RESUMEN
PURPOSE: To determine safety, pharmacokinetics, and anti-inflammatory effects of intraocular celecoxib. METHODS: The right eye of animals was injected with 1.5, 3, or 6 mg celecoxib prepared in dimethyl sulfoxide (DMSO). Left eyes served as controls and received 0.1 mL DMSO. Electroretinograms (ERG) were obtained at baseline and at 1, 4, and 12 weeks, and eyes were enucleated afterward for histopathologic analysis. For pharmacokinetics, 3 mg celecoxib was injected, and vitreous and retina/choroid drug levels were then analyzed at specific time points. For efficacy, 1 µg lipopolysaccharide was injected to induce inflammation; the right eye was then injected with 3 mg celecoxib (six eyes) or 2 mg triamcinolone acetonide (six eyes) and the left eye with saline. Twenty-four hours later, aqueous fluid was removed, and total leukocyte concentration and prostaglandin E2 (PGE2) concentration were determined. RESULTS: Histologic and ERG studies demonstrated no signs of retinal or optic nerve toxicity. After a single 3-mg injection, vitreous (0.06 µg/mL) and retina/choroid (132.31 µg/g) celecoxib concentrations at 8 weeks exceeded median inhibitory concentration. Treatment with celecoxib and triamcinolone significantly reduced total leukocyte count by 40% (P = 0.02) and 31% (P = 0.01), respectively. Reduction in PGE2 levels paralleled reduction in leukocyte counts (P < 0.05). There was no increase in intraocular pressure, but cataract formation was observed at higher concentrations. CONCLUSIONS: Intraocular injection of celecoxib appeared to be nontoxic and demonstrated excellent penetration into the retina/choroid and sustained drug levels out to 8 weeks. Celecoxib demonstrated potent anti-inflammatory effects, but there was an association with cataract formation at higher doses.
Asunto(s)
Pirazoles/farmacocinética , Retina/efectos de los fármacos , Sulfonamidas/farmacocinética , Uveítis/tratamiento farmacológico , Animales , Humor Acuoso/metabolismo , Celecoxib , Inhibidores de la Ciclooxigenasa 2/administración & dosificación , Inhibidores de la Ciclooxigenasa 2/farmacocinética , Modelos Animales de Enfermedad , Electrorretinografía , Inyecciones Intravítreas , Masculino , Pirazoles/administración & dosificación , Conejos , Retina/patología , Retina/fisiopatología , Sulfonamidas/administración & dosificación , Resultado del Tratamiento , Uveítis/metabolismo , Uveítis/patologíaRESUMEN
Since there is paucity of information on solute transporters in human ocular tissues, the aim of this study was immunohistochemical and functional characterization of peptide transporters (PEPT), organic cation transporters (OCTs), neutral and basic amino acid transporters (ATB(0,+)), and monocarboxylate transporters (MCTs) in human ocular barriers. Immunohistochemical localization of transporters was achieved using 5-µm-thick paraffin-embedded sections of whole human eyes. In vitro transport studies were carried out across human cornea and sclera-choroid-retinal pigment epithelium (SCRPE) using a cassette of specific substrates in the presence and absence of inhibitors to determine the role of transporters in transtissue solute delivery. Immunohistochemistry showed the expression of PEPT-1, PEPT-2, ATB(0,+), OCT-1, OCT-2, MCT-1, and MCT-3 in human ocular tissues. PEPT-1, PEPT-2, OCT-1, MCT-1, and ATB(0,+) expression was evident in the cornea, conjunctiva, ciliary epithelium, and neural retina. Expression of PEPT-1, PEPT-2, and OCT-1 was evident in choroid tissue as well. OCT-2 expression could be seen in the corneal and conjunctival epithelia, whereas MCT-3 expression was confined to the RPE layer. OCT-2 expression was evident in conjunctival blood vessel walls, whereas PEPT-1, PEPT-2, and OCT-1 were expressed in the choroid. Preliminary transport studies indicated inward transport of Gly-Sar (PEPT substrate), 1-methyl-4-phenylpyridinium (MPP+) (OCT substrate), and l-tryptophan (ATB(0,+) substrate) across cornea as well as SCRPE. For phenylacetic acid (MCT substrate), transporter-mediated inward transport across the cornea and outward transport across SCRPE were evident. Thus, PEPT, OCT, and ATB(0,+) are influx transporters present in human ocular barriers, and they can potentially be used for transporter-guided retinal drug delivery after topical, transscleral, and systemic administrations.