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PLGA microspheres for improving stability and efficacy of rhGH
Accepted Manuscript Title: Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH Author: Chenhui Wang Changhui Yu Jiaxin Liu Lesheng Teng Fengying Sun Youxin Li PII: DOI: Reference:
S0378-5173(15)30270-2 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.003 IJP 15258
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
10-7-2015 14-9-2015 3-10-2015
Please cite this article as: Wang, Chenhui, Yu, Changhui, Liu, Jiaxin, Teng, Lesheng, Sun, Fengying, Li, Youxin, Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH Chenhui Wang, Changhui Yu, Jiaxin Liu, Lesheng Teng, Fengying Sun* [email protected], Youxin Li* [email protected] School of Life Sciences, Jilin University, Changchun 130012, China *Corresponding author: Tel.: +86-431-85155320, Fax: +86-431-85155320.
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Graphical Abstract
2
Abstract The goal of this research is to prepare poly(cyclohexane-1,4 diyl acetone dimethylene ketal) (PCADK) / poly(D,L-lactide-co-glycolide) (PLGA) blend microspheres loaded with recombinant human growth hormone (rhGH). The effect of PCADK degradation products on the structural integrity, secondary and tertiary structure and pharmacodynamics of rhGH was evaluated by Native-polyacrylamide gel electrophoresis
(Native-PAGE),
size-exclusion
high
performance
liquid
chromatography (SEC-HPLC), circular dichroism (CD), fluorescence spectroscopy and in hypophysectomized rat models. Compared with PLGA degradation products, rhGH was found to be more stable in the presence of PCADK degradation products. PCADK/PLGA blend microspheres were then prepared and the morphology, encapsulation efficiency, release behavior and rhGH stability were investigated. PCADK/PLGA microspheres had regular shapes and smooth surfaces when the proportion of PCADK was less than 50%. The late-releasable amount of rhGH in PCADK/PLGA microspheres was greater than that in PLGA microspheres. In addition, the PCADK/PLGA microspheres showed larger AUC and improved therapeutic effects on rats than PLGA microspheres. Furthermore, the pH inside the microspheres was detected by CLSM to explain the improved rhGH stability in the PCADK/PLGA microspheres. In conclusion, PCADK/PLGA blend microspheres showed potential to improve rhGH stability and the efficacy of sustained-release of rhGH compared with PLGA microspheres. 3
Keywords: Recombinant Human Growth Hormone (rhGH); Microspheres; PCADK; Compatibility; Efficacy; pH.
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1. Introduction The recombinant human growth hormone (rhGH) is a 22-kDa endocrine hormone that is 191 amino acids in length and is synthesized and stored in the anterior pituitary gland. RhGH plays an important role in stimulating growth and cell reproduction and is clinically used to treat short stature caused by growth hormone deficiency (GHD), Turner’s
syndrome,
Prader–Willi
syndrome
and
chronic
renal
insufficiency(Cazares-Delgadill et al., 2011; Kim et al., 2005). Unfortunately, the short half-life of rhGH causes those using this clinical therapy to suffer the burden of daily injection, which results in poor patient compliance and renal toxicity (Ma, 2014). Thus, reducing injection frequency by constructing an rhGH sustained-release formulation is a universally desired goal. The most common method applied to overcome this problem is the development of an rhGH controlled-release system that could prolong the time of drug action and improve the bioavailability. Using the biodegradable polymer PLGA to encapsulate rhGH for controlled release has received tremendous interest in the last 20 years due to the approval of this molecule for use in drug formulations by the Food and Drug Administration (FDA) and the excellent properties of this molecule (Yang et al., 1997). However, the development of rhGH-loaded PLGA microspheres is accompanied by many problems, such as unstable scale-up manufacturing, high initial burst release, delay of release after the initial burst and the acidic microenvironment inside the microspheres (Yuen and Amin, 2011). The acidic intra-microsphere environment is caused by the accumulation of 5
lactic acid (LA) and glycolic acid (GA), which are generated by PLGA degradation and lead to a conformational change of rhGH, incomplete release of the hormone, chemical degradation, and loss of activity (Ding and Schwendeman, 2008; Zhu et al., 2000). It has been reported that the pH in buffer dropped to as low as 3 when the microsphere buffer was not exchanged and an environment with a minimum pH of 1.5 was formed within the aqueous pores of the PLGA microspheres (Fu et al., 2000). One way to overcome the problem is to add basic components such as magnesium hydroxide and calcium hydroxide into the microspheres. These formulations would be capable of neutralizing the acid and improving the stability of the encapsulated protein drug (Kang and Schwendeman, 2002; Zhu and Schwendeman, 2000). However, the coexistence of basic additives and the drug in the microspheres will result in a partially alkaline environment, which also influences the stability and release rate of the drug. Therefore, it is desirable to develop a new microsphere system that reduces the acidity of the intra-sphere environment without requiring the addition of potentially harmful substances. Unlike PLGA, PCADK degradation products do not create an acidic environment, which should provide the benefit of avoiding damage to the protein drug. PCADK is a hydrophobic polymer with ketal linkages in its backbone. PCADK has excellent biocompatibility
and
biodegradability
properties.
PCADK
degrades
into
1,4-cyclohexanedimethanol (1,4-CHDM) and acetone, both of which are non-toxic, neutral compounds. 1, 4-CHDM is widely used in food packaging and acetone is a 6
compound on the FDA Generally Recognized as Safe (GRAS) list (Lee et al., 2007). Currently, PCADK is mainly used for the treatment of inflammatory reactions and targeted therapy because of its acid sensitivity (Seshadri et al., 2010); however, it cannot be utilized in common subcutaneously injected microspheres because it does not substantially degrade at physiological pH. This property also limits the application of PCADK in drug delivery. Based on the results from a previous study, we are committed to developing a novel PCADK/PLGA blend microsphere system for use in subcutaneous injections to reduce the acidic environment and enhance the rhGH efficacy within microspheres. In this study, the effects of PCADK degradation products on rhGH were investigated. PCADK/PLGA-blend
microspheres
were
prepared
using
a
water/oil/water
emulsion-solvent evaporation method. The properties of the blend microspheres, including
their
morphology,
encapsulation
efficiency,
release
profiles,
pharmacokinetics and pharmacodynamics were systematically characterized. Finally, the pH of the microclimate (μpH) within the microspheres was investigated to explain the improvement of rhGH stability in PCADK/PLGA microspheres.
2. Materials and methods 2.1 Materials The polymers PLGA (5050 high, Mw 54336 Da) and PCADK (6463 Da), which were used for the preparation of the microspheres, were donated from Luye Pharmaceutical 7
Co. Ltd. (Shandong, China). LA (85-90%, AR), acetone (>99.5%, AR), disodium hydrogen phosphate dodecahydrate (>99%, AR), hydrochloric acid (36-68%, AR), and dichloromethane (>99.5%, AR) were purchased from Beijing Chemical Works (Beijing, China). GA (>98%) and 1,4-CHDM (>99%) were purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). Polyvinyl alcohol (PVA) (87–89% hydrolyzed, MW 13,000–23,000 Da) was purchased from Sigma-Aldrich (USA). The fluorescent pH-sensitive dye SNARF-1 dextran (MW 10 kDa) was purchased from Molecular Probes (USA). RhGH (MW 22 kDa) was kindly supplied by GeneScience Pharmaceuticals Co., Ltd. (Changchun, China). Protein molecular weight standards were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). All other chemicals were of analytical grade.
2.2 rhGH structural stability when incubated with PCADK or PLGA degradation products RhGH was incubated in PCADK degradation products to determine the structural stability in this environment. The 0.1 M of PCADK degradation product solution was prepared by dissolving acetone and 1,4-CHDM in deionized water and filtering (0.22 μm). Equally, the 0.1 M of PLGA degradation product solution was obtained by dissolving LA and GA in deionized water and filtering (0.22 μm). RhGH was dissolved in the solutions described above and the final concentration of all groups was 15 mg/ml. The same concentration of rhGH in PBS solution was prepared as a 8
control. After incubating at 37 °C for 7 days, all the samples were diluted 15 times with PBS to bring the pH of the different groups to a consistent value. The structural integrity of rhGH was analyzed by Native-PAGE and SEC-HPLC. To confirm the state of the secondary structure, rhGH samples were detected by CD and the spectra (190-260 nm) were collected on a Jasco J-810 instrument with a 0.1 cm quartz cell at 25 °C. Using the Jasco software, the background signal was subtracted from the spectrum of each protein solution, and the data were transformed to represent mean residue ellipticity. Fluorescence spectroscopy was used to monitor the changes in the tertiary structure. Fluorescence spectra were collected on a Shimadzu RF-5301 instrument. The emission spectrum (305–550 nm) of each sample was collected at an excitation wavelength of 280 nm. All analyses were carried out in triplicate and averaged (Determan et al., 2006).
