hGH Release Research Articles

Acromegaly: The Impacts of HGH Release on Patient Progression

Hypersecretion of human Growth Hormone (hGH) is the primary mechanism driving Acromegaly, a disease characterised by abnormal growth of extremities and disfigured facial features. This article will discuss hGH secretion, its effect on Acromegaly, and potential treatments for the disease, among other key topics.

Sleep and muscle recovery – Current concepts and empirical evidence

Generally, sleep is thought of as a state of rest that helps to recover the body and brain from the physical and cognitive activities during wakefulness. The main characteristics of sleep in contrast to wakefulness support this idea: a barely present motor activity and a very low reactivity to both internal and external stimuli. The long-term atonic state of skeletal muscles is taken as an indication that important restorative processes on the musculoskeletal system take place during sleep – and the state of unconsciousness as well as inadequate responsiveness suggests that the central nervous system pauses to engage in important synaptic plasticity processes. About 40 years ago, therefore, in sleep research, the idea was that especially NREM sleep fosters synthetic processes of growth and repair in the body, and REM sleep was believed to be directed towards increases in synthetic processes within the brain, including the consolidation of memory (Horne, 1979). The opinion that SWS is mainly for body restitution was encouraged by the observation that after falling asleep during the early night period, human growth hormone (hGH) is present in relatively large amounts in the plasma (Born et al., 1988). Additionally, it was shown that after high loads of physical activity during the day, in the following night the percentage of SWS increased (Shapiro et al., 1981). Moreover, sleep deprivation seems to weaken muscle recovery by increasing protein breakdown, which adversely affects protein synthesis and promotes muscle atrophy (Dattilo et al., 2011, 2012). Muscle recovery would potentially be impaired because this process is strongly regulated by the anabolic and catabolic hormones mentioned above, which are strongly influenced by sleep. On the other hand, the heart muscle and the diaphragm (the main muscle for respiratory activity) prove that the vital contractile elements of a muscle maintain their function without rest over the lifespan. And if they did, it seems more likely to be the motor unit of the supporting and target muscles as a whole (including the reflex pathways) that needs rest (Fitts, 2008). Furthermore, the muscle protein synthetic response following resistance exercise seems rather time-dependent than state-dependent, e.g. amino acids are taken up in skeletal muscle tissue within hours independent of being awake or asleep (McGlory et al., 2017). However, the aforementioned release of hGH during the early hours of sleep and the results from sleep deprivation studies point in the direction that some regeneration processes are dependent on sleep, and most likely on SWS (Dattilo et al., 2011). The proposed hypothesis that bodily recovery and regeneration are sleep-dependent is widely used, but often lacks extensive supportive experimental evidence. In this presentation we will discuss the current concepts of muscle recovery during sleep and review the studies in that field.

Silica-collagen nanoformulations with extended human growth hormone release

Growth hormone deficiency has been treated by the daily administration of recombinant human growth hormone (hGH) for decades. Patient compliance to this treatment is generally incomplete due to challenges including dose frequency and lack of perceived benefits. This stimulates the research on new formulations to reduce the number of periodic administrations. In this study silica nanoparticles and silica-collagen nanocomposites were evaluated for hGH loading and release. Bare nanoparticles showed higher hGH adsorption capacity than thiol- and isobutyl-bearing particles of similar diameters. Monitoring of bound protein conformation changes indicated hGH structure retention when adsorbed on bare silica nanoparticles and suggested no alterations on protein activity. Protein-loaded particles incorporated into collagen matrices (silica-collagen nanocomposites) showed a progressive protein release profile different from the observed for hGH-loaded silica nanoparticles and hGH-loaded collagen matrices. While both the collagen and the silica nanoparticle systems reached a 100 % release after 4 and 7 days respectively, silica-collagen nanocomposites showed a bi-phasic prolonged hGH release reaching approximately an 80 % after 15 days. These findings suggest that biocompatible silica-collagen nanocomposites could be used as vehicles for the prolonged delivery of hGH which could lead to a potential reduction in the number of periodic administrations.

