Life Sciences 96 (2014) 1–6
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Polymeric delivery systems for dexamethasone Justyna Urbańska, Anna Karewicz ⁎, Maria Nowakowska Faculty of Chemistry, Jagiellonian University, 30-060 Kraków, Ingardena 3, Poland
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Article history: Received 7 October 2013 Accepted 13 December 2013 Keywords: Glucocorticoids Drug Carrier
a b s t r a c t Glucocorticoids (GCs) are broadly used in the treatment of inflammation and in suppressing hyperactivity of the immune system expressed in allergies, asthma, autoimmune diseases and sepsis. They are pleiotropic in nature, showing a wide range of diverse effects, including those which are harmful for the organism. Dexamethasone (DEX) is one of the most frequently used GCs and is considered as one of the safest. Still serious side-effects have been observed for this drug, mostly due to its hydrophobicity and low bioavailability. The potentially promising polymeric carrier systems to deliver DEX effectively are revised. © 2013 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Molecular mechanisms of glucocorticoid action . . . . . . . . Polymer–dexamethasone conjugates and other polymeric carriers Poly(ethylene glycol) conjugates (PEG) . . . . . . . . . Poly(D,L-lactic-co-glycolic acid) (PLGA) . . . . . . . . . Chitosan . . . . . . . . . . . . . . . . . . . . . . . Chitosan scaffolds . . . . . . . . . . . . . . . . . . . Chitosan films . . . . . . . . . . . . . . . . . . . . Chitosan microspheres . . . . . . . . . . . . . . . . . Chitosan derivatives . . . . . . . . . . . . . . . . . . Other polymer DEX nanocarriers . . . . . . . . . . . . DEX–polymer conjugates . . . . . . . . . . . . . . . Safety considerations . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction Glucocorticoids (GCs) are steroid hormones effecting immunological and metabolic functions of the human body by their binding to the glucocorticoid receptor (GR). Their main action is related to restraining the inflammatory reactions while providing immunosuppressant effect. Dexamethasone (Fig. 1) was obtained by modifying the structure of cortisol through the introduction of the 9-α-fluoro group and a 16-α⁎ Corresponding author at: Ingardena 3, 30-060 Kraków, Poland. Tel.: +48 12 663 20 83; fax: +48 12 634 05 15. E-mail address: [email protected] (A. Karewicz). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.020
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methyl substituent and an extra double bond between carbon 1 and 2 in the A-ring. DEX binds to GR more efficiently than cortisol; the presence of the fluorine atom makes it more lipophilic, while the methyl group bound to the carbon C-16 increases its affinity to the mineralocorticoid receptor (Rang et al., 2012). Dexamethasone is currently used in many biomedical applications, such as: cell culture (to promote differentiation of the mesenchymal stem cells) (Koehler et al., 2013), ophthalmology (for the treatment of acute and chronic inflammatory posterior segment eye diseases, i.e. uveitis) (Chennamaneni et al., 2013), proliferative vitreoretinopathy, subretinal neovascularization, and diabetic macular edema. DEX is also used in allergology, due to its ability to inhibit functions of lymphocytes,
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Fig. 1. Structure of dexamethasone (DEX).
fibroblasts, macrophages and other immune cells. It also found its use in pediatric hematology in acute lymphoblastic leukemia treatment due to its ability to induce the apoptosis of lymphocytes B and T (Fratoddi et al., 2012; Gómez-Gaete et al., 2007). Despite many advantages, using dexamethasone in pharmaceutical formulations has also an essential limitation —it is highly hydrophobic in nature and high doses are necessary to reach its therapeutic level. That results in the undesired side effects, such as osteoporosis (Hurson et al., 2007), high sugar concentrations in the blood, hypertension (Goodwin et al., 2011), stomach and intestinal bleeding due to ulcers, and fluid retention (Rhen and Cidlowski, 2005). Application of DEX in ophthalmology, as drops and eye ointments, requires frequent use, causing inconvenience for patients. Moreover, in the case of an unskilled application, it may lead to corneal micro-trauma. Sodium salt of DEX was recommended as the hydrophilic alternative, but its use can lead to sodium overdosing. Broad spectrum of the side effects mentioned above shows clearly the necessity of finding a new, better DEX formulation, even more so, when one takes into consideration that DEX is still one of the safest GC drugs. A diversity of delivery systems was proposed, majority of them based on both natural and synthetic polymers. Some of the most important will be discussed in this short review.
(Stahn et al., 2007; Buckingham, 2006). Significant part of endogenic glucocorticoids released to blood vessels binds with the specific globulins while the exogenic GCs injected intravenously are connecting mostly with albumins (Stahn et al., 2007; Torpy and Ho, 2007). Two molecular mechanisms of GC action are known: the classical genomic mechanism and the non-genomic one. Genomic mechanism of GC action starts from binding of the molecule with the receptor, which leads to receptor's dissociation from the chaperone complex and translocation into the nucleus. There it is acting as positive or negative transcription factor (Stahn et al., 2007; Rang et al., 2012). Translocation of glucocorticoid–receptor complex from cytosol to nucleus can be inhibited by phosphorylation, ubiquitination, SUMOylation or acetylation (Fratoddi et al., 2012). Non-genomic mechanism of glucocorticoid action is presumably based on its nonspecific interaction with cell membrane. That results in the changes in the biomembranes' physicochemical properties —permeability of the membrane is changing, lipid peroxidation occurs, the circulation of Na+ and Ca2+ slows down, and proton outflow from mitochondria increases (Stahn et al., 2007). An alternative hypothesis regarding non-genomic mechanism of GC action assumes specific binding of glucocorticoid to the membrane receptor (Fig. 3). GR can promote apoptosis or survival in a cell-specific manner. It is able to induce apoptosis in lymphocytes, leukemic, lymphoma, and multiple myeloma cells (Distelhorst, 2002). In the same time it can inhibit apoptosis in other cell types such as hepatocytes, vascular endothelial cells, osteoclasts or mammary epithelial cells (Wu et al., 2004). Results suggest that GR agonists can promote expression of multiple factors (SGK1, MKP1, PKCε) awarding apoptosis protection (Aziz et al., 2012). DEX is a well-known activator of many signal transduction and molecular gene-regulation pathways. It frequently involves nuclear receptors, including pregnane X receptor (PXR) and progesterone receptor (PR) (Leo et al., 2004). It is also efficient at activating the adrenal steroid (AS) receptor, more so in the case of the type II than the type I AS receptor. This may explain the difference between the high affinity of DEX for the type I receptor in vitro, and its low efficacy as a type I agonist in vivo.
