Evaluation of cellulose based films comprising tea tree oil against dermatophytes and yeasts.

BACKGROUND: Onychomycosis is defined as infection caused by nondermatophytic molds and yeasts: tinea unguium is caused by dermatophytes. PURPOSE: Within this study, hydroxyethyl cellulose (HEC) as an important non-ionic, water-soluble cellulose derivative was chosen to develop formulations containing tea tree oil as active antifungal agent were developed and evaluated for their potential in the treatment of onychomycosis. METHODS: Two polymeric films based on HEC (HEC-B-04 and HEC-E-10) were obtained by solvent evaporation method and characterized in terms of appearance, disintegration, stickiness, elongation, rheological behavior and adhesiveness. Moreover, different strains of dermatophytes such as Trichophyton rubrum and yeasts as Candida albicans were treated with polymeric films containing tea tree oil (0.5 - 2 % v/v) in order to determine their antifungal potential by the inhibition zone assay. RESULTS: HEC-B-04 and HEC-E-10 were investigated by SEM measurements resulting in confluent surface morphology. HEC-B-04 and HEC-E-10 showed disintegration after 32.7 min and 34.0 min, respectively. Furthermore, HEC-E-10 revealed a moisture index of 1.74 and underpinned adhesive properties in terms of required detachment force with 4.86 N. HEC-E-10 pointed to the most antifungal one among the others against Trichophyton rubrum and Candida albicans. CONCLUSION: Taking these findings in consideration, promising adhesive onychial formulations were developed as forthcoming approach in treatment of nail infections.

In vivo evaluation of an oral self-emulsifying drug delivery system (SEDDS) for exenatide.

BACKGROUND: The aim of the study was to develop an oral self-emulsifying drug delivery system (SEDDS) for exenatide and to evaluate its in vivo efficacy. METHODS: Exenatide was lipidised via hydrophobic ion pairing with sodium docusate (DOC) and incorporated in SEDDS consisting of 35% Cremophor EL, 25% Labrafil 1944, 30% Capmul-PG 8 and 10% propylene glycol. Exenatide/DOC was characterized in terms of lipophilicity evaluating the octanol/water phase distribution (logP). Exenatide/DOC SEDDS were characterized via droplet size analysis, drug release characteristics (log DSEDDS/release medium determination) and mucus permeation studies. Furthermore, the impact of orally administered exenatide/DOC SEDDS on blood glucose level was investigated in vivo on healthy male Sprague-Dawley rats. RESULTS: Hydrophobic ion pairing in a molar ratio of 1:4 (exenatide:DOC) increased the effective logP of exenatide from -1.1 to 2.1. SEDDS with a payload of 1% exenatide/DOC had a mean droplet size of 45.87 ±â€¯2.9 nm and a Log DSEDDS/release medium of 1.9 ±â€¯0.05. Permeation experiments revealed 2.7-fold improved mucus diffusion for exenatide/DOC SEDDS compared to exenatide in solution. Orally administered exenatide/DOC SEDDS showed a relative bioavailability (versus s.c.) of 14.62% ±â€¯3.07% and caused a significant (p < .05) 20.6% decrease in AUC values of blood glucose levels. CONCLUSION: According to these results, hydrophobic ion pairing in combination with SEDDS represents a promising tool for oral peptide delivery.

Protease-functionalized mucus penetrating microparticles: In-vivo evidence for their potential.

The focus of the current study was to explore whether immobilization of proteases to microparticles could result in their enhanced penetration into mucus. The proteases papain (PAP) and bromelain (BROM) were covalently attached to a polyacrylate (PAA; Carbopol 971P) via amide bond formation based on carbodiimide reaction. Microparticles containing these conjugates were generated via ionic gelation with calcium chloride and were characterized regarding size, surface charge, enzymatic activity and fluorescein diacetate (FDA) loading efficiency. Furthermore, mucus penetration potential of these microparticles was evaluated in-vitro on freshly collected porcine intestinal mucus, on intact intestinal mucosa and in-vivo in Sprague-Dawley rats. Results showed mean diameter of microparticles ranging between 2-3µm and surface charge between -8 to -18mV. The addition of PAA-microparticles to porcine intestinal mucus led to a 1.39-fold increase in dynamic viscosity whereas a 3.10- and 2.12-fold decrease was observed in case of PAA-PAP and PAA-BROM microparticles, respectively. Mucus penetration studies showed a 4.27- and 2.21- fold higher permeation of FDA loaded PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. Extent of mucus diffusion determined via silicon tube assay illustrated 3.96- fold higher penetration for PAA-PAP microparticles and 1.99- fold for PAA-BROM microparticles. An in-vitro analysis on porcine intestinal mucosa described up to 16- and 7.35-fold higher degree of retention and furthermore, during in-vivo evaluation in Sprague-Dawley rats a 3.35- and 2.07-fold higher penetration behavior was observed in small intestine for PAA-PAP and PAA-BROM microparticles as compared to PAA microparticles, respectively. According to these results, evidence for microparticles decorated with proteases in order to overcome the mucus barrier and to reach the absorption lining has been provided that offers wide ranging applications in mucosal drug delivery.

