RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Controlled-release injectable containing Terbinafine/PLGA microspheres for Onychomycosis Treatment MURALIKRISHNAN ANGAMUTHU,1 SHIVAKUMAR H. NANJAPPA,2 VIJAYASANKAR RAMAN,3 SEONGBONG JO,1 PHANIRAJ CEGU,4 S. NARASIMHA MURTHY1,2 1
Department of Pharmaceutics, The University of Mississippi, Mississippi 38677 Institute for Drug Delivery and Biomedical Research, Bangalore, Karnataka, India 3 National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, Mississippi 38677 4 Walgreens , Memphis, Tennessee 38133 2
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Received 5 September 2013; revised 8 January 2014; accepted 13 January 2014 Published online 4 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23887 ABSTRACT: Controlled-release drug delivery systems based on biodegradable polymers have been extensively evaluated for use in localized drug delivery. In the present study, intralesionally injectable poly (lactide-co-glycolide) (PLGA) microspheres for controlled release of terbinafine hydrochloride (TH) was developed for treating fungal toe/finger nail infections. TH–PLGA microspheres were formulated using O/W emulsification and modified solvent extraction/evaporation technique. Microspheres were evaluated for particle size and size distribution, encapsulation efficiency, surface, and morphology. The in vitro drug release profile was studied in aqueous media as well as in 1% agar gel. Microspheres system was also evaluated in excised cadaver toe model, and extent of TH accumulation in nail bed, nail plate, and nail matrix was measured at different time points. Microspheres were found to provide consistent and sustained TH release. Intralesional administration of controlled-release microspheres can be a potential alternative mode of treating fungus-infected toe and/or C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1178–1183, 2014 finger nails. Keywords: controlled release; injectables; PLGA; microspheres; polymeric drug delivery systems; onychomycosis
INTRODUCTION Onychomycosis is a collective clinical condition, defining the fungal nail infections caused by dermatophytes (Tricophyton, Microsporum, and Epidermophyton species), yeasts (Candida species), and molds (Scytalidium, Fusarium, Aspergillus, Acremonium, and Scopulariopsis species). Dermatophytes, especially Tricophyton rubrum, is the cause for majority of infections (90%) occurring in finger and toe nail apparatus, a condition referred to as Tinea unguium.1,2 Onychomycosis is highly prevalent accounting for approximately 50% of all nail diseases and affecting almost 26% of the general population in the world.3 The risk of infection is high in people with predisposing factors such as age, diabetes, psoriasis, immunodeficiency, and about 14%–28% of geriatric population suffer from onychomycosis.4 Depending on the clinical manifestations and route of infection, onychomycosis is classified as distal subungual, proximal subungual, white superficial, and total dystrophic onychomycosis. Recent studies suggest that people with diabetes are approximately three times more prone to onychomycosis, which could lead to diabetic foot ulcers.5 Foot ulcers in diabetics cause considerable disability, morbidity, and is the major reason for foot amputations around the world.6,7 Onychomycosis has a profound impact on health, psychological, and social wellbeing of the infected individuals. Pain, disability, social isolation, and embarrassment are few among various comorbidities affecting the quality of life of those patients.
Correspondence to: S. Narasimha Murthy (Telephone: +662-915-5164; Fax: +662-915-1177; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 1178–1183 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
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Onychomycosis is termed as a “clinically stubborn disease” and intervention through pharmacotherapy is the best medical management available. Prolonged maintenance of effective drug levels in nail apparatus is crucial in determining the efficacy of therapy. Among current treatment modalities, topical antifungal therapy lacks robustness because of high resistance offered by nail plate. Therefore, topically administered drugs seldom reach the infected site to sustain therapeutic drug levels. Whereas oral antifungal therapy is considered superior to topical therapy but its prolonged administration is associated with increased risk of several adverse effects.8 Usually, high oral doses are administered for a prolonged time even though the minimum effective drug levels required at the site of action are minimal. Treatment failure (20%–30%) is fairly common in both systemic and topical antifungal therapy often because of low drug bioavailability in the infected tissues.9–11 This also poses a serious risk of treatment relapse as it is indicated that immediate eradication of pathogen is vital for an overall better cure.12 From pharmacoeconomic standpoint, the overall cost of treating onychomycosis is very high.2 To offer a better therapeutic intervention, compared with conventional therapies, many novel drug delivery techniques such as combination therapy, nail lacquers, iontophoresis across nail plate, and nail folds have been proposed in the recent past. In this context, we have developed a controlled-release intralesional injection for the treatment of onychomycosis.4,13–17 Intralesional administration of therapeutics has been extensively evaluated as a potential mode of treatment in various complex diseases such as Peyronie’s disease, nail psoriasis, metastatic melanomas, plantar fascitis, laryngeal papilloma, gastrointestinal strictures, glioblastoma, and keratocanthoma.18–24 The rationale behind such use is to establish a tissue depot and to avoid any physiological barrier that limits achieving effective drug levels at
Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
the site of action. Intralesional corticosteroid therapy is the gold standard treatment available for the chronic treatment of nail psoriasis. The localized introduction of corticosteroids in the nail apparatus helps in maintaining effective drug levels for a fairly long time, which prevents potential treatment relapse. In our current work, controlled-release microspheres containing terbinafine hydrochloride (TH), as a model drug, was formulated using poly (lactide-co-glycolide) (PLGA) polymer. The rationale behind choosing TH for our study is because oral terbinafine therapy is the first-line treatment indicated for onychomycosis and is regarded more effective.25 PLGA polymer is both biocompatible and biodegradable and would be ideal for prolonged residence in the biological tissues.
to remove traces of PVA and remnant organic solvents. Following lyophilization, TH microspheres were obtained as a fine dry powder. Physical Characterization of Microspheres Drug Loading and Encapsulation Efficiency. Microspheres (50 mg) was dissolved in 10 mL of tetrahydrofuran and sonicated for 10 min. THF was added to precipitate PLGA and dissolve TH. Precipitated polymer was filtered using membrane filter (0.45 :m; Fisher scientific, Pittsburgh, Pennsylvania) and amount of TH present in the filtrate was analyzed. Encapsulation efficiency was determined as reported by Fu et al.26 .