Molecular mechanisms of glucocorticoid action Glucocorticoid's action mechanism is based on binding the molecule to the intracellular nuclear receptor (ligand-dependent transcription factor) (Stahn et al., 2007; Germain et al., 2006). Glucocorticoid receptor (GR) is composed of N-terminal transactivation domain (NTD) containing the domain which takes part in regulation of the transcription process — Activation Function domain 1 (AF-1), responsible for dimerization and binding of receptor to DNA and DNA-binding domain, possessing two cysteine-rich zinc fingers. Ligand binding domain (LBD) is responsible for translocation of the receptor–ligand complex into the nucleus. One of LBD's components is a domain responsible for binding ligands, aiding in dimerization and interacting with the protecting proteins —Activation Function domain 2-(AF-2) (Fig. 2). Binding the ligand by GR is followed by the change in AF-2 conformation, which enables interactions with other factors taking part in both: the activation and the inhibition of transcription. Glucocorticoids can also activate mineralocorticoids' receptors
Fig. 2. Scheme of basic domains of glucocorticoid binding receptor.
Fig. 3. Schematic illustration of the proposed molecular mechanisms of GC action. A—genomic effect: binding of GC molecule with receptor. A1—receptor's dissociation from the chaperone complex. A2—translocation of the GC–receptor complex into nucleus. B and C —non-genomic effect: B—binding of GC molecule with membrane receptor. C—non-specific reaction of GC with membrane.
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This may also be a reason why DEX is a much more potent glucocorticoid than cortisol, even though in vitro affinity of both compounds for the type II receptor is similar (Spencer et al., 1990). DEX has no apparent in vivo mineralocorticoid receptor agonistic activity (Rafiq et al., 2011). Polymer–dexamethasone conjugates and other polymeric carriers Poly(ethylene glycol) conjugates (PEG) The main advantage of PEG–drug conjugates is related to the fact that they are approved by FDA to be used in the clinical applications. However, a linear PEG polymer possesses only terminal functional groups at the end of the polymer chain, which leads to low concentration of the conjugated drug. In order to overcome this limitation the synthesis of branched and multi-arm PEGs has been proposed. Such macromolecules can be loaded with three-fold more drug molecules compared to the linear PEG, and additionally they exhibit increased biodegradability and solubility in the aqueous media (Larson and Ghandehari, 2012; Liu et al., 2010). A linear multifunctional PEG–DEX conjugate for the treatment of rheumatoid arthritis was synthesized using a click reaction. DEX was conjugated to the click PEG via an acid-labile hydrazine bond to allow the drug release under the pathological conditions. The biological studies were performed using a rat model. The in vitro release profile of DEX (135 mg of DEX per 1 g of conjugate) determined at pH = 5.0 (characteristic for inflammation) was linear. About 9% of drug was released after 17 days (~ 0.5% per day), as demonstrated using HPLC technique. However, at pH = 7.4 (characteristic for healthy tissues) no release of DEX was observed even after 17 days (Fig. 4). Studies carried out in vivo have shown that BMD (bone mineral density) indicator for rats with rheumatoid arthritis, which have been receiving PEG–DEX treatment, was much better than the BMD of the control groups, receiving either placebo or free (not conjugated) DEX. These results were confirmed by histological analysis, clearly demonstrating the therapeutic activity of PEG–DEX (Liu et al., 2010). The branched multimeric PEG was also used to form a conjugate bearing two drugs of the synergic effect: dexamethasone and theophylline (Zacchigna et al., 2009). A slow degradation rate in simulated gastro-intestinal fluids was reported for this conjugate, while in plasma a more rapid breakdown was observed. By using a photo-initiated thiol–ene polymerization chemistry (Hoyle and Bowman, 2010) it is possible to covalently tether glucocorticoids onto the synthetic scaffolds (e.g. peptide functionalized PEG hydrogels (Yang et al., 2012)) that provide enzymatic release of the GC. DEX-conjugated 6-lauroxyhexyl ornithinate lipid was used to form gene-loaded cationic solid lipid nanoparticles (SLNs), which were subsequently surface-modified with polyethylene glycolphosphatidylethanolamine transferrin conjugate (Tf-PEG-PE) (Wang
Fig. 4. Cumulative DEX release from the PEG–DEX conjugate at various pH values. Reprinted with permission from Liu et al. (2010). Copyright (2010) American Chemical Society.