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Ojo/metabolismo , Inmunohistoquímica , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Simportadores/metabolismo , Anciano , Anciano de 80 o más Años , Sistemas de Transporte de Aminoácidos Básicos/antagonistas & inhibidores , Sistemas de Transporte de Aminoácidos Neutros/antagonistas & inhibidores , Transporte Biológico , Coroides/metabolismo , Córnea/metabolismo , Ojo/efectos de los fármacos , Femenino , Humanos , Cinética , Masculino , Moduladores del Transporte de Membrana/farmacología , Persona de Mediana Edad , Transportadores de Ácidos Monocarboxílicos/antagonistas & inhibidores , Proteínas de Transporte de Catión Orgánico/antagonistas & inhibidores , Adhesión en Parafina , Epitelio Pigmentado de la Retina/metabolismo , Esclerótica/metabolismo , Simportadores/antagonistas & inhibidoresRESUMEN
In this work, we aim to design and synthesize prodrugs of gatifloxacin targeting organic cation transporter (OCT), monocarboxylate transporter (MCT), and ATB (0, +) transporters and to identify a prodrug with enhanced delivery to the back of the eye. Dimethylamino-propyl, carboxy-propyl, and amino-propyl(2-methyl) derivatives of gatifloxacin (GFX), DMAP-GFX, CP-GFX, and APM-GFX, were designed and synthesized to target OCT, MCT, and ATB (0, +) transporters, respectively. An LC-MS method was developed to analyze drug and prodrug levels in various studies. Solubility and log D (pH 7.4) were measured for prodrugs and the parent drug. The permeability of the prodrugs was determined in the cornea, conjunctiva, and sclera-choroid-retinal pigment epitheluim (SCRPE) and compared with gatifloxacin using an Ussing chamber assembly. Permeability mechanisms were elucidated by determining the transport in the presence of transporter specific inhibitors. 1-Methyl-4-phenylpyridinium iodide (MPP+), nicotinic acid sodium salt, and α-methyl-DL-tryptophan were used to inhibit OCT, MCT, and ATB (0, +) transporters, respectively. A prodrug selected based on in vitro studies was administered as an eye drop to pigmented rabbits, and the delivery to various eye tissues including vitreous humor was compared with gatifloxacin dosing. DMAP-GFX exhibited 12.8-fold greater solubility than GFX. All prodrugs were more lipophilic, with the measured log D (pH 7.4) values ranging from 0.05 to 1.04, when compared to GFX (log D: -1.15). DMAP-GFX showed 1.4-, 1.8-, and 1.9-fold improvement in permeability across the cornea, conjunctiva, and SCRPE when compared to GFX. Moreover, it exhibited reduced permeability in the presence of MPP+ (competitive inhibitor of OCT), indicating OCT-mediated transport. CP-GFX showed 1.2-, 2.3-, and 2.5-fold improvement in permeability across the cornea, conjunctiva, and SCRPE, respectively. In the presence of nicotinic acid (competitive inhibitor of MCT), the permeability of CP-GFX was reduced across the conjunctiva. However, the cornea and SCRPE permeability of CP-GFX was not affected by nicotinic acid. APM-GFX did not show any improvement in permeability when compared to GFX across the cornea, conjunctiva, and SCRPE. Based on solubility and permeability, DMAP-GFX was selected for in vivo studies. DMAP-GFX showed 3.6- and 1.95-fold higher levels in vitreous humor and CRPE compared to that of GFX at 1 h after topical dosing. In vivo conversion of DMAP-GFX prodrug to GFX was quantified in tissues isolated at 1 h after dosing. The parent drug-to-prodrug ratio was 8, 70, 24, 21, 29, 13, 55, and 60% in the cornea, conjunctiva, iris-ciliary body, aqueous humor, sclera, CRPE, retina, and vitreous humor, respectively. In conclusion, DMAP-GFX prodrug enhanced solubility, log D, as well as OCT mediated delivery of gatifloxacin to the back of the eye.
