood samples were collected before injection, and after injection at 0.25, 0.5, 1, 2, 4, 8, and 24 h and 3, 6, 9, 13, 16, 20, 23, 28, 37, 48, 56, and 69 days. For the rhGH loaded microspheres groups, blood samples were collected before injection, and after injection at 4 and 8 h and 1, 3, 6, 9, 13, 16, 20, 23, 28, 37, 48, 56, and 69 days. Plasma was separated via centrifugation (Sigma 3K30, German, rotor 12158H) at 8800 g for 5 min, then stored at −70 to −80 °C in the ultralow temperature freezer (SANYO, Japan) until assay. Two biomarkers, IGF-1 and IGFBP-3, are linked to the normal function of growth hormone in vivo. Therefore, their plasma levels were also monitored. The concentration of rhGH, IGF-1, and IGFBP-3 in rat plasma was analyzed by commercial enzyme-linked immunosorbent assay kits: hGH, IGF-1, and IGFBP-3 ELISA kits (Cusabio Biotech Co., Ltd. USA), respectively. Pharmacokinetic and pharmacodynamic parameters such as maximum plasma concentration (Cmax) and the time to reach the maximum concentration (Tmax) were determined directly from the observed data. The area under the plasma concentration−time curve (AUC) from time zero to day 1 (AUC0−1d) for rhGH and the AUC for rhGH from time zero to the last day of sampling (AUC0−56d) were calculated using standard methods.41 The area under the curve (AUC) was determined by the linear trapezoidal rule. 2.4.2. Measurement of rhGH Pharmacological Activity. Increases in body weight and the width of the tibial growth plate in hypophysectomized (Hpx) rats have been utilized as indices of the pharmacological efficacy of rhGH.17,22,42 Herein, growth-promoting tests were performed using the developmental hypophysectomized male SD rat model to confirm the pharmacological activity of rhGH released from PELA microspheres. Male SD rats (4 weeks old, weighing approximately 60−80 g) were hypophysectomized and transferred to individual animal cages two weeks after surgery. The animals were kept in a room with constant humidity (50 ± 10% RH (relative humidity)) and temperature (24 ± 2 °C) with controlled lighting (12 h light followed by 12 h dark) and fed with a standard pellet diet and water. They were randomly assigned into five treatment groups (n = 6 per group): control group (no treatment), daily rhGH, rhGH-PELA, rhGH-PLA, and rhGH-PLGA microspheres. Each group consisted of rats with balanced equal mean initial body weight prior to treatments. On day 21 after surgery, the control group and daily rhGH injection group received subcutaneous injection of either saline or rhGH at a dose of 1.0 mg/mL/kg for 6 consecutive days. The rhGH loaded microspheres group received a single subcutaneous injection of a suspension of PELA, PLA, or PLGA microspheres at an rhGH dose of 6 mg/ kg. The microsphere suspensions were prepared by suspending the microspheres in an aqueous vehicle (0.9% NaCl, 0.1% Tween 20). The samples were placed in an incubator (Sukun, SKY-200B) at 4 °C and shaken at 200 rpm for 5 min before use. After administration, the weights of rats were monitored every other day. The width of the tibial growth plate in Hpx, another indicator of the pharmacological activity of rhGH,43 was measured as follows. Right hind limbs were extirpated and fixed in 10% neutral buffered formalin. The fixed tibias were decalcified in 30% formic acid solution and were subsequently split at the proximal end in the frontal plane. The tibias were embedded in paraffin, and 8 μm sagittal sections were cut. The bone sections were then stained with hematoxylin and eosin.