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et al., 2012). Transferrin is a well-studied ligand for delivering anticancer drugs/genes, due to the increased number of its receptors found on tumor cells. A model gene, pEGFP (enhanced green fluorescence protein) plasmid, was incorporated into Tf-PEG-PE-modified SLNs and its expression in HepG2 solid tumor was examined in mice. Authors have shown that the presence of both: transferrin and dexamethasone significantly enhances transfection efficiency both in vitro and in vivo. A similar, lipidbased, PEG-containing system was successfully applied by Mao et al. (2013) for therapeutic DEX delivery in CD74+ B-cell malignancies. Distearoylphosphatidylethanolamine–methoxypolyethylene glycol– maleimide (DSPE–PEG–Mal) liposomes were incubated with DEX and then reacted to anti-CD74-SH to yield CD74-targeted liposomal dexamethasone. The system showed high in vitro and in vivo efficiency, superior to the non-targeted and control formulations. A microparticulate delivery system for DEX was also reported in literature, where poly(ethylene glycol) was used as a plasticizer. The microcores consisting of the dexamethasone adsorbed onto the colloidal silicone dioxide were coated via spray-drying with Eudragit-based nanoparticles either in the presence or in the absence of PEG. That resulted in obtaining nanoparticle-coated microparticles (NpCMp) schematically shown in Fig. 5. The microparticulate system synthetized in the presence of PEG showed more internal localization of DEX and slower release rates (Beck et al., 2007). Poly(D,L-lactic-co-glycolic acid) (PLGA) PLGA is biocompatible and biodegradable, can exhibit a wide range of possible degradation times, has tunable mechanical properties and most importantly, is a FDA approved polymer. PLGA nanoparticles have been most frequently used for ocular drug delivery due to their low ocular toxicity. DEX can be encapsulated in PLGA nanoparticles via solvent emulsion–evaporation technique. Studies have shown that these nanocapsules are releasing the DEX load within around 4 hours (Gómez-Gaete et al., 2007). It has been also demonstrated that it is possible to constantly, slowly release DEX for over 30 days from the PLGA scaffolds fabricated by a gas-foaming/salt-leaching method, while preserving the anti-inflammatory activity of the drug, and inhibiting the proliferation of smooth muscle cells cultured within scaffolds (Yoon et al. 2003) The release of DEX from PLGA nanoparticles embedded in alginate hydrogel was also investigated. It has been shown that impedance's amplitude of microelectrodes coated with DEX–PLGA nanoparticles in alginate matrix was maintained up to 3 weeks, nearly at its initial level, and DEX was sustainably released over that period of time, proving the usefulness of such system to locally administer DEX in neural electrodes (Kim and Martin, 2006). PLGA microcapsules loaded with bupivacaine and dexamethasone (approximately 72% free base with 0.04% of DEX) were also prepared and tested on human volunteers (Kopacz et al., 2003a). Dexamethasone has been found to prolong the duration of local anesthetic action of bupivacaine in human (Kopacz et al., 2003a,b). It was concluded that subcutaneous injection of dexamethasone and bupivacaine-containing PLGA microcapsules induced a prolonged duration of skin anesthesia and analgesia in humans (Kopacz et al., 2003b).
Fig. 5. Structure of NpCMp.
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Studies on localized delivery of growth factors using implantable drug delivery systems proved that concurrent release of DEX and VEGF from PLGA microspheres/PVA hydrogel composites effectively minimize inflammation, inhibit fibrosis and promote neoangiogenesis at the implant site at a fraction of the typical oral parental intravascular bolus doses (Patil et al., 2007). Moreover the PLGA microspheres loaded with DEX and growth factors provided an ideal environment for the proliferation of transplanted mesenchymal stem cells Park et al. (2009). A more complex system comprising of DEX-loaded PLGA microspheres coated with heparin-bound bFGF-loaded nanoparticles was evaluated as a cell carrier for the regeneration of intervertebral disk (Liang et al., 2012). Both bioactive factors, DEX and bFGF, were released continuously from this system for 4 weeks. The microbeads showed no cytotoxicity to rat mesenchymal stem cells (rMSCs) and promoted rMSC growth and differentiation into nucleus pulposus-like cells. Additionally they also reduced the inflammatory response. The system shows potential as an implantable scaffold for the intervertebral disk regeneration, a step further compared to the current short-term therapies addressed mostly to the treatment of symptoms. PLGA, and some other polymers e.g. 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) and hyaluronic acid (HA) can be used in Trojan particle formation, from which drug is released in three different steps demonstrated schematically in Fig. 6. The release from such a system is considerably slower than that from the regular polymer nanocapsules (Gómez-Gaete et al., 2008). In 2010 dexamethasone intravitreal implant containing PLGA (Chan et al., 2011), Ozurdex®, was officially approved to be used in all EU countries as a sustained-release for the treatment of macular edema, following branch retinal vein occlusion (BRVO) or central retinal vein occlusion (CRVO). Based on in vivo research done by Chang-Lin et al. (2011) on 34 male monkeys DEX released from the implant was detected in the retina and vitreous humor for 6 months without a need for reinjections. Further clinical trials have shown that, in the short term, the implant improves vision and decreases the risk of vision loss already after first application, however, for most CRVO patients repeat intervention is necessary (Chan et al., 2011).
Chitosan scaffolds Chitosan can be used in tissue engineering for the preparation of scaffolds. DEX was encapsulated in chitosan-based bone scaffolds in order to reduce local inflammation or to induce the differentiation of mesenchymal stem cells into osteoblasts. As an alternative, DEX can be also immobilized on the chitosan scaffold's surface by covalent bond formation (Chiang et al., 2012). DEX was also trapped inside the chitosan scaffolds obtained using supercritical fluid and its sustained release from these materials was observed (Duarte et al., 2009). The yield of impregnation using supercritical fluid was not very high but the concentration of the released DEX was found to be sufficient for tissue engineering applications.
Chitosan films Studies on the chitosan films loaded with DEX have indicated that interactions between drug molecules and polymer chains are more efficient for a bilayer than for a monolayer. Drug release occurred faster in the monolayer —after 8 hours only 10.4% of the drug was left in the material, while in the case of bilayer 16% of the drug still remained in the material after four weeks. For both types of chitosan films the release was much slower than for the conventionally used ophthalmic steroid drops (Rodrigues et al., 2009).
Chitosan microspheres Dexamethasone sodium phosphate encapsulated in chitosan microspheres was tested in brain edema after cold injury using a female rat model. The experiments confirmed the effectiveness of DEX in the treatment of brain edema after thermal trauma. Moreover, encapsulating DEX in the chitosan microspheres allowed increasing therapeutic effect and minimizing side effects due to prolonged release and lower local concentration of DEX (Turkoglu et al., 2005).
