RESUMEN
High concentration formulations have become an important pre-requisite in the development of biological drugs, particularly in the case of subcutaneous administration where limited injection volume negatively affects the administered dose. In this study, we propose to develop high concentration formulations of biologics using a reversible protein-polyelectrolyte complex (RPC) approach. First, the versatility of RPC was assessed using different complexing agents and formats of therapeutic proteins, to define the optimal conditions for complexation and dissociation of the complex. The stability of the protein was investigated before and after complexation, as well as upon a 4-week storage period at various temperatures. Subsequently, two approaches were selected to develop high concentration RPC formulations: first, using up-concentrated RPC suspensions in aqueous buffers, and second, by generating spray-dried RPC and further resuspension in non-aqueous solvents. Results showed that the RPC concept is applicable to a wide range of therapeutic protein formats and the complexation-dissociation process did not affect the stability of the proteins. High concentration formulations up to 200 mg/mL could be achieved by up-concentrating RPC suspensions in aqueous buffers and RPC suspensions in non-aqueous solvents were concentrated up to 250 mg/mL. Although optimization is needed, our data suggests that RPC may be a promising avenue to achieve high concentration formulations of biologics for subcutaneous administration.
Asunto(s)
Productos Biológicos , Composición de Medicamentos , Proteínas , Excipientes , SolventesRESUMEN
Impaired wound healing in patients receiving glucocorticoid therapy is a serious clinical concern: mineralocorticoid receptor (MR) antagonists can counter glucocorticoid-induced off-target activation of MR receptors. The aim of this study was to investigate the cutaneous delivery of the potent MR antagonist, spironolactone (SPL), from polymeric micelle nanocarriers, prepared using a biodegradable copolymer, methoxy-poly(ethylene glycol)-di-hexyl-substituted-poly(lactic acid). Immunofluorescent labelling of the MR showed that it was principally located in the pilosebaceous unit (PSU), justifying the study rationale since polymeric micelles accumulate preferentially in appendageal structures. Cutaneous biodistribution studies under infinite and finite dose conditions, demonstrated delivery of pharmacologically relevant amounts of SPL to the epidermis and upper dermis. Preferential PSU targeting was confirmed by comparing amounts of SPL in PSU-containing and PSU-free skin biopsies: SPL nanomicelles showed 5-fold higher delivery of SPL in the PSU-containing biopsies, 0.54 ± 0.18 ng/mm2vs. 0.10 ± 0.03 ng/mm2, after application of a hydrogel in finite conditions. Canrenone, an active metabolite of SPL, was also quantified in skin samples. In addition to being used for the treatment of delayed cutaneous wound healing by site-specific antagonism of the MR, the formulation might also be used to treat pilosebaceous androgen-related skin diseases, e.g. acne vulgaris, since SPL is a potent androgen receptor antagonist.
Asunto(s)
Micelas , Espironolactona , Glucocorticoides , Humanos , Receptores de Mineralocorticoides/metabolismo , Distribución Tisular , Cicatrización de HeridasRESUMEN
Sustained-release formulations for ocular delivery are of increasing interest given their potential to significantly improve treatment efficacy and patient adherence. The objectives of this study were (i) to develop a sustained-release formulation of spironolactone (SPL) using a biodegradable and injectable polymer, hexyl-substituted poly-lactic acid (hexPLA) and (ii) to investigate the ocular biodistribution and tolerability of SPL and its metabolites in rats in vivo over 1 month following a single intravitreal injection (IVT inj). The concentrations of SPL and its two principal active metabolites, 7α-thiomethylspironolactone and canrenone (CAN), in the different ocular compartments were determined at different time points (3, 7, and 31 days after IVT inj) using a validated ultra-high-performance liquid chromatography-mass spectrometry method. Systemic exposure following a single IVT inj of 5% SPL-hexPLA formulation was evaluated by quantifying SPL and its metabolites in the plasma. Ocular tolerability of the formulation was evaluated using in vivo retinal imaging and histology. In vitro release studies revealed a sustained release of SPL from 5% SPL-hexPLA for up to 65 days. In vivo studies showed that SPL and its metabolites were detected in all ocular tissues at 3 and 7 days post-IVT inj. At 31 days post-IVT inj, SPL and CAN were mainly detected in the retina. These results also highlighted the clearance pathway of SPL and its metabolite involving the anterior and posterior routes in the first week (days 3 and 7), then mainly the posterior segment in the last week (day 31). This study showed that a single IVT inj of 5% SPL-hexPLA in rats enabled sustained delivery of therapeutic amounts of SPL for up to 1 month to the retina without systemic exposure. This formulation may be of interest for the local treatment of diseases involving overactivation of the mineralocorticoid receptor in the chorioretina such as chronic central serous chorioretinopathy.