2.3 rhGH pharmacological activity upon incubation with PCADK or PLGA degradation products RhGH solutions (1 mg/ml) were incubated in a series of PCADK and PLGA degradation products similar to the case described in Section 2.2. After incubating at 37 °C for 7 days, all the rhGH samples were diluted to 0.1 mg/ml with PBS to bring the pH of the different groups to a consistent value. The efficacy of rhGH incubated with PCADK degradation products was assessed using hypophysectomized (Hpx) rats (average weight 100 g). To confirm the Hpx rats model were successful, Hpx rats 9
were allowed to acclimate for one week, during which the changes in body weight were monitored. Hpx rats that demonstrated greater than 10% increase in body weight were considered to be incompletely hypophysectomized and were excluded from the study. Hpx rats were randomly divided into four treatment groups (n = 7 rats per group). Three experimental groups received a daily subcutaneous injection of rhGH incubated in PBS, rhGH incubated in PCADK degradation products or rhGH incubated in PLGA degradation products for 7 days. The control group received a subcutaneous injection of PBS. All injections were given in a volume of 0.25 ml. Increases in the body weight of Hpx rats have been utilized as indices of the pharmacological efficacy of rhGH.
2.4 Microspheres preparation Microspheres were prepared by the W/O/W double-emulsion technique. Two-hundred microliters of 50 mg/ml rhGH was emulsified in 2 ml of methylene chloride containing 500 mg of PLGA or a PCADK/PLGA blend (10/0, 2/8, 5/5, 8/2, 0/10) using a homogenizer at 5000 rpm for 90 s in an ice bath. The stabilized primary emulsion was immediately injected into 100 ml of an aqueous solution containing 1% (w/v) PVA and 4% (w/v) NaCl and homogenized at 5000 rpm for 120 s. The secondary emulsion was placed in magnetic stirring conditions for 3 h. The hardened microspheres were washed three times and then freeze-dried (Cleland et al., 1997).
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2.5 Characterization of PCADK/PLGA microspheres The encapsulation efficiency of rhGH in microspheres were determined by dissolving 10 mg of microspheres in 1 ml of 1 M NaOH for 1 day, after which 2 ml of 1 M HCl was added and the mixture was incubated at 37 ℃ for another day. Then, the solution was neutralized with NaOH. A micro-BCA assay was used to analyze the rhGH content in the clear solution. The surface morphology of microspheres was investigated by SEM (JXA-840, JEOL, Japan). The SEM samples were mounted on the surfaces of specimen stubs and coated with platinum under vacuum. The accelerating voltage used was 3 kV.
2.6 In vitro release of rhGH from microspheres Ten milligrams of the microspheres were suspended in 5 ml of 20 mM PBS (pH 7.4) containing 0.05% (w/v) sodium azide and placed in an incubator at 100 rpm and 37 °C. At predetermined time intervals, the buffer was withdrawn and fresh buffer solution was added to the microspheres. The amount of the released protein was determined using the Micro-BCA Protein Assay Kit (ComWin Biotech Co., China).
2.7 Confocal imaging of the microclimate pH inside microspheres The microclimate pH change inside the PCADK/PLGA microspheres was measured using a quantitative ratio metric method based on confocal laser scanning microscopy (CLSM). Microspheres containing SNARF-1 dextran (a fluorescent pH-sensitive dye) 11
were suspended in 2 ml PBS (pH 7.4) containing 0.01% (w/v) sodium azide and placed into an incubator at 100 rpm and 37 ℃. At predetermined time intervals, microspheres were collected and a small amount of microspheres was used to obtain CLSM images. The fluorescent dye encapsulated in the microspheres was excited at 488 nm by an Ar/He laser, and two images at different wavelengths (580 and 640 nm) were taken. The two images were overlapped to observe the microclimate pH change (Wei et al., 2011).
2.8 Pharmacokinetics of rhGH in SD rats The animal experiments were approved by the Experimental Animal Ethics Committee in the School of Life Sciences, Jilin University. The pharmacokinetics of rhGH PCADK/PLGA microspheres was assessed in SD rats (6 weeks old, average weight: 200 g). The PCADK/PLGA microspheres group and the PLGA microspheres group were subcutaneously injected with PCADK/PLGA microspheres and PLGA microspheres at 5 mg/kg, respectively. The microspheres were suspended in a vehicle consisting of 0.5% w/v carboxymethyl cellulose, 5.0% w/v mannitol and 0.1% v/v Tween 80 in an aqueous solution. The rhGH solution group received a single subcutaneous injection of fresh rhGH solution at the same dose. At the reservation time, blood samples were collected into heparinized tubes and centrifuged at 10,000 rpm for 10 min. Then, plasma samples were collected and the rhGH levels were determined by an rhGH ELISA kit (Jordan et al., 2010). 12
2.9 In vivo efficacy study For the in vivo efficacy study, 6-week-old SD rats (average weight: 200 g) were subcutaneously injected with 5 mg/kg PCADK/PLGA microspheres, 5 mg/kg PLGA microspheres, or saline. A 0.2 ml blood sample was collected from each rat using a heparinized syringe at reservation time after administration. IGF-1 levels were determined using an IGF-1 ELISA kit (R&D Systems, Inc. China) (Wei et al., 2012).
2.10 Measurement of rhGH pharmacological activity in hypophysectomized rats The rhGH pharmacological activity of PLGA microspheres and PCADK/PLGA microspheres was compared by subcutaneously injecting Hpx rats (average weight: 100 g) with each microspheres formulation or native rhGH. The efficacy of rhGH PCADK/PLGA microspheres was assessed by the increase in the body weight of Hpx rats. The Hpx rats were randomly assigned to four groups (5 rats per group) and were subcutaneously injected with PCADK/PLGA microspheres, PLGA microspheres, fresh rhGH solution or saline. Rats in the rhGH solution group received daily subcutaneous injections of fresh rhGH solution at a dose of 0.25 mg/kg/day for 14 days (for a total dose of 3.5 mg/kg). Rats in the microsphere formulations groups received a single subcutaneous injection of PCADK/PLGA microspheres or PLGA microspheres at 3.5 mg/kg. Rats in the negative control group received daily subcutaneous injections of saline. Weight gain in Hpx rats was determined every other day for 14 days(Kang, 2014; Kwak et al., 2009). 13
3. Results and discussion 3.1 rhGH stability upon incubation with PCADK or PLGA degradation products To study the biocompatibility between rhGH and PCADK, electrophoresis, SEC-HPLC, CD and fluorescence spectroscopy were used to detect the structural changes in rhGH following incubation in PCADK degradation products. Floccule appeared in the rhGH solution incubated in PLGA degradation products while rhGH solution incubated in PCADK degradation products was still clear (Fig.S1), indicating that the rhGH insoluble aggregations were produced by PLGA degradation products. To detect the component of the precipitation in PLGA-incubation group, the precipitation was washed three times and SDS or β-mercaptoethanol solution was added in the tube. The supernatant was detected by Native-PAGE and the result was shown in Fig.S2. The precipitation dissolved in SDS and the supernatant showed diffuse band below native rhGH band in Native-PAGE gel, indicating that the precipitation was produced by non-covalent bond. The supernatant was collected by centrifugation and used for further detection. Fig. 1 shows the SEC-HPLC chromatograms of rhGH after incubation. The rhGH monomer peak appeared at 18 min. The peaks characterized by shorter retention times (9 min) and longer retention times (21 min) were identified as aggregation and fragment products, respectively. The rhGH that had been incubated for 2 days in the degradation products of PCADK retained the main peak at 18 min, while the rhGH that had incubated for 2 days in the degradation products of PLGA generated aggregation and fragment peaks. Continued 14
incubation of rhGH in the degradation products of PCADK for 5 days maintained the major peak albeit with a “shoulder” after the monomer peak. Incubation of rhGH for 7 days in the PLGA degradation products resulted in a monomer peak that was very weak and a major fragment peak. The Native-PAGE results were shown in Fig. 2. The PCADK group retained the band of fresh rhGH, with very light degradation band below the rhGH monomer. However, heavy fragment band of rhGH was observed in the PLGA-incubation group. The result was consistent with the SEC-HPLC chromatogram which indicating that PCADK maintains the integrity of the rhGH basically while PLGA leads to rhGH fragment seriously. Fig. 3 shows the circular dichroism spectra. RhGH incubated in PCADK degradation products showed a similar spectrum to that of fresh rhGH, maintaining significant circular dichroic activity with two peaks between 210 and 230 nm. However, the CD signal intensity of PCADK group was a little smaller than that of fresh rhGH and the negative band at 208 nm showed bathochromic effect, indicating that the PCADK degradation products had slight effect on the rhGH secondary structure. It is worth noting that the CD signal of rhGH in PLGA degradation products was very weak and the characteristic 208 and 222 nm peaks disappeared completely, demonstrating that the PLGA degradation products resulted in destruction of the rhGH secondary structure (Kim and Tg., 1999). Fig. 4 shows the fluorescence spectrum of rhGH; while the peak positions of rhGH incubated in PCADK and PLGA degradation products were not changed, the peak intensities changed compared with that of fresh rhGH. The 15
fluorescence spectrum peak decreased slightly to 331 in the PCADK group, while the peak from the PLGA group decreased greatly to an intensity of 107. The decrease of the fluorescence intensity indicated the change of the rhGH tertiary structure. The degradation products of PCADK have little effect on the tertiary structure of rhGH, while the degradation products of PLGA exert a larger influence on rhGH. These results provide evidence that PCADK degradation products have better compatibility with rhGH than the PLGA degradation products.