Sustained transdermal delivery of human growth hormone from niosomal gel: in vitro and in vivo studies

Human growth hormone (hGH) is widely used to treat several diseases for decades. However, the current treatment regime requires frequent injections via subcutaneous route due to short in vivo half-life, which leads to pain and poor patient compliance. In this study, a novel transdermal (non-invasive) hGH loaded niosomes gel was prepared to reduce the frequency of subcutaneous injections and to improve the patient compliance. Niosomes were prepared by film hydration technique at three levels of cholesterol. The particle size and entrapment efficiency increases with an increase in the level of cholesterol. Transmission electron microscopy images confirmed the spherical shape of niosomes without aggregation. Texture profiles analysis indicates that the niosomal gel has the required mechanical properties for transdermal application. The ex vivo permeation profile showed sustain hGH release for 4 days from the niosomal gel compared to 24 h from the control gel without niosomes. A rabbit skin irritation study showed no sign of irritation after application of niosomal gel. The pharmacokinetic parameters in the rat model showed 7.22-fold higher bioavailability with niosomal gel compared to control gel. In conclusion, the study demonstrated the potential of niosomal gel as an effective long-term sustained release strategy for hGH delivery to replace traditional subcutaneous injections.

Mechanism for the Increase in Human Growth Hormone with Administration of a Novel Test Supplement and Results Indicating Improved Physical Fitness and Sleep Efficiency.

An oral test supplement increases serum human growth hormone (hGH) levels after acute administration in healthy adults. We investigated the mechanism for the increase in hGH and the effect of continued daily administration of the test supplement on measures of physical fitness and sleep efficiency. In Study 1, serum triiodothyronine (T3) was measured in samples from a prior placebo-controlled, double-blind study in which 16 healthy participants received both placebo and the test supplement in a crossover design; treatment order was randomized, and treatments were separated by a 1-week washout. In Study 2, physical fitness (VO2 max) was measured at baseline and after 2 weeks of daily administration of the test supplement (N = 12 healthy participants). Study 3 assessed daily sleep onset latency and time awake during 3 weeks of daily administration of the test supplement (N = 15 healthy participants). A fall from baseline in T3 was observed with placebo (-6.1 ± 8.5 ng/dL, P = .01). Of note, the change in T3 was smaller with the test supplement (-3.3 ± 10.7 ng/dL, P = not significant) but was not statistically different from placebo. Mean VO2 max increased by 6% from baseline after 2 weeks (P = .02). Sleep-onset latency and time awake during the night were reduced from baseline to week 3 by 22% and 65%, respectively (P = .01 and P = .02). The conservation of T3 levels suggests that the mechanism for increased hGH secretion by the test supplement is through somatostatin inhibition. Furthermore, pilot studies indicated that daily administration of the supplement improved physical fitness and sleep efficiency from baseline, effects consistent with increased endogenous hGH release. Clinical Trial Registration No. NCT02987868.

Release of human growth hormone from an in-situ implant modulated by poly(ethylene glycol) dimethyl ether and tris(hydroxymethyl) aminomethane

A patient-friendly delivery system to release human growth hormone (hGH) is very desirable. In situ forming implant systems (ISIs) can provide a long acting and effective protein delivery. In these systems, solvents and additives play major roles in drug release. In this study, four groups of PLGA-based ISIs containing hGH were prepared in N-methyl-2-pyrrolidone (NMP) and poly(ethylene glycol) dimethyl ether (PEG-DME) as solvents with and without tris(hydroxymethyl) aminomethane (Tris) as stabilizer. Several analyses were used to investigate the implants, which include release profile, viscosity, contact angle, gel permeation chromatography (GPC), scanning electron microscopy (SEM), hGH and IGF-1 serum measurements and histopathology. In in vitro release experiments, the hGH cumulative release from PEG-DME system was twice that from NMP system during 14 days, and hGH release was tripled in the presence of Tris. With the addition of Tris to the ISIs containing PEG-DME, the water penetration, interconnectivity of pores and inner channels, surface pores and hydrophilicity were increased. Moreover, the effect of Tris on the hGH stabilization synergized its positive effects and increased the hGH final cumulative release. Results of the ISIs containing PEG-DME and Tris injection in rabbits demonstrated a reduced tissue inflammation. Moreover, the 14-days serum levels of the hGH and IGF-1 of this system in recipient rabbits were comparable to those of the commercial daily injection samples.