Asunto(s)
Poliésteres/química , Retina/metabolismo , Espironolactona/administración & dosificación , Espironolactona/farmacocinética , Animales , Canrenona/química , Cromatografía Liquida , Preparaciones de Acción Retardada/química , Preparaciones de Acción Retardada/farmacocinética , Fondo de Ojo , Inyecciones Intravítreas , Espectrometría de Masas , Ratas , Ratas Wistar , Retina/citología , Retina/efectos de los fármacos , Espironolactona/análogos & derivados , Espironolactona/química , Espironolactona/toxicidad , Factores de Tiempo , Distribución Tisular , Tomografía de Coherencia ÓpticaRESUMEN
Glucocorticoids are a mainstay for the treatment of immune-mediated conditions and inflammatory diseases. However, their chronic use causes numerous side-effects including delays in corneal and cutaneous wound healing. This is attributed to off-target agonism of the mineralocorticoid receptor, which can be reduced by co-administration of a mineralocorticoid receptor antagonist such as spironolactone. The aim of this study was to develop a fast, selective and sensitive UHPLC-ESI-MS method for the simultaneous quantification of spironolactone, its active metabolites (7α-thiomethylspironolactone and canrenone), the latter's water-soluble prodrug potassium canrenoate and the synthetic glucocorticoid, dexamethasone, in corneal samples (17α-methyltestosterone served as an internal standard). A one-step extraction procedure using MeOH-H2 O (1:1) was validated and employed to recover the analytes from the corneal tissue. Extracts were centrifuged and the supernatant analyzed under isocratic conditions. Compounds were detected using selected ion recording mode. The method satisfied US Food and Drug Administration guidelines with respect to selectivity, precision and accuracy and displayed linearity from 5 to 1000 ng/mL for all of the analytes. The lower limit of quantitation of the method was 5 ng/mL, making it sufficiently sensitive for quantification of the analytes in samples from in vivo studies.
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Cromatografía Líquida de Alta Presión/métodos , Córnea/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Espironolactona/análisis , Espironolactona/metabolismo , Animales , Córnea/metabolismo , Estabilidad de Medicamentos , Límite de Detección , Modelos Lineales , Reproducibilidad de los Resultados , Espironolactona/química , Porcinos , Espectrometría de Masas en Tándem/métodosRESUMEN
The objective was to investigate whether mineralocorticoid receptor antagonism using a novel topical micellar formulation of spironolactone could prevent glucocorticoid-induced delayed corneal wound healing in New Zealand white rabbits. Spironolactone micelles (0.1%, w/v) with a mean number weighted diameter of 20 nm were prepared using a pegylated copolymer (mPEG-dihexPLA) and showed a preliminary stability of at least 12 months at 5 °C. Preclinical studies in New Zealand white rabbits demonstrated that the 0.1% spironolactone micellar formulation was well-tolerated since no reaction was observed in the cornea following multiple daily instillation over 5 days. As expected, the preclinical studies also confirmed that dexamethasone significantly delayed epithelial wound healing as compared to untreated control (percentage re-epithelialization after day 4: 84.6 ± 13.9% versus 99.5 ± 1.0% for the control, p < 0.05). However, the addition of the 0.1% spironolactone micellar formulation significantly improved the extent of re-epithelialization, countering the dexamethasone induced delayed wound healing with a percentage re-epithelialization that was statistically equivalent to the control (96.9 ± 7.3% versus 99.5 ± 1.0%, p > 0.05). The biodistribution study provided insight into the ocular metabolism of spironolactone and hence the relative contributions of the parent molecule and its two principal metabolites, 7α-thiomethylspironolactone and canrenone, to the observed pharmacological effects. Comparison of the efficacies of spironolactone and potassium canrenoate (a water-soluble precursor of canrenone) in overcoming the dexamethasone-induced delayed wound healing confirmed that the former had greater efficacy. The results pointed to the greater potency of 7α-thiomethylspironolactone over canrenone as a mineralocorticoid receptor antagonist, which explained its superior ability in countering the glucocorticoid-induced overactivation that was responsible for the delayed wound healing. In conclusion, the preliminary results supported the above-mentioned hypothesis suggesting that coadministration of mineralocorticoid receptor antagonists to patients under glucocorticoid therapy might prevent the deleterious effects of glucocorticoids on complex corneal wound healing processes.