3.2 rhGH pharmacological activity upon incubation with PCADK or PLGA degradation products To evaluate the effect of PCADK degradation products on rhGH pharmacodynamics, hypophysectomized rats were given daily injections of growth hormone and body weights of the rats were monitored (Fig. 5). After 7 days of drug injection, three treatment groups of hypophysectomized rats all showed gains in body weight. The average weight gain of the fresh-rhGH group was 32.1 g. The average weight gain for rats in the PCADK group was 12.1 g, indicating that PCADK degradation products affects a part of rhGH pharmacological activity. The PLGA-group rats showed only an average increase in body weight of 5.6 g, far lower than that of the fresh rhGH group. The results showed that PCADK degradation products cause smaller losses in the biological activity of rhGH than PLGA degradation products and confirm that PCADK presents better biocompatibility with rhGH than PLGA. PCADK is known to 16
degrade into 1, 4-CHDM and acetone, both of which are neutral products that likely act mildly on rhGH. PLGA, however, degrades into LA and GA, and the resulting acidic environment has an irreparable effect on rhGH activity.
3.3 PCADK/PLGA microspheres PCADK/PLGA microspheres with different PCADK proportions were prepared, as shown in Fig. 6. Microspheres were generally spherical with smooth surfaces when PCADK was present in a proportion lower than 50%. With further PCADK increases, however, the microspheres became irregularly shaped, showing rough surfaces and apparently more holes. The low molecular weight of PCADK leads to relatively weak mechanical properties, and when the proportion of PCADK is over 50%, the balling property of the polymer mixture worsens. The encapsulation efficiencies of the PCADK/PLGA microspheres that contained 0%, 20% and 50% PCADK were 65.8%, 62.1% and 60.6%, respectively. Once PCADK increased to 80%, the encapsulation efficiency significantly decreased to 40.3%; even worse, the encapsulation efficiency of the PCADK-only microsphere was only 22.7% (Table 1). The drug is more liable to escape from microspheres during solvent evaporation when there was a large amount of holes on the microspheres, so the large proportion of PCADK in microspheres caused very low encapsulation efficiency.
3.4 In vitro release of rhGH from microspheres 17
To detect the release behavior of PCADK/PLGA microspheres, microspheres with different proportions of PCADK were maintained at 37 °C in an incubator at 100 rpm (Fig. 7). After 14 days of release, microspheres with 0% and 20% PCADK reached total release amounts of 15% and 37%, respectively, while microspheres with 50%, 80% and 100% PCADK reached approximately 65% of release. The results indicated that the addition of PCADK improved the total release of rhGH to a certain degree. PLGA microspheres almost stopped releasing rhGH after the initial burst release, while PCADK/PLGA microspheres released rhGH at a later period (total release － burst release) to a different degree. The later release of microspheres increased from 16% to 43% when the proportion of PCADK was increased from 20% to 50%. However, microspheres with 80% and 100% PCADK only presented 26% and 16% later release due to the enormous burst release. There are two mechanisms by which the drug can be released from sustained-release microspheres: the diffusion mechanism and the degradation mechanism. The dissolution and diffusion of the drug is the main source of burst release. Water-soluble drugs prefer the external water phase and the permeation of the inner rhGH phase to the external water phase brings the drug to the microsphere surface. The release medium enters microspheres through the holes and forms a concentration gradient, which creates an osmotic pressure difference and causes the drug inside the microspheres to permeate outside. Therefore, when the proportion of PCADK increases to 80% and 100%, additional holes on the surfaces of microspheres lead to larger burst releases. In the later period of release, 18
water-soluble channels could accelerate drug release and the high concentration gradient in microspheres induces drug diffusion to the external environment. Microspheres with less than 50% of PCADK were shown to be morphologically strong at the early stages of release. PLGA degraded into LA and GA during the incubation period, which led to an acid microenvironment. PCADK degraded rapidly in the acidic microenvironment, leading to a loose structure in the PCADK/PLGA microspheres. The loose structure acted as similar to water-soluble channels by accelerating drug release. This result indicates that PCADK/PLGA microspheres have better release properties than PLGA microspheres.
3.5 Pharmacokinetics of rhGH in SD rats By investigating the morphology, encapsulation and in vitro release characteristics of PCADK/PLGA microspheres, we found that when the proportion of PCADK is 50%, microspheres maintained excellent morphological features and encapsulation efficiency and showed improved in vitro release properties. Therefore, in the pharmacokinetics study, we applied PCADK/PLGA microspheres with 50% of PCADK. We examined the in vivo release of rhGH from PLGA microspheres and PCADK/PLGA microspheres. Fig. 8 shows the plasma rhGH concentration-time release profiles for PCADK/PLGA and PLGA microspheres. The results showed that PCADK/PLGA and PLGA microspheres underwent rapid release of rhGH in the first day followed by release of rhGH for up to two weeks. The AUC (0-14 d) values were 19
calculated by the software DAS 3.0 (Drug and Statistics Software, Mathematical Pharmacology Professional Committee of China, Shanghai, China). AUC (0-14 d) values of PCADK/PLGA and PLGA microspheres were 219.76 and 106.02 µg/L*d, respectively. The AUC value of the PCADK/PLGA microspheres was increased 2-fold compared to that of the PLGA microspheres. These results suggest that the addition of PCADK effectively increases the bioavailability of rhGH.
3.6 Pharmacodynamic study IGF-1 is an endocrine hormone and the plasma concentration of IGF-1 is associated with rhGH function. Therefore, IGF-1 can be used as a biological marker of rhGH pharmacodynamics (Mathews et al., 1986). As shown in Fig. 9, PCADK/PLGA microspheres showed a more significant IGF-1 response than PLGA microspheres. Both PCADK/PLGA microspheres and PLGA microspheres achieved significant increases in IGF-1 levels after administration. However, the concentration of IGF-1 in the PLGA microspheres group decreased rapidly and afterward tended to match with the blank group. On the other hand, the concentration of IGF-1 in plasma remained elevated for 2 weeks after administration of PCADK/PLGA microspheres. The IGF-1 concentration associated with PCADK/PLGA microspheres was always higher than that of PLGA microspheres, indicating the superiority of PCADK/PLGA microspheres in vivo. AUC (0-14 d) values of PCADK/PLGA microspheres were 9038.86 µg/L*d, approximately 50% greater than that observed for PLGA 20
microspheres (6439.24 µg/L*d). The results suggested that the PCADK/PLGA microspheres released a greater amount of bioactive rhGH in rats.
3.7 Pharmacological efficacy study in Hpx rats To compare the in vivo efficacy of PLGA and PCADK/PLGA microspheres, Hpx rats were subcutaneously injected with microsphere formulations (3.5 mg/kg) or native rhGH (0.25 mg/kg/day for a total dose of 3.5 mg/kg over 14 days). As shown in Fig. 9, after the 14-day injection period Hpx rats that had been injected with normal saline showed no changes in body weight, while the three rhGH treatment groups showed apparent increases in body weight. Rats in the rhGH solution group maintained a well-proportioned increase in body weight of 55 g over the 2-week course of treatment. The PCADK/PLGA group showed a significant increase in body weight over the 2 weeks after administration. However, the PLGA group showed a weight increase only in the first 5 days after administration, after which point the weight of the rats remained constant. The results suggested that the PCADK/PLGA microspheres had better pharmacological efficacy than the PLGA microspheres due to the prolongation of release and improvement of rhGH efficacy. During the preparation of the microspheres with the W/O/W method, the oil-water contact caused the denaturation of rhGH, resulting in a loss in efficacy of both PCADK/PLGA and PLGA microspheres.