(Christ plus2-4, USA) for 2 days. rhGH was also loaded into PLA and PLGA microspheres using the same methods above. 2.3. Characterization of Microspheres. The surface morphology of microspheres was observed by a JSM-6700F (JEOL, Japan) scanning electron microscope (SEM). The microspheres were sputter-coated with gold in 20 mA for 120 s (JFC-1600, JEOL, Japan), then observed by SEM. The volumemean diameter of microspheres dispersed in distilled water was measured by laser diffraction using Mastersizer 2000 (Malvern, U.K.). The uniformity of microspheres was expressed as a span value. A smaller value of span means a more narrow size distribution of microspheres. The total encapsulation efficiency of rhGH in PELA, PLA, and PLGA microspheres was determined by dissolving 20 mg of the vacuum freeze-dried microspheres in 1 mL of 1 M NaOH. The loaded amount of rhGH was determined by micro bicinchoninic acid (microBCA) assay (Pierce, USA). The encapsulation efficiency (EE) was calculated by the following equation: m EE = × 100% m0 where m0 is total mass of rhGH added and m is the mass of rhGH loaded in the microspheres. The loading efficiency (LE) was calculated by the following equation: a LE = × 100% b where b is total mass of microspheres (including mass of polymer and rhGH loaded) and a is the mass of rhGH loaded in the microspheres. All analyses were carried out in triplicate (n = 3) and are presented as means ± SD. A one-way ANOVA (OriginPro, Version 8.0) was used to determine statistical significance, and the difference is considered significant when p < 0.05. 2.4. Animal Studies. 2.4.1. Pharmacokinetic and Pharmacodynamic Study of rhGH in SD Rats. The animal experiments were carried out in accordance with the guidelines set by the National Institutes of Health (NIH publication no. 85-23, revised 1985) and were approved by the Experimental Animal Ethics Committee in Beijing. Male Sprague−Dawley (SD) rats (6 weeks old) of narrow weight range (200−220 g) were from the Department of Laboratory Animal Science, Peking University Health Science Center (China). The animals were kept in a room with constant humidity (50 ± 10% RH (relative humidity)) and temperature (24 ± 2 °C) with controlled lighting (12 h light followed by 12 h dark) and fed with a standard pellet diet and water. Thirty male SD rats were randomly assigned into five treatment groups (n = 6 per group): control group (physiological saline), daily rhGH, rhGH-PELA, rhGH-PLA, and rhGH-PLGA microspheres. The control group and daily rhGH injection group received subcutaneous injection of either saline or rhGH at a dose of 1.0 mg/mL/kg for 6 consecutive days. The rhGH loaded microspheres group received a single subcutaneous injection of a suspension of PELA, PLA, or PLGA microspheres at an rhGH dose of 6 mg/kg. The microsphere suspensions were prepared by suspending the microspheres in an aqueous vehicle (0.9% NaCl, 0.1% Tween 20). The samples were placed in an incubator (Sukun, SKY-200B) at 4 °C and shaken at 200 rpm for 5 min before use. Blood samples (approximately 0.3 mL) were collected into microtainer tubes with K2EDTA via the orbit for 69 days. For 2041

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Figure 1. SEM images of rhGH-loaded microspheres prepared by different polymers: (a) PELA, (b) PLA, and (c) PLGA; (d) size distributions of three microspheres.

subcutaneous tissue sample was used as a negative control. Photomicrographs of the histological slices were taken and digitally stored using an Olympus microscope at a 40× magnification. 2.4.5. Detection of Toxic Effects of rhGH-PELA Microspheres. In order to evaluate potential toxic effects of rhGHPELA microspheres on cardiac, hepatic, and renal functions of experiment animals, biochemical analyses were performed on rat serum. Experimental animal models, animal grouping, and drug administration were as described in section 2.4.1. For all groups (control, daily rhGH, and rhGH loaded microspheres groups), blood samples were collected at 30 and 60 days after the injection. The plasma levels of the following relevant substances were determined: creatine kinase (CK) and lactate dehydrogenase (LDH) for cardiac function; glutamic-oxaloacetic transaminase (GOT), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total protein (TP), and albumin (Alb) for hepatic function; blood urea nitrogen (BUN), uric acid (UA), and creatinine (Cre) for renal function. All analyses were carried out using Analyzer Medical System (AUTO LAB, Italy). 2.4.6. Statistics Analysis. All analyses were carried out in sixplicate (n = 6) and were presented as means ± SD. A oneway ANOVA (OriginPro, Version 8.0) was used to determine statistical significance, and the difference is considered significant when p < 0.05.

