Onychomycosis also known as dermatophytic onychomycosis is the fungal infection of the toenails and fingernails, characterized by discoloration and thickening of the nail and involves the nail plate, nail bed and nail folds. The disease is more than a cosmetic problem, as it severely impacts the patient's quality of life. Onychomycosis is an opportunistic infection in special subpopulations of patients suffering from diabetes, psoriasis, HIV/AIDS etc. The current treatment strategies involve systemic delivery of oral antifungal agents including azoles (e.g. itraconazole) and allylamines (e.g. terbinafine hydrochloride) which are delivered to the nail plate from the nail bed. More recently, topical delivery of drugs including amorolfine and bifonazole/urea (available outside the United States) and Penlac® nail lacquer (ciclopirox) topical solution, 8%, available in the US are an alternative treatment option to the oral antifungal agents. Topical delivery of antifungal agents through the human nail offer several advantages over oral therapy including lower incidence of adverse events and lower potential for drug-drug interaction with drugs used to treat diabetes, HIV/AIDS and psoriasis. The objectives of this project were to: 1) To determine the critical factors affecting the delivery of ciclopirox olamine across the human nail, 2) To screen and select penetration enhancer(s) specific for ciclopirox olamine delivery into the target tissue(s) and 3) To develop a novel transungual formulation containing ciclopirox olamine (CPO) and penetration enhancer(s) for transungual delivery. Ciclopirox olamine, the salt form of the free acid of ciclopirox was used in the study to develop a novel transungual patch formulation and skin and nail permeation from the patch formulation was compared to Penlac® nail lacquer. Various factors such as drug partitioning into the healthy and infected toenail, drug-keratin binding, lateral diffusion, drug-epidermal binding and the formulation components, all play a role in achieving optimum drug penetration and permeation through the nail. Understanding the interplay of these factors helped in the development of an effective topical formulation which was observed to be superior to Penlac® nail lacquer in the in vitro studies. Most cases of onychomycosis show infection and inflammation of the nail folds (skin surrounding the nails). Therefore for an efficient treatment of OM, the antifungal drugs must be delivered to two target tissues - human nail and the nail folds. The major challenges in developing a topical formulation for treatment on OM are: a) Achieving antifungal drug minimum inhibitory concentration (MIC) in the epidermis of the nail folds. b) Enhancing penetration and permeation of the antifungal drug across the human nail to reach the nail bed and achieve the necessary MIC (tissue underneath the nail). Twelve chemical penetration enhancers (PEs) were screened for their ability to enhance ciclopirox olamine accumulation into the nail folds and permeation through the nail. Propylene glycol (PG) enhanced the levels of the drug in the epidermis of the skin while limiting its permeation across the skin. Thiourea (TU) was selected as the best enhancer to increase ciclopirox olamine penetration into the nail. The diffusion of the antifungal drug across the human nail was studied in vitro using human cadaver toenails mounted in Franz diffusion cells. Pressure sensitive adhesives (PSA) belonging to the polyisobutylene, polysiloxane and polyacrylate classes of adhesives were screened to develop a monolithic drug-in-adhesive-type nail patch. The in vitro release of CPO from the PSA patches were limited and did not improve in presence of hydrophilic plasticizer (propylene glycol) and hydrophobic plasticizers (triacetin and triethyl citrate). Increasing the concentration of TU from 1 % to 10 %, lead to its crystallization in the dry patches. Therefore a change in the patch design was recommended. Other hydrophilic polymers including Polyoxyethylene (POLYOX®) and hydroxyl propyl methyl cellulose (HPMC) were also screened to develop a modified drug-in-hydrophilic matrix patch design. The patch was designed to incorporate CPO, PG and TU in the polymer matrix overlaid on a non-occlusive backing membrane cast with polyacrylate PSA. The HPMC films showed the best drug release profile with 80 % release in 2 to 4 hours using a USP apparatus 5. These patches were characterized for drug