Chitosan This biodegradable natural polymer is widely used in biomedical sciences, especially in bioengineering due to its ability to form films, scaffolds and microparticles, which are also applicable in drug delivery (Duarte et al., 2009). Some modifications are also widely studied, such as sulfonated chitosan (Bulwan et al., 2009), quaternary ammonium–chitosan (Karewicz et al., 2013), thiolated quaternary ammonium–chitosan (Zambito and Di Colo, 2010) or carboxymethylchitosan/poly(amidoamine) dendrimer (Oliveira et al., 2009, 2010).
Chitosan derivatives Thiolated quaternary ammonium–chitosan was found to be a better DEX carrier in ophthalmologic formulations due to the enhanced transcorneal absorption (Zambito and Di Colo, 2010) while the carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles loaded with dexamethasone were shown to promote the osteogenic differentiation of rat bone marrow stroma cells (RBMSCs) due to higher calcium deposition (Oliveira et al., 2009, 2010).
Fig. 6. Schematic representation of DEX release from Trojan particles: A —Trojan particles before hydration, B —fast release of the free fraction of DEX associated to the DPPC-HA matrix and slow release of the PLGA nanoparticles loaded with DEX, and C —free PLGA nanoparticles release DEX while DPPC-HA matrix slowly releases these PLGA nanoparticles which are still trapped.
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Other polymer DEX nanocarriers Poly(ε-caprolactone) may be used to create intravitreal implants loaded with DEX. The in vitro release profile does not show any significant burst release, which often occurs for other polymeric nanoparticles loaded with DEX (Fialho et al., 2008). Nanoparticles of amphiphilic monomethoxy poly(ethylene glycol)-blockpoly(trimethylene carbonate) (mPEG–PTMC) were prepared by either salting out or emulsion polymerization. DEX loading efficiency was found to be 88% for those obtained by salting out and 72% for the nanoparticles produced by the emulsion method. The release of DEX from these nanoparticles was shown to be diffusion-controlled and was sustained for up to 60 days. Depending on the nature of the polymer employed and the preparation method, dexamethasone diffusion coefficients varied between 4.8 × 10−18 and 22.6 × 10−18 cm2/s (Zhang et al., 2006). Biodegradable nanocapsules consisting of a hydroxyethylated glucose polymer (hydroxyethyl starch) shell with encapsulated DEX were recently synthesized and it was shown that they can deliver drug in a targeted manner to the selected liver cells. When they were incorporated into the Kupffer cells the significant suppression of the release of inflammatory cytokines was observed (Fichter et al., 2013). DEX–polymer conjugates Water-soluble DEX–polymer conjugates can be also obtained. Dexamethasone-21-mesylate was attached to the low molecular weight (2 kDa) polyethylenimine (PEI) forming water-soluble lipopolymer. The resulting conjugate showed no toxicity to HEK 293 and HepG2 cells in the 10–180 μg/ml concentration range. The conjugate acted as a prodrug, with PEI chain enhancing the solubility of dexamethasone. The obtained lipopolymer was tested as a nucleus-targeting gene carrier (Bae et al., 2007). DEX can dilate the nuclear pore complexes and translocate into the nucleus when it is bound to its glucocorticoid receptor, thus facilitating the transport of DNA into the nucleus. In vitro transfection experiments have shown that indeed PEI–DEX/DNA complex had higher gene delivery efficiency than PEI/DNA. (Linear PEI)–DEX conjugates were also found to be useful in non-viral gene therapy applications (Kim et al., 2010). DEX conjugated with polyamidoamine (PAMAM) dendrimer was also reported (Choi et al., 2006). PAMAM–DEX, similarly to PEI–DEX, showed no toxicity to HEK 93 cells and enhanced transfection efficiency. Safety considerations With few exceptions, the polymers used in the discussed drug delivery systems for dexamethasone were already well known for their biocompatibility and lack of side effects. The only concern could involve the use of PEI and dendrimers, however the in vitro tests revealed no significant toxicity of these formulations against HEK 293 cells. Unfortunately no in vivo experiments with these systems were performed yet. It then came as no surprise that no toxicity or serious side-effects of the discussed new DEX delivery systems were reported so far. Several reports on polylactic acid (PLA) (Li et al., 2013), poly-ε-caprolactone (PCL) (Fialho et al., 2008) and PLGA (Peng et al., 2010) implants releasing dexamethasone confirmed the safety of their application in vivo, pointing out only at mild, transient inflammatory effects caused by the implantation procedure. Summary A wide variety of polymeric carriers have been used to deliver dexamethasone in a controlled manner, including micro and nanocapsules, scaffolds, hydrogels and conjugates. Most of the systems discussed in this review were designed to provide a sustained release of dexamethasone leading to an increase in its therapeutic effect while minimizing side-effects. The formulation used depended mostly on the requested
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delivery profile and administration route. Effectiveness of several polymeric carriers has been proven in in vivo experiments —the designed delivery systems were characterized by superior and longer-lasting effects as well as favorable safety profile compared with systemically administered free DEX. No toxicity or serious side-effects of the new DEX delivery systems were reported. Currently the most successful applications of DEX delivery systems were developed in the field of ophthalmology, as intravitreal implants. One of the proposed solutions, PLGA implant, is already in regular clinical use at the commercial name Ozurdex® to treat adults with swelling of the macula (macular edema) following BRVO or CRVO or noninfectious inflammation of the uvea (uveitis) affecting the back segment of the eye. Conjugates with PEI and dendrimers were proposed as efficient non-viral gene delivery devices, though the studies on these formulations have not reached yet the clinical trial phase. The problem of targeted DEX delivery was also addressed. Conflict of interest statement The authors declare that there are no conflicts of interest.
