New trends towards glaucoma treatment: Topical osmoprotective microemulsions loaded with latanoprost.

The chronic use of hypotensive agents eventually leads to ocular surface damage and poor patient compliance during glaucoma management. Thus, new sustained drug delivery systems are needed. This work aimed to develop osmoprotective latanoprost-loaded microemulsion formulations as new potential glaucoma treatments with ocular surface protective properties. The microemulsions were characterized and latanoprost encapsulation efficacy determined. In-vitro tolerance, osmoprotective efficacy, cell internalization as well as cell-microemulsion interactions and distribution were performed. In vivo hypotensive activity was conducted in rabbits to assess intraocular pressure reduction and relative ocular bioavailability. Physicochemical characterization showed nanodroplet sizes within 20-30 nm, being in vitro tolerance within 80 and 100% viability in corneal and conjunctival cells. Besides, microemulsions exhibited higher protection under hypertonic conditions than untreated cells. Cell fluorescence lasted for 11 days after short exposure to coumarin-loaded microemulsions (5 min) showing extensive internalization in different cell compartments by electronic microscopy. In vivo studies exhibited that a single instillation of latanoprost-loaded microemulsions reduced the intraocular pressure for several days (4-6 days without polymer and 9-13 days with polymers). Relative ocular bioavailability was 4.5 and 19 times higher than the marketed formulation. These findings suggest the use of these microemulsions as potential combined strategies for extended surface protection and glaucoma treatment.

Development of an osmoprotective microemulsion as a therapeutic platform for ocular surface protection.

Self-emulsified osmoprotective ophthalmic microemulsions (O/A) were prepared by combining betaine/leucine, clusterin/oleanolic acid, and hyaluronic acid or Dextran. The microemulsions contained an internal oily phase (1.2%), an external aqueous phase (96.3%), cosolvents (1%), and surfactants (1.5%). Physicochemical characterization and in vivo and in vitro tolerance were analyzed. The formulations' osmoprotective in vitro activity was assayed in a hyperosmolar model in human corneal cells. Average internal phase sizes were 16-26 nm for the microemulsions including Dextran. Addition of hyaluronic acid increased the size range (25-39 nm). Addition of osmoprotectants did not change nanodroplet size. The formulations were isotonic (280-290 mOsm/L) with neutral pH (≈7) and zeta potential (-10 to 0 mV), low surface tension (≈35-40mN·m-1), and low viscosity (≈1 mPa·s), except for the microemulsions containing hyaluronic acid (≈4-5 mPa·s). SEM and cryo-TEM showed that all formulations exhibited sphere-shaped morphology with good cell tolerance (≈100%) and were stable at 8 °C for 9 months. Osmoprotective formulations were well tolerated in vitro and in vivo, protecting cells from hypertonic stress. We therefore developed stable microemulsions compatible with the ocular surface that could constitute a novel tool for treatment of ophthalmic diseases.

Combined hyperosmolarity and inflammatory conditions in stressed human corneal epithelial cells and macrophages to evaluate osmoprotective agents as potential DED treatments.

PURPOSE: To develop an easy-to-perform combined model in human corneal epithelial cells (HCECs) and Balb/c mice macrophages J774.A1 (MP) for preliminary screening of potential ophthalmic therapeutic substances. METHODS: HCECs were exposed to different osmolarities (350-500 mOsm/L) and MTT assay was employed for cell survival and flow cytometry to assess apoptosis-necrosis and relative cell size (RCS) distribution. Effectiveness of Betaine, L-Carnitine, Taurine at different concentrations (ranging from 20 mM to 200 mM) was studied. Also, mucoadhesive polymers such as Hyaluronic acid (HA) and Hydroxypropylmethylcellulose (HPMC) (0.4 and 0.8%) were evaluated. Cells were pre-incubated with the compounds (8h) and then exposed to hyperosmotic stress (470 mOsm/L) for 16h. Moreover, anti-inflammatory activity was performed in LPS-stimulated MP. RESULTS: Exposure to hyperosmotic solutions between 450 and 500 mOsm/L promoted the highest cell death after 16h exposures (p < 0.0001) with a drop in viability to 34.96% ± 11.77 for 470 mOsm/L. Pre-incubation with Betaine at 150 mM and 200 mM provided the highest cell survival against hyperosmolarity (66.01% ± 3.65 and 65.90% ± 0.78 respectively) while HA 0.4% was the most effective polymer in preventing cell death (42.2% ± 3.60). Flow cytometry showed that Betaine and Taurine at concentrations between 150-200 mM and 20-80 mM respectively presented the highest anti-apoptotic activity. Also, HA and HPMC polymers reduced apoptotic-induced cell death. All osmoprotectants modified RCS, and polymers increased their value over 100%. L-Carnitine 50 mM, Taurine 40 mM and HA 0.4% presented the highest TNF-α inhibition activity (60%) albeit all of them showed anti-inflammatory inhibition percentages higher than 20% CONCLUSIONS: HCECs hyperosmolar model combined with inflammatory conditions in macrophages allows the screening of osmoprotectants by simulating chronic hyperosmolarity (16h) and inflammation (24h).