penetration into the skin and nail permeation. Penlac® nail lacquer was used as the comparator control product. The prototype HPMC K15M patch containing 10 %w/w each of the drug and TU and 150 % w/w of PG showed 2.8 fold increase in CPO accumulation in epidermis compared to Penlac® nail lacquer in 24 hours. The skin permeation was found to be similar to that of Penlac®. The HPMC K15M patch formulation showed 2.7 fold increase in CPO concentration within the nail and 4.2 fold increase in transungual flux compared to Penlac®. The patch delivered higher levels of ciclopirox olamine into the target tissues with a lower permeation lag-time. The novel nail patch delivery system had the following properties: a) Ease of application, b) Contact with the nail surface, c) Increased concentration of drug in dissolved form within the patch, d) Presence of enhancers. The novel nail patch formulation has shown increased efficiency in topical and transungual drug delivery for treatment of OM, when compared to the commercial formulation, Penlac® nail lacquer in the in vitro studies. The physical characterization of the patch using Scanning Electron Microscopy, Polarized Light Microscopy, Optical Light Microscopy, Differential Scanning Calorimetry, X-Ray Diffractometer and Fourier Transform Infrared Spectroscopy show that ciclopirox olamine exists at a sub-saturation level in a non-crystalline form in the patch without any significant drug-polymer interaction. In conclusion, all the objectives of the study were met by successfully selecting penetration enhancers for CPO delivery into the nail folds and across the nail plate, evaluating the interaction between CPO and target tissues, developing a transungual patch system and characterizing the novel transungual patch. / Pharmaceutical Sciences

Submitted by Fabio Sobreira Campos da Costa (fabio.sobreira@ufpe.br) on 2017-02-13T13:18:57Z No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE colação de grau.pdf: 1971286 bytes, checksum: 680d0e24d2f0efbe0313ab8752f1cc93 (MD5) / Made available in DSpace on 2017-02-13T13:18:57Z (GMT). No. of bitstreams: 2 license_rdf: 1232 bytes, checksum: 66e71c371cc565284e70f40736c94386 (MD5) TESE colação de grau.pdf: 1971286 bytes, checksum: 680d0e24d2f0efbe0313ab8752f1cc93 (MD5) Previous issue date: 2013-02-22 / CNPq / A criptococose é uma infecção fúngica predominantemente oportunista cujos principais agentes etiológicos são Cryptococcus neoformans e C. gattii. O tratamento de escolha para a micose é a anfotericina B associada ou não a 5-fluorocitosina seguido de terapia de manutenção com fluconazol. Contudo, falhas no tratamento associadas à toxicidade e ao aparecimento de resistência aos fármacos têm sido relatadas, o que torna essencial a descoberta de novas alternativas terapêuticas, como a ciclopirox olamina (CPO). Neste contexto, o objetivo deste estudo foi caracterizar e avaliar a ação in vitro e in vivo da CPO livre e encapsulada em lipossomas frente a amostras de Cryptococcus neoformans para futura aplicação no tratamento da criptococose sistêmica. Foram obtidas 30 amostras de Cryptococcus neoformans provenientes de pacientes imunocomprometidos. A preparação dos lipossomas convencionais e furtivos de CPO foi realizada pelo método da hidratação do filme lipídico e a caracterização foi realizada avaliando os seguintes parâmetros: tamanho de partícula, Índice de Polidispersão (PDI) e taxa de encapsulação (EE%). Para otimização dos constituintes lipídicos, foi realizado um planejamento fatorial fracionado a 24-1 a partir da melhor formulação obtida nos estudos de pré-formulação. A cinética de liberação in vitro foi conduzida para avaliar e comparar estatisticamente o perfil de liberação dos sistemas convencional e furtivo. Adicionalmente, testes de susceptibilidade antifúngica foram realizados de acordo com Clinical and Laboratotry Standards Institute (CLSI). Para caracterização molecular dos isolados, PCR fingerprinting foi conduzida utilizando os primers M13 e URA5. O estudo in vivo foi conduzido com camundongos imunossuprimidos, infectados com Cryptococcus neoformans (106 cels/mL) e tratados com CPO lipossomal (Lipo-CPO) (0.5 mg/Kg). As concentrações de CPO utilizadas na forma livre e encapsuladas em lipossomas convencionais e furtivos variaram de 0,30 a 625 µg/mL. Os resultados do planejamento fatorial mostraram que o ponto central apresentou características proeminentes com redução do tamanho de partícula em 17,1%; melhora