In-vitro Release Evaluation of Growth Hormone from an Injectable In-Situ Forming Gel Using PCL-PEG-PCL Thermosensitive Triblock.

An injectable long acting In-Situ Forming Gel (ISFG) of human Growth Hormone (hGH) was prepared by using triblock PCL-PEG-PCL (Mw 1500-1500-1500). Ring-Opening Polymerization (ROP) of triblock using microwave was applied. The BCA protein assay Kit was used to determine the concentration of hGH in the in-vitro release medium. Finally, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE) tests and Circular Dichroism (CD) spectrum were done to approve the stability of released hGH. The result of ROP demonstrated that the proportion of PCL to PEG accorded with the initial molar ratio of the monomers. The cross-section of the Surface Electron Microscopy (SEM) indicated the porous framework of the hydrogel could load the drug into its tridimensional matrixes structure. There is the low initial burst release of hGH from the supramolecular hydrogel. The maximum in-vitro release of hGH was 71.2 % ± 1.5 that were due to hGH degrading after this time (21 days). The CD spectrum and SDS-PAGE results confirmed the stability of hGH during invitro release evaluation. The results suggest that the sustained-release formulation using PCL-PEG-PCL can be applied to control the release of hGH.

Increased Human Growth Hormone After Oral Consumption of an Amino Acid Supplement: Results of a Randomized, Placebo-Controlled, Double-Blind, Crossover Study in Healthy Subjects.

Human growth hormone (hGH) is best known for influencing bone and muscle growth, as well as body composition, but the use of recombinant hGH is controversial. Amino acids are a potentially safer alternative; however, preliminary investigations of the effects of oral amino acids on hGH release have been inconclusive. Therefore, we tested the effects of a novel blend of amino acids optimized to increase hGH release. Does an investigational amino acid supplement affect hGH release? This was a randomized, placebo-controlled, double-blind, crossover study that included 16 (12 men, 4 women; age 32 ± 14 years; body mass index 26.4 ± 5.0 kg/m) healthy participants. All participants received both placebo and the amino acid supplement after an overnight fast and completed all study visits. Treatment order was randomized, and each treatment was separated by a 1-week washout period. The primary outcomes were the percent change in hGH from baseline to 120 minutes and the area under the curve of hGH over baseline. Serum hGH was measured using enzyme-linked immunosorbent assay at baseline and 15, 30, 60, 90, and 120 minutes. At 120 minutes, hGH levels increased by 682% (8-fold) from baseline and were significantly higher than placebo (P = 0.01). In addition, a significantly higher mean area under the curve was observed for the amino acid supplement compared with the placebo [20.4 (95% confidence interval, 19.9-21.0 ng/mL) vs. 19.7 (95% confidence interval, 18.7-20.6 ng/mL); P = 0.04]. These results show that a single dose of the oral amino acid supplement was sufficient to significantly increase hGH levels in healthy adult men and women. CLINICAL TRIAL REGISTRY:: clinicaltrials.gov NCT01540773.

Poly(amino carbonate urethane)-based biodegradable, temperature and pH-sensitive injectable hydrogels for sustained human growth hormone delivery.

In this study, a new pH-/temperature-sensitive, biocompatible, biodegradable, and injectable hydrogel based on poly(ethylene glycol)-poly(amino carbonate urethane) (PEG-PACU) copolymers has been developed for the sustained delivery of human growth hormone (hGH). In aqueous solutions, PEG-PACU-based copolymers existed as sols at low pH and temperature (pH 6.0, 23 °C), whereas they formed gels in the physiological condition (pH 7.4, 37 °C). The physicochemical characteristics, including gelation rate, mechanical strength and viscosity, of the PEG-PACU hydrogels could be finely tuned by varying the polymer weight, pH and temperature of the copolymer. An in vivo injectable study in the back of Sprague-Dawley (SD) rats indicated that the copolymer could form an in situ gel, which exhibited a homogenous porous structure. In addition, an in vivo biodegradation study of the PEG-PACU hydrogels showed controlled degradation of the gel matrix without inflammation at the injection site and the surrounding tissue. The hGH-loaded PEG-PACU copolymer solution readily formed a hydrogel in SD rats, which subsequently inhibited the initial hGH burst and led to the sustained release of hGH. Overall, the PEG-PACU-based copolymers prepared in this study are expected to be useful biomaterials for the sustained delivery of hGH.