Encapsulation efficiency =
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Experimental drug loading ×100% Theoretical drug loading
Materials Terbinafine hydrochloride was obtained from Uquifa (Jiutepac, Mexico), PLGA with a lactide–glycolide ratio 50:50 (ester terminated end group, inherent viscosity = 0.6 dl/g in hexafluoroisopropanol) was obtained from LACTEL (Birmingham, Alabama), poly vinyl alcohol (PVA, 98%–99% hydrolyzed, medium viscosity grade) was obtained from Alfa Aesar (Ward Hill, Massachusetts), Agarose type II was obtained from Sigma–Aldrich (St. Louis, Missouri), and all other chemicals and solvents used were of analytical grade obtained from Fisher scientific (Fairway, New Jersey). Healthy human cadaver toes of both male and female were procured from Science Care (Phoenix, Arizona) and stored at −20◦ C until use. All solutions, dilutions, and release medium were prepared using highly purified deionized water (resistivity ≥18.2 M cm; Barnstead Nanopure TM Diamond , Barrington, Illinois). Methods Validated HPLC method was used to quantify TH in all the samples. The HPLC system (Waters Corporation, Milford, Massachusetts) consists of Waters 1525 pump equipped with an autosampler (Waters 717 plus) and a UV absorbance detector (Waters 2487) with analytical wavelength set at 254 nm. The analytical column was a reversed-phase symmetry C18 (4.6 × 150 mm2 ; 5 m:) and the mobile phase consists of aqueous solution (containing 0.096 M triethylamine and 0.183 M orthophosphoric acid) and acetonitrile in the ratio (60:40, v/v) and was eluted in isocratic mode at a flow rate set at 1 mL/min. The chromatograph was analyzed using Breeze software system (Waters Corporation, Milford, Massachusetts). The range for the calibration curve was 2–1000 ng/mL (R2 = 0.98). R
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Formulation of TH–PLGA Microspheres Conventional O/W emulsification technique was employed to encapsulate TH in PLGA, using PVA as stabilizer. The oil phase constitutes 500 mg of PLGA and 50 mg of TH dissolved in a mixture of solvent containing 4.5 mL methylene chloride and 0.5 mL methanol. The oil phase was homogenized in 100 ml of aqueous phase containing 1% PVA using an Ultra-Turrax (T25 basic; IKA-Werke, Staufen, Germany) at 13,000 rpm for 10 min. The resultant emulsion was stirred at 600 rpm on a hot plate stirrer, at 25◦ C for about 6 h. Microspheres formed were collected by centrifugation and washed subsequently in distilled water DOI 10.1002/jps.23887
Particle Size Distribution. Particle size distribution was analyzed for different batches of microspheres using laser diffraction technique. Aliquots were prepared by uniformly suspending a small amount of microspheres in deionized water before analyzing using Mastersizer (Malvern instruments, Malvern, UK).
Scanning Electron Microscopy. Surface morphology of TH microspheres was investigated using scanning electron microscopy. The samples were mounted on aluminum stubs and fixed using glued carbon tapes. Further, it was coated with gold using Hummer 6.2 sputter coater (Anatech USA, Union City, California). The sputter coater chamber was supplied with argon gas throughout the coating process. Photomicrographs of the microsphere specimens were prepared using a model JSM5600 scanning electron microscopy (JEOL Ltd., Tokyo, Japan). Drug Release Studies In Vitro Drug Release in Water Medium. Microspheres equivalent to 8 mg TH content was suspended in 5 mL deionized water (adjusted to pH 3.0 to maintain “sink” conditions) in jacketed glass cells maintained at 37◦ C using a circulating water bath. Deionized water was preferred for release testing of microspheres as it provides better sink conditions compared with other media such as phosphate buffer. The release medium was stirred at a constant speed of 60 rpm using a magnetic stirrer to avoid microsphere aggregation as well as to ensure uniform mixing of the medium. A sample of volume 250 :L was withdrawn from the release medium at predetermined time points. Samples obtained were centrifuged at 14,000g for 2 min using Galaxy 14 D Microcentrifuge (VWR international, Chicago, Illinois). The supernatant was collected and stored for further analysis while microsphere sediment from centrifuge tubes were redispersed in 250 :L of fresh medium and introduced back into the release medium.
In Vitro Drug Release in Agar Gel Blocks. Another in vitro model consisting of agar gel block (1% agar content) was used to analyze TH release from microsphere formulation depot. The formulation depot was achieved by injecting microspheres equivalent to 0.4 mg of TH content (reconstituted in 200 :L Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
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Figure 1. The transverse section of toe model showing the site of injection of microspheres.
deionized water) in agar gel blocks using a 25 gauge needle syringe. Agar blocks were previously prepared by slowly dispersing 1 g of agar powder in 100 mL of deionized water (pH 3.0) maintained at 60◦ C. From the above medium, 2 mL solution was transferred to glass vials and allowed to cool at room temperature for solidification. At predetermined time points, agar gel block was homogenized with 0.5 N HCl to extract TH released from microsphere depot. The extracted medium was filtered using 0.45 :m filter discs and quantified for TH content. Ex Vivo Drug Release in Excised Cadaver Toe Model. Terbinafine hydrochloride release from microspheres was also evaluated in an excised cadaver toe following microspheres injection in proximal nail folds (PNF) (Fig. 1). In order to prepare the cadaver toes for drug release testing, it was allowed to thaw for 2 h at room temperature. The toe was washed and the excess tissue was excised and removed leaving only the intact toe. The toe was soaked in normal physiological saline containing 0.5% gentamycin. Following microspheres injection, the cadaver toes were stored at −20◦ C. For the analysis of drug release at each time point, the toes were removed from storage and allowed to thaw for 2 h. The toenail was then separated using a scalpel and a forceps followed by nail bed and the nail matrix. TH loaded in the tissues was extracted using the standard protocol and then analyzed for the drug content. The method for isolation of individual tissues and drug extraction was carried out as demonstrated by Nair et al.27 . The rationale behind choosing PNF as a potential injection site was because of its anatomical proximity to nail matrix and other soft tissues in nail apparatus. Microspheres equivalent to 0.4 mg of TH content (reconstituted in 200 :L of deionized water) was injected using a 25 gauge needle syringe. Similarly, a control study was carried out by administering TH (0.4 mg/ 200 :L, dispersed in deionized water) in the excised toes. Analytical Method Statistical Analysis GraphPad InStat 3 (GraphPad Software, Inc., La Jolla, California) was used for statistical analysis. One-way ANOVA was used to determine the level of significance for correlation between parameters and p < 0.05 was considered as the acceptable level of significance. Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
Figure 2. Particle size distribution of TH–PLGA microspheres.