The width of the tibial growth plate, which was defined as the distance from the undifferentiated layer to the hypertrophic layer, was measured using a microscope. An average of five measurements for each sample was adopted as the width of the tibial growth plate. Photomicrographs of the tibial growth plate were taken and digitally stored using an Olympus microscope at a 20× magnification. 2.4.3. Measurement of Plasma Anti-hGH Antibodies. The immunogenicity of rhGH-PELA, rhGH-PLA, and rhGH-PLGA microspheres was evaluated by measurement of anti-hGH antibodies. Experimental animal models, animal grouping, and drug administration were as described in section 2.4.1. AntihGH antibody formation was evaluated at 0, 3, 9, 16, 23, 37, 48, and 56 days after administration and analyzed via rat antihuman growth hormone antibody (IgG) ELISA kit (Cusabio Biotech Co., Ltd., USA). Briefly, the microtiter plate provided in this kit has been precoated with specific antigen. Plasma samples and control were then added to the appropriate microtiter plate wells, followed by addition of horseradish peroxidase (HRP)-conjugated anti-rat IgG to each well and incubation. Finally, a TMB (3,3′,5,5′-tetramethylbenzidine) substrate solution was added to each well. The enzyme− substrate reaction was terminated by the addition of a sulfuric acid solution, and the color change was measured spectrophotometrically at a wavelength of 450 nm. Plasma samples with OD values that were less than twice the average of the negative control at a dilution of 1:100 were scored as negative; all other OD values were scored as positive.44 2.4.4. Histological Examination. Histological study was used to evaluate the safety of PELA microspheres in vivo. Experimental animal models, animal grouping, and drug administration were as described in section 2.4.1. Subcutaneous tissues associated with the injected microspheres were removed from the injection site after two and eight weeks. The tissue samples from the various rats were fixed in 10% formalin, and sections were immersed in paraffin and cut using a microtome. The tissues were stained with hematoxylin and eosin. Untreated

3. RESULTS AND DISCUSSION 3.1. Characterization of Microspheres. The PELA microspheres encapsulating rhGH with narrow size distribution were successfully prepared by a double emulsion method followed by premix membrane emulsification. In our previous study, we found that the size of drug-loaded microspheres played an important role in the drug cumulative release. It was shown that the smaller the microcapsule size was, the faster the drug released from the microcapsules. This was largely due to the larger specific surface between the microcapsules and the 2042

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outer water phase.39 In the in vitro release experiment, PELA microspheres around 2 μm were confirmed to exhibit low initial and last release during the whole period.35 Herein, we used PELA microspheres with 2.0 μm for administration. For comparison, PLA and PLGA microspheres were also prepared using the same process. As shown in Figure 1, the three rhGHloaded microspheres with spherical structure and smooth surface were observed by SEM. PELA, PLA, and PLGA microspheres had mean diameters of 1.96, 2.36, and 2.22 μm, and span values of 0.610, 0.709, and 0.776 (Figure 1d), respectively. Because of the difference in polymer composition, the emulsions prepared by SPG membrane shrank differently to form microspheres during solidification, and there is a fine distinction among the particle sizes of different microspheres. Nevertheless, the closeness of size distributions for the three microsphere preparations around 2 μm clearly indicates good process reproducibility even for different types of polymers, and prognosticates repeatable release behavior. As shown in Table 1, the encapsulation efficiencies (EE) of PELA, PLA, and PLGA

Tmax of 0.5 h, and declined to baseline values within 8 h of administration. The plasma concentration of rhGH from PLA and PLGA microspheres began to decline to 5 ng/mL after 23 days. However, PELA microspheres can continuously release drug at a higher rate, leading to a plasma concentration of rhGH above 13 ng/mL for 28 days. After 40 days, PELA microspheres released rhGH at a similar rate with PLA or PLGA microspheres. After 56 days, no rhGH could be detected from all three microspheres groups (but there is a data point around 70 days), and pharmacokinetic parameters were estimated based on a period of 56 days. Table 2 lists the pharmacokinetic parameters of rats. The plasma concentrations of rhGH from all microsphere

Table 1. Characterization of rhGH-Loaded PELA, PLA, and PLGA Microspheres

Cmax (ng/mL) Tmax (h) AUC0−1d (ng·d /mL) AUC0−56d (ng·d /mL)

a

polymer

particle size (μm)

span value

LE,a %

EE,b %

PELA PLA PLGA

1.96 2.35 2.22

0.610 0.709 0.776

5.81 4.23 3.79

90.8 66.1 59.2

Table 2. Pharmacokinetic Parameters of rhGH Solution and Different Microspheres in SD Ratsa microspheres rhGH solution 773 ± 35 0.5 65.2 ± 5.5

PELA

PLA

PLGA

109 ± 9*

127 ± 9*

127 ± 7*

57.4 ± 3.6

83.2 ± 8.8 *,† 588 ± 19†

83.8 ± 4.6 *,† 474 ± 19†

834 ± 22

a Cmax, maximum concentration; Tmax, time of maximal drug concentration; AUC, area under drug concentration vs time curve; d, day. Values are expressed as the group mean ± SD. *p < 0.05, vs rhGH solution. †p < 0.05, vs PELA.