Human growth hormone‐loaded nanogels composed of cinnamoyl alginate, cinnamoyl Pluronic F127, and cinnamoyl poly(ethylene glycol)

ABSTRACTCinnamoyl alginate (CinAlg), cinnamoyl Pluronic F127 (CinPlu), and cinnamoyl poly(ethylene glycol) (CinPEG) could be self‐assembled into nanogels, possibly due to hydrophobic interaction among cinnamic acid (CA) residues. The nanogels were nearly spherical particles on TEM photographs. The photodimerization of CA residue had little effect on the integrity and the size of nanogel. The stability of the nanogels in blood plasma for 48 h at 37°C did not depend on UV irradiation, however they were stable in deoxycholate solution (1 and 10 mM) only when they were UV‐treated. Human growth hormone (hGH) could readily be loaded in the nanogel under an acidic condition possibly due to the electrostatic interaction between hGH and CinAlg. hGH hardly released from the nanogel at body temperature (37°C) when the release medium was at acidic condition (e.g., pH 3.0, pH 5.0; <10% in 170 h). However, it readily released at the same temperature when the medium was at physiological pH (e.g., pH 7.4; around 45% in 170 h) and at an alkaline condition (e.g., pH 9.0; >50% in 170 h). However, slow release of hGH was observed for 100 h possibly due to the layer of thermally condensed Pluronic F127 chains. In addition, initial burst release was suppressed as the CinPlu content increased. For example, the release degree at pH 9 in 60 min decreased from 15.3 to 5.9% as the CinPlu content increased from 20 to 60%. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42446.

Nanostructure controlled sustained delivery of human growth hormone using injectable, biodegradable, pH/temperature responsive nanobiohybrid hydrogel.

The clinical efficacy of a therapeutic protein, the human growth hormone (hGH), is limited by its short plasma half-life and premature degradation. To overcome this limitation, we proposed a new protein delivery system by the self-assembly and intercalation of a negatively charged hGH onto a positively charged 2D-layered double hydroxide nanoparticle (LDH). The LDH-hGH ionic complex, with an average particle size of approximately 100 nm, retards hGH diffusion. Nanobiohybrid hydrogels (PAEU/LDH-hGH) were prepared by dispersing the LDH-hGH complex into a cationic pH- and temperature-sensitive injectable PAEU copolymer hydrogel to enhance sustained hGH release by dual ionic interactions. Biodegradable copolymer hydrogels comprising poly(β-amino ester urethane) and triblock poly(ε-caprolactone-lactide)-poly(ethylene glycol)-poly-(ε-caprolactone-lactide) (PCLA-PEG-PCLA) were synthesized and characterized. hGH was self-assembled and intercalated onto layered LDH nanoparticles through an anion exchange technique. X-ray diffraction and zeta potential results showed that the LDH-hGH complex was prepared successfully and that the PAEU/LDH-hGH nanobiohybrid hydrogel had a disordered intercalated nanostructure. The biocompatibility of the nanobiohybrid hydrogel was confirmed by an in vitro cytotoxicity test. The in vivo degradation of pure PAEU and its nanobiohybrid hydrogels was investigated and it showed a controlled degradation of the PAEU/LDH nanobiohybrids compared with the pristine PAEU copolymer hydrogel. The LDH-hGH loaded injectable hydrogels suppressed the initial burst release of hGH and extended the release period for 13 days in vitro and 5 days in vivo. The developed nanohybrid hydrogel has the potential for application as a protein carrier to improve patient compliance.