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Polymeric delivery systems for dexamethasone Justyna Urbańska, Anna Karewicz ⁎, Maria Nowakowska Faculty of Chemistry, Jagiellonian University, 30-060 Kraków, Ingardena 3, Poland
a r t i c l e
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Article history: Received 7 October 2013 Accepted 13 December 2013 Keywords: Glucocorticoids Drug Carrier
a b s t r a c t Glucocorticoids (GCs) are broadly used in the treatment of inflammation and in suppressing hyperactivity of the immune system expressed in allergies, asthma, autoimmune diseases and sepsis. They are pleiotropic in nature, showing a wide range of diverse effects, including those which are harmful for the organism. Dexamethasone (DEX) is one of the most frequently used GCs and is considered as one of the safest. Still serious side-effects have been observed for this drug, mostly due to its hydrophobicity and low bioavailability. The potentially promising polymeric carrier systems to deliver DEX effectively are revised. © 2013 Elsevier Inc. All rights reserved.
Contents Introduction . . . . . . . . . . . . . . . . . . . . . . . . Molecular mechanisms of glucocorticoid action . . . . . . . . Polymer–dexamethasone conjugates and other polymeric carriers Poly(ethylene glycol) conjugates (PEG) . . . . . . . . . Poly(D,L-lactic-co-glycolic acid) (PLGA) . . . . . . . . . Chitosan . . . . . . . . . . . . . . . . . . . . . . . Chitosan scaffolds . . . . . . . . . . . . . . . . . . . Chitosan films . . . . . . . . . . . . . . . . . . . . Chitosan microspheres . . . . . . . . . . . . . . . . . Chitosan derivatives . . . . . . . . . . . . . . . . . . Other polymer DEX nanocarriers . . . . . . . . . . . . DEX–polymer conjugates . . . . . . . . . . . . . . . Safety considerations . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest statement . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .
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Introduction Glucocorticoids (GCs) are steroid hormones effecting immunological and metabolic functions of the human body by their binding to the glucocorticoid receptor (GR). Their main action is related to restraining the inflammatory reactions while providing immunosuppressant effect. Dexamethasone (Fig. 1) was obtained by modifying the structure of cortisol through the introduction of the 9-α-fluoro group and a 16-α⁎ Corresponding author at: Ingardena 3, 30-060 Kraków, Poland. Tel.: +48 12 663 20 83; fax: +48 12 634 05 15. E-mail address: [email protected] (A. Karewicz). 0024-3205/$ – see front matter © 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.lfs.2013.12.020
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methyl substituent and an extra double bond between carbon 1 and 2 in the A-ring. DEX binds to GR more efficiently than cortisol; the presence of the fluorine atom makes it more lipophilic, while the methyl group bound to the carbon C-16 increases its affinity to the mineralocorticoid receptor (Rang et al., 2012). Dexamethasone is currently used in many biomedical applications, such as: cell culture (to promote differentiation of the mesenchymal stem cells) (Koehler et al., 2013), ophthalmology (for the treatment of acute and chronic inflammatory posterior segment eye diseases, i.e. uveitis) (Chennamaneni et al., 2013), proliferative vitreoretinopathy, subretinal neovascularization, and diabetic macular edema. DEX is also used in allergology, due to its ability to inhibit functions of lymphocytes,
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Fig. 1. Structure of dexamethasone (DEX).
fibroblasts, macrophages and other immune cells. It also found its use in pediatric hematology in acute lymphoblastic leukemia treatment due to its ability to induce the apoptosis of lymphocytes B and T (Fratoddi et al., 2012; Gómez-Gaete et al., 2007). Despite many advantages, using dexamethasone in pharmaceutical formulations has also an essential limitation —it is highly hydrophobic in nature and high doses are necessary to reach its therapeutic level. That results in the undesired side effects, such as osteoporosis (Hurson et al., 2007), high sugar concentrations in the blood, hypertension (Goodwin et al., 2011), stomach and intestinal bleeding due to ulcers, and fluid retention (Rhen and Cidlowski, 2005). Application of DEX in ophthalmology, as drops and eye ointments, requires frequent use, causing inconvenience for patients. Moreover, in the case of an unskilled application, it may lead to corneal micro-trauma. Sodium salt of DEX was recommended as the hydrophilic alternative, but its use can lead to sodium overdosing. Broad spectrum of the side effects mentioned above shows clearly the necessity of finding a new, better DEX formulation, even more so, when one takes into consideration that DEX is still one of the safest GC drugs. A diversity of delivery systems was proposed, majority of them based on both natural and synthetic polymers. Some of the most important will be discussed in this short review.
(Stahn et al., 2007; Buckingham, 2006). Significant part of endogenic glucocorticoids released to blood vessels binds with the specific globulins while the exogenic GCs injected intravenously are connecting mostly with albumins (Stahn et al., 2007; Torpy and Ho, 2007). Two molecular mechanisms of GC action are known: the classical genomic mechanism and the non-genomic one. Genomic mechanism of GC action starts from binding of the molecule with the receptor, which leads to receptor's dissociation from the chaperone complex and translocation into the nucleus. There it is acting as positive or negative transcription factor (Stahn et al., 2007; Rang et al., 2012). Translocation of glucocorticoid–receptor complex from cytosol to nucleus can be inhibited by phosphorylation, ubiquitination, SUMOylation or acetylation (Fratoddi et al., 2012). Non-genomic mechanism of glucocorticoid action is presumably based on its nonspecific interaction with cell membrane. That results in the changes in the biomembranes' physicochemical properties —permeability of the membrane is changing, lipid peroxidation occurs, the circulation of Na+ and Ca2+ slows down, and proton outflow from mitochondria increases (Stahn et al., 2007). An alternative hypothesis regarding non-genomic mechanism of GC action assumes specific binding of glucocorticoid to the membrane receptor (Fig. 3). GR can promote apoptosis or survival in a cell-specific manner. It is able to induce apoptosis in lymphocytes, leukemic, lymphoma, and multiple myeloma cells (Distelhorst, 2002). In the same time it can inhibit apoptosis in other cell types such as hepatocytes, vascular endothelial cells, osteoclasts or mammary epithelial cells (Wu et al., 2004). Results suggest that GR agonists can promote expression of multiple factors (SGK1, MKP1, PKCε) awarding apoptosis protection (Aziz et al., 2012). DEX is a well-known activator of many signal transduction and molecular gene-regulation pathways. It frequently involves nuclear receptors, including pregnane X receptor (PXR) and progesterone receptor (PR) (Leo et al., 2004). It is also efficient at activating the adrenal steroid (AS) receptor, more so in the case of the type II than the type I AS receptor. This may explain the difference between the high affinity of DEX for the type I receptor in vitro, and its low efficacy as a type I agonist in vivo.