RESULTS AND DISCUSSION Formulation of PLGA Microspheres Poly (lactide-co-glycolide) is a biodegradable polymer extensively used in various biomedical applications especially in the prolonged delivery of therapeutic agents. In order to check the feasibility of developing a controlled-release injection for treating onychomycosis, microsphere formulation was prepared utilizing PLGA (mol wt ∼60,000 Da) and TH. Methanol present in the organic phase acts as a cosolvent and eases the extraction of methylene chloride from oil globules uniformly, during the solvent extraction/evaporation phase, which is essential for the formation of less porous and homogenous particles.28 The drug loading and encapsulation efficiency were found to be 39.56 ± 3.56%. Employment of 50 mg of terbinafine and 500 mg of PLGA (drug–polymer ratio; 1:10) was an attempt to achieve a prototype formulation to carry out the proof of concept studies. However, various formulation and process parameters can be tailored to further optimize the drug loading/dose in microspheres, which is beyond the scope of the present study. The operating parameters such as homogenization rate, time, and bulk volume were optimized to prepare microspheres of desired size range between 5 and 15 :m. Microspheres possessed a mean diameter of 12.75 :m, as shown in Figure 2. The size of microspheres in the above range was assumed to be ideal because of the combination of critical factors such as (1) volumetric limit of injectable fluids in the nail tissues, (2) efficient reconstitution of microspheres with the limited volume of injectable fluids, (3) use of finer needles to avoid pain at the site of injection, and (4) ease of microspheres injection through those finer needles. The use of nanospheres for intralesional application might look more appealing while taking into consideration the above limiting factors. However, the nanosized carriers are more likely to be cleared off from the injection site by circulation. The size of the TH–PLGA microspheres is several orders of magnitude larger than the average size of the pores in the capillary endothelium that makes the microspheres less amenable to clearance into systemic circulation from the site of injection. However, one of the potential mechanisms of clearance of microspheres may be through lymphatic pathway. Any of these clearance mechanisms were absent in the evaluation models DOI 10.1002/jps.23887
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Figure 3. TH release from PLGA microspheres at 37◦ C in pH 3.0 water and agar media. The data represent an average of n = 3 ± SD Inset figure: Correlation of cumulative percent TH release from PLGA microspheres at 37◦ C in pH 3.0 water and agar media. The data represent an average of n = 3 ± SD.
used in this project. Although lymphatic system present in the normal human nail apparatus encompass various components (CD4+, CD8+ T cells, Langerhans cells, macrophages, natural killer, and mast cells), those cells are differentially distributed in and around the nail apparatus. Some of the cell-mediated immune mechanisms were reported to be scarcely present or impaired in those nail apparatus.29 Therefore, an answer to how long the formulation would be retained in the injected site of the fungus infected nail apparatus could be found out only after a clinical pharmacokinetic study. Although less likely, if lymphatic clearance is found to be a major determinant of retention time, the microsphere could be PEGylated or linked to polar moieties to minimize their phagocytosis. In Vitro Release Studies in Water Medium Usually, the drug release from PLGA formulations has been reported to follow three release phases, an initial burst followed by a lag phase and then a secondary apparent zero-order phase. TH release from PLGA microspheres did not exhibit a “typical” triphasic-release profile, rather a lag phase was observed for initial 3 days followed by a steady-state phase (Fig. 3). Predominant factors that are responsible for initial burst release are high porosity of PLGA microspheres and the presence of any surface-adsorbed drug. Generally, porous nature of the microspheres expose larger surface area to the surrounding medium and drug diffusion from pores are not essentially polymer controlled (pores or microchannels are formed in the microspheres during the solvent evaporation phase).30 Lack of initial burst release in case of TH-loaded microspheres can be attributed to less porosity (scanning electron microscopy images; Fig. 4) and lack of any surface-associated drug because of low drug– polymer ratio.31 High concentration (10%) of PLGA used in oil phase along with slow rate of extraction of organic solvent from the O/W emulsion could be the predominant reasons for formation of less porous microspheres.30,32–41 DOI 10.1002/jps.23887
In order to simulate TH release from microsphere system in soft tissues of nail compartment, we developed an in vitro testing model consisting agar gel that simulate the subungual soft tissues. From our previous studies, agar gel (1%) was found to be a valuable in vitro tool for studying subungual drug delivery.13,42 Identical pH and temperature were maintained while performing release testing in both water and agar mediums. The extent of correlation between cumulative percent drug release in agar and water media was assessed (Fig. 3; R2 = 0.79, inset figure). It was observed that the cumulative percent terbinafine release in agar blocks is approximately five times more than the release in water. The reason for such accelerated TH release in a static medium (agar blocks) compared with that of a dynamic medium (water being stirred continuously) is unknown. It is likely that the accumulation of acidic byproducts (because of polymer degradation) around the microspheres accelerates the drug release in agar medium.43 Whereas the byproducts would be instantaneously diluted when water was used as medium, which is under constant stirring. However, an acceptable correlation between the two sets of data suggests that either of them could be used to screen the