LE: loading efficiency. bEE: encapsulation efficiency.

microspheres were 90.8%, 66.1%, and 59.2%, respectively, with corresponding loading efficiencies 5.81%, 4.23%, and 3.79%. The higher encapsulation efficiency of PELA could reasonably be attributed to the stable interfacial layer at the oil and water interface which prevented the protein in the inner droplets from merging into the external water phase, minimizing the loss of encapsulated protein during the fabrication process.35 3.2. Pharmacokinetic Study. Different types of microspheres were evaluated by measuring the rhGH plasma concentrations for several weeks after a single subcutaneous injection at an rhGH dose of 6 mg/mL/kg in comparison with daily subcutaneous injection of rhGH solution in SD rats. As shown in Figure 2, the plasma concentration of rhGH from daily injection group rapidly reached Cmax of 773 ng/mL with a

preparations at the first sampling point (i.e., 0.5 h) were significantly lower than Cmax observed for rhGH solution, despite administration of a significantly greater dose of rhGH with microsphere preparations. Among the three microsphere preparations, PELA showed the lowest Cmax. Moreover, the initial burst of rhGH from microspheres was reflected by the AUC0−1d. The AUC0−1d of rhGH from PELA, PLA, and PLGA microspheres was 57.4, 83.2, and 83.8 ng·d/mL, respectively. The AUC0−1d of rhGH from PLA and PLGA microspheres were approximately 44.9% and 46.0% higher than the values observed for PELA microspheres. In contrast, the AUC0−56d of rhGH from PELA microspheres was approximately 41.8% and 75.9% greater than the values observed for PLA and PLGA microspheres, respectively. These results indicated that PELA microspheres controlled the burst release of rhGH and improved its release in vivo better than PLA and PLGA microspheres. It was reported that the incomplete release was frequently encountered for many proteins entrapped in PLA or PLGA microspheres mainly due to nonspecific protein adsorption occurring within the microspheres.45 We interpret the greater amount of releasable rhGH from PELA microspheres as a result of the added mPEG sequences preventing the protein from contacting with the hydrophobic PLA regions. Moreover, the addition of hydrophilic sequences into the hydrophobic PLA can enhance water penetrability and consequently change the degradation rate of the polymer. Thus, the PELA microsphere preparation is a more favorable system than PLA and PLGA microspheres for rhGH delivery. 3.3. Pharmacodynamic Study. As mentioned before, the plasma levels of IGF-1 and IGFBP-3 are associated with the function of rhGH released from microspheres, and thus monitored. The initial values of IGF-1 and IGFBP-3 concentrations in plasma were determined before adminis-

Figure 2. Average plasma rhGH concentrations in rats treated with rhGH solution and different rhGH-loaded microspheres for 69 days. Data are means ± SD for six rats per group. 2043

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tration. As shown in Figure 3, the IGF-1 concentration in the PELA microspheres group was higher than the initial value

Figure 4. The average plasma IGFBP-3 concentrations in rats treated with rhGH solution and different rhGH-loaded microspheres. Data are means ± SD for six rats per group. Figure 3. The average plasma IGF-1 concentrations in rats treated with rhGH solution and different rhGH-loaded microspheres. Data are means ± SD for six rats per group.