Asunto(s)
Sistemas de Liberación de Medicamentos , Ojo/efectos de los fármacos , Fluoroquinolonas/química , Transportadores de Ácidos Monocarboxílicos/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Profármacos/administración & dosificación , Administración Tópica , Animales , Transporte Biológico , Cromatografía Liquida , Gatifloxacina , Permeabilidad , Profármacos/síntesis química , Profármacos/farmacocinética , Conejos , Solubilidad , Espectrometría de Masas en Tándem , Distribución TisularRESUMEN
The enormous success of antibiotics is seriously threatened by the development of resistance to most of the drugs available on the market. Thus, novel antibiotics are needed that are less prone to bacterial resistance and are directed toward novel biological targets. Antimicrobial peptides (AMPs) have attracted considerable attention due to their unique mode of action and broad spectrum activity. However, these agents suffer from liability to proteases and the high cost of manufacturing has impeded their development. Previously, we have reported on a novel class of benzophenone-based antibiotics and early studies suggested that these agents might target the bacterial membrane. In this study, we present our work on the mechanism of action of these novel membrane targeted antibiotics. These compounds have good affinities to polyanionic components of the cell wall such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS). We found that these agents release potassium ions from treated bacteria; thus, resulting in disruption of the bacterial membrane potential. Benzophenone-based membrane targeted antibiotics (BPMTAs) cause membrane disruption in synthetic lipid vesicles that mimic Gram-positive or Gram-negative bacteria. The compounds display no hemolytic activity up to a concentration that is 100 times the MIC values and they are capable of curing mice of a lethal MRSA infection. Repeated attempts to develop a mutant resistant to these agents has failed. Taken together, BPMTAs represent a promising new class of membrane-targeted antibacterial agents.
Asunto(s)
Antibacterianos/química , Antibacterianos/uso terapéutico , Benzofenonas/química , Benzofenonas/uso terapéutico , Farmacorresistencia Bacteriana , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Infecciones Estafilocócicas/tratamiento farmacológico , Animales , Antibacterianos/farmacología , Benzofenonas/farmacología , Hemólisis/efectos de los fármacos , Humanos , Liposomas/metabolismo , Staphylococcus aureus Resistente a Meticilina/metabolismo , Ratones , Potasio/metabolismo , Ovinos , Infecciones Estafilocócicas/metabolismoRESUMEN
The increase in the incidence of antibiotic-resistant infections is a major concern to healthcare workers and requires the development of novel antibacterial agents. Recently, we described a series of benzophenone-containing antibiotics which displayed activity against antibiotic-resistant bacteria. We have shown that these agents function by disrupting the bacterial membrane. To further explore these compounds, a practical and efficient solution-phase parallel synthesis method was developed which allowed us to prepare combinatorial libraries of these agents. Using this method, we prepared 218 compounds in 58 reactions. All of the compounds were characterized by HPLC and MALDI-TOF mass spectrometry. Analysis of this library for antibacterial activity identified six compounds which displayed MIC values of 2.0 mg/L against Staphylococcus aureus. Examination of the structure-function relationships of these agents revealed that cationic groups were required and that cyclic, aliphatic amines were crucial for activity. Using the information generated here, we speculate on how the various structural features of the molecule are necessary for the interaction with the bacterial membrane.
Asunto(s)
Antibacterianos/síntesis química , Técnicas Químicas Combinatorias/métodos , Diseño de Fármacos , Membranas/efectos de los fármacos , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/química , Antibacterianos/farmacología , Benzofenonas/química , Cromatografía Líquida de Alta Presión , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Soluciones/química , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
The increase in the incidence of both hospital- and community-acquired antibiotic-resistant infections is a major concern to the healthcare community. There have been only two new classes of antibiotics approved by the FDA over the past 40 years, and clearly there is a growing need for additional antimicrobial agents. In this paper, we present our work on the discovery of a class of benzophenone containing compounds that possess good activity against MRSA, VISA, VRSA, and VRE and moderate activity against E. coli. These compounds display MIC values in the 0.5-2.0 mg/L range and are not cytotoxic against mammalian cells. Extensive structure-activity relationship studies revealed that the benzophenone was absolutely essential for antibacterial activity as was the presence of a cationic group. Although these agents display DNA binding activity, we observed that these compounds do not inhibit any macromolecular synthesis reliant upon DNA nor do they inhibit lipid or cell wall biosynthesis. Instead, we found that these agents cause membrane depolarization, indicating that the bacterial membrane was the primary site of action for these agents. Our studies suggest that caution should be taken in assigning the mechanism of action for DNA binding antibiotics.