intralesional drug delivery systems. Evaluation of Microspheres in Excised Cadaver Toe Model In the present study, ex vivo cadaver toe model was utilized to investigate the ability of microspheres to affect a controlled drug delivery in nail apparatus. However, this model lacks the physiological clearance of drugs, which would be one of the major determinants of localized TH tissue levels in vivo. However, owing to the similarity in anatomical structure with in vivo toe, the use of excised cadaver toe model could be a useful tool to perform preliminary investigation to assess the ability of microspheres to control the release of drug in a simulated structural environment. The tissue drug levels in excised cadaver toe following microspheres injection is depicted in Figure 5. On day 1, quantifiable amount of TH was found only in nail matrix, but not in nail bed or nail plate because of the proximity of the former with the site of injection. However, the data for 10th and 30th days showed significant TH accumulation in nail bed and nail plate, which is likely because of the diffusion of accumulated drug from nail matrix. A cumulative amount of approximately 2 and 3 :g of TH was observed in all soft tissues combined (nail bed, nail plate, and nail matrix) at the end of day 10 and 30, respectively, and drug levels in individual nail compartments were as follows: approximately 50 ng/mg (day 10) and approximately 60 ng/mg (day 30) in nail matrix; more than 3 ng/mg in nail bed and more than 3 ng/mg in nail plate. It is to be noted that TH is a potent antifungal drug that has low-minimum inhibitory concentration (∼1–10 ng/mL) and low-minimum fungicidal concentration (∼3–6 ng/mL).44 Unlike microspheres, control injection (drug dispersed in deionized water), led to relatively higher TH levels in nail matrix, nail plate, and nail bed on day 1 itself, as expected. But the drug levels on 10th day (in case of nail matrix) was either same or significantly less than day 1 (Fig. 5: inset). Contrastingly, microspheres system profoundly increased TH levels in nail matrix after day 1. The tissue drug level–time profile affected from microspheres injection clearly differs from that of control, demonstrating the ability of microspheres to provide a controlled drug delivery to the sites of interest in the nail Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
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Figure 4. Scanning electron microscopy photographs of TH–PLGA microspheres: (a) ×2200, (b) ×5000.
Figure 5. Amount of TH (ng/mg weight of tissue) found in nail bed, nail plate, and nail matrix after microspheres injection in excised cadaver toe model. The data represent an average of n = 3 ± SD. Inset figure: Amount of TH (ng/mg weight of tissue) found in nail bed, nail plate, and nail matrix after control injection (TH in deionized water) in excised cadaver toe model. The data represent an average of n = 3 ± SD.
apparatus, especially to nail matrix. Sustaining TH levels in nail matrix is crucial as it remains a potential site for fungal infection in onychomycosis because fungus infects the entire nail bed and underside of the nail plate through nail matrix (nail matrix houses stem cells called onychocytes that grow laterally into newly formed nail plate).44 It is possible to achieve higher rate and extent of TH release to compensate for the loss because of clearance by simply increasing TH loading in microspheres or by administering larger amount of microspheres within anatomically acceptable limits.
CONCLUSIONS Terbinafine hydrochloride-loaded PLGA microspheres delivery system for controlled-release intralesional application was formulated using simple emulsification technique. Lyophilization Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
of microspheres resulted in a fine powder and was reconstitutable in limited fluid volume (0.2 mL) available for injection. The experimental drug loading was found to be moderate; however, drug can be increased by controlling critical formulation and process parameters. In vitro release testing in water indicated that microspheres-controlled TH release for more than 45 days and no dose dumping was observed. Release testing in agar medium showed an accelerated TH release compared with water medium. This phenomenon was because of autocatalytic degradation of PLGA polymer. Drug release testing in ex vivo cadaver toe model indicated that a single dose of microspheres delivered and sustained TH levels above minimum inhibitory/fungicidal concentration levels in soft tissues in the nail apparatus. Also, it was inferred that microspheres system controlled the drug release in nail apparatus that potentially sustained the drug levels, especially in nail matrix. Sustenance of TH levels in nail unit is an essential prerequisite to treat onychomycosis without treatment relapse and disease recurrence. Further, clinical pharmacokinetic studies may be required to optimize the microspheres dose for intralesional therapy to deliver clinically effective dose of TH to individual soft tissues in nail apparatus.
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iontophoretic ungual delivery of terbinafine. Pharm Res 26(9):2194– 2201. 28. Tuncay M, Calis S, Kas HS, Ercan MT, Peksoy I, Hincal AA. 2000. Diclofenac sodium incorporated PLGA (50:50) microspheres: Formulation considerations and in vitro/in vivo evaluation. Int J Pharm 195(1– 2):179–188. 29. Ito T, Ito N, Saathoff M, Stampachiacchiere B, Bettermann A, Bulfone-Paus S, Takigawa M, Nickoloff BJ, Paus R. 2005. Immunology of the human nail apparatus: The nail matrix is a site of relative immune privilege. J Invest Dermatol 125(6):1139–1148. 30. Mao S, Xu J, Cai C, Germershaus O, Schaper A, Kissel T. 2007. Effect of WOW process parameters on morphology and burst release of FITC-dextran loaded PLGA microspheres. Int J Pharm 334(1–2):137– 148. 31. Hickey T, Kreutzer D, Burgess DJ, Moussy F. 2002. Dexamethasone/PLGA microspheres for continuous delivery of an antiinflammatory drug for implantable medical devices. Biomaterials 23(7):1649–1656. 