can maintain the bioactivity of encapsulated rhGH better than the rest of the preparations. This was because mPEG-PLA block copolymer possessed a function similar to surfactants which had an intrinsic characteristic of oriented localization on the biphasic interface. The function can effectively minimize the contact of protein with oil/water interfaces and decreases the protein aggregates. Furthermore, no molecular hydrolysis and covalent aggregation were detected during the preparation and release process.35 3.4. Pharmacological Efficacy Study. Biological efficacy of rhGH released from PELA microspheres was examined in a hypophysectomized rat model by measuring weight gain and compared with negative control, daily injection of rhGH solution, and PLA and PLGA microspheres groups. rhGH solution was administered daily for 6 days, while PELA, PLA, and PLGA microspheres were administered once (on day 0). As shown in Figure 5b, the cumulative weight gains of hypophysectomized rats treated by negative control, daily injection of rhGH solution, and PELA, PLA, and PLGA microspheres were 41.4, 80.8, 124.7, 88.8, and 92.1 g, respectively. From day 0 to day 7, body weights significantly increased in all treatment groups compared with no treatment (negative controls received 0.9% NaCl solution daily for 6 days). A key observation was that the weight gain associated with PELA microspheres was steady and comparable to that seen with daily rhGH treatment for the first seven days (Figure 5a). The daily rhGH treatment group showed weight gain most significantly for a total of 14 days during and after drug administration. In contrast, the group treated with PELA microspheres showed a steady weight gain during the entire monitoring period of 56 days, whereas the groups treated with

(359 ng/mL) at all time points, with a maximum of 514 ng/mL at 23 days. The initial concentrations of IGF-1 were 406 ng/mL and 312 ng/mL for PLA and PLGA microspheres, respectively. After injection, the IGF-1 concentrations of the two types of microspheres were always lower than that of PELA microspheres, indicating a superiority of PELA microspheres in vivo. The AUC0−56d of IGF-1 for the daily injection group and the PELA, PLA, and PLGA microspheres groups were 19800 ng·d/ mL, 24200 ng·d/mL, 18900 ng·d/mL, and 19700 ng·d/mL, respectively (Table 3). The AUC0−56d of IGF-1 from PELA microspheres was approximately 21.9%, 27.7%, and 22.8% greater than those for the daily injection group and the PLA and PLGA microspheres groups, respectively. The results suggested that PELA microspheres released the greatest amount of bioactive rhGH. As shown in Figure 4, the initial IGFBP-3 concentrations of the daily injection group and the PELA, PLA, and PLGA microspheres groups were 880 ng/mL, 836 ng/mL, 890 ng/ mL, and 690 ng/mL, respectively. The PELA microspheres group maintained highest level among all groups during the release period. As shown in Table 3, the AUC0−56d of IGFBP-3 for the daily injection group and the PELA, PLA, and PLGA microspheres groups were 43900 ng·d/mL, 62700 ng·d/mL, 42800 ng·d/mL, and 43900 ng·d/mL, respectively. The PELA microspheres group showed the largest AUC0−56d, which was 42.8%, 46.5%, and 42.9% greater than those from the daily injection group and the PLA and PLGA microspheres groups, respectively. This strongly suggests that PELA microspheres

Table 3. Pharmacodynamic Parameters of rhGH Solution and Different rhGH-Loaded Microspheres in SD Ratsa microspheres rhGH solution

PELA

PLA

PLGA

366 ± 20 19800 ± 600

514 ± 23* 24200 ± 700*

400 ± 33 18900 ± 500†

375 ± 44 19700 ± 600†

1130 ± 170 43900 ± 1100

1440 ± 130 62700 ± 1500*

1120 ± 120 42800 ± 1200†

906 ± 104† 43900 ± 1100†

IGF-1 Cmax (ng/mL) AUC0−56d (ng·d/mL) IGFBP-3 Cmax (ng/mL) AUC0−56d (ng·d/mL) a

Cmax, maximum concentration; Tmax, time of maximal drug concentration; AUC, area under drug concentration vs time curve; d, day. Values are expressed as the group mean ± SD. *p < 0.05, vs rhGH solution. †p < 0.05, vs PELA. 2044

