Irene T. Molina-Martínez

Retinal ganglion cells survival in a glaucoma model by GDNF/Vit E PLGA microspheres prepared according to a novel microencapsulation procedure.

The present experimental work describes the use of a novel protein encapsulation method to achieve protection of the biological factor during the microencapsulation procedure. With this aim, the protein is included in poly(lactic-co-glycolic acid) (PLGA) microspheres without any preliminary manipulation, in contrast to the traditional S/O/W (solid-in-oil-in-water) method where the bioactive substance is first dissolved and then freeze-dried in the presence of lyoprotectors. Furthermore, the presented technique involves the use of an oily additive, vitamin E (Vit E), useful from a technological point of view, by promoting additional protein protection and also from a pharmacological point of view, because of its antioxidant and antiproliferative properties. Application of this microencapsulation technique has been performed for GDNF (glial cell line-derived neurotrophic factor) designed for the treatment of optic nerve degenerative diseases, such as glaucoma, the second leading cause of blindness in the western world. The protein was released in vitro in its bioactive form for more than three months, demonstrated by the survival of their potential target cells (photoreceptors and retinal ganglion cells (RGC)). Moreover, the intravitreal injection of GDNF/Vit E PLGA microspheres in an experimental animal model of glaucoma significantly increased RGC survival compared with GDNF, Vit E or blank microspheres (p<0.01). This effect was present for at least eleven weeks, which suggests that the formulation prepared may be clinically useful as a neuroprotective tool in the treatment of glaucomatous optic neuropathy.

The potential of using biodegradable microspheres in retinal diseases and other intraocular pathologies

Pathologies affecting the posterior segment of the eye are one of the major causes of blindness in developed countries and are becoming more prevalent due to the increase in society longevity. Successful therapy of diseases affecting the back of the eye requires effective concentrations of the active substance maintained during a long period of time in the intraocular target site. Treatment of vitreoretinal diseases often include repeated intravitreous injections that are associated with adverse effects. Local administration of biodegradable microspheres offers an excellent alternative to multiple administrations, as they are able to deliver the therapeutic molecule in a controlled fashion. Furthermore, injection of microparticles is performed without the need for surgical procedures. As most of the retinal diseases are multifactorial, microspheres result especially promising because they can be loaded with more than one active substance and complemented with the inclusion of additives with pharmacological properties. Personalized therapy can be easily achieved by changing the amount of administered microspheres. Contrary to non-biodegradable devices, biodegradable PLA and PLGA microspheres disappear from the site of administration after delivering the drug. Furthermore, microspheres prepared from these mentioned biomaterials are well tolerated after periocular and intravitreal injections in animals and humans. After injection, PLA and PLGA microspheres suffer aggregation behaving like an implant. Biodegradable microspheres are potential tools in regenerative medicine for retinal repair. According to the reported results, presumably a variety of microparticulate formulations for different ophthalmic therapeutic uses will be available in the clinical practice in the near future.

Sterilized ibuprofen-loaded poly(D,L-lactide-co-glycolide) microspheres for intra-articular administration: effect of γ-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) (13 137 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 γ-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 physico-chemical 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°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 (12 957 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.

Ophthalmic formulations of the intraocular hypotensive melatonin agent 5-MCA-NAT

Melatonin is a hormone responsible for the regulation of circadian and seasonal rhythms. This hormone is synthesised in many tissues in the body including the eye, where it regulates important processes. During the recent years, the role of melatonin in the control of IOP has been investigated and it has been demonstrated that melatonin receptors are present and involved in the dynamics of the aqueous humour. 5-Methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT) is a selective MT3 melatonin receptor agonist. Topical application of this product produces a clear reduction in intraocular pressure (IOP) in New Zealand white rabbits and in glaucomatous monkeys. In this work, the potent ocular hypotensive 5-MCA-NAT has been dissolved in excipients used in currently marketed drug formulations. Until now, this melatonin analogue had been dissolved in either DMSO or ethanol neither of which is suitable for ocular topical application in humans. Solubility assays in the different solvents were performed by the observation of the presence of drug crystals under optical microscopy. 5-MCA-NAT was completely dissolved in propylene glycol (PG) and polyethylene glycol 300 (PEG 300) within 24h. Ophthalmic formulations were prepared from different ratios of PG:PBS and the commercialized Systane product. Quantification of 5-MCA-NAT in the vehicles was assessed by HPLC. In vitro cytotoxicity of the formulations was evaluated by the MTT method and in vivo tolerance of 5-MCA-NAT in the solvents was analyzed by biomicroscopy and specular microscopy. Systane and proportions of PG:PBS up to 10% of PG did not show cytotoxicity in human corneal limbal epithelial cells (HCLE). In vivo experiments showed that the higher the ocular tolerance, the less amount of PG present. The ocular hypotensive effect of 5-MCA-NAT dissolved in the new formulations was checked measuring IOP for 8h after instillation of the substance. The best effect lowering IOP was obtained with 5-MCA-NAT dissolved in PG and diluted with PBS (PG 1.43%) in which 5-MCA-NAT produced a reduction of 28.11+/-2.0% and the effect lasted about 7h. In conclusion, new formulations accepted for ocular topical treatments different from DMSO or ethanol were capable of dissolving the melatonin analogue 5-MCA-NAT, preserving its ocular hypotensive ability. Therefore, the use of 5-MCA-NAT may be possible in the treatment of ocular hypertension and glaucoma.