Studying the release of hGH from gamma-irradiated PLGA microparticles using ATR-FTIR imaging

Attenuated total reflection-Fourier transform infrared (ATR-FTIR) imaging has been applied for the first time to monitor the redistribution and release of hGH from a range of PLGA/PLA microparticles during a set of dissolution experiments at 37°C in D2O. The effect of gamma-irradiation, a common sterilisation method, on hGH release kinetics from such systems has been demonstrated. Increasing the gamma dose was shown to have a profound influence on the nature of the release mechanism, with higher gamma doses leading to a dramatic increase in the initial burst release followed by a retardation in the sustained release and a lower total level of hGH release over the dissolution experiment. These changes were shown to be the result of a combination of factors; firstly, via scanning electron microscopy (SEM), gamma-irradiation was shown to strongly influence the morphology of the PLGA/PLA microparticles; reducing their overall porosity and reducing the available surface area, whilst forcing some of the entrapped hGH to the microparticle surface. Secondly, from FTIR measurements, gamma-irradiation was shown to increase the number of oxygenated components in the Poloxamer 407 excipient, by a process of chain scission, thereby increasing the strength of interaction between the microparticle and the entrapped hGH.

Dual ionic interaction system based on polyelectrolyte complex and ionic, injectable, and thermosensitive hydrogel for sustained release of human growth hormone

A dual ionic interaction system composed of a positively charged polyelectrolyte complex (PEC) containing human growth hormone (hGH) and anionic thermosensitive hydrogel has been suggested for sustained delivery of bioactive hGH. The PEC was prepared by ionic interaction between negatively charged hGH and positively charged protamine sulfate (PS) to suppress diffusion of hGH. Moreover, we loaded the positively charged PEC into an anionic, injectable, and thermosensitive poly(organophosphazene) hydrogel to enhance sustained release of hGH by dual ionic interactions. PS formed a spherical complex with hGH, and their ionic interaction grew stronger with increasing amounts of PS. From a weight ratio of 0.5, the PS/hGH complex had a size and zeta-potential that were constantly maintained around 500 nm and +8 mV, respectively, in 0.9% NaCl. The PEC-loaded hydrogels suppressed the initial burst release of hGH and extended the release period in vitro and in vivo. In a pharmacokinetic study in rats, the PEC-loaded anionic hydrogel extended half-life 13-fold with similar area under the curve (AUC) compared to hGH solution. Furthermore, single injection of PEC-loaded anionic hydrogel showed a more increased growth rate than daily injection of hGH solution for 7 days in hypophysectomized rats, demonstrating its potential as an injectable, sustained delivery system that can release bioactive hGH.

The biological efficiency and bioavailability of human growth hormone delivered using injectable, ionic, thermosensitive poly(organophosphazene)-polyethylenimine conjugate hydrogels

We have endeavored to develop injectable, thermosensitive, biodegradable hydrogels that prolong human growth hormone (hGH) release, improving bioavailability through introducing balanced ionic interactions. Cationic poly(organophosphazene)-polyethylenimine (PEI, 1.8 kDa) conjugate hydrogels were synthesized as those hydrogels for sustained delivery of anionic hGH with proper ionic strength of association/dissociation. We have additionally prepared different chain lengths of α-amino-ω-methoxy-poly(ethylene glycol) (AMPEG550 and AMPEG750) for the synthesis of conjugates as a means to control hydrogel degradation rates. All Aqueous solutions of PEI-conjugates became hydrogels hydrolyzable in proportion to AMPEG molecular weight at body temperature; these PEI-conjugates complexed with hGH and extended hGH release in vitro. In pharmacokinetic studies of hGH behavior in SD rats, hydrogels of PEI-conjugate/hGH complexes could suppress the initial burst-phase, and extend the duration, of release, as well as increasing of area under the curve (AUC) compared to controls including hGH solution or non-ionic hydrogel. In a hypophysectomized rat model, the biological efficacy of hGH delivered from PEI-conjugate/hGH complex hydrogels was equivalent to that from daily administration over four days based on body weight gain and width of the tibial growth plate. These results suggest that ionic, thermosensitive, poly(organophosphazene)-PEI-conjugate hydrogel demonstrates potential as an injectable depot for sustained delivery of bioavailable hGH.

