RESEARCH ARTICLE – Pharmaceutics, Drug Delivery and Pharmaceutical Technology
Design and In Vivo Anti-Inflammatory Effect of Ketoprofen Delayed Delivery Systems ANDREA CERCIELLO, GIULIA AURIEMMA, SILVANA MORELLO, ALDO PINTO, PASQUALE DEL GAUDIO, PAOLA RUSSO, RITA P. AQUINO Department of Pharmacy, University of Salerno, I-84084, Fisciano, (SA), Italy Received 13 May 2015; accepted 29 May 2015 Published online 18 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24554 ABSTRACT: For the treatment of inflammatory-based diseases affected by circadian rhythms, the development of once-daily dosage forms is required to target early morning symptoms. In this study, Zn–alginate beads containing ketoprofen (K) were developed by a tandem technique prilling/ionotropic gelation. The effect of main critical variables on particles micromeritics, inner structure as well as on drug loading and in vitro drug release was studied. The in vivo anti-inflammatory efficacy was evaluated using a modified protocol of carrageenan-induced edema in rat paw administering beads to rats by oral gavage at 0, 3, or 5 h before edema induction. Good drug loading and desired particle size and morphology were obtained for the optimized formulation F20. In vitro dissolution studies showed that F20 had a gastroresistant behavior and delayed release of the drug in simulated intestinal fluid. The in vitro delayed release pattern was clearly reflected in the prolonged anti-inflammatory effect in vivo of F20, compared to pure ketoprofen; F20, administered 3 h before edema induction, showed a significant anti-inflammatory activity, reducing maximum paw volume in response to carrageenan injection, whereas no response was observed for ketoprofen. The designed beads appear a promising platform suitable for a delayed release of C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3451–3458, 2015 anti-inflammatory drugs. Keywords: oral drug delivery; microencapsulation; solid dosage form; polymeric drug delivery systems; alginate; microparticles; dissolution
INTRODUCTION Ketoprofen is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties indicated for the symptomatic treatment of pain and inflammation in osteoarthritis and rheumatoid arthritis. However, its efficacy is countered by a large number of side effects, especially those related to gastrointestinal complications. Moreover, its short biological half-life (t1/2 = 2.1 h) and high frequency of dosing (2–4 times daily) increase health risks precluding a long-term use, as required in chronic inflammations and namely to relieve early morning pain in the management of osteoarticular pathologies.1 For these reasons, ketoprofen is a good candidate for the development of controlled release formulations, able to provide drug release at predetermined rate and time, targeting intestine, and reducing the well-known adverse effect of NSAIDs.2–5 In the last few years, different technologies have been proposed for the development of controlled and delayed drug delivery systems.6–8 Prilling (also known as laminar jet breakup) producing gel beads through vibrating nozzle extrusion is a time-saving technique, which offers several advantages compared with other microencapsulation technologies such as mild operative conditions (i.e., aqueous feed solutions, ambient temperature, and pressure) and an easy scale-up process.9–11 In the present study, a gel beads formulation was designed for delayed delivery of ketoprofen using prilling as a microencapsulation technique and alginate as a polymer carrier. in vivo studies were conducted to fully characterize formulation Correspondence to: Giulia Auriemma (Telephone: +39-089969395; Fax: +39089969602; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 3451–3458 (2015) C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
ability to delay in vivo absorption. Delay in ketoprofen release and in vivo absorption may be of interest to treat chronic inflammatory and painful diseases showing a circadian pattern with symptoms exacerbation in early morning.12 The main critical variables of the prilling process, i.e., composition of the aqueous feed solutions (sodium alginate and ketoprofen in different ratio), cross-linking conditions, frequency of vibration and flow rate, as well as beads micromeritics, were related to formulation performance. In vitro dissolution/release performances were assessed in conditions simulating the gastrointestinal environment (USP 36). The delayed anti-inflammatory effectiveness was evaluated in vivo using a carrageenan-induced acute edema in rat paw (male Wistar rats) protocol.