The Use of Mucoadhesive Polymers to Enhance the Hypotensive Effect of a Melatonin Analogue, 5-MCA-NAT, in Rabbit Eyes

PURPOSE 5-Methoxy-carbonylamino-N-acetyltryptamine (5-MCA-NAT, a melatonin receptor agonist) produces a clear intraocular pressure (IOP) reduction in New Zealand White rabbits and glaucomatous monkeys. The goal of this study was to evaluate whether the hypotensive effect of 5-MCA-NAT was enhanced by the presence of cellulose derivatives, some of them with bioadhesive properties, as well as to determine whether these formulations were well tolerated by the ocular surface. METHODS Formulations were prepared with propylene glycol (0.275%), carboxymethyl cellulose (CMC, 0.5% and 1.0%) of low and medium viscosity and hydroxypropylmethyl cellulose (0.3%). Quantification of 5-MCA-NAT (100 μM) was assessed by HPLC. In vitro tolerance was evaluated by the MTT method in human corneal-limbal epithelial cells and normal human conjunctival cells. In vivo tolerance was analyzed by biomicroscopy and specular microscopy in rabbit eyes. The ocular hypotensive effect was evaluated measuring IOP for 8 hours in rabbit eyes. RESULTS All the formulations demonstrated good in vitro and in vivo tolerance. 5-MCA-NAT in CMC medium viscosity 0.5% was the most effective at reducing IOP (maximum IOP reduction, 30.27%), and its effect lasted approximately 7 hours. CONCLUSIONS The hypotensive effect of 5-MCA-NAT was increased by using bioadhesive polymers in formulations that are suitable for the ocular surface and also protective of the eye in long-term therapies. The use of 5-MCA-NAT combined with bioadhesive polymers is a good strategy in the treatment of ocular hypertension and glaucoma.

Design and characterization of an ocular topical liposomal preparation to replenish the lipids of the tear film.

PURPOSE Dry eye (DE) includes a group of diseases related to tear film disorders. Current trends for DE therapy focus on providing lipid components to replace the damaged lipid layer. Formulations that contain aqueous and mucin-like compounds may have additional therapeutic benefits for DE patients. The aim of this work was to design and evaluate novel formulations having the potential to become topical treatment for DE. METHODS Unpreserved liposomal formulations composed of phosphatidylcholine (PC), cholesterol, and α-tocopherol (vit E) were prepared by the thin-film hydration technique. Formulations were characterized in terms of liposome size, pH, surface tension, osmolarity, and viscosity. In vitro tolerance assays were performed on macrophage, human corneal, and conjunctival cell lines at short- and long-term exposures. In vivo ocular tolerance was studied after instillation of the formulation. RESULTS The mean liposome size was less than 1 μm and surface tension < 30 mN/m for all formulations. The final liposomal formulation (PC-cholesterol-vit E in a ratio of 8:1:0.8) had physiological values of pH (6.45 ± 0.09), osmolarity (289.43 ± 3.28 mOsm), and viscosity (1.82 ± 0.02 mPa · s). Cell viability was greater than 80% in the corneal and conjunctival cells. This formulation was well tolerated by experimental animals. CONCLUSIONS The unpreserved liposomal formulation has suitable properties to be administered by a topical ophthalmic route. The liposome-based artificial tear had good in vitro and in vivo tolerance responses. This formulation, composed of a combination of liposomes and bioadhesive polymers, may be used successfully as a tear film substitute in DE therapy.