Transgenic mice expressing the human growth hormone gene provide a model system to study human growth hormone synthesis and secretion in non-tumor-derived pituitary cells: Differential effects of dexamethasone and thyroid hormone

Growth hormone (GH) is regulated by pituitary and hypothalamic factors as well as peripheral endocrine factors including glucocorticoids and thyroid hormone. Studies on human GH are limited largely to the assessment of plasma levels in endocrine disorders. Thus, insight into the regulation of synthesis versus secretion has come mainly from studies done on non-human GH and/or pituitary tumor cells. However, primate and non-primate GH gene loci have differences in their structure and, by extension, regulation. We generated transgenic (171hGH/CS-TG) mice containing the intact hGH1 gene and locus control region, including sequences required for integration-independent and preferential pituitary expression. Here, we show hGH co-localizes with mouse (m) GH in somatotrophs in situ and in primary pituitary cells. Dexamethasone treatment increased hGH and mGH, as well as GH releasing hormone (GHRH) receptor RNA levels, and hGH release was stimulated by GHRH treatment. By contrast, triiodothyronine decreased or had no effect on hGH and mGH production, respectively, and the negative effect on hGH was also seen in the presence of dexamethasone. Thus, 171hGH/CS-TG mouse pituitary cultures represent a model system to investigate hormonal control of hGH synthesis and secretion.

Effects Of 24-h Fasting On Human Growth Hormone And Cytokine Responses After Resistance Exercise

PURPOSE: This study was to investigate effects of 24-hour fasting on human growth hormone (hGH) and cytokine responses after resistance exercise METHODS: Ten male students (Age; 24.7 ± 1.2 yr, Height; 174.1 ± 2.0 cm, Body Weight; 70.4 ± 1.5 kg) were randomly received to the control treatment (CON) and 24hrs fasting treatment (24-FT). The CON ingested the typical diet (about 400 kcal) prior to 3 hours and performed the resistance exercise, while 24-FT was fasting for 24 hours. The resistance exercise session consisted of squat exercise with 5 sets (8 RM). Blood samples were drawn immediately before (pre) and after (0) exercise and at 15min, 30 min, 60 min, 120 min, 180 min, and 48hrs after exercise. Plasma hGH, IGF-1, total testosterone, cortisol, free fatty acid (FFA), insulin and glucose levels, CK activity, cytokine (IL-6, IL-10, IL-1 B) were determined. RESULTS: Plasma hGH level in 24-FT was significantly increased at 0, 15, 30, 60 min (0.69±0.18, 2.32±0.71, 1.62±0.45, 1.29±0.43 μg/L, respectively) after exercise compared with the CON (0.31±0.04, 0.90±0.31, 0.41±0.08, 0.42±0.14 μg/L, respectively) (p<0.05), but hGH in 24-FT (0.23±0.05 μg/L) was significantly lower than CON at 180 min(0.72±0.23 μg/L) (p<0.05). IL-6 in the 24-FT (48.3±3.6 pg/ml) was significantly increased at 48h after exercise as compared with the CON (39.7±1.5 pg/ml) (p<0.05). CONCLUSIONS: We observed that the fasting did affect hGH releases for 60 min after exercise compared to typical diet in exercise, but this effect in hGH release was disappeared 3 hours after exercise. In addition, we assumed that IL-6 elevation at 48 h after exercise might be due to muscle injury which was caused by the fasting.

Development of mass transport resistance in poly(lactide-co-glycolide) films and particles – A mechanistic study

Poly( d, l-lactide-co-glycolide) (PLG) is the most frequently used biodegradable polymer in the controlled release of an encapsulated drug. The purpose of this work was to explain the surprisingly slow diffusion through this polymer, and locate the major source of mass transport resistance. Diffusion of human growth hormone (hGH) and glucose through PLG films was undetectable (using a diffusion cell), although the degraded polymer contained several times more water than polymer mass. In vitro release of hGH from PLG-coated particles also showed a surprisingly slow rate of release. Non-porous regions inside the PLG films were detected after three weeks of degradation using dextran-coupled fluorescent probes and confocal microscopy. The findings were supported by scanning electron microscopy. Diffusion through PLG films degraded for five weeks was significantly increased when the porosity of both surfaces was increased due to the presence of ZnCl 2 in the buffer the last 3 days of the degradation period. The results indicated high mass transport resistance inside the films after three weeks of degradation, and at the surfaces after five weeks of degradation. These results should also be applicable to microparticles of different sizes. Knowledge of the reason for transport resistance is important in the development of pharmaceuticals and when modifying the rate of drug release.