MATERIALS AND METHODS Materials Sodium alginate (European Pharmacopoeia XII, molecular weight ࣈ280 kDa) was purchased from Carlo Erba (Milan, Italy); cross-linking agent, zinc acetate dehydrate, was purchased from Sigma–Aldrich (Milan, Italy). Ketoprofen was kindly donated by Domp`e (Aquila, Italy). All other chemicals and reagents were obtained from Sigma–Aldrich and used as supplied. Drug-Loaded Beads Preparation An appropriate amount of sodium alginate was dissolved in deionized water at room conditions under gentle stirring in order to produce 100 mL of polymer solution with concentrations ranging between 1.5% and 2.0% (w/w). Different amounts of solid ketoprofen, crystalline form, were suspended into the
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alginate solutions and vigorously stirred for 3 h in order to obtain different drug/polymer ratios (1:5, 1:10, and 1:20). Ketoprofen-loaded beads were obtained by a vibrating nozzle device (Nisco Encapsulator Unit, Var D; Nisco Engineering Inc., Zurich, Switzerland), injecting ketoprofen–alginate suspension through a nozzle of 400 :m in diameter. Feed solutions were pumped through the nozzle using a syringe pump (Model 200 Series, Kd Scientific Inc., Boston, MA).13 Prilling experiments were performed at various volumetric flow rates between 5 and 10 mL/min. Breakup of the feed solution coming through the nozzle was obtained by setting vibration frequency between 250 and 350 Hz and with 100% vibration amplitude. A stroboscopic lamp was set at the same frequency amplitude to visualize the generated falling droplets. Droplets were gelled in a zinc acetate aqueous solution (10% w/v, pH 1.5) at room temperature and under gentle stirring. Distance between the vibrating nozzle and the gelling bath was fixed at 25 cm. After recovering, beads were rinsed with deionized water and dried at room temperature by exposure to air (22°C; 67% relative humidity, RH) for several hours (10–16 h) until constant weight was reached. Blank beads were also produced by prilling with the same experimental condition to be used as a control. Beads Size, Morphology, and Inner Structure Size distribution of hydrated beads was measured by an optical microscope (Citoval 2; Alessandrini, Milan, Italy) equipped with a camera, whereas dried beads were analyzed by scanning electron microscopy (SEM) using a Carl Zeiss EVO MA 10 microscope with a secondary electron detector (Carl Zeiss SMT Ltd, Cambridge, U.K.), equipped with a LEICA EMSCD005 ˚ metallization system producing a deposition of a 200–400 A thick gold layer. Analysis was conducted at 20 keV. Image analysis using ImageJ software (Wayne Rasband, National Institute of Health, Bethesda, MD) was performed on beads microphotographs to obtain projection diameter of both hydrated and dried beads. A minimum of 100 beads images were analyzed for each batch, and relative SD for at least three different prilling processes was calculated. Perimeter and projection surface area obtained by image analysis were used to calculate beads coefficient of sphericity (SC) by the following equation14 :
SC =
4BA P2
(1)
where A is the projected bead surface area and P is its perimeter. Beads inner structure images were obtained by cryofracture of the samples and further analysis by fluorescent microscopy (FM) through a Zeiss Axiophot microscope, equipped with a 20 × 1.4 NA no-oil immersion plan Apochromat objective ¨ (Carl Zeiss Vision, Munchen-Hallbergmoos, Germany) using standard 4 ,6-diamidino-2-phenylindole optics that adsorb violet radiation (maximum 372 nm) and emit a blue fluorescence (maximum 456 nm). In brief, freeze-fracture of the beads was performed by plunging the particles into liquid nitrogen for 1 min, then fracturing the frozen beads by means of two needles. Split particles were attached to aluminum stab, covered with gold, and analyzed. Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
Calorimetric Analysis Raw materials, blank, and drug-loaded beads were analyzed by differential scanning calorimetry on an indium calibrated Mettler Toledo DSC 822e (Mettler Toledo, Columbus, OH). Thermograms were recorded by placing accurately weighed quantities of each sample in a 40 :L aluminum pan, which was sealed and pierced. The samples were heated from 25°C to 350°C at a scanning rate of 10°C/min in nitrogen atmosphere at a flow rate of 100 mL/min. Melting temperature (Tm ) and peaks intensity (mW) were measured for all samples and compared with each other. The analyses were carried out in triplicate. Drug Content and Encapsulation Efficiency About 10 mg of beads from each batch was dissolved under vigorous stirring in fresh PBS buffer (100 mM; pH 7.0). Actual drug content (ADC) was determined by UV spectroscopy at 8 = 260 nm (Evolution 201 UV/VIS Spectrometer; Thermo Scientific, Waltham, MA). Encapsulation efficiency (EE) was calculated as the percentage ratio of actual to theoretical drug content. Each analysis was performed in triplicate; results were expressed in terms of mean ± SD. Drug Release Preliminary in vitro dissolution/release tests of ketoprofen from beads were carried out through a USP dissolution Apparatus 2 (Sotax AT7 Smart; Sotax, Allschwil, CH, Switzerland): paddle, 75 rpm, 37°C online with a UV spectrophotometer (Lambda 25 UV/VIS Spectrometer; PerkinElmer, Waltham, MA). A classic pH-change assay, method A, according to monograph “The Dissolution Procedure: Development and Validation” (USP 36), was used. So, in order to respect sink conditions, an accurately weighed aliquot of beads containing about 10 mg of drug was plunged into 750 mL of 0.1 M HCl for 2 h (acid stage); then, 250 mL of 0.2 M Na3 PO4 was added and pH was adjusted to 6.8 (buffer stage). Optimized formulations were also tested using USP dissolution Apparatus 4 (Sotax CE 7Smart; Sotax): flow-through cell for powders and granulates, open loop, 16 mL/min, 37°C, R PLUS online with a UV/VIS spectrophotometer (SPECORD Spectrometer; Analytik Jena, Jena, Germany). As a dissolution procedure, method B reported in USP 36 monograph was used. Acid stage was conducted as before described; after 2 h, buffer stage was performed in a phosphate buffer (pH 6.8) and prepared mixing three volumes of 0.1 M HCl with one volume of 0.2 M Na3 PO4 solution. Data were analyzed spectrophotometrically at the wavelength of maximum absorbance of ketoprofen in each medium (8 258 nm in acid and 8 260 nm in buffer medium, respectively). Dissolution tests were conducted on six different batches of particles, and mean values and SD were evaluated. Animals and Carrageenan Edema Induction Male Wistar rats (180–220 g) were purchased from Charles River Laboratories (Calco, Italy). Rats were anesthetized with isoflurane, and edema was induced by injecting 100 :L of carrageenan 1% (w/v) in the right hind paw as previously reported.15 Paw volume was measured plethysmographycally (2Biological-Instruments, Besozzo, Italy) at 0 h, each hour for 6 h, and at 24 h. Data were expressed as mean ± SD. Statistical differences were evaluated by two-way ANOVA. pValue of 0.90), whereas F5 presented SC value around 0.88. Overall results from particle size and SC analysis suggest the formation of more compact cross-linked matrix according to the content of drug into the gel polymeric network. Inner Structure Analysis To better understand the influence of drug content on polymeric matrix structure, beads were cryofractured and successively analyzed by both SEM and FM. Results from SEM showed that F20 had a more compact polymeric matrix than other formulation (Figs. 2c and 2d). Pure ketoprofen arose as a crystalline material with blue fluorescence consisting of small crystals (d50 = 3.1 :m) forming aggregates with a larger size (d50 = 20.5 :m) (Fig. 3a), while blank particles cross-sections (F) showed a pale yellow and off-white fluorescence (Fig. 3b). FM images of cryofractured ketoprofenloaded beads F20 displayed fluorescent spots due to ketoprofen crystals homogeneously embedded within the Zn–alginate matrix (Fig. 3c). As highlighted by FM images (Fig. 3), ketoprofen seems to keep its crystallinity after prilling and drying process, and crystal clusters of drug were homogeneously embedded within the polymeric Zn–alginate matrix (Figs 2d and 3c). Calorimetric Analysis The DSC thermal profiles of ketoprofen raw material and Zn– alginate beads, both blank (F) and ketoprofen loaded (F5–F20), are shown in Figure 4. Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
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Figure 1. SEM microphotographs of dried Zn–alginate beads formulations: (a) F, (b) F5, (c) F10, and (d) F20.