Simultaneous co-delivery of neuroprotective drugs from multi-loaded PLGA microspheres for the treatment of glaucoma.

Glaucoma is a multifactorial neurodegenerative disorder and one of the leading causes of irreversible blindness globally and for which intraocular pressure is the only modifiable risk factor. Although neuroprotective therapies have been suggested to have therapeutic potential, drug delivery for the treatment of ocular disorders such as glaucoma remains an unmet clinical need, further complicated by poor patient compliance with topically applied treatments. In the present study we describe the development of multi-loaded PLGA-microspheres (MSs) incorporating three recognised neuroprotective agents (dexamethasone (DX), melatonin (MEL) and coenzyme Q10 (CoQ10)) in a single formulation (DMQ-MSs) to create a novel sustained-release intraocular drug delivery system (IODDS) for the treatment of glaucoma. MSs were spherical, with a mean particle size of 29.04 ±â€¯1.89 µm rendering them suitable for intravitreal injection using conventional 25G-32G needles. >62% incorporation efficiency was achieved for the three drug cargo and MSs were able to co-deliver the encapsulated active compounds in a sustained manner over 30-days with low burst release. In vitro studies showed DMQ-MSs to be neuroprotective in a glutamate-induced cytotoxicity model (IC50 10.00 ±â€¯0.94 mM versus 6.89 ±â€¯0.82 mM in absence of DMQ-MSs) in R28 cell line. In vivo efficacy studies were performed using a well-established rodent model of chronic ocular hypertension (OHT), comparing single intravitreal injections of microspheres of DMQ-MSs to their equivalent individual single-drug loaded MSs mixture (MSsmix), empty MSs, no-treatment OHT only and naïve groups. Twenty one days after OHT induction, DMQ-MSs showed a significantly neuroprotective effect on RGCs compared to OHT only controls. No such protective effect was observed in empty MSs and single-drug MSs treated groups. This work suggests that multi-loaded PLGA MSs present a novel therapeutic approach in the management of retinal neurodegeneration conditions such as glaucoma.

Pharmaceutical microscale and nanoscale approaches for efficient treatment of ocular diseases.

Efficient treatment of ocular diseases can be achieved thanks to the proper use of ophthalmic formulations based on emerging pharmaceutical approaches. Among them, microtechnology and nanotechnology strategies are of great interest in the development of novel drug delivery systems to be used for ocular therapy. The location of the target site in the eye as well as the ophthalmic disease will determine the route of administration (topical, intraocular, periocular, and suprachoroidal administration) and the most adequate device. In this review, we discuss the use of colloidal pharmaceutical systems (nanoparticles, liposomes, niosomes, dendrimers, and microemulsions), microparticles (microcapsules and microspheres), and hybrid systems (combination of different strategies) in the treatment of ophthalmic diseases. Emphasis has been placed in the therapeutic significance of each drug delivery system for clinical translation.

Novel Water-Soluble Mucoadhesive Carbosilane Dendrimers for Ocular Administration.