32. Huang X, Brazel CS. 2001. On the importance and mechanisms of burst release in matrix-controlled drug delivery systems. J Control Release 73(2–3):121–136. 33. Zolnik BS, Leary PE, Burgess DJ. 2006. Elevated temperature accelerated release testing of PLGA microspheres. J Control Release 112(3):293–300. 34. Zolnik BS, Burgess DJ. 2008. Evaluation of in vivo-in vitro release of dexamethasone from PLGA microspheres. J Control Release 127(2):137–145. 35. Park TG, Cohen S, Langer R. 1992. Controlled protein release from polyethyleneimine-coated poly(L-lactic acid)/pluronic blend matrices. Pharm Res 9(1):37–39. 36. Jalil R, Nixon JR. 1990. Biodegradable poly(lactic acid) and poly(lactide-co-glycolide) microcapsules: Problems associated with preparative techniques and release properties. J Microencapsul 7(3):297–325. 37. Ahmed AR, Dashevsky A, Bodmeier R. (2000). Reduced burst effect in drug release with solvent-treated microparticles prepared by the solvent evaporation method. Control Release Bioact Mater 27:6112. 38. Park TG, Cohen S, Langer R. 1992. Poly (L-lactic acid)/pluronic blends: Characterization of phase separation behavior, degradation, and morphology and use as protein-releasing matrixes. Macromolecules 25(1):116–122. 39. van de Weert M, van’t Hof R, van der Weerd J, Heeren R, Posthuma G, Hennink WE, Crommelin DJ. 2000. Lysozyme distribution and conformation in a biodegradable polymer matrix as determined by FTIR techniques. J Control Release 68(1):31–40. 40. Yang Y-Y, Chia H-H, Chung T-S. 2000. Effect of preparation temperature on the characteristics and release profiles of PLGA microspheres containing protein fabricated by double-emulsion solvent extraction/evaporation method. J Control Release 69(1):81. 41. Wang H, Schmitt E, Flanagan D, Linhardt R. 1991. Influence of formulation methods on the in vitro controlled release of protein from poly (ester) microspheres. J Control Release 17(1):23–31. 42. Nair AB, Kim HD, Chakraborty B, Singh J, Zaman M, Gupta A, Friden PM, Murthy SN. 2009. Ungual and trans-ungual iontophoretic delivery of terbinafine for the treatment of onychomycosis. J Pharm Sci 98(11):4130–4140. 43. von Burkersroda F, Schedl L, Gopferich A. 2002. Why degradable polymers undergo surface erosion or bulk erosion. Biomaterials 23(21):4221–4231. 44. Takeo M, Chou WC, Sun Q, Lee W, Rabbani P, Loomis C, Taketo MM, Ito M. 2013. Wnt activation in nail epithelium couples nail growth to digit regeneration. Nature 499(7457):228–232.
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Controlled-release injectable containing Terbinafine/PLGA microspheres for Onychomycosis Treatment MURALIKRISHNAN ANGAMUTHU,1 SHIVAKUMAR H. NANJAPPA,2 VIJAYASANKAR RAMAN,3 SEONGBONG JO,1 PHANIRAJ CEGU,4 S. NARASIMHA MURTHY1,2 1
Department of Pharmaceutics, The University of Mississippi, Mississippi 38677 Institute for Drug Delivery and Biomedical Research, Bangalore, Karnataka, India 3 National Center for Natural Products Research, School of Pharmacy, The University of Mississippi, Mississippi 38677 4 Walgreens , Memphis, Tennessee 38133 2
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Received 5 September 2013; revised 8 January 2014; accepted 13 January 2014 Published online 4 February 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.23887 ABSTRACT: Controlled-release drug delivery systems based on biodegradable polymers have been extensively evaluated for use in localized drug delivery. In the present study, intralesionally injectable poly (lactide-co-glycolide) (PLGA) microspheres for controlled release of terbinafine hydrochloride (TH) was developed for treating fungal toe/finger nail infections. TH–PLGA microspheres were formulated using O/W emulsification and modified solvent extraction/evaporation technique. Microspheres were evaluated for particle size and size distribution, encapsulation efficiency, surface, and morphology. The in vitro drug release profile was studied in aqueous media as well as in 1% agar gel. Microspheres system was also evaluated in excised cadaver toe model, and extent of TH accumulation in nail bed, nail plate, and nail matrix was measured at different time points. Microspheres were found to provide consistent and sustained TH release. Intralesional administration of controlled-release microspheres can be a potential alternative mode of treating fungus-infected toe and/or C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 103:1178–1183, 2014 finger nails. Keywords: controlled release; injectables; PLGA; microspheres; polymeric drug delivery systems; onychomycosis
INTRODUCTION Onychomycosis is a collective clinical condition, defining the fungal nail infections caused by dermatophytes (Tricophyton, Microsporum, and Epidermophyton species), yeasts (Candida species), and molds (Scytalidium, Fusarium, Aspergillus, Acremonium, and Scopulariopsis species). Dermatophytes, especially Tricophyton rubrum, is the cause for majority of infections (90%) occurring in finger and toe nail apparatus, a condition referred to as Tinea unguium.1,2 Onychomycosis is highly prevalent accounting for approximately 50% of all nail diseases and affecting almost 26% of the general population in the world.3 The risk of infection is high in people with predisposing factors such as age, diabetes, psoriasis, immunodeficiency, and about 14%–28% of geriatric population suffer from onychomycosis.4 Depending on the clinical manifestations and route of infection, onychomycosis is classified as distal subungual, proximal subungual, white superficial, and total dystrophic onychomycosis. Recent studies suggest that people with diabetes are approximately three times more prone to onychomycosis, which could lead to diabetic foot ulcers.5 Foot ulcers in diabetics cause considerable disability, morbidity, and is the major reason for foot amputations around the world.6,7 Onychomycosis has a profound impact on health, psychological, and social wellbeing of the infected individuals. Pain, disability, social isolation, and embarrassment are few among various comorbidities affecting the quality of life of those patients.