Figure 2. SEM microphotographs at different magnifications of cryofractured beads: (a and b) F10 and (c and d) F20.
According to previous observations,23 thermal profile of crystalline ketoprofen raw material (Fig. 4a) exhibited a very narrow melting peak at 97°C with an intensity of −13.88 mW. The thermogram of blank beads F (Fig. 4b) showed two endothermic events, well separated in temperature values, at 107°C and 190°C. The first event exhibited its onset at 84°C typical of the loss of hydration water from the polymer matrix24,25 and an intensity of −9.54 mW; the second event at 190°C was related to the endothermic melting process of the Zn–alginate matrix and had an intensity of −20.81 mW. This peak is followed by a ramp-like event ranging from 230°C to 252°C referable to polymeric matrix degradation. All ketoprofen-loaded beads (Figs. 4c–4e) showed a similar thermal trend with the endothermic events shifted at lower temperature. In fact, the first endothermic peak was found between 91°C and 94°C, for F5 and F20, respectively. The intensity of this peak increased with formulation drug content and has been related to two simultaneous events overlapping in this Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
range, namely, the loss of water and the melting of residual ketoprofen crystals, which were not encapsulated in the egg-box structure. The second endothermic melting process, between 155°C and 160°C, also showed an increased intensity related to ketoprofen content. This behavior may be due to the physical interaction, which occurs between the polymer and the drug after loading into the polymeric matrix. Drug Release Pilot dissolution tests of all formulations were performed by conventional vessel methods (USP Apparatus 2, paddle)26 using a classic pH change method, commonly used to provide essential information and recommended in various guidelines27,28 as a first choice for the in vitro dissolution testing of controlled/modified release formulations.29 Results showed that during the permanence of the formulations in acidic medium (2 h), F5 and F10 released 43.7% and DOI 10.1002/jps.24554
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Figure 3. Fluorescent microscopy images of (a) ketoprofen raw material, (b) cryofractured blank Zn–alginate beads F, and (c) cryofractured ketoprofen-loaded formulation F20.
32.8% of the loaded ketoprofen, respectively. After that, pH was changed and a drug release rate increased very rapidly, leading to a complete release of ketoprofen in simulated intestinal fluid (SIF) in about 1 h, whereas pure ketoprofen exhibited lower release rate (Fig. 5). On the contrary, ketoprofen release from F20 was greatly reduced (around 20%) in simulated gastric fluid (SGF) in 2 h. Thus, F20 acts as a gastroresistant oral dosage form; complete drug release was achieved in SIF after pH change in about 1.5–2 h, with a dissolution rate lower than those observed for F5 and F10. Although previous papers reported no significant effects on drug release under simulated gastrointestinal environment by varying the ketoprofen concentration in Ca–alginate beads,2,30 in this study ketoprofen in vitro release rate from Zn–alginate beads resulted strongly is influenced by the amount of drug loaded. F20, formulated with the highest amount of ketoprofen, exhibited a gastroresistant profile followed by a slow and extended release of the drug in SIF (100% ketoprofen release at about 3.5 h, Fig. 5). These data matched with SEM microphotographs analyses of the cryofractured F20 beads (Figs. 2c and 2d). In fact, the loading of high amount of drug into
Figure 4. Differential scanning calorimetry thermograms of ketoprofen raw material (a) and Zn–alginate beads, both blank F (b) and ketoprofen-loaded F5 (c), F10 (d), and F20 (e). DOI 10.1002/jps.24554
Figure 5. Release profiles of ketoprofen raw material (full triangle) and ketoprofen-loaded formulations F5 (empty circle), F10 (full square), and F20 (empty diamond), performed by USP Apparatus 2 using a pHchange assay. Mean ± SD; (n = 6).