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.

Comparison of the In Vitro Tolerance and In Vivo Efficacy of Traditional Timolol Maleate Eye Drops versus New Formulations with Bioadhesive Polymers

PURPOSE To assess the in vitro tolerance and in vivo efficacy of new unpreserved formulations of timolol maleate (TM) in aqueous solutions of bioadhesive polymers used for dry eye treatment and to compare them with three traditional TM formulations: unpreserved Timabak (Thea, Madrid, Spain), benzalkonium chloride (BAK)-preserved Timoftol (Frosst Laboratories, Madrid, Spain), and BAK-preserved Timolol Sandoz (Frosst Laboratories). METHODS New formulations were composed of TM (0.5%) and carboxymethyl cellulose (0.5%), hyaluronic acid (0.2%), or hydroxypropylmethyl cellulose (0.3% or 0.5%). In vitro tolerance was determined in human corneal-limbal epithelial cells and normal human conjunctival cells. The ocular hypotensive effect was evaluated measuring IOP in rabbit eyes for 8 hours. RESULTS In all cases, cell survival after exposure to the formulations was greater in the new unpreserved TM formulations than in the traditional TM solutions (BAK-preserved and unpreserved). In addition, the new formulations were demonstrated to maintain the hypotensive effect of TM in different magnitudes. The maximum hypotensive effect was reached by TM 0.5% in carboxymethyl cellulose 0.5% (32.37%). CONCLUSIONS The results demonstrated that new unpreserved formulations of TM with bioadhesive polymers decreased IOP in rabbits and reached values closer to those reached by traditional solutions. Furthermore, new formulations presented a significantly higher in vitro tolerance than the same compound in traditional formulations. Although unpreserved formulations are usually more expensive, preservative-free antiglaucoma eye drops should improve compliance and adherence in the medical treatment of glaucoma. Bioadhesive polymers could be part of antiglaucoma formulations to reduce ocular toxicity, improve drug efficacy, and protect the ocular surface in long-term therapies.

PLA and PLGA microparticles for intravitreal drug delivery : an overview

Disorders affecting the posterior segment of the eye are one of the major causes of blindness in western countries. Treatments of these pathologies often require multiple intravitreal injections to achieve effective concentrations in the vitreous cavity. However, repeated injections are poorly tolerated and are frequently associated with cataract formation, retinal detachment, and endophthalmitis. Furthermore, the risk of adverse effects increases with the frequency of intravitreal administration. Injectable microparticles offer a good alternative to multiple injections because they can be administered as conventional intraocular formulations. Among them, microspheres prepared from biodegradable polymers such as poly(lactic) (PLA) acid and poly(lactic-co-glycolic)acid (PLGA) have the advantage of disappearing from the site of administration once the active substance has been delivered. This work describes relevant in vitro and in vivo parameters and variables related to the intravitreal administration of PLA and PLGA microspheres for the treatment of diseases affecting the posterior segment.

Hybrid Formulations of Liposomes and Bioadhesive Polymers Improve the Hypotensive Effect of the Melatonin Analogue 5-MCA-NAT in Rabbit Eyes

For the treatment of chronic ocular diseases such as glaucoma, continuous instillations of eye drops are needed. However, frequent administrations of hypotensive topical formulations can produce adverse ocular surface effects due to the active substance or other components of the formulation, such as preservatives or other excipients. Thus the development of unpreserved formulations that are well tolerated after frequent instillations is an important challenge to improve ophthalmic chronic topical therapies. Furthermore, several components can improve the properties of the formulation in terms of efficacy. In order to achieve the mentioned objectives, we have developed formulations of liposomes (150–200 nm) containing components similar to those in the tear film and loaded with the hypotensive melatonin analog 5-methoxycarbonylamino-N-acetyltryptamine (5-MCA-NAT, 100 µM). These formulations were combined with mucoadhesive (sodium hyaluronate or carboxymethylcellulose) or amphiphilic block thermosensitive (poloxamer) polymers to prolong the hypotensive efficacy of the drug. In rabbit eyes, the decrease of intraocular pressure with 5-MCA-NAT-loaded liposomes that were dispersed with 0.2% sodium hyaluronate, 39.1±2.2%, was remarkably higher compared to other liposomes formulated without or with other bioadhesive polymers, and the effect lasted more than 8 hours. According to the results obtained in the present work, these technological strategies could provide an improved modality for delivering therapeutic agents in patients with glaucoma.