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3.8 Confocal imaging of the microclimate pH inside microspheres To detect the pH inside the PCADK/PLGA microspheres, we co-encapsulated rhGH and SNARF-1 dextran into microspheres and observed the color change during incubation by laser scanning confocal microscope. At the beginning of the incubation, both PCADK/PLGA and PLGA microspheres appeared yellow, indicating that the internal environment was neutral. After 2 days of incubation, both PCADK/PLGA and PLGA microspheres turned orange, indicating that the internal environment was becoming acidic due to the accumulation of PLGA degradation products. As the time went on, however, the orange color presented by PLGA microspheres became darker while that of the PCADK/PLGA microspheres became a light orange. The results showed that the acidity of the internal environment of PLGA microspheres became stronger due to the accumulation of PLGA degradation products. However, the internal environment of PCADK/PLGA microspheres was more neutral due to the addition of PCADK. At the initial stages of the incubation, the PLGA within the PCADK/PLGA microspheres degraded into LA and GA, leading to the decreased pH within the microspheres. The acidic environment promoted the degradation of PCADK due to the acid-sensitive property, leading to the formation of holes on the surface of PCADK/PLGA microspheres (Fig.S3), which provided channels for protein diffusion and release. Autocatalysis degradation mechanism of PCADK/PLGA microspheres guarantees
the
application
of
PCADK/PLGA 22
microspheres
in
common
subcutaneously injection. At the later stages of the incubation, the holes also provide channels for exhausting of PLGA degradation products and the diffusion of buffer into the microspheres, which neutralizes the acid internal microenvironment. Moreover,
PCADK
degradation
products
are
neutral
and
present
better
biocompatibility with rhGH. Therefore, the more neutral microenvironment of PCADK/PLGA microspheres is a significant reason for the higher activity of rhGH in PCADK/PLGA than in PLGA microspheres. 4. Conclusion In this study, PCADK degradation products are shown to possess better biocompatibility with rhGH than PLGA degradation products. PCADK/PLGA microspheres with different proportions of PCADK were prepared using W/O/W method and loaded with rhGH. The 5/5 PCADK/PLGA microspheres were finally chosen by investigating the drug loading capacity, microsphere morphologies and release behaviors. Compared with PLGA microspheres, PCADK/PLGA microspheres presented improved release behavior and larger AUC in rats. Moreover, we found that PCADK/PLGA microspheres had better efficacy than PLGA microspheres in hypophysectomized rats. While PCADK/PLGA microspheres are imperfect with regard to rhGH activity caused by the W/O/W method used during preparation, these microspheres have an apparent advantage over PLGA microspheres prepared using the same method. Thus PCADK/PLGA microspheres have great potential for use in the sustained release of rhGH. 23
Reference Cazares-Delgadill, J., Ganem-Rondero, A., Kalia, Y.N., 2011. Human growth hormone: New delivery systems, alternative routes of administration, and their pharmacological relevance. Eur. J. Pharm. Biopharm. 78, 278-288. 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., 1997. Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect. J. Control. Release 49, 193-205. Determan, A.S., Wilson, J.H., Kipper, M.J., Wannemuehler, M.J., Narasimhan, B., 2006. Protein stability in the presence of polymer degradation products: Consequences for controlled release formulations. Biomaterials 27, 3312-3320. Ding, A.G., Schwendeman, S.P., 2008. Acidic microclimate pH distribution in PLGA microspheres monitored by confocal laser scanning microscopy. Pharm. Res. 25, 2041-2052. Fu, K., Pack, D.W., Klibanov, A.M., Langer, R., 2000. Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm. Res. 17, 100-106. Jordan, F., Naylor, A., Kelly, C.A., Howdle, S.M., Lewis, A., Illum, L., 2010. Sustained release hGH microsphere formulation produced by a novel supercritical fluid technology: In vivo studies. J. Control. Release 141, 153-160. 24
Kang, J., 2014. Development of Recombinant Human Growth Hormone (rhGH) sustained-release microspheres by a low temperature aqueous phase/aqueous phase emulsion method. Eur. J. Pharm. Sci. 62, 141–147. Kang, J.C., Schwendeman, S.P., 2002. Comparison of the effects of Mg(OH)2 and sucrose on the stability of bovine serum albumin encapsulated in injectable poly(D,L-lactide-co-glycolide) implants. Biomaterials 23, 239-245. Kim, H.K., Tg., P., 1999. Microencapsulation of human growth hormone within biodegradable polyester microspheres: Protein aggregation stability and incomplete release mechanism. Biotechnol. Bioeng. 65, 659–667. Kim, S.J., Hahn, S.K., Kim, M.J., Kim, D.H., Lee, Y.P., 2005. Development of a novel sustained release formulation of recombinant human growth hormone using sodium hyaluronate microparticles. J. Control. Release 104, 323-335. 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., 2009. Development of a sustained-release recombinant human growth hormone formulation. J. Control. Release 137, 160-165. Lee, S., Yang, S.C., Heffernan, M.J., Taylor, W.R., Murthy, N., 2007. Polyketal microparticles: A new delivery vehicle for superoxide dismutase. Bioconjugate Chem. 18, 4-7. Ma, G., 2014. Microencapsulation of protein drugs for drug delivery: Strategy, preparation, and applications. J. Control. Release 193, 324-340. 25
Mathews, L.S., Norstedt, G., Palmiter, R.D., 1986. Regulation of insulin-like growth factor I gene expression by growth hormone. P. Natl. Acad. Sci. USA. 83, 9343-9347. Seshadri, G., Sy, J.C., Brown, M., Dikalov, S., Yang, S.C., Murthy, N., Davis, M.E., 2010. The delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury. Biomaterials 31, 1372-1379. Wei, Y., Wang, Y.-X., Wang, W., Ho, S.V., Wei, W., Ma, G.-H., 2011. mPEG-PLA microspheres with narrow size distribution increase the controlled release effect of recombinant human growth hormone. J. Mater. Chem. 21, 12691-12699. Wei, Y., Wang, Y., Kang, A., Wang, W., Ho, S.V., Gao, J., Ma, G., Su, Z., 2012. A Novel Sustained-Release Formulation of Recombinant Human Growth Hormone and Its Pharmacokinetic, Pharmacodynamic and Safety Profiles. Mol. Pharm. 9, 2039-2048. Yang, J., Wilson, M., Celniker, A.C., 1997. Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect. J. Control. Release 49, 193-205(113). Yuen, K.C.J., Amin, R., 2011. Developments in administration of growth hormone treatment: focus on Norditropin (R) Flexpro (R). Patient Prefer. Adher. 5, 117-124. Zhu, G.Z., Mallery, S.R., Schwendeman, S.P., 2000. Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide). Nat. Biotechnol. 18, 52-57. Zhu, G.Z., Schwendeman, S.P., 2000. Stabilization of proteins encapsulated in 26
Accepted Manuscript Title: Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH Author: Chenhui Wang Changhui Yu Jiaxin Liu Lesheng Teng Fengying Sun Youxin Li PII: DOI: Reference:
S0378-5173(15)30270-2 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.003 IJP 15258
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
10-7-2015 14-9-2015 3-10-2015
Please cite this article as: Wang, Chenhui, Yu, Changhui, Liu, Jiaxin, Teng, Lesheng, Sun, Fengying, Li, Youxin, Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Figure Captions
Fig. 1. SEC-HPLC chromatograms of rhGH incubated in the presence of PCADK and PLGA degradation products. a: pure rhGH, b: rhGH incubated in PCADK degradation products for 2 days, c: rhGH incubated in PLGA degradation products for 2 days, d: rhGH incubated in PCADK degradation products for 7 days, e: rhGH incubated in PLGA degradation products for 7 days.
28
Fig.2 Native-PAGE analysis of rhGH incubated in the presence of PCADK and PLGA degradation products for 7 days. Lane 1: fresh rhGH, lane 2: rhGH in PLGA degradation products, lanes 3: rhGH in PCADK degradation products.
29
Fig. 3. CD spectra of rhGH after incubation in the presence of PCADK and PLGA degradation products for 7 days.
30
Fig. 4. Fluorescence spectroscopy of rhGH incubated in the presence of PCADK and PLGA degradation products for 7 days.
31
Fig. 5. Pharmacological activity of rhGH incubated in the presence of PCADK or PLGA degradation products for 7 days.
32
Fig. 6. SEM micrographs of PCADK/PLGA microspheres with different PCADK proportions. a: 0% PCADK b: 20% PCADK c: 50% PCADK d: 80% PCADK e: 100% PCADK.
33
Fig. 7. In vitro release of rhGH from PCADK/PLGA microspheres.
34
Fig. 8. In vivo release of rhGH from PCADK/PLGA microspheres.
35
Fig. 9. The IGF-1 concentrations in rats treated with rhGH-loaded microspheres.
36
Fig. 10. The weight gains in hypophysectomized rats treated with rhGH-loaded microspheres.
37
Fig. 11 Confocal imaging of microclimate pH inside microspheres
38
Tables Table 1 Encapsulation efficiency of PCADK/PLGA microspheres with different ratios of PCADK
PCADK/PLGA
0/10
2/8
5/5
8/2
10/10
Encapsulation Efficiency (%)
65.8±2.4
62.1±1.9
60.6±2.6
40.3±3.1
22.7±1.7
39
S0378-5173(15)30270-2 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.003 IJP 15258
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
10-7-2015 14-9-2015 3-10-2015
Please cite this article as: Wang, Chenhui, Yu, Changhui, Liu, Jiaxin, Teng, Lesheng, Sun, Fengying, Li, Youxin, Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH Chenhui Wang, Changhui Yu, Jiaxin Liu, Lesheng Teng, Fengying Sun* [email protected], Youxin Li* [email protected] School of Life Sciences, Jilin University, Changchun 130012, China *Corresponding author: Tel.: +86-431-85155320, Fax: +86-431-85155320.