feed solutions promote intermolecular interactions between the polymeric chains enhancing polymer entanglement during the gelling phase.21 This effect combined to the high cross-linking degree due to zinc ions provides tougher polymer matrix, especially in F20, leading to a stronger resistance to drug diffusion. F20 dissolution behavior was further investigated using the USP apparatus 4 (flow-through, open-loop configuration), which is reported to closely mimic in vivo hydrodynamics and thus to better predict in vivo performance of solid oral dosage forms.31–33 As shown in Figure 6, the apparatus 4 confirmed that F20 had enteric release behavior (19% of the drug released in 2 h
Figure 6. Release profiles of F20 formulation, performed by USP Apparatus 2 (empty diamond) and USP Apparatus 4 (full diamond), using a pH-change assay. Mean ± SD; (n = 6). Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
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In Vivo Studies
Figure 7. Dose–response curves of pure ketoprofen (1, 3, and 10 mg/kg doses) administered per os to rats 0.5 h before carrageenan injection; mean ± SD (n = 8). **p ࣘ 0.01, ***p ࣘ 0.001 compared with control.
at pH 1) and in SIF showed a slower rate than that obtained using the USP paddle method. Differences between the two apparatus may be explained by the different hydrodynamic conditions that characterize the flow-through cell system, where no agitation mechanisms occur and the dosage form is continuously exposed to a uniform laminar flow, similar to the natural environment of the gastrointestinal tract.34,35 F20 performance may be explained by considering alginate pH-dependent solubility, zinc cross-linking properties, and high structural density of matrix in F20; beads did not swell or erode in SGF and still keep intact matrix, whereas in SIF (at pH 6.8) they started to swell and further erode due to the ion exchange.36
The anti-inflammatory activity of the formulation F20 in rats was evaluated by a carrageenan-induced edema model and compared with the activity of pure drug. Rat paw edema reached the maximum value (paw volume, mL) between 2 and 4 h after carrageenan injection, as shown in Figure 7. First, a dose–response curve of pure ketoprofen (1, 3, and 10 mg/kg per os doses) was performed following a conventional protocol, which prescribes drug administration 0.5 h before the injection of the phlogistic agent. As expected, the administration of pure ketoprofen caused a significant reduction of edema after the first hour from carrageenan injection, compared with control at all tested doses. The anti-inflammatory effect of pure ketoprofen persisted even in the late phase of carrageenaninduced rat paw edema (24 h). These preliminary experiments suggest to select the dose of 3 mg/kg of ketoprofen as it achieved optimal anti-inflammatory effects (Fig. 7). With the aim to verify a corresponded delayed in vivo antiinflammatory effect, the optimized formulation F20 (ketoprofen equivalent dose of 3 mg/kg) was administered orally following the same protocol at different time points (5, 3, or 0.5 h) before edema induction. Interestingly, F20 showed a prolonged anti-inflammatory effect in terms of paw edema inhibition after oral administration to rats compared with control. Pure ketoprofen (3 mg/kg) was efficient in reducing rat paw edema only at t = 0 (0.5 h before phlogistic agent injection) (Fig. 7), whereas no response was observed when it was administered at 3 or 5 h before carrageenan injection. Blank Zn–alginate beads (F) did not significantly affect the paw edema when administered to rats at the same time points before edema induction (data not shown), thus, indicating that Zn–alginate matrix do not interfere with the inflammatory process. It is interesting to point out that F20 administered 3 h or 5 h before edema induction still showed a significant anti-inflammatory activity by reducing maximum paw volume
Figure 8. Edema volume reduction obtained by administering F20 and pure ketoprofen per os to rats 5 and 3 h before carrageenan injection compared with control; mean ± SD (n = 8). **p ࣘ 0.01, ***p ࣘ 0.001 compared with control. Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
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(3–4 h) in response to carrageenan injection, therefore reflecting its drug delayed release (Fig. 8). The strong delay in drug anti-inflammatory activity appreciated in vivo may be due to a further lowering of the drug absorption in the GIT as an effect of the mucoadhesive properties of alginate beads37 , which may guarantee a much more intimate and sustained contact with the absorption site.38
CONCLUSIONS In many inflammatory-based pathologies, clinical symptoms are commonly most severe in the early morning, closely following the circadian rhythm of the proinflammatory mediators such as cytokines and interleukins. A delayed release dosage form administered prior to sleep and able to deliver NSAID several hours after oral administration may be optimally effective to treat early morning symptoms. The present study suggested that prilling by the accurate selection of biopolymer, the opportune set-up of process parameters and gelling conditions allows to produce interesting delayed drug delivery systems. Particularly, Zn2+ as gelling/curing cross-linker seems to affect physicochemical characteristics and in vivo performance of beads by a combination of effects. Technological properties of Zn–alginate-based beads, such as morphology, hardness of cross-linked matrix, mucoadhesion and, consequently, in vitro and in vivo release behavior resulted strongly influenced by the amount of loaded ketoprofen. The optimized formulation F20 acts as a chronotherapeutic system that is able to delay in vivo ketoprofen release and absorption to 6–7 h. By opportunely timing the administration of the oral dosage form, it is possible to match the disease rhythms and to better control early morning symptoms, particularly in osteoarthritis and rheumatoid arthritis therapy.
ACKNOWLEDGMENTS The authors thank Luigi De Lucia (University of Salerno) for his excellent technical support during the in vivo experiments. This work was supported by PON R&S 2007–2013 codex PON02 00029 3203241 (POLIFARMA) as part of the research project “Progettazione e Sviluppo di Sistemi Terapeutici per il Rilascio Controllato di Famaci Attivi nelle Early Morning Pathologies” and authors would like to thank MIUR (Ministero dell’Istruzione, dell’Universita` e della Ricerca) for the provided financial support.