PKC‐dependent inhibition of CA2+‐dependent exocytosis from astrocytes

Astrocytes release various bioactive substances via Ca(2+) - and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)-dependent exocytosis; however the regulatory mechanisms of glial exocytosis are still poorly understood. In the present study, we investigated the effect of protein kinase C (PKC) on exocytosis in glial cells using primary cultured astrocytes and clonal rat glioma C6 cells. Mass spectrometry and Western blot analysis using phospho-specific antibodies revealed that phorbol 12-myristate 13-acetate (PMA) treatment induced the phosphorylation of synaptosomal-associated protein of 23 kDa (SNAP-23) on Ser(95), Ser(120), and Ser(160) in cultured astrocytes and C6 cells. Phosphorylation at these sites was suppressed by treatment with the PKC inhibitor, bisindolylmaleimide I (BIS). In contrast, Ser(110) of SNAP-23 was constitutively phosphorylated in these cells and was dephosphorylated in a PKC-dependent manner. Exogenously expressed human growth hormone (hGH) accumulated in cytoplasmic granular structures in cultured astrocytes, and its release after ATP-treatment was Ca(2+) - and SNARE-dependent. PMA treatment suppressed the ATP-induced hGH release from astrocytes and this inhibition was reversed by BIS. We also observed PMA-dependent suppression and an attenuation of that suppression by BIS in ionomycin-induced hGH release from C6 cells. These results suggest that intracellular activation of PKC suppresses Ca(2+) - and SNARE-dependent exocytosis in astroglial cells.

Sustained delivery of human growth hormone using a polyelectrolyte complex-loaded thermosensitive polyphosphazene hydrogel

A combined system of polyelectrolyte complex (PEC) and injectable, biodegradable and thermosensitive poly(organophosphazene) hydrogel has been suggested as an injectable depot for a controlled and sustained delivery of human growth hormone (hGH) to improve patient compliance. PEC was prepared by mixing polycations with hGH to suppress diffusion of hGH from the hydrogel through an enlargement of the hydrodynamic size of hGH. Among the polycations, poly- l-arginine (PLA) formed a large complex with hGH and its size increased as the amount of PLA increased. When PLA and/or zinc were added to hGH, the time-dependent stability of hGH increased more than that of native-hGH. The polymer solution containing PECs formed a gel at 37 °C. PLA decreased the initial release rate of hGH in proportion to the amount of PLA in vitro and in vivo. Zinc increased the released amount of hGH from the PEC-loaded hydrogel in vitro and in vivo. In a pharmacokinetic study in rats, a single administration of PEC-loaded hydrogel resulted in the sustained release of hGH for 5 days. These results suggest that injectable, biodegradable, and thermosensitive PEC-loaded poly(organophosphazene) hydrogel has great potential to be used as an effective delivery system for a sustained release of hGH with improved patient compliance.

Single-File Diffusion of Protein Drugs through Cylindrical Nanochannels

A new drug delivery device using cylindrical block copolymer nanochannels was successfully developed for controlled protein drug delivery applications. Depending on the hydrodynamic diameter of the protein drugs, the pore size in cylindrical nanochannels could be controlled precisely down to 6 nm by Au deposition. Zero-order release of bovine serum albumin (BSA) and human growth hormone (hGH) by single-file diffusion, which has been observed for gas diffusion through zeolite pores, was realized up to 2 months without protein denaturation. Furthermore, a nearly constant in vivo release of hGH from the drug delivery nanodevice implanted to Sprague-Dawley (SD) rats was continued up to 3 weeks, demonstrating the feasibility for long-term controlled delivery of therapeutic protein drugs.