Correspondence to: S. Narasimha Murthy (Telephone: +662-915-5164; Fax: +662-915-1177; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 103, 1178–1183 (2014) C 2014 Wiley Periodicals, Inc. and the American Pharmacists Association
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Onychomycosis is termed as a “clinically stubborn disease” and intervention through pharmacotherapy is the best medical management available. Prolonged maintenance of effective drug levels in nail apparatus is crucial in determining the efficacy of therapy. Among current treatment modalities, topical antifungal therapy lacks robustness because of high resistance offered by nail plate. Therefore, topically administered drugs seldom reach the infected site to sustain therapeutic drug levels. Whereas oral antifungal therapy is considered superior to topical therapy but its prolonged administration is associated with increased risk of several adverse effects.8 Usually, high oral doses are administered for a prolonged time even though the minimum effective drug levels required at the site of action are minimal. Treatment failure (20%–30%) is fairly common in both systemic and topical antifungal therapy often because of low drug bioavailability in the infected tissues.9–11 This also poses a serious risk of treatment relapse as it is indicated that immediate eradication of pathogen is vital for an overall better cure.12 From pharmacoeconomic standpoint, the overall cost of treating onychomycosis is very high.2 To offer a better therapeutic intervention, compared with conventional therapies, many novel drug delivery techniques such as combination therapy, nail lacquers, iontophoresis across nail plate, and nail folds have been proposed in the recent past. In this context, we have developed a controlled-release intralesional injection for the treatment of onychomycosis.4,13–17 Intralesional administration of therapeutics has been extensively evaluated as a potential mode of treatment in various complex diseases such as Peyronie’s disease, nail psoriasis, metastatic melanomas, plantar fascitis, laryngeal papilloma, gastrointestinal strictures, glioblastoma, and keratocanthoma.18–24 The rationale behind such use is to establish a tissue depot and to avoid any physiological barrier that limits achieving effective drug levels at
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the site of action. Intralesional corticosteroid therapy is the gold standard treatment available for the chronic treatment of nail psoriasis. The localized introduction of corticosteroids in the nail apparatus helps in maintaining effective drug levels for a fairly long time, which prevents potential treatment relapse. In our current work, controlled-release microspheres containing terbinafine hydrochloride (TH), as a model drug, was formulated using poly (lactide-co-glycolide) (PLGA) polymer. The rationale behind choosing TH for our study is because oral terbinafine therapy is the first-line treatment indicated for onychomycosis and is regarded more effective.25 PLGA polymer is both biocompatible and biodegradable and would be ideal for prolonged residence in the biological tissues.
to remove traces of PVA and remnant organic solvents. Following lyophilization, TH microspheres were obtained as a fine dry powder. Physical Characterization of Microspheres Drug Loading and Encapsulation Efficiency. Microspheres (50 mg) was dissolved in 10 mL of tetrahydrofuran and sonicated for 10 min. THF was added to precipitate PLGA and dissolve TH. Precipitated polymer was filtered using membrane filter (0.45 :m; Fisher scientific, Pittsburgh, Pennsylvania) and amount of TH present in the filtrate was analyzed. Encapsulation efficiency was determined as reported by Fu et al.26 .
Encapsulation efficiency =
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Experimental drug loading ×100% Theoretical drug loading
Materials Terbinafine hydrochloride was obtained from Uquifa (Jiutepac, Mexico), PLGA with a lactide–glycolide ratio 50:50 (ester terminated end group, inherent viscosity = 0.6 dl/g in hexafluoroisopropanol) was obtained from LACTEL (Birmingham, Alabama), poly vinyl alcohol (PVA, 98%–99% hydrolyzed, medium viscosity grade) was obtained from Alfa Aesar (Ward Hill, Massachusetts), Agarose type II was obtained from Sigma–Aldrich (St. Louis, Missouri), and all other chemicals and solvents used were of analytical grade obtained from Fisher scientific (Fairway, New Jersey). Healthy human cadaver toes of both male and female were procured from Science Care (Phoenix, Arizona) and stored at −20◦ C until use. All solutions, dilutions, and release medium were prepared using highly purified deionized water (resistivity ≥18.2 M cm; Barnstead Nanopure TM Diamond , Barrington, Illinois). Methods Validated HPLC method was used to quantify TH in all the samples. The HPLC system (Waters Corporation, Milford, Massachusetts) consists of Waters 1525 pump equipped with an autosampler (Waters 717 plus) and a UV absorbance detector (Waters 2487) with analytical wavelength set at 254 nm. The analytical column was a reversed-phase symmetry C18 (4.6 × 150 mm2 ; 5 m:) and the mobile phase consists of aqueous solution (containing 0.096 M triethylamine and 0.183 M orthophosphoric acid) and acetonitrile in the ratio (60:40, v/v) and was eluted in isocratic mode at a flow rate set at 1 mL/min. The chromatograph was analyzed using Breeze software system (Waters Corporation, Milford, Massachusetts). The range for the calibration curve was 2–1000 ng/mL (R2 = 0.98). R
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Formulation of TH–PLGA Microspheres Conventional O/W emulsification technique was employed to encapsulate TH in PLGA, using PVA as stabilizer. The oil phase constitutes 500 mg of PLGA and 50 mg of TH dissolved in a mixture of solvent containing 4.5 mL methylene chloride and 0.5 mL methanol. The oil phase was homogenized in 100 ml of aqueous phase containing 1% PVA using an Ultra-Turrax (T25 basic; IKA-Werke, Staufen, Germany) at 13,000 rpm for 10 min. The resultant emulsion was stirred at 600 rpm on a hot plate stirrer, at 25◦ C for about 6 h. Microspheres formed were collected by centrifugation and washed subsequently in distilled water DOI 10.1002/jps.23887
Particle Size Distribution. Particle size distribution was analyzed for different batches of microspheres using laser diffraction technique. Aliquots were prepared by uniformly suspending a small amount of microspheres in deionized water before analyzing using Mastersizer (Malvern instruments, Malvern, UK).
Scanning Electron Microscopy. Surface morphology of TH microspheres was investigated using scanning electron microscopy. The samples were mounted on aluminum stubs and fixed using glued carbon tapes. Further, it was coated with gold using Hummer 6.2 sputter coater (Anatech USA, Union City, California). The sputter coater chamber was supplied with argon gas throughout the coating process. Photomicrographs of the microsphere specimens were prepared using a model JSM5600 scanning electron microscopy (JEOL Ltd., Tokyo, Japan). Drug Release Studies In Vitro Drug Release in Water Medium. Microspheres equivalent to 8 mg TH content was suspended in 5 mL deionized water (adjusted to pH 3.0 to maintain “sink” conditions) in jacketed glass cells maintained at 37◦ C using a circulating water bath. Deionized water was preferred for release testing of microspheres as it provides better sink conditions compared with other media such as phosphate buffer. The release medium was stirred at a constant speed of 60 rpm using a magnetic stirrer to avoid microsphere aggregation as well as to ensure uniform mixing of the medium. A sample of volume 250 :L was withdrawn from the release medium at predetermined time points. Samples obtained were centrifuged at 14,000g for 2 min using Galaxy 14 D Microcentrifuge (VWR international, Chicago, Illinois). The supernatant was collected and stored for further analysis while microsphere sediment from centrifuge tubes were redispersed in 250 :L of fresh medium and introduced back into the release medium.