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DOI 10.1002/jps.24554
Design and In Vivo Anti-Inflammatory Effect of Ketoprofen Delayed Delivery Systems ANDREA CERCIELLO, GIULIA AURIEMMA, SILVANA MORELLO, ALDO PINTO, PASQUALE DEL GAUDIO, PAOLA RUSSO, RITA P. AQUINO Department of Pharmacy, University of Salerno, I-84084, Fisciano, (SA), Italy Received 13 May 2015; accepted 29 May 2015 Published online 18 June 2015 in Wiley Online Library (wileyonlinelibrary.com). DOI 10.1002/jps.24554 ABSTRACT: For the treatment of inflammatory-based diseases affected by circadian rhythms, the development of once-daily dosage forms is required to target early morning symptoms. In this study, Zn–alginate beads containing ketoprofen (K) were developed by a tandem technique prilling/ionotropic gelation. The effect of main critical variables on particles micromeritics, inner structure as well as on drug loading and in vitro drug release was studied. The in vivo anti-inflammatory efficacy was evaluated using a modified protocol of carrageenan-induced edema in rat paw administering beads to rats by oral gavage at 0, 3, or 5 h before edema induction. Good drug loading and desired particle size and morphology were obtained for the optimized formulation F20. In vitro dissolution studies showed that F20 had a gastroresistant behavior and delayed release of the drug in simulated intestinal fluid. The in vitro delayed release pattern was clearly reflected in the prolonged anti-inflammatory effect in vivo of F20, compared to pure ketoprofen; F20, administered 3 h before edema induction, showed a significant anti-inflammatory activity, reducing maximum paw volume in response to carrageenan injection, whereas no response was observed for ketoprofen. The designed beads appear a promising platform suitable for a delayed release of C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association J Pharm Sci 104:3451–3458, 2015 anti-inflammatory drugs. Keywords: oral drug delivery; microencapsulation; solid dosage form; polymeric drug delivery systems; alginate; microparticles; dissolution
INTRODUCTION Ketoprofen is a nonsteroidal anti-inflammatory drug (NSAID) with analgesic and antipyretic properties indicated for the symptomatic treatment of pain and inflammation in osteoarthritis and rheumatoid arthritis. However, its efficacy is countered by a large number of side effects, especially those related to gastrointestinal complications. Moreover, its short biological half-life (t1/2 = 2.1 h) and high frequency of dosing (2–4 times daily) increase health risks precluding a long-term use, as required in chronic inflammations and namely to relieve early morning pain in the management of osteoarticular pathologies.1 For these reasons, ketoprofen is a good candidate for the development of controlled release formulations, able to provide drug release at predetermined rate and time, targeting intestine, and reducing the well-known adverse effect of NSAIDs.2–5 In the last few years, different technologies have been proposed for the development of controlled and delayed drug delivery systems.6–8 Prilling (also known as laminar jet breakup) producing gel beads through vibrating nozzle extrusion is a time-saving technique, which offers several advantages compared with other microencapsulation technologies such as mild operative conditions (i.e., aqueous feed solutions, ambient temperature, and pressure) and an easy scale-up process.9–11 In the present study, a gel beads formulation was designed for delayed delivery of ketoprofen using prilling as a microencapsulation technique and alginate as a polymer carrier. in vivo studies were conducted to fully characterize formulation Correspondence to: Giulia Auriemma (Telephone: +39-089969395; Fax: +39089969602; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 104, 3451–3458 (2015) C 2015 Wiley Periodicals, Inc. and the American Pharmacists Association
ability to delay in vivo absorption. Delay in ketoprofen release and in vivo absorption may be of interest to treat chronic inflammatory and painful diseases showing a circadian pattern with symptoms exacerbation in early morning.12 The main critical variables of the prilling process, i.e., composition of the aqueous feed solutions (sodium alginate and ketoprofen in different ratio), cross-linking conditions, frequency of vibration and flow rate, as well as beads micromeritics, were related to formulation performance. In vitro dissolution/release performances were assessed in conditions simulating the gastrointestinal environment (USP 36). The delayed anti-inflammatory effectiveness was evaluated in vivo using a carrageenan-induced acute edema in rat paw (male Wistar rats) protocol.