Molecular mechanisms of glucocorticoid action Glucocorticoid's action mechanism is based on binding the molecule to the intracellular nuclear receptor (ligand-dependent transcription factor) (Stahn et al., 2007; Germain et al., 2006). Glucocorticoid receptor (GR) is composed of N-terminal transactivation domain (NTD) containing the domain which takes part in regulation of the transcription process — Activation Function domain 1 (AF-1), responsible for dimerization and binding of receptor to DNA and DNA-binding domain, possessing two cysteine-rich zinc fingers. Ligand binding domain (LBD) is responsible for translocation of the receptor–ligand complex into the nucleus. One of LBD's components is a domain responsible for binding ligands, aiding in dimerization and interacting with the protecting proteins —Activation Function domain 2-(AF-2) (Fig. 2). Binding the ligand by GR is followed by the change in AF-2 conformation, which enables interactions with other factors taking part in both: the activation and the inhibition of transcription. Glucocorticoids can also activate mineralocorticoids' receptors
Fig. 2. Scheme of basic domains of glucocorticoid binding receptor.
Fig. 3. Schematic illustration of the proposed molecular mechanisms of GC action. A—genomic effect: binding of GC molecule with receptor. A1—receptor's dissociation from the chaperone complex. A2—translocation of the GC–receptor complex into nucleus. B and C —non-genomic effect: B—binding of GC molecule with membrane receptor. C—non-specific reaction of GC with membrane.
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This may also be a reason why DEX is a much more potent glucocorticoid than cortisol, even though in vitro affinity of both compounds for the type II receptor is similar (Spencer et al., 1990). DEX has no apparent in vivo mineralocorticoid receptor agonistic activity (Rafiq et al., 2011). Polymer–dexamethasone conjugates and other polymeric carriers Poly(ethylene glycol) conjugates (PEG) The main advantage of PEG–drug conjugates is related to the fact that they are approved by FDA to be used in the clinical applications. However, a linear PEG polymer possesses only terminal functional groups at the end of the polymer chain, which leads to low concentration of the conjugated drug. In order to overcome this limitation the synthesis of branched and multi-arm PEGs has been proposed. Such macromolecules can be loaded with three-fold more drug molecules compared to the linear PEG, and additionally they exhibit increased biodegradability and solubility in the aqueous media (Larson and Ghandehari, 2012; Liu et al., 2010). A linear multifunctional PEG–DEX conjugate for the treatment of rheumatoid arthritis was synthesized using a click reaction. DEX was conjugated to the click PEG via an acid-labile hydrazine bond to allow the drug release under the pathological conditions. The biological studies were performed using a rat model. The in vitro release profile of DEX (135 mg of DEX per 1 g of conjugate) determined at pH = 5.0 (characteristic for inflammation) was linear. About 9% of drug was released after 17 days (~ 0.5% per day), as demonstrated using HPLC technique. However, at pH = 7.4 (characteristic for healthy tissues) no release of DEX was observed even after 17 days (Fig. 4). Studies carried out in vivo have shown that BMD (bone mineral density) indicator for rats with rheumatoid arthritis, which have been receiving PEG–DEX treatment, was much better than the BMD of the control groups, receiving either placebo or free (not conjugated) DEX. These results were confirmed by histological analysis, clearly demonstrating the therapeutic activity of PEG–DEX (Liu et al., 2010). The branched multimeric PEG was also used to form a conjugate bearing two drugs of the synergic effect: dexamethasone and theophylline (Zacchigna et al., 2009). A slow degradation rate in simulated gastro-intestinal fluids was reported for this conjugate, while in plasma a more rapid breakdown was observed. By using a photo-initiated thiol–ene polymerization chemistry (Hoyle and Bowman, 2010) it is possible to covalently tether glucocorticoids onto the synthetic scaffolds (e.g. peptide functionalized PEG hydrogels (Yang et al., 2012)) that provide enzymatic release of the GC. DEX-conjugated 6-lauroxyhexyl ornithinate lipid was used to form gene-loaded cationic solid lipid nanoparticles (SLNs), which were subsequently surface-modified with polyethylene glycolphosphatidylethanolamine transferrin conjugate (Tf-PEG-PE) (Wang
Fig. 4. Cumulative DEX release from the PEG–DEX conjugate at various pH values. Reprinted with permission from Liu et al. (2010). Copyright (2010) American Chemical Society.