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than those of other groups including daily rhGH solution injection. There was no significant difference among the daily injection rhGH and PLA and PLGA microspheres groups. These results demonstrated that the rhGH released from PELA microspheres maintained its biological activity and the rhGH encapsulated in the PELA microspheres showed better effects than rhGH solution due to the sustained release. 3.5. Immunogenicity and Safety of rhGH-PELA Microspheres. The immunogenicity of protein-based drugs is an important safety issue that must be considered during development of these therapeutic agents. The immunogenicity rhGH solution and rhGH-PELA microspheres were evaluated by measurement of anti-hGH antibodies. Non-native proteins, such as aggregated rhGH, produced during manufacturing or release from the microparticles, may be immunogenic. The absorbance at 450 nm for the plasma samples from all groups (rhGH solution, rhGH-PELA, rhGH-PLA, and rhGH-PLGA microspheres) were below the cutoff value (i.e., twice the average of the negative control), indicating that antibody formation in response to administered rhGH preparations was negligible. Release of acidic monomeric units resulting from PLGA degradation may lead to an inflammatory response.16 In a previous study, we have employed pH-sensitive dye to monitor the microclimate pH within different microspheres, indicating that the rhGH encapsulated in the PELA microspheres was in a less acidic microenvironment.35 The local environment surrounding the injected microspheres in vivo still needed to be examined at 2 and 8 weeks to further investigate the safety of PELA microspheres. As shown in Figure 7, at two weeks, there was no inflammatory cell reaction observed in tissues of rats administered with PELA microspheres. However, a typical inflammatory response at initial stages (vasodilatation and plasma exudation) was clearly seen in the PLA and PLGA microspheres groups. After eight weeks, the inflammatory response to PELA microspheres was minimal while a severe inflammatory response to the PLA and PLGA microspheres (many lymphocytes and neutrophils appeared in fibrous tissue) could be observed. It is likely that amphiphilic PELA has a lower percentage of polyester for degradation into acidic products, minimizing pH drop inside the microspheres and tissue incompatibility. Thus, PELA microspheres would be superior to PLA and PLGA microspheres for injection with respect to minimization of an inflammatory response. To further demonstrate the safety of rhGH-PELA microspheres, potential toxic effects on heart, liver, and kidney were evaluated by analyzing the levels of several key substances in rat serum 4 weeks and 8 weeks after drug administration as described before (Tables 4 and 5). In comparison with the PLA

Figure 5. The weight gains in hypophysectomized rats treated with rhGH solution and different rhGH-loaded microspheres during the first seven days (a) and 56 days (b). Data are means ± SD for six rats per group.

the PLA or PLGA microspheres showed a significantly lower weight gain in the same period than that of PELA microspheres. In addition, compared to daily injection of the rhGH solution group, only the PELA microspheres group showed significant differences based on cumulative weight gains (p < 0.05). This result indicates that PELA microspheres are a more efficacious system for sustained delivery of rhGH than the other two types of microspheres. To confirm the biological activity of the released rhGH, we measured tibial growth plates at 30 days from the start of the administration. As shown in Figure 6, the widths of the tibial growth plate for rats treated by negative control, daily rhGH solution injection, and PELA, PLA, and PLGA microspheres were 261, 289, 361, 286, and 302 μm, respectively. Growth plates of rhGH-PELA treated group were significantly wider

Figure 6. Representative sections of the proximal tibial growth plate from rats of negative control (a), and after injection of rhGH solution (b), PELA microspheres (c), PLA microspheres (d), and PLGA microspheres (e) (20× magnification). White line and arrow mark the width of proximal tibial growth plate. 2045

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Figure 7. Rats were administrated with rhGH-loaded PELA, PLA, and PLGA microspheres, respectively. At 2 and 8 weeks, the rats were sacrificed and the injection site was dissected, processed, and stained used hematoxylin and eosin (H&E). Top panel is the negative control, which is normal tissue and can be seen in untreated tissue sections. Left, middle, and right panels show samples from rats treated with PLEA, PLA, and PLGA microspheres, respectively (40× magnification).

Table 4. The Relative Effects of Different rhGH-Loaded Microspheres on Cardiac, Renal, and Hepatic Functions in SD Rats Examined at 4 Weeksa microspheres negative control cardiac function CK (U/L) LDH (U/L) hepatic function GOT (U/L) ALT (U/L) ALP(U/L) TP (g/dL) Alb (g/L) renal function BUN (mmol/L) UA (μmol/L) Cre (mg/dL) a