do PDI em 15,34% e da quantidade de fármaco encapsulado (25%). A cinética do lipossoma furtivo apresentou uma velocidade de liberação mais controlada quando comparada ao lipossoma convencional. Com relação ao teste de susceptibilidade, todos os inóculos foram susceptíveis a CPO livre, com atividade fungistática entre 0,30 e 0,61 g/mL e fungicida entre 1,22 e 4,88 g/mL. Não houve diferença relacionada à atividade antifúngica entre as formulações lipossomais convencionais e furtivas. A atividade fungistática dos lipossomas foi observada em concentrações variando de 1,22 e 2,44 g/mL. A faixa das concentrações fungicidas foi de 1,22 a 9,76 g/mL. O padrão de bandas do URA5 revelou que todos os isolados apresentam genótipo VNI, característico de C. neoformans. Lipo-CPO apresentou eficácia antifúngica comparada à anfotericina B após 14 dias de infecção, reduzindo a carga fúngica em aproximadamente 8% no baço, 41% no fígado, 63% no pulmão e 89% no cérebro. O exame histológico evidenciou infiltrado celular no fígado dos grupos tratados com Lipo-CPO e anfotericina B, porém com menor intensidade quando comparado ao grupo controle. O estudo sugere que a CPO encapsulada em lipossomas apresenta significativa ação antimicótica frente às amostras sistêmicas de C. neoformans, reforçando seu potencial na terapêutica da criptococose. / Cryptococcosis is an opportunistic fungal infection whose the main ethiological agents are Cryptococcus neoformans and C. gattii. The treatment of choice for this mycosis is amphotericin B combined or not with 5-fluorocytosine, followed by maintenance therapy with fluconazole. However treatment failures associated with toxicity and drug resistance has been reported, which makes it essential to the discovery of new therapies, such as ciclopirox olamine (CPO). The purpose of this study was to characterize and evaluate the in vitro and in vivo antifungal activity of ciclopirox olamine in its free form and encapsulated in liposomes against thirty Cryptococcus neoformans isolates obtained from immunocompromised patients for future application in systemic cryptococcosis treatment. Preparation of conventional and stealth liposomes was performed to define particle size, polydispersity index (PDI), CPO amount of encapsulated and efficiency of encapsulation (EE%). For optimization of liposomal lipid constituents, a 24-1 fractional factorial design was carried out from the prominent formulation obtained in pre-characterization studies. In vitro release kinetics was conducted to evaluate and compare statistically the release profile of conventional and stealth liposomes. Antifungal susceptibility testing was conducted in accordance with the reference method. Regarding molecular characterization, PCR fingerprinting was carried out by using MT3 and URA5 primers. Concentrations of CPO used in free form and encapsulated into stealth and conventional liposomes ranged from 625 to 0.3 g.mL-1. Despite of the central point of the factorial design have increased the total lipids amount in 35.52%, it showed prominent characteristics when compared with the L4 formulation with improvement of the mean size in 17.1%, PDI in 15.34% and CPO amount in 25%. The minimum concentrations of stearylamine to obtain the stable formulation for one month was 5.88 mM. . Kinetics of stealth liposomes showed a release profile more controlled as compared to conventional liposomes. All inoculations were susceptible to CPO in its free form, presenting fungistatic activity between 0.3 and 0.61 g.mL-1 and fungicidal activity between 1.22 and 4.88 g.mL-1. There was no difference with respect to antimycotic activity between conventional and stealth liposomal formulations. Fungistatic activity of liposomes was observed at concentrations ranging from 1.22 and 2.44 g.mL-1. Fungicidal concentration range was 1.22-9.76 g.mL-1. The URA5 profile analized demonstrated all isolates are VNI genotype (C. neoformans). The treatment with Lipo-CPO showed a reduction of 8% of the C. neoformans population in spleen, 40.8% in liver, 63% in lungs and 89% in brain after 14 days of infection. Histological examination revealed cell infiltrate either Lipo-CPO or Amphotericin B treated groups, but less intense when compared to control group. The results suggest that Ciclopirox olamine loaded-liposomes have significant antimycotic activity against Cryptoccocus spp, reinforcing its potential for in vivo studies and its application in cryptococcosis treatment.