1
Graphical Abstract
2
Abstract The goal of this research is to prepare poly(cyclohexane-1,4 diyl acetone dimethylene ketal) (PCADK) / poly(D,L-lactide-co-glycolide) (PLGA) blend microspheres loaded with recombinant human growth hormone (rhGH). The effect of PCADK degradation products on the structural integrity, secondary and tertiary structure and pharmacodynamics of rhGH was evaluated by Native-polyacrylamide gel electrophoresis
(Native-PAGE),
size-exclusion
high
performance
liquid
chromatography (SEC-HPLC), circular dichroism (CD), fluorescence spectroscopy and in hypophysectomized rat models. Compared with PLGA degradation products, rhGH was found to be more stable in the presence of PCADK degradation products. PCADK/PLGA blend microspheres were then prepared and the morphology, encapsulation efficiency, release behavior and rhGH stability were investigated. PCADK/PLGA microspheres had regular shapes and smooth surfaces when the proportion of PCADK was less than 50%. The late-releasable amount of rhGH in PCADK/PLGA microspheres was greater than that in PLGA microspheres. In addition, the PCADK/PLGA microspheres showed larger AUC and improved therapeutic effects on rats than PLGA microspheres. Furthermore, the pH inside the microspheres was detected by CLSM to explain the improved rhGH stability in the PCADK/PLGA microspheres. In conclusion, PCADK/PLGA blend microspheres showed potential to improve rhGH stability and the efficacy of sustained-release of rhGH compared with PLGA microspheres. 3
Keywords: Recombinant Human Growth Hormone (rhGH); Microspheres; PCADK; Compatibility; Efficacy; pH.
4
1. Introduction The recombinant human growth hormone (rhGH) is a 22-kDa endocrine hormone that is 191 amino acids in length and is synthesized and stored in the anterior pituitary gland. RhGH plays an important role in stimulating growth and cell reproduction and is clinically used to treat short stature caused by growth hormone deficiency (GHD), Turner’s
syndrome,
Prader–Willi
syndrome
and
chronic
renal
insufficiency(Cazares-Delgadill et al., 2011; Kim et al., 2005). Unfortunately, the short half-life of rhGH causes those using this clinical therapy to suffer the burden of daily injection, which results in poor patient compliance and renal toxicity (Ma, 2014). Thus, reducing injection frequency by constructing an rhGH sustained-release formulation is a universally desired goal. The most common method applied to overcome this problem is the development of an rhGH controlled-release system that could prolong the time of drug action and improve the bioavailability. Using the biodegradable polymer PLGA to encapsulate rhGH for controlled release has received tremendous interest in the last 20 years due to the approval of this molecule for use in drug formulations by the Food and Drug Administration (FDA) and the excellent properties of this molecule (Yang et al., 1997). However, the development of rhGH-loaded PLGA microspheres is accompanied by many problems, such as unstable scale-up manufacturing, high initial burst release, delay of release after the initial burst and the acidic microenvironment inside the microspheres (Yuen and Amin, 2011). The acidic intra-microsphere environment is caused by the accumulation of 5
lactic acid (LA) and glycolic acid (GA), which are generated by PLGA degradation and lead to a conformational change of rhGH, incomplete release of the hormone, chemical degradation, and loss of activity (Ding and Schwendeman, 2008; Zhu et al., 2000). It has been reported that the pH in buffer dropped to as low as 3 when the microsphere buffer was not exchanged and an environment with a minimum pH of 1.5 was formed within the aqueous pores of the PLGA microspheres (Fu et al., 2000). One way to overcome the problem is to add basic components such as magnesium hydroxide and calcium hydroxide into the microspheres. These formulations would be capable of neutralizing the acid and improving the stability of the encapsulated protein drug (Kang and Schwendeman, 2002; Zhu and Schwendeman, 2000). However, the coexistence of basic additives and the drug in the microspheres will result in a partially alkaline environment, which also influences the stability and release rate of the drug. Therefore, it is desirable to develop a new microsphere system that reduces the acidity of the intra-sphere environment without requiring the addition of potentially harmful substances. Unlike PLGA, PCADK degradation products do not create an acidic environment, which should provide the benefit of avoiding damage to the protein drug. PCADK is a hydrophobic polymer with ketal linkages in its backbone. PCADK has excellent biocompatibility
and
biodegradability
properties.
PCADK
degrades
into
1,4-cyclohexanedimethanol (1,4-CHDM) and acetone, both of which are non-toxic, neutral compounds. 1, 4-CHDM is widely used in food packaging and acetone is a 6
compound on the FDA Generally Recognized as Safe (GRAS) list (Lee et al., 2007). Currently, PCADK is mainly used for the treatment of inflammatory reactions and targeted therapy because of its acid sensitivity (Seshadri et al., 2010); however, it cannot be utilized in common subcutaneously injected microspheres because it does not substantially degrade at physiological pH. This property also limits the application of PCADK in drug delivery. Based on the results from a previous study, we are committed to developing a novel PCADK/PLGA blend microsphere system for use in subcutaneous injections to reduce the acidic environment and enhance the rhGH efficacy within microspheres. In this study, the effects of PCADK degradation products on rhGH were investigated. PCADK/PLGA-blend
microspheres
were
prepared
using
a
water/oil/water
emulsion-solvent evaporation method. The properties of the blend microspheres, including
their
morphology,
encapsulation
efficiency,
release
profiles,
pharmacokinetics and pharmacodynamics were systematically characterized. Finally, the pH of the microclimate (μpH) within the microspheres was investigated to explain the improvement of rhGH stability in PCADK/PLGA microspheres.
2. Materials and methods 2.1 Materials The polymers PLGA (5050 high, Mw 54336 Da) and PCADK (6463 Da), which were used for the preparation of the microspheres, were donated from Luye Pharmaceutical 7
Co. Ltd. (Shandong, China). LA (85-90%, AR), acetone (>99.5%, AR), disodium hydrogen phosphate dodecahydrate (>99%, AR), hydrochloric acid (36-68%, AR), and dichloromethane (>99.5%, AR) were purchased from Beijing Chemical Works (Beijing, China). GA (>98%) and 1,4-CHDM (>99%) were purchased from Aladdin Chemistry Co. Ltd (Shanghai, China). Polyvinyl alcohol (PVA) (87–89% hydrolyzed, MW 13,000–23,000 Da) was purchased from Sigma-Aldrich (USA). The fluorescent pH-sensitive dye SNARF-1 dextran (MW 10 kDa) was purchased from Molecular Probes (USA). RhGH (MW 22 kDa) was kindly supplied by GeneScience Pharmaceuticals Co., Ltd. (Changchun, China). Protein molecular weight standards were purchased from Dingguo Changsheng Biotechnology Co., Ltd. (Beijing, China). All other chemicals were of analytical grade.
2.2 rhGH structural stability when incubated with PCADK or PLGA degradation products RhGH was incubated in PCADK degradation products to determine the structural stability in this environment. The 0.1 M of PCADK degradation product solution was prepared by dissolving acetone and 1,4-CHDM in deionized water and filtering (0.22 μm). Equally, the 0.1 M of PLGA degradation product solution was obtained by dissolving LA and GA in deionized water and filtering (0.22 μm). RhGH was dissolved in the solutions described above and the final concentration of all groups was 15 mg/ml. The same concentration of rhGH in PBS solution was prepared as a 8
control. After incubating at 37 °C for 7 days, all the samples were diluted 15 times with PBS to bring the pH of the different groups to a consistent value. The structural integrity of rhGH was analyzed by Native-PAGE and SEC-HPLC. To confirm the state of the secondary structure, rhGH samples were detected by CD and the spectra (190-260 nm) were collected on a Jasco J-810 instrument with a 0.1 cm quartz cell at 25 °C. Using the Jasco software, the background signal was subtracted from the spectrum of each protein solution, and the data were transformed to represent mean residue ellipticity. Fluorescence spectroscopy was used to monitor the changes in the tertiary structure. Fluorescence spectra were collected on a Shimadzu RF-5301 instrument. The emission spectrum (305–550 nm) of each sample was collected at an excitation wavelength of 280 nm. All analyses were carried out in triplicate and averaged (Determan et al., 2006).