The purpose of this research was to determine the potential use of water-soluble anionic and cationic carbosilane dendrimers (generations 1-3) as mucoadhesive polymers in eyedrop formulations. Cationic carbosilane dendrimers decorated with ammonium -NH3(+) groups were prepared by hydrosylilation of Boc-protected allylamine and followed by deprotection with HCl. Anionic carbosilane dendrimers with terminal carboxylate groups were also employed in this study. In vitro and in vivo tolerance studies were performed in human ocular epithelial cell lines and rabbit eyes respectively. The interaction of dendrimers with transmembrane ocular mucins was evaluated with a surface biosensor. As proof of concept, the hypotensive effect of a carbosilane dendrimer eyedrop formulation containing acetazolamide (ACZ), a poorly water-soluble drug with limited ocular penetration, was tested after instillation in normotensive rabbits. The methodology used to synthesize cationic dendrimers avoids the difficulty of obtaining neutral -NH2 dendrimers that require harsher reaction conditions and also present high aggregation tendency. Tolerance studies demonstrated that both prototypes of water-soluble anionic and cationic carbosilane dendrimers were well tolerated in a range of concentrations between 5 and 10 µM. Permanent interactions between cationic carbosilane dendrimers and ocular mucins were observed using biosensor assays, predominantly for the generation-three (G3) dendrimer. An eyedrop formulation containing G3 cationic carbosilane dendrimers (5 µM) and ACZ (0.07%) (289.4 mOsm; 5.6 pH; 41.7 mN/m) induced a rapid (onset time 1 h) and extended (up to 7 h) hypotensive effect, and led to a significant increment in the efficacy determined by AUC0(8h) and maximal intraocular pressure reduction. This work takes advantage of the high-affinity interaction between cationic carbosilane dendrimers and ocular transmembrane mucins, as well as the tensioactive behavior observed for these polymers. Our results indicate that low amounts of cationic carbosilane dendrimers are well tolerated and able to improve the hypotensive effect of an acetazolamide solution. Our results suggest that carbosilane dendrimers can be used in a safe range of concentrations to enhance the bioavailability of drugs topically administered in the eye.

Preservation of biological activity of glial cell line-derived neurotrophic factor (GDNF) after microencapsulation and sterilization by gamma irradiation.

A main issue in controlled delivery of biotechnological products from injectable biodegradable microspheres is to preserve their integrity and functional activity after the microencapsulation process and final sterilization. The present experimental work tested different technological approaches to maintain the biological activity of an encapsulated biotechnological product within PLGA [poly (lactic-co-glycolic acid)] microspheres (MS) after their sterilization by gamma irradiation. GDNF (glial cell line-derived neurotrophic factor), useful in the treatment of several neurodegenerative diseases, was chosen as a labile model protein. In the particular case of optic nerve degeneration, GDNF has been demonstrated to improve the damaged retinal ganglion cells (RGC) survival. GDNF was encapsulated in its molecular state by the water-in-oil-in-water (W/O/W) technique or as solid according to the solid-in-oil-in-water (S/O/W) method. Based on the S/O/W technique, GDNF was included in the PLGA microspheres alone (S/O/W 1) or in combination with an antioxidant (vitamin E, Vit E) (S/O/W 2). Microspheres were sterilized by gamma-irradiation (dose of 25 kGy) at room and low (-78 °C) temperatures. Functional activity of GDNF released from the different microspheres was evaluated both before and after sterilization in their potential target cells (retinal cells). Although none of the systems proposed achieved with the goal of totally retain the structural stability of the GDNF-dimer, the protein released from the S/O/W 2 microspheres was clearly the most biologically active, showing significantly less retinal cell death than that released from either W/O/W or S/O/W 1 particles, even in low amounts of the neurotrophic factor. According to the results presented in this work, the biological activity of biotechnological products after microencapsulation and sterilization can be further preserved by the inclusion of the active molecule in its solid state in combination with antioxidants and using low temperature (-78 °C) during gamma irradiation exposure.

Interfacial interaction between transmembrane ocular mucins and adhesive polymers and dendrimers analyzed by surface plasmon resonance.