In Vitro Drug Release in Agar Gel Blocks. Another in vitro model consisting of agar gel block (1% agar content) was used to analyze TH release from microsphere formulation depot. The formulation depot was achieved by injecting microspheres equivalent to 0.4 mg of TH content (reconstituted in 200 :L Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
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Figure 1. The transverse section of toe model showing the site of injection of microspheres.
deionized water) in agar gel blocks using a 25 gauge needle syringe. Agar blocks were previously prepared by slowly dispersing 1 g of agar powder in 100 mL of deionized water (pH 3.0) maintained at 60◦ C. From the above medium, 2 mL solution was transferred to glass vials and allowed to cool at room temperature for solidification. At predetermined time points, agar gel block was homogenized with 0.5 N HCl to extract TH released from microsphere depot. The extracted medium was filtered using 0.45 :m filter discs and quantified for TH content. Ex Vivo Drug Release in Excised Cadaver Toe Model. Terbinafine hydrochloride release from microspheres was also evaluated in an excised cadaver toe following microspheres injection in proximal nail folds (PNF) (Fig. 1). In order to prepare the cadaver toes for drug release testing, it was allowed to thaw for 2 h at room temperature. The toe was washed and the excess tissue was excised and removed leaving only the intact toe. The toe was soaked in normal physiological saline containing 0.5% gentamycin. Following microspheres injection, the cadaver toes were stored at −20◦ C. For the analysis of drug release at each time point, the toes were removed from storage and allowed to thaw for 2 h. The toenail was then separated using a scalpel and a forceps followed by nail bed and the nail matrix. TH loaded in the tissues was extracted using the standard protocol and then analyzed for the drug content. The method for isolation of individual tissues and drug extraction was carried out as demonstrated by Nair et al.27 . The rationale behind choosing PNF as a potential injection site was because of its anatomical proximity to nail matrix and other soft tissues in nail apparatus. Microspheres equivalent to 0.4 mg of TH content (reconstituted in 200 :L of deionized water) was injected using a 25 gauge needle syringe. Similarly, a control study was carried out by administering TH (0.4 mg/ 200 :L, dispersed in deionized water) in the excised toes. Analytical Method Statistical Analysis GraphPad InStat 3 (GraphPad Software, Inc., La Jolla, California) was used for statistical analysis. One-way ANOVA was used to determine the level of significance for correlation between parameters and p < 0.05 was considered as the acceptable level of significance. Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
Figure 2. Particle size distribution of TH–PLGA microspheres.
RESULTS AND DISCUSSION Formulation of PLGA Microspheres Poly (lactide-co-glycolide) is a biodegradable polymer extensively used in various biomedical applications especially in the prolonged delivery of therapeutic agents. In order to check the feasibility of developing a controlled-release injection for treating onychomycosis, microsphere formulation was prepared utilizing PLGA (mol wt ∼60,000 Da) and TH. Methanol present in the organic phase acts as a cosolvent and eases the extraction of methylene chloride from oil globules uniformly, during the solvent extraction/evaporation phase, which is essential for the formation of less porous and homogenous particles.28 The drug loading and encapsulation efficiency were found to be 39.56 ± 3.56%. Employment of 50 mg of terbinafine and 500 mg of PLGA (drug–polymer ratio; 1:10) was an attempt to achieve a prototype formulation to carry out the proof of concept studies. However, various formulation and process parameters can be tailored to further optimize the drug loading/dose in microspheres, which is beyond the scope of the present study. The operating parameters such as homogenization rate, time, and bulk volume were optimized to prepare microspheres of desired size range between 5 and 15 :m. Microspheres possessed a mean diameter of 12.75 :m, as shown in Figure 2. The size of microspheres in the above range was assumed to be ideal because of the combination of critical factors such as (1) volumetric limit of injectable fluids in the nail tissues, (2) efficient reconstitution of microspheres with the limited volume of injectable fluids, (3) use of finer needles to avoid pain at the site of injection, and (4) ease of microspheres injection through those finer needles. The use of nanospheres for intralesional application might look more appealing while taking into consideration the above limiting factors. However, the nanosized carriers are more likely to be cleared off from the injection site by circulation. The size of the TH–PLGA microspheres is several orders of magnitude larger than the average size of the pores in the capillary endothelium that makes the microspheres less amenable to clearance into systemic circulation from the site of injection. However, one of the potential mechanisms of clearance of microspheres may be through lymphatic pathway. Any of these clearance mechanisms were absent in the evaluation models DOI 10.1002/jps.23887
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Figure 3. TH release from PLGA microspheres at 37◦ C in pH 3.0 water and agar media. The data represent an average of n = 3 ± SD Inset figure: Correlation of cumulative percent TH release from PLGA microspheres at 37◦ C in pH 3.0 water and agar media. The data represent an average of n = 3 ± SD.