2.3 rhGH pharmacological activity upon incubation with PCADK or PLGA degradation products RhGH solutions (1 mg/ml) were incubated in a series of PCADK and PLGA degradation products similar to the case described in Section 2.2. After incubating at 37 °C for 7 days, all the rhGH samples were diluted to 0.1 mg/ml with PBS to bring the pH of the different groups to a consistent value. The efficacy of rhGH incubated with PCADK degradation products was assessed using hypophysectomized (Hpx) rats (average weight 100 g). To confirm the Hpx rats model were successful, Hpx rats 9
were allowed to acclimate for one week, during which the changes in body weight were monitored. Hpx rats that demonstrated greater than 10% increase in body weight were considered to be incompletely hypophysectomized and were excluded from the study. Hpx rats were randomly divided into four treatment groups (n = 7 rats per group). Three experimental groups received a daily subcutaneous injection of rhGH incubated in PBS, rhGH incubated in PCADK degradation products or rhGH incubated in PLGA degradation products for 7 days. The control group received a subcutaneous injection of PBS. All injections were given in a volume of 0.25 ml. Increases in the body weight of Hpx rats have been utilized as indices of the pharmacological efficacy of rhGH.
2.4 Microspheres preparation Microspheres were prepared by the W/O/W double-emulsion technique. Two-hundred microliters of 50 mg/ml rhGH was emulsified in 2 ml of methylene chloride containing 500 mg of PLGA or a PCADK/PLGA blend (10/0, 2/8, 5/5, 8/2, 0/10) using a homogenizer at 5000 rpm for 90 s in an ice bath. The stabilized primary emulsion was immediately injected into 100 ml of an aqueous solution containing 1% (w/v) PVA and 4% (w/v) NaCl and homogenized at 5000 rpm for 120 s. The secondary emulsion was placed in magnetic stirring conditions for 3 h. The hardened microspheres were washed three times and then freeze-dried (Cleland et al., 1997).
10
2.5 Characterization of PCADK/PLGA microspheres The encapsulation efficiency of rhGH in microspheres were determined by dissolving 10 mg of microspheres in 1 ml of 1 M NaOH for 1 day, after which 2 ml of 1 M HCl was added and the mixture was incubated at 37 ℃ for another day. Then, the solution was neutralized with NaOH. A micro-BCA assay was used to analyze the rhGH content in the clear solution. The surface morphology of microspheres was investigated by SEM (JXA-840, JEOL, Japan). The SEM samples were mounted on the surfaces of specimen stubs and coated with platinum under vacuum. The accelerating voltage used was 3 kV.
2.6 In vitro release of rhGH from microspheres Ten milligrams of the microspheres were suspended in 5 ml of 20 mM PBS (pH 7.4) containing 0.05% (w/v) sodium azide and placed in an incubator at 100 rpm and 37 °C. At predetermined time intervals, the buffer was withdrawn and fresh buffer solution was added to the microspheres. The amount of the released protein was determined using the Micro-BCA Protein Assay Kit (ComWin Biotech Co., China).
2.7 Confocal imaging of the microclimate pH inside microspheres The microclimate pH change inside the PCADK/PLGA microspheres was measured using a quantitative ratio metric method based on confocal laser scanning microscopy (CLSM). Microspheres containing SNARF-1 dextran (a fluorescent pH-sensitive dye) 11
were suspended in 2 ml PBS (pH 7.4) containing 0.01% (w/v) sodium azide and placed into an incubator at 100 rpm and 37 ℃. At predetermined time intervals, microspheres were collected and a small amount of microspheres was used to obtain CLSM images. The fluorescent dye encapsulated in the microspheres was excited at 488 nm by an Ar/He laser, and two images at different wavelengths (580 and 640 nm) were taken. The two images were overlapped to observe the microclimate pH change (Wei et al., 2011).
2.8 Pharmacokinetics of rhGH in SD rats The animal experiments were approved by the Experimental Animal Ethics Committee in the School of Life Sciences, Jilin University. The pharmacokinetics of rhGH PCADK/PLGA microspheres was assessed in SD rats (6 weeks old, average weight: 200 g). The PCADK/PLGA microspheres group and the PLGA microspheres group were subcutaneously injected with PCADK/PLGA microspheres and PLGA microspheres at 5 mg/kg, respectively. The microspheres were suspended in a vehicle consisting of 0.5% w/v carboxymethyl cellulose, 5.0% w/v mannitol and 0.1% v/v Tween 80 in an aqueous solution. The rhGH solution group received a single subcutaneous injection of fresh rhGH solution at the same dose. At the reservation time, blood samples were collected into heparinized tubes and centrifuged at 10,000 rpm for 10 min. Then, plasma samples were collected and the rhGH levels were determined by an rhGH ELISA kit (Jordan et al., 2010). 12
2.9 In vivo efficacy study For the in vivo efficacy study, 6-week-old SD rats (average weight: 200 g) were subcutaneously injected with 5 mg/kg PCADK/PLGA microspheres, 5 mg/kg PLGA microspheres, or saline. A 0.2 ml blood sample was collected from each rat using a heparinized syringe at reservation time after administration. IGF-1 levels were determined using an IGF-1 ELISA kit (R&D Systems, Inc. China) (Wei et al., 2012).
2.10 Measurement of rhGH pharmacological activity in hypophysectomized rats The rhGH pharmacological activity of PLGA microspheres and PCADK/PLGA microspheres was compared by subcutaneously injecting Hpx rats (average weight: 100 g) with each microspheres formulation or native rhGH. The efficacy of rhGH PCADK/PLGA microspheres was assessed by the increase in the body weight of Hpx rats. The Hpx rats were randomly assigned to four groups (5 rats per group) and were subcutaneously injected with PCADK/PLGA microspheres, PLGA microspheres, fresh rhGH solution or saline. Rats in the rhGH solution group received daily subcutaneous injections of fresh rhGH solution at a dose of 0.25 mg/kg/day for 14 days (for a total dose of 3.5 mg/kg). Rats in the microsphere formulations groups received a single subcutaneous injection of PCADK/PLGA microspheres or PLGA microspheres at 3.5 mg/kg. Rats in the negative control group received daily subcutaneous injections of saline. Weight gain in Hpx rats was determined every other day for 14 days(Kang, 2014; Kwak et al., 2009). 13
3. Results and discussion 3.1 rhGH stability upon incubation with PCADK or PLGA degradation products To study the biocompatibility between rhGH and PCADK, electrophoresis, SEC-HPLC, CD and fluorescence spectroscopy were used to detect the structural changes in rhGH following incubation in PCADK degradation products. Floccule appeared in the rhGH solution incubated in PLGA degradation products while rhGH solution incubated in PCADK degradation products was still clear (Fig.S1), indicating that the rhGH insoluble aggregations were produced by PLGA degradation products. To detect the component of the precipitation in PLGA-incubation group, the precipitation was washed three times and SDS or β-mercaptoethanol solution was added in the tube. The supernatant was detected by Native-PAGE and the result was shown in Fig.S2. The precipitation dissolved in SDS and the supernatant showed diffuse band below native rhGH band in Native-PAGE gel, indicating that the precipitation was produced by non-covalent bond. The supernatant was collected by centrifugation and used for further detection. Fig. 1 shows the SEC-HPLC chromatograms of rhGH after incubation. The rhGH monomer peak appeared at 18 min. The peaks characterized by shorter retention times (9 min) and longer retention times (21 min) were identified as aggregation and fragment products, respectively. The rhGH that had been incubated for 2 days in the degradation products of PCADK retained the main peak at 18 min, while the rhGH that had incubated for 2 days in the degradation products of PLGA generated aggregation and fragment peaks. Continued 14
incubation of rhGH in the degradation products of PCADK for 5 days maintained the major peak albeit with a “shoulder” after the monomer peak. Incubation of rhGH for 7 days in the PLGA degradation products resulted in a monomer peak that was very weak and a major fragment peak. The Native-PAGE results were shown in Fig. 2. The PCADK group retained the band of fresh rhGH, with very light degradation band below the rhGH monomer. However, heavy fragment band of rhGH was observed in the PLGA-incubation group. The result was consistent with the SEC-HPLC chromatogram which indicating that PCADK maintains the integrity of the rhGH basically while PLGA leads to rhGH fragment seriously. Fig. 3 shows the circular dichroism spectra. RhGH incubated in PCADK degradation products showed a similar spectrum to that of fresh rhGH, maintaining significant circular dichroic activity with two peaks between 210 and 230 nm. However, the CD signal intensity of PCADK group was a little smaller than that of fresh rhGH and the negative band at 208 nm showed bathochromic effect, indicating that the PCADK degradation products had slight effect on the rhGH secondary structure. It is worth noting that the CD signal of rhGH in PLGA degradation products was very weak and the characteristic 208 and 222 nm peaks disappeared completely, demonstrating that the PLGA degradation products resulted in destruction of the rhGH secondary structure (Kim and Tg., 1999). Fig. 4 shows the fluorescence spectrum of rhGH; while the peak positions of rhGH incubated in PCADK and PLGA degradation products were not changed, the peak intensities changed compared with that of fresh rhGH. The 15
fluorescence spectrum peak decreased slightly to 331 in the PCADK group, while the peak from the PLGA group decreased greatly to an intensity of 107. The decrease of the fluorescence intensity indicated the change of the rhGH tertiary structure. The degradation products of PCADK have little effect on the tertiary structure of rhGH, while the degradation products of PLGA exert a larger influence on rhGH. These results provide evidence that PCADK degradation products have better compatibility with rhGH than the PLGA degradation products.