PURPOSE: Development of the first in vitro method based on biosensor chip technology designed for probing the interfacial interaction phenomena between transmembrane ocular mucins and adhesive polymers and dendrimers intended for ophthalmic administration. METHODS: The surface plasmon resonance (SPR) technique was used. A transmembrane ocular mucin surface was prepared on the chip surface and characterized by QCM-D (Quartz Crystal Microbalance with Dissipation) and XPS (X-ray photoelectron spectroscopy). The mucoadhesive molecules tested were: hyaluronic acid (HA), carboxymethyl cellulose (CMC), hydroxypropylmethyl cellulose (HPMC), chitosan (Ch) and polyamidoamine dendrimers (PAMAM). RESULTS: While Ch originated interfacial interaction with ocular transmembrane mucins, for HA, CMC and HPMC, chain interdiffusion seemed to be mandatory for bioadherence at the concentrations used in ophthalmic clinical practise. Interestingly, PAMAM dendrimers developed permanent interfacial interactions with transmembrane ocular mucins whatever their surface chemical groups, showing a relevant importance of co-operative effect of these multivalent systems. Polymers developed interfacial interactions with ocular membrane-associated mucins in the following order: Ch(1 %) > G4PAMAM-NH(2)(2 %) = G4PAMAM-OH(2 %) > G3.5PAMAM-COOH(2 %)>> CMC(0.5 %) = HA(0.2 %) = HPMC(0.3 %). CONCLUSIONS: The method proposed is useful to discern between the mucin-polymer chemical interactions at molecular scale. Results reinforce the usefulness of chitosan and dendrimers as polymers able to increase the retention time of drugs on the ocular surface and hence their bioavailability.

Aplicación de la metodología de aprendizaje cooperativo a la materia de Biofarmacia y Farmacocinética / Implementation of the cooperative learning methodology to the Biopharmaceutics and Pharmacokinetics subject

La convergencia dentro del marco Europeo de Educación Superior plantea la necesidad de introducir cambios en el sistema educativo universitario. En este sentido, la formación en la universidad debe asegurar el desarrollo integral y continuo de los nuevos profesionales. El modelo tradicional de enseñanza ligado a conocimientos disciplinares ha de sustituirse por una formación en competencias ligadas al desempeño profesional y a un saber hacer cualificado para cada situación concreta. Metodologías activas como el aprendizaje cooperativo (AC) son reconocidas como estrategias idóneas para alcanzar estas competencias.En este entorno se plantea el objetivo de este trabajo como una experiencia de aprendizaje cooperativo que se está llevando a cabo con un grupo de alumnos de la asignatura de Biofarmacia y Farmacocinética en la Licenciatura de Farmacia. Este estudio forma parte del desarrollo de un proyecto de Innovación y Mejora de la Calidad Docente de la Universidad Complutense de Madrid (UCM 2009-276). Dentro del programa de la asignatura se han elegido aquellos temas que resultan más adecuados para los objetivos de esta modalidad de aprendizaje. El grupo en el que se ha llevado a cabo esta experiencia, es un grupo piloto (adscripción voluntaria para los alumnos) que cuenta con 63 alumnos. Se han formado 9 grupos de trabajo con la participación de 7 especialistas por grupo. Para la comunicación con los alumnos y la entrega de documentación de trabajo se ha utilizado el Campus Virtual de la UCM que utiliza la plataforma WebCT(AU)

The convergence in the European Higher Education Framework presents the need to make changes in the University Educational System. In this sense, the education in the University must ensure the all-round and continuous development of new professionals. The traditional model of education related to the knowledge of subjects must be substituted by the education in competences related to professional performance and qualification know-how for each particular situation. Active methodologies such as cooperative learning are recognized as suitable strategies to get those competences. In this environment, the objective of this work is presented as a cooperative learning experience that is carrying out with a group of students of Biopharmacy and Pharmacokinetic subject of the Pharmacy Grade. This study is part of the development of a Project of Innovation and Improvement of the Educational Quality in the Complutense University of Madrid. The topics that have been chosen from the whole program of the subject are the most suitable to reach the objectives of this learning method. This experience has been tested in a pilot group integrated by 63 voluntary students. Nine work groups have been formed with the participation of seven specialists in each group. The UCM Virtual Campus website (based on WebCT platform) has been used for communication with the students and documentation purposes(AU)

Biocompatibility of elastin-like polymer poly(VPAVG) microparticles: in vitro and in vivo studies.