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et al., 2012). Transferrin is a well-studied ligand for delivering anticancer drugs/genes, due to the increased number of its receptors found on tumor cells. A model gene, pEGFP (enhanced green fluorescence protein) plasmid, was incorporated into Tf-PEG-PE-modified SLNs and its expression in HepG2 solid tumor was examined in mice. Authors have shown that the presence of both: transferrin and dexamethasone significantly enhances transfection efficiency both in vitro and in vivo. A similar, lipidbased, PEG-containing system was successfully applied by Mao et al. (2013) for therapeutic DEX delivery in CD74+ B-cell malignancies. Distearoylphosphatidylethanolamine–methoxypolyethylene glycol– maleimide (DSPE–PEG–Mal) liposomes were incubated with DEX and then reacted to anti-CD74-SH to yield CD74-targeted liposomal dexamethasone. The system showed high in vitro and in vivo efficiency, superior to the non-targeted and control formulations. A microparticulate delivery system for DEX was also reported in literature, where poly(ethylene glycol) was used as a plasticizer. The microcores consisting of the dexamethasone adsorbed onto the colloidal silicone dioxide were coated via spray-drying with Eudragit-based nanoparticles either in the presence or in the absence of PEG. That resulted in obtaining nanoparticle-coated microparticles (NpCMp) schematically shown in Fig. 5. The microparticulate system synthetized in the presence of PEG showed more internal localization of DEX and slower release rates (Beck et al., 2007). Poly(D,L-lactic-co-glycolic acid) (PLGA) PLGA is biocompatible and biodegradable, can exhibit a wide range of possible degradation times, has tunable mechanical properties and most importantly, is a FDA approved polymer. PLGA nanoparticles have been most frequently used for ocular drug delivery due to their low ocular toxicity. DEX can be encapsulated in PLGA nanoparticles via solvent emulsion–evaporation technique. Studies have shown that these nanocapsules are releasing the DEX load within around 4 hours (Gómez-Gaete et al., 2007). It has been also demonstrated that it is possible to constantly, slowly release DEX for over 30 days from the PLGA scaffolds fabricated by a gas-foaming/salt-leaching method, while preserving the anti-inflammatory activity of the drug, and inhibiting the proliferation of smooth muscle cells cultured within scaffolds (Yoon et al. 2003) The release of DEX from PLGA nanoparticles embedded in alginate hydrogel was also investigated. It has been shown that impedance's amplitude of microelectrodes coated with DEX–PLGA nanoparticles in alginate matrix was maintained up to 3 weeks, nearly at its initial level, and DEX was sustainably released over that period of time, proving the usefulness of such system to locally administer DEX in neural electrodes (Kim and Martin, 2006). PLGA microcapsules loaded with bupivacaine and dexamethasone (approximately 72% free base with 0.04% of DEX) were also prepared and tested on human volunteers (Kopacz et al., 2003a). Dexamethasone has been found to prolong the duration of local anesthetic action of bupivacaine in human (Kopacz et al., 2003a,b). It was concluded that subcutaneous injection of dexamethasone and bupivacaine-containing PLGA microcapsules induced a prolonged duration of skin anesthesia and analgesia in humans (Kopacz et al., 2003b).
Fig. 5. Structure of NpCMp.
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Studies on localized delivery of growth factors using implantable drug delivery systems proved that concurrent release of DEX and VEGF from PLGA microspheres/PVA hydrogel composites effectively minimize inflammation, inhibit fibrosis and promote neoangiogenesis at the implant site at a fraction of the typical oral parental intravascular bolus doses (Patil et al., 2007). Moreover the PLGA microspheres loaded with DEX and growth factors provided an ideal environment for the proliferation of transplanted mesenchymal stem cells Park et al. (2009). A more complex system comprising of DEX-loaded PLGA microspheres coated with heparin-bound bFGF-loaded nanoparticles was evaluated as a cell carrier for the regeneration of intervertebral disk (Liang et al., 2012). Both bioactive factors, DEX and bFGF, were released continuously from this system for 4 weeks. The microbeads showed no cytotoxicity to rat mesenchymal stem cells (rMSCs) and promoted rMSC growth and differentiation into nucleus pulposus-like cells. Additionally they also reduced the inflammatory response. The system shows potential as an implantable scaffold for the intervertebral disk regeneration, a step further compared to the current short-term therapies addressed mostly to the treatment of symptoms. PLGA, and some other polymers e.g. 1,2-dipalmitoyl-sn-glycero-3phosphatidylcholine (DPPC) and hyaluronic acid (HA) can be used in Trojan particle formation, from which drug is released in three different steps demonstrated schematically in Fig. 6. The release from such a system is considerably slower than that from the regular polymer nanocapsules (Gómez-Gaete et al., 2008). In 2010 dexamethasone intravitreal implant containing PLGA (Chan et al., 2011), Ozurdex®, was officially approved to be used in all EU countries as a sustained-release for the treatment of macular edema, following branch retinal vein occlusion (BRVO) or central retinal vein occlusion (CRVO). Based on in vivo research done by Chang-Lin et al. (2011) on 34 male monkeys DEX released from the implant was detected in the retina and vitreous humor for 6 months without a need for reinjections. Further clinical trials have shown that, in the short term, the implant improves vision and decreases the risk of vision loss already after first application, however, for most CRVO patients repeat intervention is necessary (Chan et al., 2011).
Chitosan scaffolds Chitosan can be used in tissue engineering for the preparation of scaffolds. DEX was encapsulated in chitosan-based bone scaffolds in order to reduce local inflammation or to induce the differentiation of mesenchymal stem cells into osteoblasts. As an alternative, DEX can be also immobilized on the chitosan scaffold's surface by covalent bond formation (Chiang et al., 2012). DEX was also trapped inside the chitosan scaffolds obtained using supercritical fluid and its sustained release from these materials was observed (Duarte et al., 2009). The yield of impregnation using supercritical fluid was not very high but the concentration of the released DEX was found to be sufficient for tissue engineering applications.
Chitosan films Studies on the chitosan films loaded with DEX have indicated that interactions between drug molecules and polymer chains are more efficient for a bilayer than for a monolayer. Drug release occurred faster in the monolayer —after 8 hours only 10.4% of the drug was left in the material, while in the case of bilayer 16% of the drug still remained in the material after four weeks. For both types of chitosan films the release was much slower than for the conventionally used ophthalmic steroid drops (Rodrigues et al., 2009).
Chitosan microspheres Dexamethasone sodium phosphate encapsulated in chitosan microspheres was tested in brain edema after cold injury using a female rat model. The experiments confirmed the effectiveness of DEX in the treatment of brain edema after thermal trauma. Moreover, encapsulating DEX in the chitosan microspheres allowed increasing therapeutic effect and minimizing side effects due to prolonged release and lower local concentration of DEX (Turkoglu et al., 2005).