rhGH soln

PELA

PLA

PLGA

795 ± 19 1771 ± 26

788 ± 18 1466 ± 19

807 ± 19 1369 ± 18

759 ± 11 1507 ± 17

573 ± 19* 1575 ± 15

164 ± 9 65 ± 4 283 ± 9 7.21 ± 0.11 40 ± 1

167 ± 7 75 ± 5 234 ± 7 7.54 ± 0.96 39 ± 2

183 ± 6 72 ± 4 200 ± 7 7.56 ± 0.58 39 ± 2

162 ± 7 99 ± 4* 446 ± 20* 8.37 ± 0.64 43 ± 2

119 ± 9 97 ± 7* 423 ± 19* 8.17 ± 0.31 43 ± 3

8.01 ± 0.25 152 ± 6 0.771 ± 0.029

9.68 ± 0.91 96 ± 1 0.806 ± 0.047

11.6 ± 0.53 159 ± 6 0.722 ± 0.035

11.3 ± 0.12 167 ± 5 0.727 ± 0.021

14.0 ± 0.32 159 ± 8 0.530 ± 0.018*

Values are expressed as the group means ± SD, n = 6. *p < 0.05, vs negative control.

and PLGA microspheres groups, the differences between the mean values of the rhGH solution group and PELA microspheres group were the least. No significant differences were observed between rats treated with PELA microspheres and the rhGH solution group in the mean values of these substances: CK and LDH (cardiac function); GOT, ALT, ALP, TP, and Alb (hepatic function); BUN, UA and Cre (renal function). This suggested that the side effects were the least for PELA microspheres among the three microsphere preparations. In addition, the results demonstrated that none of the rats treated with the PELA microspheres developed detectable toxic effects on heart, liver, and kidney during the whole monitoring period. It is probably because amphiphilic PELA has a lower percentage of polyester for degradation into acidic products,

minimizing pH drop around tissue and decreasing side effects to organs. The evaluation of toxic effects in combination with immunogenicity and histopathology evaluation indicated that rhGH-PELA microspheres were safer and better tolerated than rhGH-loaded PLA and PLGA microspheres.

4. CONCLUSION The combined in vivo results demonstrated that PELA microspheres with narrow size distribution have clear advantages over the daily injection formulation and other conventional PLA and PLGA microspheres. First of all, the PELA microspheres delivery system may reduce significantly the frequency of injection, improving patient acceptance and compliance. More importantly, any possible side effects related 2046

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Table 5. The Relative Effects of Different rhGH-Loaded Microspheres on Cardiac, Renal, and Hepatic Functions in SD Rats Examined at 8 Weeksa microspheres negative control cardiac function CK (U/L) LDH (U/L) hepatic function GOT (U/L) ALT (U/L) ALP(U/L) TP (g/dL) Alb (g/L) renal function BUN (mmol/L) UA (μmol/L) Cre (mg/dL) a