Poly(L-valine-L-proline-L-alanine-L-valine-L-glycine) (VPAVG) is a new kind of proteinaceous polymer belonging to the Elastin-like family. These polymers are based on the recurrence of certain short peptide monomers that are considered as "building blocks" in the natural elastin. This smart thermoresponsive polymer has the ability to self-associate at physiological temperature to form aggregates with about 60% in water. This ability can be harnessed to prepare microparticles loaded with an active substance. The aim of this report is to evaluate, from the results of the experiment conducted, the biocompatibility of microparticles prepared from poly(VPAVG). We have studied the cytotoxic effects of microparticles, edema formation after subcutaneous injection (1 and 2.5 mg) in rats (n = 6), and also intraocular tolerance after the intravitreal injection of 2.5 mg of poly(VPAVG) microparticles into pigmented rabbits (n = 12). The polymer did not induce any cytotoxicity or nonspecific depression of cellular respiration on macrophages under the range of polymer concentrations investigated in this study (20, 30, 40, and 60 mg/mL). We observed no inflammatory response to microparticles after subcutaneous injection in the hind-paw of rats, with no significant differences between the control group (PBS) and experimental groups. Anterior and posterior segment signs were evaluated after intraocular injection of poly(VPAVG) microparticles. Only a few eyes (2/11) of the experimental group presented inflammation signs at day 28 postinjection. Nevertheless, 45% (5/11) of the eyes receiving microparticles showed tractional retinal detachment. The results observed in this work suggested certain fibroblastic activity induced by poly(VPAVG) microparticles after their intraocular injection.

Sistemas de cesión controlada por vía intraocular / Intraocular drug delivery systems

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Self-assembled particles of an elastin-like polymer as vehicles for controlled drug release.

Elastin-like polymers (ELPs) are a new kind of protein-based polymers showing interesting properties in the biomaterial field. This work explored the use of self-assembled poly(VPAVG) micro- and nanoparticles as vehicles for the controlled release of the model drug dexamethasone phosphate (DMP). Poly(VPAVG) has shown to form stable particles with a size below 3 mum as a water or PBS polymer solution was warmed above its transition temperature ( approximately 30 degrees C). Due to the peculiar composition of the monomer, the formation and redissolution of the self-assembled microparticles shows an interesting hysteresis behaviour by which the particles are formed at this temperature but do not redissolve until a strong undercooling of approximately 12-15 degrees C is achieved. Therefore, the particles, once formed, are stable either at room or body temperature. These self-assembled particles are able to encapsulate significant amounts of the model drug when self-assembling was carried out in a co-solution polymer-DMP. The release profiles showed a sustained DMP release for about 30 days. Being the potential of this new polymeric carrier high, further research is being conducted to functionalise the poly(VPAVG) base as a way to induce a stronger polymer-drug binding and, accordingly, a more sustained release.

Biodegradable ibuprofen-loaded PLGA microspheres for intraarticular administration. Effect of Labrafil addition on release in vitro.

The objective of this study was the development and optimisation of biodegradable PLGA microspheres loaded with ibuprofen destined for intraarticular administration. The formulation was designed to provide "in vitro" therapeutic concentrations of ibuprofen (8 microg/ml) for as long as possible. The solvent evaporation method based on an o/w emulsion was used to form the microparticles. The polymer used was Poly (D,L-lactide-co-glicolide) 50:50 (PLGA), of different molecular weights (Mw) (34,000, 48,000 and 80,000 Da). In order to get a more controlled release rate of ibuprofen, a biodegradable oil, Labrafil M1944CS, polyethylene glycol 300 derivative, was used as an additive. The formulation was optimised by means of an experimental design, 2(3) being the variables: X(1) = PLGA Mw; X(2) = initial ibuprofen:polymer ratio; X(3) = percentage of Labrafil. The theoretical profile yielding in vitro "therapeutic" concentrations of ibuprofen (8 microg/ml) was calculated. The experimental profiles obtained for the formulations tested were compared with the theoretical one by means of the difference factor (f(1)). In all cases, the addition of Labrafil lowered the initial ibuprofen burst, prolonging the release rate of the drug from 24 h (without additive) up to 8 days incorporating the oil. The microspheres made from the PLGA (Mw = 34,000 Da) with Labrafil addition (10%) and ibuprofen:polymer (15%) ratio (formulation 1) yielded the most suitable release profiles. Forty milligram of the selected formulation (formulation 1), was sufficient to provide in vitro "therapeutic" concentrations of ibuprofen (8 microg/ml) up to 8 days. Labrafil modulates the release rate of donor-acceptor substances such as ibuprofen.