MATERIALS AND METHODS Materials Sodium alginate (European Pharmacopoeia XII, molecular weight ࣈ280 kDa) was purchased from Carlo Erba (Milan, Italy); cross-linking agent, zinc acetate dehydrate, was purchased from Sigma–Aldrich (Milan, Italy). Ketoprofen was kindly donated by Domp`e (Aquila, Italy). All other chemicals and reagents were obtained from Sigma–Aldrich and used as supplied. Drug-Loaded Beads Preparation An appropriate amount of sodium alginate was dissolved in deionized water at room conditions under gentle stirring in order to produce 100 mL of polymer solution with concentrations ranging between 1.5% and 2.0% (w/w). Different amounts of solid ketoprofen, crystalline form, were suspended into the
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alginate solutions and vigorously stirred for 3 h in order to obtain different drug/polymer ratios (1:5, 1:10, and 1:20). Ketoprofen-loaded beads were obtained by a vibrating nozzle device (Nisco Encapsulator Unit, Var D; Nisco Engineering Inc., Zurich, Switzerland), injecting ketoprofen–alginate suspension through a nozzle of 400 :m in diameter. Feed solutions were pumped through the nozzle using a syringe pump (Model 200 Series, Kd Scientific Inc., Boston, MA).13 Prilling experiments were performed at various volumetric flow rates between 5 and 10 mL/min. Breakup of the feed solution coming through the nozzle was obtained by setting vibration frequency between 250 and 350 Hz and with 100% vibration amplitude. A stroboscopic lamp was set at the same frequency amplitude to visualize the generated falling droplets. Droplets were gelled in a zinc acetate aqueous solution (10% w/v, pH 1.5) at room temperature and under gentle stirring. Distance between the vibrating nozzle and the gelling bath was fixed at 25 cm. After recovering, beads were rinsed with deionized water and dried at room temperature by exposure to air (22°C; 67% relative humidity, RH) for several hours (10–16 h) until constant weight was reached. Blank beads were also produced by prilling with the same experimental condition to be used as a control. Beads Size, Morphology, and Inner Structure Size distribution of hydrated beads was measured by an optical microscope (Citoval 2; Alessandrini, Milan, Italy) equipped with a camera, whereas dried beads were analyzed by scanning electron microscopy (SEM) using a Carl Zeiss EVO MA 10 microscope with a secondary electron detector (Carl Zeiss SMT Ltd, Cambridge, U.K.), equipped with a LEICA EMSCD005 ˚ metallization system producing a deposition of a 200–400 A thick gold layer. Analysis was conducted at 20 keV. Image analysis using ImageJ software (Wayne Rasband, National Institute of Health, Bethesda, MD) was performed on beads microphotographs to obtain projection diameter of both hydrated and dried beads. A minimum of 100 beads images were analyzed for each batch, and relative SD for at least three different prilling processes was calculated. Perimeter and projection surface area obtained by image analysis were used to calculate beads coefficient of sphericity (SC) by the following equation14 :
SC =
4BA P2
(1)
where A is the projected bead surface area and P is its perimeter. Beads inner structure images were obtained by cryofracture of the samples and further analysis by fluorescent microscopy (FM) through a Zeiss Axiophot microscope, equipped with a 20 × 1.4 NA no-oil immersion plan Apochromat objective ¨ (Carl Zeiss Vision, Munchen-Hallbergmoos, Germany) using standard 4 ,6-diamidino-2-phenylindole optics that adsorb violet radiation (maximum 372 nm) and emit a blue fluorescence (maximum 456 nm). In brief, freeze-fracture of the beads was performed by plunging the particles into liquid nitrogen for 1 min, then fracturing the frozen beads by means of two needles. Split particles were attached to aluminum stab, covered with gold, and analyzed. Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
Calorimetric Analysis Raw materials, blank, and drug-loaded beads were analyzed by differential scanning calorimetry on an indium calibrated Mettler Toledo DSC 822e (Mettler Toledo, Columbus, OH). Thermograms were recorded by placing accurately weighed quantities of each sample in a 40 :L aluminum pan, which was sealed and pierced. The samples were heated from 25°C to 350°C at a scanning rate of 10°C/min in nitrogen atmosphere at a flow rate of 100 mL/min. Melting temperature (Tm ) and peaks intensity (mW) were measured for all samples and compared with each other. The analyses were carried out in triplicate. Drug Content and Encapsulation Efficiency About 10 mg of beads from each batch was dissolved under vigorous stirring in fresh PBS buffer (100 mM; pH 7.0). Actual drug content (ADC) was determined by UV spectroscopy at 8 = 260 nm (Evolution 201 UV/VIS Spectrometer; Thermo Scientific, Waltham, MA). Encapsulation efficiency (EE) was calculated as the percentage ratio of actual to theoretical drug content. Each analysis was performed in triplicate; results were expressed in terms of mean ± SD. Drug Release Preliminary in vitro dissolution/release tests of ketoprofen from beads were carried out through a USP dissolution Apparatus 2 (Sotax AT7 Smart; Sotax, Allschwil, CH, Switzerland): paddle, 75 rpm, 37°C online with a UV spectrophotometer (Lambda 25 UV/VIS Spectrometer; PerkinElmer, Waltham, MA). A classic pH-change assay, method A, according to monograph “The Dissolution Procedure: Development and Validation” (USP 36), was used. So, in order to respect sink conditions, an accurately weighed aliquot of beads containing about 10 mg of drug was plunged into 750 mL of 0.1 M HCl for 2 h (acid stage); then, 250 mL of 0.2 M Na3 PO4 was added and pH was adjusted to 6.8 (buffer stage). Optimized formulations were also tested using USP dissolution Apparatus 4 (Sotax CE 7Smart; Sotax): flow-through cell for powders and granulates, open loop, 16 mL/min, 37°C, R PLUS online with a UV/VIS spectrophotometer (SPECORD Spectrometer; Analytik Jena, Jena, Germany). As a dissolution procedure, method B reported in USP 36 monograph was used. Acid stage was conducted as before described; after 2 h, buffer stage was performed in a phosphate buffer (pH 6.8) and prepared mixing three volumes of 0.1 M HCl with one volume of 0.2 M Na3 PO4 solution. Data were analyzed spectrophotometrically at the wavelength of maximum absorbance of ketoprofen in each medium (8 258 nm in acid and 8 260 nm in buffer medium, respectively). Dissolution tests were conducted on six different batches of particles, and mean values and SD were evaluated. Animals and Carrageenan Edema Induction Male Wistar rats (180–220 g) were purchased from Charles River Laboratories (Calco, Italy). Rats were anesthetized with isoflurane, and edema was induced by injecting 100 :L of carrageenan 1% (w/v) in the right hind paw as previously reported.15 Paw volume was measured plethysmographycally (2Biological-Instruments, Besozzo, Italy) at 0 h, each hour for 6 h, and at 24 h. Data were expressed as mean ± SD. Statistical differences were evaluated by two-way ANOVA. pValue of 0.90), whereas F5 presented SC value around 0.88. Overall results from particle size and SC analysis suggest the formation of more compact cross-linked matrix according to the content of drug into the gel polymeric network. Inner Structure Analysis To better understand the influence of drug content on polymeric matrix structure, beads were cryofractured and successively analyzed by both SEM and FM. Results from SEM showed that F20 had a more compact polymeric matrix than other formulation (Figs. 2c and 2d). Pure ketoprofen arose as a crystalline material with blue fluorescence consisting of small crystals (d50 = 3.1 :m) forming aggregates with a larger size (d50 = 20.5 :m) (Fig. 3a), while blank particles cross-sections (F) showed a pale yellow and off-white fluorescence (Fig. 3b). FM images of cryofractured ketoprofenloaded beads F20 displayed fluorescent spots due to ketoprofen crystals homogeneously embedded within the Zn–alginate matrix (Fig. 3c). As highlighted by FM images (Fig. 3), ketoprofen seems to keep its crystallinity after prilling and drying process, and crystal clusters of drug were homogeneously embedded within the polymeric Zn–alginate matrix (Figs 2d and 3c). Calorimetric Analysis The DSC thermal profiles of ketoprofen raw material and Zn– alginate beads, both blank (F) and ketoprofen loaded (F5–F20), are shown in Figure 4. Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
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Figure 1. SEM microphotographs of dried Zn–alginate beads formulations: (a) F, (b) F5, (c) F10, and (d) F20.