Chitosan This biodegradable natural polymer is widely used in biomedical sciences, especially in bioengineering due to its ability to form films, scaffolds and microparticles, which are also applicable in drug delivery (Duarte et al., 2009). Some modifications are also widely studied, such as sulfonated chitosan (Bulwan et al., 2009), quaternary ammonium–chitosan (Karewicz et al., 2013), thiolated quaternary ammonium–chitosan (Zambito and Di Colo, 2010) or carboxymethylchitosan/poly(amidoamine) dendrimer (Oliveira et al., 2009, 2010).
Chitosan derivatives Thiolated quaternary ammonium–chitosan was found to be a better DEX carrier in ophthalmologic formulations due to the enhanced transcorneal absorption (Zambito and Di Colo, 2010) while the carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles loaded with dexamethasone were shown to promote the osteogenic differentiation of rat bone marrow stroma cells (RBMSCs) due to higher calcium deposition (Oliveira et al., 2009, 2010).
Fig. 6. Schematic representation of DEX release from Trojan particles: A —Trojan particles before hydration, B —fast release of the free fraction of DEX associated to the DPPC-HA matrix and slow release of the PLGA nanoparticles loaded with DEX, and C —free PLGA nanoparticles release DEX while DPPC-HA matrix slowly releases these PLGA nanoparticles which are still trapped.
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Other polymer DEX nanocarriers Poly(ε-caprolactone) may be used to create intravitreal implants loaded with DEX. The in vitro release profile does not show any significant burst release, which often occurs for other polymeric nanoparticles loaded with DEX (Fialho et al., 2008). Nanoparticles of amphiphilic monomethoxy poly(ethylene glycol)-blockpoly(trimethylene carbonate) (mPEG–PTMC) were prepared by either salting out or emulsion polymerization. DEX loading efficiency was found to be 88% for those obtained by salting out and 72% for the nanoparticles produced by the emulsion method. The release of DEX from these nanoparticles was shown to be diffusion-controlled and was sustained for up to 60 days. Depending on the nature of the polymer employed and the preparation method, dexamethasone diffusion coefficients varied between 4.8 × 10−18 and 22.6 × 10−18 cm2/s (Zhang et al., 2006). Biodegradable nanocapsules consisting of a hydroxyethylated glucose polymer (hydroxyethyl starch) shell with encapsulated DEX were recently synthesized and it was shown that they can deliver drug in a targeted manner to the selected liver cells. When they were incorporated into the Kupffer cells the significant suppression of the release of inflammatory cytokines was observed (Fichter et al., 2013). DEX–polymer conjugates Water-soluble DEX–polymer conjugates can be also obtained. Dexamethasone-21-mesylate was attached to the low molecular weight (2 kDa) polyethylenimine (PEI) forming water-soluble lipopolymer. The resulting conjugate showed no toxicity to HEK 293 and HepG2 cells in the 10–180 μg/ml concentration range. The conjugate acted as a prodrug, with PEI chain enhancing the solubility of dexamethasone. The obtained lipopolymer was tested as a nucleus-targeting gene carrier (Bae et al., 2007). DEX can dilate the nuclear pore complexes and translocate into the nucleus when it is bound to its glucocorticoid receptor, thus facilitating the transport of DNA into the nucleus. In vitro transfection experiments have shown that indeed PEI–DEX/DNA complex had higher gene delivery efficiency than PEI/DNA. (Linear PEI)–DEX conjugates were also found to be useful in non-viral gene therapy applications (Kim et al., 2010). DEX conjugated with polyamidoamine (PAMAM) dendrimer was also reported (Choi et al., 2006). PAMAM–DEX, similarly to PEI–DEX, showed no toxicity to HEK 93 cells and enhanced transfection efficiency. Safety considerations With few exceptions, the polymers used in the discussed drug delivery systems for dexamethasone were already well known for their biocompatibility and lack of side effects. The only concern could involve the use of PEI and dendrimers, however the in vitro tests revealed no significant toxicity of these formulations against HEK 293 cells. Unfortunately no in vivo experiments with these systems were performed yet. It then came as no surprise that no toxicity or serious side-effects of the discussed new DEX delivery systems were reported so far. Several reports on polylactic acid (PLA) (Li et al., 2013), poly-ε-caprolactone (PCL) (Fialho et al., 2008) and PLGA (Peng et al., 2010) implants releasing dexamethasone confirmed the safety of their application in vivo, pointing out only at mild, transient inflammatory effects caused by the implantation procedure. Summary A wide variety of polymeric carriers have been used to deliver dexamethasone in a controlled manner, including micro and nanocapsules, scaffolds, hydrogels and conjugates. Most of the systems discussed in this review were designed to provide a sustained release of dexamethasone leading to an increase in its therapeutic effect while minimizing side-effects. The formulation used depended mostly on the requested
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delivery profile and administration route. Effectiveness of several polymeric carriers has been proven in in vivo experiments —the designed delivery systems were characterized by superior and longer-lasting effects as well as favorable safety profile compared with systemically administered free DEX. No toxicity or serious side-effects of the new DEX delivery systems were reported. Currently the most successful applications of DEX delivery systems were developed in the field of ophthalmology, as intravitreal implants. One of the proposed solutions, PLGA implant, is already in regular clinical use at the commercial name Ozurdex® to treat adults with swelling of the macula (macular edema) following BRVO or CRVO or noninfectious inflammation of the uvea (uveitis) affecting the back segment of the eye. Conjugates with PEI and dendrimers were proposed as efficient non-viral gene delivery devices, though the studies on these formulations have not reached yet the clinical trial phase. The problem of targeted DEX delivery was also addressed. Conflict of interest statement The authors declare that there are no conflicts of interest.
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