3.2 rhGH pharmacological activity upon incubation with PCADK or PLGA degradation products To evaluate the effect of PCADK degradation products on rhGH pharmacodynamics, hypophysectomized rats were given daily injections of growth hormone and body weights of the rats were monitored (Fig. 5). After 7 days of drug injection, three treatment groups of hypophysectomized rats all showed gains in body weight. The average weight gain of the fresh-rhGH group was 32.1 g. The average weight gain for rats in the PCADK group was 12.1 g, indicating that PCADK degradation products affects a part of rhGH pharmacological activity. The PLGA-group rats showed only an average increase in body weight of 5.6 g, far lower than that of the fresh rhGH group. The results showed that PCADK degradation products cause smaller losses in the biological activity of rhGH than PLGA degradation products and confirm that PCADK presents better biocompatibility with rhGH than PLGA. PCADK is known to 16
degrade into 1, 4-CHDM and acetone, both of which are neutral products that likely act mildly on rhGH. PLGA, however, degrades into LA and GA, and the resulting acidic environment has an irreparable effect on rhGH activity.
3.3 PCADK/PLGA microspheres PCADK/PLGA microspheres with different PCADK proportions were prepared, as shown in Fig. 6. Microspheres were generally spherical with smooth surfaces when PCADK was present in a proportion lower than 50%. With further PCADK increases, however, the microspheres became irregularly shaped, showing rough surfaces and apparently more holes. The low molecular weight of PCADK leads to relatively weak mechanical properties, and when the proportion of PCADK is over 50%, the balling property of the polymer mixture worsens. The encapsulation efficiencies of the PCADK/PLGA microspheres that contained 0%, 20% and 50% PCADK were 65.8%, 62.1% and 60.6%, respectively. Once PCADK increased to 80%, the encapsulation efficiency significantly decreased to 40.3%; even worse, the encapsulation efficiency of the PCADK-only microsphere was only 22.7% (Table 1). The drug is more liable to escape from microspheres during solvent evaporation when there was a large amount of holes on the microspheres, so the large proportion of PCADK in microspheres caused very low encapsulation efficiency.
3.4 In vitro release of rhGH from microspheres 17
To detect the release behavior of PCADK/PLGA microspheres, microspheres with different proportions of PCADK were maintained at 37 °C in an incubator at 100 rpm (Fig. 7). After 14 days of release, microspheres with 0% and 20% PCADK reached total release amounts of 15% and 37%, respectively, while microspheres with 50%, 80% and 100% PCADK reached approximately 65% of release. The results indicated that the addition of PCADK improved the total release of rhGH to a certain degree. PLGA microspheres almost stopped releasing rhGH after the initial burst release, while PCADK/PLGA microspheres released rhGH at a later period (total release － burst release) to a different degree. The later release of microspheres increased from 16% to 43% when the proportion of PCADK was increased from 20% to 50%. However, microspheres with 80% and 100% PCADK only presented 26% and 16% later release due to the enormous burst release. There are two mechanisms by which the drug can be released from sustained-release microspheres: the diffusion mechanism and the degradation mechanism. The dissolution and diffusion of the drug is the main source of burst release. Water-soluble drugs prefer the external water phase and the permeation of the inner rhGH phase to the external water phase brings the drug to the microsphere surface. The release medium enters microspheres through the holes and forms a concentration gradient, which creates an osmotic pressure difference and causes the drug inside the microspheres to permeate outside. Therefore, when the proportion of PCADK increases to 80% and 100%, additional holes on the surfaces of microspheres lead to larger burst releases. In the later period of release, 18
water-soluble channels could accelerate drug release and the high concentration gradient in microspheres induces drug diffusion to the external environment. Microspheres with less than 50% of PCADK were shown to be morphologically strong at the early stages of release. PLGA degraded into LA and GA during the incubation period, which led to an acid microenvironment. PCADK degraded rapidly in the acidic microenvironment, leading to a loose structure in the PCADK/PLGA microspheres. The loose structure acted as similar to water-soluble channels by accelerating drug release. This result indicates that PCADK/PLGA microspheres have better release properties than PLGA microspheres.
3.5 Pharmacokinetics of rhGH in SD rats By investigating the morphology, encapsulation and in vitro release characteristics of PCADK/PLGA microspheres, we found that when the proportion of PCADK is 50%, microspheres maintained excellent morphological features and encapsulation efficiency and showed improved in vitro release properties. Therefore, in the pharmacokinetics study, we applied PCADK/PLGA microspheres with 50% of PCADK. We examined the in vivo release of rhGH from PLGA microspheres and PCADK/PLGA microspheres. Fig. 8 shows the plasma rhGH concentration-time release profiles for PCADK/PLGA and PLGA microspheres. The results showed that PCADK/PLGA and PLGA microspheres underwent rapid release of rhGH in the first day followed by release of rhGH for up to two weeks. The AUC (0-14 d) values were 19
calculated by the software DAS 3.0 (Drug and Statistics Software, Mathematical Pharmacology Professional Committee of China, Shanghai, China). AUC (0-14 d) values of PCADK/PLGA and PLGA microspheres were 219.76 and 106.02 µg/L*d, respectively. The AUC value of the PCADK/PLGA microspheres was increased 2-fold compared to that of the PLGA microspheres. These results suggest that the addition of PCADK effectively increases the bioavailability of rhGH.
3.6 Pharmacodynamic study IGF-1 is an endocrine hormone and the plasma concentration of IGF-1 is associated with rhGH function. Therefore, IGF-1 can be used as a biological marker of rhGH pharmacodynamics (Mathews et al., 1986). As shown in Fig. 9, PCADK/PLGA microspheres showed a more significant IGF-1 response than PLGA microspheres. Both PCADK/PLGA microspheres and PLGA microspheres achieved significant increases in IGF-1 levels after administration. However, the concentration of IGF-1 in the PLGA microspheres group decreased rapidly and afterward tended to match with the blank group. On the other hand, the concentration of IGF-1 in plasma remained elevated for 2 weeks after administration of PCADK/PLGA microspheres. The IGF-1 concentration associated with PCADK/PLGA microspheres was always higher than that of PLGA microspheres, indicating the superiority of PCADK/PLGA microspheres in vivo. AUC (0-14 d) values of PCADK/PLGA microspheres were 9038.86 µg/L*d, approximately 50% greater than that observed for PLGA 20
microspheres (6439.24 µg/L*d). The results suggested that the PCADK/PLGA microspheres released a greater amount of bioactive rhGH in rats.
3.7 Pharmacological efficacy study in Hpx rats To compare the in vivo efficacy of PLGA and PCADK/PLGA microspheres, Hpx rats were subcutaneously injected with microsphere formulations (3.5 mg/kg) or native rhGH (0.25 mg/kg/day for a total dose of 3.5 mg/kg over 14 days). As shown in Fig. 9, after the 14-day injection period Hpx rats that had been injected with normal saline showed no changes in body weight, while the three rhGH treatment groups showed apparent increases in body weight. Rats in the rhGH solution group maintained a well-proportioned increase in body weight of 55 g over the 2-week course of treatment. The PCADK/PLGA group showed a significant increase in body weight over the 2 weeks after administration. However, the PLGA group showed a weight increase only in the first 5 days after administration, after which point the weight of the rats remained constant. The results suggested that the PCADK/PLGA microspheres had better pharmacological efficacy than the PLGA microspheres due to the prolongation of release and improvement of rhGH efficacy. During the preparation of the microspheres with the W/O/W method, the oil-water contact caused the denaturation of rhGH, resulting in a loss in efficacy of both PCADK/PLGA and PLGA microspheres.