Figure 2. SEM microphotographs at different magnifications of cryofractured beads: (a and b) F10 and (c and d) F20.
According to previous observations,23 thermal profile of crystalline ketoprofen raw material (Fig. 4a) exhibited a very narrow melting peak at 97°C with an intensity of −13.88 mW. The thermogram of blank beads F (Fig. 4b) showed two endothermic events, well separated in temperature values, at 107°C and 190°C. The first event exhibited its onset at 84°C typical of the loss of hydration water from the polymer matrix24,25 and an intensity of −9.54 mW; the second event at 190°C was related to the endothermic melting process of the Zn–alginate matrix and had an intensity of −20.81 mW. This peak is followed by a ramp-like event ranging from 230°C to 252°C referable to polymeric matrix degradation. All ketoprofen-loaded beads (Figs. 4c–4e) showed a similar thermal trend with the endothermic events shifted at lower temperature. In fact, the first endothermic peak was found between 91°C and 94°C, for F5 and F20, respectively. The intensity of this peak increased with formulation drug content and has been related to two simultaneous events overlapping in this Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
range, namely, the loss of water and the melting of residual ketoprofen crystals, which were not encapsulated in the egg-box structure. The second endothermic melting process, between 155°C and 160°C, also showed an increased intensity related to ketoprofen content. This behavior may be due to the physical interaction, which occurs between the polymer and the drug after loading into the polymeric matrix. Drug Release Pilot dissolution tests of all formulations were performed by conventional vessel methods (USP Apparatus 2, paddle)26 using a classic pH change method, commonly used to provide essential information and recommended in various guidelines27,28 as a first choice for the in vitro dissolution testing of controlled/modified release formulations.29 Results showed that during the permanence of the formulations in acidic medium (2 h), F5 and F10 released 43.7% and DOI 10.1002/jps.24554
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Figure 3. Fluorescent microscopy images of (a) ketoprofen raw material, (b) cryofractured blank Zn–alginate beads F, and (c) cryofractured ketoprofen-loaded formulation F20.
32.8% of the loaded ketoprofen, respectively. After that, pH was changed and a drug release rate increased very rapidly, leading to a complete release of ketoprofen in simulated intestinal fluid (SIF) in about 1 h, whereas pure ketoprofen exhibited lower release rate (Fig. 5). On the contrary, ketoprofen release from F20 was greatly reduced (around 20%) in simulated gastric fluid (SGF) in 2 h. Thus, F20 acts as a gastroresistant oral dosage form; complete drug release was achieved in SIF after pH change in about 1.5–2 h, with a dissolution rate lower than those observed for F5 and F10. Although previous papers reported no significant effects on drug release under simulated gastrointestinal environment by varying the ketoprofen concentration in Ca–alginate beads,2,30 in this study ketoprofen in vitro release rate from Zn–alginate beads resulted strongly is influenced by the amount of drug loaded. F20, formulated with the highest amount of ketoprofen, exhibited a gastroresistant profile followed by a slow and extended release of the drug in SIF (100% ketoprofen release at about 3.5 h, Fig. 5). These data matched with SEM microphotographs analyses of the cryofractured F20 beads (Figs. 2c and 2d). In fact, the loading of high amount of drug into
Figure 4. Differential scanning calorimetry thermograms of ketoprofen raw material (a) and Zn–alginate beads, both blank F (b) and ketoprofen-loaded F5 (c), F10 (d), and F20 (e). DOI 10.1002/jps.24554
Figure 5. Release profiles of ketoprofen raw material (full triangle) and ketoprofen-loaded formulations F5 (empty circle), F10 (full square), and F20 (empty diamond), performed by USP Apparatus 2 using a pHchange assay. Mean ± SD; (n = 6).