Sterilized ibuprofen-loaded poly(D,L-lactide-co-glycolide) microspheres for intra-articular administration: effect of gamma-irradiation and storage.

The aim of this study was to prepare and characterize a controlled-release system (microspheres) loaded with ibuprofen, for intra-articular administration, to extend its anti-inflammatory effect in the knee joint cavity. Among the bioresorbable polymers employed, poly(D,L-lactic-co-glycolic) acid (PLGA) (13137 Da) was chosen because of its high biocompatiblity. Microspheres were produced by the solvent evaporation process from an O/W emulsion. Labrafil M 1944 CS was included in the formulation as an additive in order to modulate the release rate of the non-steroidal anti-inflammatory drug (NSAID). Once prepared, the microspheres were sobre-sterilized by gamma-irradiation. The effect of the irradiation dose (25 kGy) exposure, at low temperature, on the formulation was evaluated. The sterilization procedure employed did not alter the physicochemical characteristics of the formulation. Dissolution profiles of formulations behaved similarly and overlapped (f2=87.23, f1=4.2) before and after sterilization. Size Exclusion Chromatography (SEC) revealed no significant changes in the polymer molecular weight. Additionally, a stability study of the sterilized formulation was carried out using microsphere storage conditions of 4 degrees C in a vacuum desiccator for 1 year. The results obtained after storing the sterilized microspheres show no significant alterations in the ibuprofen release rate (f2 = 85.06, f1 = 4.32) or in the molecular weight of the PLGA (12957 Da). The employment of low molecular weight PLGA polymers resulted as advantageous, due to the practical absence of degradation after gamma irradiation (25 kGy) exposure at low temperature.

Consideraciones generales sobre los estudios de farmacocinética ocular / General considerations related to ocular pharmacokinetic studies

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Pharmacokinetics of intravenous luxabendazole in rabbits: influence of the enterohepatic circulation.

Luxabendazole (LBZ) is a new benzimidazole carbamate chemotherapeutic agent, which has proved to be very effective against adult and immature stages of the major gastrointestinal nematodes, trematodes and cestodes. While information on the efficacy of LBZ in several animal species is available, there seems to be no published information describing the disposition kinetics in any of them. As a part of the clinical development of luxabendazole, the pharmacokinetics of a single intravenous dose was investigated in parasite-free rabbits. Serial blood samples were collected at timed intervals for 12 h following administration of the dose, and concentrations in plasma were determined by a sensitive and specific HPLC method. Published data on LBZ point to the possible existence of an enterohepatic cycle (EHC), and so, it seemed appropriate to carry out two different forms of test. In the first, the possibility of intestinal reabsorption of LBZ excreted via the bile was allowed for (Treatment 1), while in the second it was interrupted by the oral administration of activated charcoal (Treatment 2). In both cases the animals were given a single dose of 10 mg kg-1 of LBZ intravenously (i.v). Comparison of the areas under the curve (AUCs) of LBZ concentrations in plasma samples taken from the animals receiving each treatment showed significant difference (p < 0.05). The given dose (10 mg kg-1) was converted in Treatment 1 to an effective dose of 13.9 mg kg-1 through recycling of LBZ. With Treatment 2 a bicompartmental distribution model for this drug was confirmed, together with high apparent distribution volumes: Vc = 1.87 L kg-1, and V beta = 7.09 L kg-1.

Bioavailability and bioequivalence of two formulations of etodolac (tablets and suppositories).

We studied the influence of administration route on the biopharmaceutical behavior of etodolac. The levels obtained in plasma when the same dose of etodolac is administered orally (tablets, dosage form A) and rectally (suppositories, dosage form B) were compared. The study was done in a crossover design with healthy volunteers of both sexes, of average build, and younger than 35 years of age. From the concentration in plasma-time data, the maximum concentration in plasma (Cmax), time to Cmax, and area under the curve up to the last measurable concentration (AUC0t) or infinity (AUC 0 infinity) were calculated and compared by analysis of variance. With the exception of Cmax, no significant differences between treatments were found in the rest of the parameters. Finally, with formulation A (tablets) as a reference, the relative bioavailability was established, on the basis of the ratio (B:A) of AUC0t and AUC 9 infinity, within the range 100 +/- 20%. The results indicate that the two routes of administration are bioequivalent and that the rectal route is an alternative administration route for etodolac.