rhGH soln

PELA

PLA

PLGA

303 ± 17 1920 ± 40

303 ± 13 1940 ± 28

290 ± 12 1880 ± 27

328 ± 18 1980 ± 46

294 ± 24 1900 ± 39

169 ± 13 75.2 ± 13.2 240 ± 14 7.43 ± 0.52 36.7 ± 0.6

189 ± 14 75.5 ± 1.9 210 ± 8 6.75 ± 0.42 34.3 ± 3

179 ± 17 80.1 ± 13.5 210 ± 9 7.35 ± 0.50 36.1 ± 3.2

205 ± 6* 119.8 ± 6.5* 270 ± 16 7.53 ± 0.46 38.9 ± 1.7

172 ± 9 91.3 ± 3.8 290 ± 18* 7.84 ± 0.50 41.3 ± 2.4

10.3 ± 0.53 187 ± 7 0.847 ± 0.054

10.4 ± 0.71 191 ± 8 0.787 ± 0.029

11.0 ± 0.53 194 ± 8 0.889 ± 0.015

9.97 ± 0.62 162 ± 9 0.795 ± 0.032

9.00 ± 0.65 142 ± 8 0.837 ± 0.016

Values are expressed as the group means ± SD, n = 6. *p < 0.05, vs negative control. (2) McGauley, G. A. Quality of life assessment before and after growth-hormone treatment in adults with growth-hormone deficiency. Acta Paediatr. Scand. 1989, 70−74. (3) Schlechter, N. L.; Russell, S. M.; Spencer, E. M.; Nicoll, C. S. Evidence suggesting that the direct growth-promoting effect of growth-hormone on cartilage in vivo is mediated by local production of somatomedin. Proc. Natl. Acad. Sci. U.S.A. 1986, 83 (20), 7932− 7934. (4) Salomon, F.; Cuneo, R. C.; Hesp, R.; Sonksen, P. H. The effects of treatment with recombinant human growth-hormone on bodycomposition and metabolism in adults with growth-hormone defficiency. N. Engl. J. Med. 1989, 321 (26), 1797−1803. (5) Fine, R. N.; Kohaut, E.; Brown, D.; Kuntze, J.; Attie, K. M. Longterm treatment of growth retarded children with chronic renal insufficiency, with recombinant human growth hormone. Kidney Int. 1996, 49 (3), 781−785. (6) Massa, G.; Dezegher, F.; Vanderschuerenlodeweyckx, M. Effect of growth-hormone therapy on thyroid status of girls with turners syndrome. Clin. Endocrinol. 1991, 34 (3), 205−209. (7) Fine, R. N.; Pykegrimm, K.; Nelson, P. A.; Boechat, M. I.; Lippe, B. M.; Yadin, O.; Kamil, E. Recombinant human growth-hormone treatment of children with chronic-renal-failure-long-term (1 year to 3 year) outcome. Pediatr. Nephrol. 1991, 5 (4), 477−481. (8) Bond, W. S.; Hussar, D. A. Detection methods and strategies for improving medication compliance. Am. J. Hosp. Pharm. 1991, 48 (9), 1978−1988. (9) Cleland, J. L.; Duenas, E.; Daugherty, A.; Marian, M.; Yang, J.; Wilson, M.; Celniker, A. C.; Shahzamani, A.; Quarmby, V.; Chu, H.; Mukku, V.; Mac, A.; Roussakis, M.; Gillette, N.; Boyd, B.; Yeung, D.; Brooks, D.; Maa, Y. F.; Hsu, C.; Jones, A. J. S. Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect. J. Controlled Release 1997, 49 (2−3), 193−205. (10) Capan, Y.; Jiang, G.; Giovagnoli, S.; Na, K. H.; DeLuca, P. P. Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone. AAPS PharmSciTech 2003, 4 (2), 509−513. (11) Takada, S.; Yamagata, Y.; Misaki, M.; Taira, K.; Kurokawa, T. Sustained release of human growth hormone from microcapsules prepared by a solvent evaporation technique. J. Controlled Release 2003, 88 (2), 229−242. (12) Kwak, H. H.; Shim, W. S.; Choi, M. K.; Son, M. K.; Kim, Y. J.; Yang, H. C.; Kim, T. H.; Lee, G. I.; Kim, B. M.; Kang, S. H.; Shim, C. K. Development of a sustained-release recombinant human growth hormone formulation. J. Controlled Release 2009, 137 (2), 160−165. (13) Rafi, M.; Singh, S. M.; Kanchan, V.; Anish, C. K.; Panda, A. K. Controlled release of bioactive recombinant human growth hormone

to the high plasma rhGH concentration from daily injection of rhGH solution would be minimized with the controlled release system. A single administration of rhGH-PELA microspheres showed greater efficacy than that of six daily administration of rhGH solution in hypophysectomized rats. In comparison with PLA and PLGA microspheres, PELA microspheres not only showed the least burst release but also released the highest amount of rhGH in two months. Because of this and possibly better protection of rhGH activity, PELA microspheres appear to be more efficacious in terms of weight gain, and elevation in IGF-1 and IGFBP-3 levels. Furthermore, rhGH PELA microspheres did not appear to be immunogenic with no or minimal inflammatory response or toxic effects in the body. In addition, the narrow size distribution of microspheres would guarantee the repeatable productivity and pharmacological efficacy. In summary, all these results strongly suggested that PELA microspheres, prepared by a combination of traditional and membrane emulsification methods, held great potential as a clinically effective and safe system for sustained delivery of rhGH.

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AUTHOR INFORMATION

Corresponding Author

*No. 1 Bei Er Tiao, Zhongguancun, Haidian District, Beijing 100190, China. Tel/fax: +86 10 82627072. E-mail: yxwang@ home.ipe.ac.cn (Y.W.); [email protected] (G.M.). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Pfizer Inc. for kindly providing the material and are thankful for the financial support of 973 project (2009CB930300) and National Natural Science Foundation of China (20820102036, 51173187).

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REFERENCES

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