feed solutions promote intermolecular interactions between the polymeric chains enhancing polymer entanglement during the gelling phase.21 This effect combined to the high cross-linking degree due to zinc ions provides tougher polymer matrix, especially in F20, leading to a stronger resistance to drug diffusion. F20 dissolution behavior was further investigated using the USP apparatus 4 (flow-through, open-loop configuration), which is reported to closely mimic in vivo hydrodynamics and thus to better predict in vivo performance of solid oral dosage forms.31–33 As shown in Figure 6, the apparatus 4 confirmed that F20 had enteric release behavior (19% of the drug released in 2 h
Figure 6. Release profiles of F20 formulation, performed by USP Apparatus 2 (empty diamond) and USP Apparatus 4 (full diamond), using a pH-change assay. Mean ± SD; (n = 6). Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
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In Vivo Studies
Figure 7. Dose–response curves of pure ketoprofen (1, 3, and 10 mg/kg doses) administered per os to rats 0.5 h before carrageenan injection; mean ± SD (n = 8). **p ࣘ 0.01, ***p ࣘ 0.001 compared with control.
at pH 1) and in SIF showed a slower rate than that obtained using the USP paddle method. Differences between the two apparatus may be explained by the different hydrodynamic conditions that characterize the flow-through cell system, where no agitation mechanisms occur and the dosage form is continuously exposed to a uniform laminar flow, similar to the natural environment of the gastrointestinal tract.34,35 F20 performance may be explained by considering alginate pH-dependent solubility, zinc cross-linking properties, and high structural density of matrix in F20; beads did not swell or erode in SGF and still keep intact matrix, whereas in SIF (at pH 6.8) they started to swell and further erode due to the ion exchange.36
The anti-inflammatory activity of the formulation F20 in rats was evaluated by a carrageenan-induced edema model and compared with the activity of pure drug. Rat paw edema reached the maximum value (paw volume, mL) between 2 and 4 h after carrageenan injection, as shown in Figure 7. First, a dose–response curve of pure ketoprofen (1, 3, and 10 mg/kg per os doses) was performed following a conventional protocol, which prescribes drug administration 0.5 h before the injection of the phlogistic agent. As expected, the administration of pure ketoprofen caused a significant reduction of edema after the first hour from carrageenan injection, compared with control at all tested doses. The anti-inflammatory effect of pure ketoprofen persisted even in the late phase of carrageenaninduced rat paw edema (24 h). These preliminary experiments suggest to select the dose of 3 mg/kg of ketoprofen as it achieved optimal anti-inflammatory effects (Fig. 7). With the aim to verify a corresponded delayed in vivo antiinflammatory effect, the optimized formulation F20 (ketoprofen equivalent dose of 3 mg/kg) was administered orally following the same protocol at different time points (5, 3, or 0.5 h) before edema induction. Interestingly, F20 showed a prolonged anti-inflammatory effect in terms of paw edema inhibition after oral administration to rats compared with control. Pure ketoprofen (3 mg/kg) was efficient in reducing rat paw edema only at t = 0 (0.5 h before phlogistic agent injection) (Fig. 7), whereas no response was observed when it was administered at 3 or 5 h before carrageenan injection. Blank Zn–alginate beads (F) did not significantly affect the paw edema when administered to rats at the same time points before edema induction (data not shown), thus, indicating that Zn–alginate matrix do not interfere with the inflammatory process. It is interesting to point out that F20 administered 3 h or 5 h before edema induction still showed a significant anti-inflammatory activity by reducing maximum paw volume
Figure 8. Edema volume reduction obtained by administering F20 and pure ketoprofen per os to rats 5 and 3 h before carrageenan injection compared with control; mean ± SD (n = 8). **p ࣘ 0.01, ***p ࣘ 0.001 compared with control. Cerciello et al., JOURNAL OF PHARMACEUTICAL SCIENCES 104:3451–3458, 2015
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(3–4 h) in response to carrageenan injection, therefore reflecting its drug delayed release (Fig. 8). The strong delay in drug anti-inflammatory activity appreciated in vivo may be due to a further lowering of the drug absorption in the GIT as an effect of the mucoadhesive properties of alginate beads37 , which may guarantee a much more intimate and sustained contact with the absorption site.38
CONCLUSIONS In many inflammatory-based pathologies, clinical symptoms are commonly most severe in the early morning, closely following the circadian rhythm of the proinflammatory mediators such as cytokines and interleukins. A delayed release dosage form administered prior to sleep and able to deliver NSAID several hours after oral administration may be optimally effective to treat early morning symptoms. The present study suggested that prilling by the accurate selection of biopolymer, the opportune set-up of process parameters and gelling conditions allows to produce interesting delayed drug delivery systems. Particularly, Zn2+ as gelling/curing cross-linker seems to affect physicochemical characteristics and in vivo performance of beads by a combination of effects. Technological properties of Zn–alginate-based beads, such as morphology, hardness of cross-linked matrix, mucoadhesion and, consequently, in vitro and in vivo release behavior resulted strongly influenced by the amount of loaded ketoprofen. The optimized formulation F20 acts as a chronotherapeutic system that is able to delay in vivo ketoprofen release and absorption to 6–7 h. By opportunely timing the administration of the oral dosage form, it is possible to match the disease rhythms and to better control early morning symptoms, particularly in osteoarthritis and rheumatoid arthritis therapy.
ACKNOWLEDGMENTS The authors thank Luigi De Lucia (University of Salerno) for his excellent technical support during the in vivo experiments. This work was supported by PON R&S 2007–2013 codex PON02 00029 3203241 (POLIFARMA) as part of the research project “Progettazione e Sviluppo di Sistemi Terapeutici per il Rilascio Controllato di Famaci Attivi nelle Early Morning Pathologies” and authors would like to thank MIUR (Ministero dell’Istruzione, dell’Universita` e della Ricerca) for the provided financial support.
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DOI 10.1002/jps.24554