used in this project. Although lymphatic system present in the normal human nail apparatus encompass various components (CD4+, CD8+ T cells, Langerhans cells, macrophages, natural killer, and mast cells), those cells are differentially distributed in and around the nail apparatus. Some of the cell-mediated immune mechanisms were reported to be scarcely present or impaired in those nail apparatus.29 Therefore, an answer to how long the formulation would be retained in the injected site of the fungus infected nail apparatus could be found out only after a clinical pharmacokinetic study. Although less likely, if lymphatic clearance is found to be a major determinant of retention time, the microsphere could be PEGylated or linked to polar moieties to minimize their phagocytosis. In Vitro Release Studies in Water Medium Usually, the drug release from PLGA formulations has been reported to follow three release phases, an initial burst followed by a lag phase and then a secondary apparent zero-order phase. TH release from PLGA microspheres did not exhibit a “typical” triphasic-release profile, rather a lag phase was observed for initial 3 days followed by a steady-state phase (Fig. 3). Predominant factors that are responsible for initial burst release are high porosity of PLGA microspheres and the presence of any surface-adsorbed drug. Generally, porous nature of the microspheres expose larger surface area to the surrounding medium and drug diffusion from pores are not essentially polymer controlled (pores or microchannels are formed in the microspheres during the solvent evaporation phase).30 Lack of initial burst release in case of TH-loaded microspheres can be attributed to less porosity (scanning electron microscopy images; Fig. 4) and lack of any surface-associated drug because of low drug– polymer ratio.31 High concentration (10%) of PLGA used in oil phase along with slow rate of extraction of organic solvent from the O/W emulsion could be the predominant reasons for formation of less porous microspheres.30,32–41 DOI 10.1002/jps.23887
In order to simulate TH release from microsphere system in soft tissues of nail compartment, we developed an in vitro testing model consisting agar gel that simulate the subungual soft tissues. From our previous studies, agar gel (1%) was found to be a valuable in vitro tool for studying subungual drug delivery.13,42 Identical pH and temperature were maintained while performing release testing in both water and agar mediums. The extent of correlation between cumulative percent drug release in agar and water media was assessed (Fig. 3; R2 = 0.79, inset figure). It was observed that the cumulative percent terbinafine release in agar blocks is approximately five times more than the release in water. The reason for such accelerated TH release in a static medium (agar blocks) compared with that of a dynamic medium (water being stirred continuously) is unknown. It is likely that the accumulation of acidic byproducts (because of polymer degradation) around the microspheres accelerates the drug release in agar medium.43 Whereas the byproducts would be instantaneously diluted when water was used as medium, which is under constant stirring. However, an acceptable correlation between the two sets of data suggests that either of them could be used to screen the intralesional drug delivery systems. Evaluation of Microspheres in Excised Cadaver Toe Model In the present study, ex vivo cadaver toe model was utilized to investigate the ability of microspheres to affect a controlled drug delivery in nail apparatus. However, this model lacks the physiological clearance of drugs, which would be one of the major determinants of localized TH tissue levels in vivo. However, owing to the similarity in anatomical structure with in vivo toe, the use of excised cadaver toe model could be a useful tool to perform preliminary investigation to assess the ability of microspheres to control the release of drug in a simulated structural environment. The tissue drug levels in excised cadaver toe following microspheres injection is depicted in Figure 5. On day 1, quantifiable amount of TH was found only in nail matrix, but not in nail bed or nail plate because of the proximity of the former with the site of injection. However, the data for 10th and 30th days showed significant TH accumulation in nail bed and nail plate, which is likely because of the diffusion of accumulated drug from nail matrix. A cumulative amount of approximately 2 and 3 :g of TH was observed in all soft tissues combined (nail bed, nail plate, and nail matrix) at the end of day 10 and 30, respectively, and drug levels in individual nail compartments were as follows: approximately 50 ng/mg (day 10) and approximately 60 ng/mg (day 30) in nail matrix; more than 3 ng/mg in nail bed and more than 3 ng/mg in nail plate. It is to be noted that TH is a potent antifungal drug that has low-minimum inhibitory concentration (∼1–10 ng/mL) and low-minimum fungicidal concentration (∼3–6 ng/mL).44 Unlike microspheres, control injection (drug dispersed in deionized water), led to relatively higher TH levels in nail matrix, nail plate, and nail bed on day 1 itself, as expected. But the drug levels on 10th day (in case of nail matrix) was either same or significantly less than day 1 (Fig. 5: inset). Contrastingly, microspheres system profoundly increased TH levels in nail matrix after day 1. The tissue drug level–time profile affected from microspheres injection clearly differs from that of control, demonstrating the ability of microspheres to provide a controlled drug delivery to the sites of interest in the nail Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
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Figure 4. Scanning electron microscopy photographs of TH–PLGA microspheres: (a) ×2200, (b) ×5000.
Figure 5. Amount of TH (ng/mg weight of tissue) found in nail bed, nail plate, and nail matrix after microspheres injection in excised cadaver toe model. The data represent an average of n = 3 ± SD. Inset figure: Amount of TH (ng/mg weight of tissue) found in nail bed, nail plate, and nail matrix after control injection (TH in deionized water) in excised cadaver toe model. The data represent an average of n = 3 ± SD.
apparatus, especially to nail matrix. Sustaining TH levels in nail matrix is crucial as it remains a potential site for fungal infection in onychomycosis because fungus infects the entire nail bed and underside of the nail plate through nail matrix (nail matrix houses stem cells called onychocytes that grow laterally into newly formed nail plate).44 It is possible to achieve higher rate and extent of TH release to compensate for the loss because of clearance by simply increasing TH loading in microspheres or by administering larger amount of microspheres within anatomically acceptable limits.
CONCLUSIONS Terbinafine hydrochloride-loaded PLGA microspheres delivery system for controlled-release intralesional application was formulated using simple emulsification technique. Lyophilization Angamuthu et al., JOURNAL OF PHARMACEUTICAL SCIENCES 103:1178–1183, 2014
of microspheres resulted in a fine powder and was reconstitutable in limited fluid volume (0.2 mL) available for injection. The experimental drug loading was found to be moderate; however, drug can be increased by controlling critical formulation and process parameters. In vitro release testing in water indicated that microspheres-controlled TH release for more than 45 days and no dose dumping was observed. Release testing in agar medium showed an accelerated TH release compared with water medium. This phenomenon was because of autocatalytic degradation of PLGA polymer. Drug release testing in ex vivo cadaver toe model indicated that a single dose of microspheres delivered and sustained TH levels above minimum inhibitory/fungicidal concentration levels in soft tissues in the nail apparatus. Also, it was inferred that microspheres system controlled the drug release in nail apparatus that potentially sustained the drug levels, especially in nail matrix. Sustenance of TH levels in nail unit is an essential prerequisite to treat onychomycosis without treatment relapse and disease recurrence. Further, clinical pharmacokinetic studies may be required to optimize the microspheres dose for intralesional therapy to deliver clinically effective dose of TH to individual soft tissues in nail apparatus.
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