21
3.8 Confocal imaging of the microclimate pH inside microspheres To detect the pH inside the PCADK/PLGA microspheres, we co-encapsulated rhGH and SNARF-1 dextran into microspheres and observed the color change during incubation by laser scanning confocal microscope. At the beginning of the incubation, both PCADK/PLGA and PLGA microspheres appeared yellow, indicating that the internal environment was neutral. After 2 days of incubation, both PCADK/PLGA and PLGA microspheres turned orange, indicating that the internal environment was becoming acidic due to the accumulation of PLGA degradation products. As the time went on, however, the orange color presented by PLGA microspheres became darker while that of the PCADK/PLGA microspheres became a light orange. The results showed that the acidity of the internal environment of PLGA microspheres became stronger due to the accumulation of PLGA degradation products. However, the internal environment of PCADK/PLGA microspheres was more neutral due to the addition of PCADK. At the initial stages of the incubation, the PLGA within the PCADK/PLGA microspheres degraded into LA and GA, leading to the decreased pH within the microspheres. The acidic environment promoted the degradation of PCADK due to the acid-sensitive property, leading to the formation of holes on the surface of PCADK/PLGA microspheres (Fig.S3), which provided channels for protein diffusion and release. Autocatalysis degradation mechanism of PCADK/PLGA microspheres guarantees
the
application
of
PCADK/PLGA 22
microspheres
in
common
subcutaneously injection. At the later stages of the incubation, the holes also provide channels for exhausting of PLGA degradation products and the diffusion of buffer into the microspheres, which neutralizes the acid internal microenvironment. Moreover,
PCADK
degradation
products
are
neutral
and
present
better
biocompatibility with rhGH. Therefore, the more neutral microenvironment of PCADK/PLGA microspheres is a significant reason for the higher activity of rhGH in PCADK/PLGA than in PLGA microspheres. 4. Conclusion In this study, PCADK degradation products are shown to possess better biocompatibility with rhGH than PLGA degradation products. PCADK/PLGA microspheres with different proportions of PCADK were prepared using W/O/W method and loaded with rhGH. The 5/5 PCADK/PLGA microspheres were finally chosen by investigating the drug loading capacity, microsphere morphologies and release behaviors. Compared with PLGA microspheres, PCADK/PLGA microspheres presented improved release behavior and larger AUC in rats. Moreover, we found that PCADK/PLGA microspheres had better efficacy than PLGA microspheres in hypophysectomized rats. While PCADK/PLGA microspheres are imperfect with regard to rhGH activity caused by the W/O/W method used during preparation, these microspheres have an apparent advantage over PLGA microspheres prepared using the same method. Thus PCADK/PLGA microspheres have great potential for use in the sustained release of rhGH. 23
Reference Cazares-Delgadill, J., Ganem-Rondero, A., Kalia, Y.N., 2011. Human growth hormone: New delivery systems, alternative routes of administration, and their pharmacological relevance. Eur. J. Pharm. Biopharm. 78, 278-288. 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., 1997. Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect. J. Control. Release 49, 193-205. Determan, A.S., Wilson, J.H., Kipper, M.J., Wannemuehler, M.J., Narasimhan, B., 2006. Protein stability in the presence of polymer degradation products: Consequences for controlled release formulations. Biomaterials 27, 3312-3320. Ding, A.G., Schwendeman, S.P., 2008. Acidic microclimate pH distribution in PLGA microspheres monitored by confocal laser scanning microscopy. Pharm. Res. 25, 2041-2052. Fu, K., Pack, D.W., Klibanov, A.M., Langer, R., 2000. Visual evidence of acidic environment within degrading poly(lactic-co-glycolic acid) (PLGA) microspheres. Pharm. Res. 17, 100-106. Jordan, F., Naylor, A., Kelly, C.A., Howdle, S.M., Lewis, A., Illum, L., 2010. Sustained release hGH microsphere formulation produced by a novel supercritical fluid technology: In vivo studies. J. Control. Release 141, 153-160. 24
Kang, J., 2014. Development of Recombinant Human Growth Hormone (rhGH) sustained-release microspheres by a low temperature aqueous phase/aqueous phase emulsion method. Eur. J. Pharm. Sci. 62, 141–147. Kang, J.C., Schwendeman, S.P., 2002. Comparison of the effects of Mg(OH)2 and sucrose on the stability of bovine serum albumin encapsulated in injectable poly(D,L-lactide-co-glycolide) implants. Biomaterials 23, 239-245. Kim, H.K., Tg., P., 1999. Microencapsulation of human growth hormone within biodegradable polyester microspheres: Protein aggregation stability and incomplete release mechanism. Biotechnol. Bioeng. 65, 659–667. Kim, S.J., Hahn, S.K., Kim, M.J., Kim, D.H., Lee, Y.P., 2005. Development of a novel sustained release formulation of recombinant human growth hormone using sodium hyaluronate microparticles. J. Control. Release 104, 323-335. 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., 2009. Development of a sustained-release recombinant human growth hormone formulation. J. Control. Release 137, 160-165. Lee, S., Yang, S.C., Heffernan, M.J., Taylor, W.R., Murthy, N., 2007. Polyketal microparticles: A new delivery vehicle for superoxide dismutase. Bioconjugate Chem. 18, 4-7. Ma, G., 2014. Microencapsulation of protein drugs for drug delivery: Strategy, preparation, and applications. J. Control. Release 193, 324-340. 25
Mathews, L.S., Norstedt, G., Palmiter, R.D., 1986. Regulation of insulin-like growth factor I gene expression by growth hormone. P. Natl. Acad. Sci. USA. 83, 9343-9347. Seshadri, G., Sy, J.C., Brown, M., Dikalov, S., Yang, S.C., Murthy, N., Davis, M.E., 2010. The delivery of superoxide dismutase encapsulated in polyketal microparticles to rat myocardium and protection from myocardial ischemia-reperfusion injury. Biomaterials 31, 1372-1379. Wei, Y., Wang, Y.-X., Wang, W., Ho, S.V., Wei, W., Ma, G.-H., 2011. mPEG-PLA microspheres with narrow size distribution increase the controlled release effect of recombinant human growth hormone. J. Mater. Chem. 21, 12691-12699. Wei, Y., Wang, Y., Kang, A., Wang, W., Ho, S.V., Gao, J., Ma, G., Su, Z., 2012. A Novel Sustained-Release Formulation of Recombinant Human Growth Hormone and Its Pharmacokinetic, Pharmacodynamic and Safety Profiles. Mol. Pharm. 9, 2039-2048. Yang, J., Wilson, M., Celniker, A.C., 1997. Recombinant human growth hormone poly(lactic-co-glycolic acid) (PLGA) microspheres provide a long lasting effect. J. Control. Release 49, 193-205(113). Yuen, K.C.J., Amin, R., 2011. Developments in administration of growth hormone treatment: focus on Norditropin (R) Flexpro (R). Patient Prefer. Adher. 5, 117-124. Zhu, G.Z., Mallery, S.R., Schwendeman, S.P., 2000. Stabilization of proteins encapsulated in injectable poly (lactide-co-glycolide). Nat. Biotechnol. 18, 52-57. Zhu, G.Z., Schwendeman, S.P., 2000. Stabilization of proteins encapsulated in 26
Accepted Manuscript Title: Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH Author: Chenhui Wang Changhui Yu Jiaxin Liu Lesheng Teng Fengying Sun Youxin Li PII: DOI: Reference:
S0378-5173(15)30270-2 http://dx.doi.org/doi:10.1016/j.ijpharm.2015.10.003 IJP 15258
To appear in:
International Journal of Pharmaceutics
Received date: Revised date: Accepted date:
10-7-2015 14-9-2015 3-10-2015
Please cite this article as: Wang, Chenhui, Yu, Changhui, Liu, Jiaxin, Teng, Lesheng, Sun, Fengying, Li, Youxin, Preparation and in vivo evaluation of PCADK/PLGA microspheres for improving stability and efficacy of rhGH.International Journal of Pharmaceutics http://dx.doi.org/10.1016/j.ijpharm.2015.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Figure Captions
Fig. 1. SEC-HPLC chromatograms of rhGH incubated in the presence of PCADK and PLGA degradation products. a: pure rhGH, b: rhGH incubated in PCADK degradation products for 2 days, c: rhGH incubated in PLGA degradation products for 2 days, d: rhGH incubated in PCADK degradation products for 7 days, e: rhGH incubated in PLGA degradation products for 7 days.
28
Fig.2 Native-PAGE analysis of rhGH incubated in the presence of PCADK and PLGA degradation products for 7 days. Lane 1: fresh rhGH, lane 2: rhGH in PLGA degradation products, lanes 3: rhGH in PCADK degradation products.
29
Fig. 3. CD spectra of rhGH after incubation in the presence of PCADK and PLGA degradation products for 7 days.
30
Fig. 4. Fluorescence spectroscopy of rhGH incubated in the presence of PCADK and PLGA degradation products for 7 days.
31
Fig. 5. Pharmacological activity of rhGH incubated in the presence of PCADK or PLGA degradation products for 7 days.
32
Fig. 6. SEM micrographs of PCADK/PLGA microspheres with different PCADK proportions. a: 0% PCADK b: 20% PCADK c: 50% PCADK d: 80% PCADK e: 100% PCADK.
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Fig. 7. In vitro release of rhGH from PCADK/PLGA microspheres.
34
Fig. 8. In vivo release of rhGH from PCADK/PLGA microspheres.
35
Fig. 9. The IGF-1 concentrations in rats treated with rhGH-loaded microspheres.
36
Fig. 10. The weight gains in hypophysectomized rats treated with rhGH-loaded microspheres.
37
Fig. 11 Confocal imaging of microclimate pH inside microspheres
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Tables Table 1 Encapsulation efficiency of PCADK/PLGA microspheres with different ratios of PCADK
PCADK/PLGA
0/10
2/8
5/5
8/2
10/10
Encapsulation Efficiency (%)
65.8±2.4
62.1±1.9
60.6±2.6
40.3±3.1
22.7±1.7
39