In Vitro and Ex Vivo Evaluation of Novel Curcumin-Loaded Excipient for Buccal Delivery.

This study aimed to develop a mucoadhesive polymeric excipient comprising curcumin for buccal delivery. Curcumin encompasses broad range of benefits such as antioxidant, anti-inflammatory, and chemotherapeutic activity. Hyaluronic acid (HA) as polymeric excipient was modified by immobilization of thiol bearing ligands. L-Cysteine (SH) ethyl ester was covalently attached via amide bond formation between cysteine and the carboxylic moiety of hyaluronic acid. Succeeded synthesis was proved by H-NMR and IR spectra. The obtained thiolated polymer hyaluronic acid ethyl ester (HA-SH) was evaluated in terms of stability, safety, mucoadhesiveness, drug release, and permeation-enhancing properties. HA-SH showed 2.75-fold higher swelling capacity over time in comparison to unmodified polymer. Furthermore, mucoadhesion increased 3.4-fold in case of HA-SH and drug release was increased 1.6-fold versus HA control, respectively. Curcumin-loaded HA-SH exhibits a 4.4-fold higher permeation compared with respective HA. Taking these outcomes in consideration, novel curcumin-loaded excipient, namely thiolated hyaluronic acid ethyl ester appears as promising tool for pharyngeal diseases.

Modified biomolecule as potential vehicle for buccal delivery of doxepin.

AIM: Doxepin is a traditional tricyclic antidepressant with analgesic and anesthetic properties when applied topically to the mucosa. Doxepin is one approach in treating insomnia and depression in Parkinson's disease. Patients with Parkinson's disease suffer difficulties in swallowing. Therefore, it was the aim of this study to develop a buccal-adhesive delivery system. METHODS: Pectin was modified with cysteine. Stability assays in form of disintegration assay according to the Ph.Eur were performed. Furthermore, bioadhesiveness on buccal mucosa was investigated incorporating the drug doxepin. RESULTS: The adhesiveness was improved 1.4-fold and revealed a sustained release over 3 h. CONCLUSION: Taking these findings into account, the modifications render this designed excipient fruitful for buccal delivery.

Design, modification and in vitro evaluation of pectin's bucco-adhesiveness.

AIM: It was the aim of this study to synthesize pectin (PEC) with sulfhydryl groups and evaluate its suitability in buccal application. MATERIALS & METHODS: Native PEC was chemically modified by covalent attachment of sulfhydryl-bearing cysteine (CYS). Stability assays in form of water uptake behavior and erosion study were performed. Additionally, mucoadhesive study on buccal mucosa was performed. RESULTS: Pectin-cysteine (PECCYS) was successfully synthesized as proved by IR and Ellman's assay exhibiting 436.59 ± 127.87 µmol thiol groups per gram polymer. Stability assay showed that PECCYS revealed a 2.27-fold improved water uptake and mucoadhesiveness augmented 3.75-fold in comparison to unmodified PEC. CONCLUSION: PECCYS might be a future suitable excipient for buccal adhesive application.

Development and in vitro evaluation of zeta potential changing self-emulsifying drug delivery systems for enhanced mucus permeation.

The aim of this study was the development of zeta potential changing self-emulsifying drug delivery systems (SEDDS). Various cationic surfactants were incorporated into a formulation consisting of 30% Cremophor EL, 30% Capmul MCM, 30% Captex 355 and 10% propylene glycol (w/w). A substrate of intestinal alkaline phosphatase (IAP), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid sodium (PA), was thereafter incorporated into SEDDS. Size, zeta potential and polydispersity index were determined. Phosphate release studies were performed using three different models, namely, isolated IAP, Caco-2 cell monolayer and rat intestinal mucosa and the amount of released phosphate was quantified by malachite green assay. Interaction of SEDDS and mucus was investigated regarding surface charges and mucus diffusion studies were performed using rotating tube technique. SEDDS were diluted 1:100 in 100mM HEPES buffer and a negative zeta potential was obtained. By addition of isolated IAP, 15% to 20% phosphate was liberated from SEDDS within 3h and a shift of zeta potential from negative to positive was observed. On Caco-2 cell monolayer and rat intestinal mucosa, 12% and 23% phosphate were released, respectively, from SEDDS diluted 1:1000 in glucose-HEPES buffer. Positively charged droplets were bound to negatively charged mucus resulting in a decrease of zeta potential, whereas negatively charged SEDDS showed no interaction. Furthermore, negatively charged SEDDS diffused faster through mucus layer as higher extent of incorporated Lumogen was present in deeper mucus segments in comparison to positively charged ones. Accordingly, zeta potential changing SEDDS provide an effective mucus permeation combined with higher cellular uptake when droplets reach absorptive epithelium membrane.

P-glycoprotein inhibitors: synthesis and in vitro evaluation of a preactivated thiomer.

The aim of this study was to synthesize the preactivated thiomer poly(acrylic acid)-cyteine-2-mercaptonicotinic acid (PAA-Cys-2MNA) and to evaluate its P-glycoprotein (P-gp) inhibitory properties. The thiomer (PAA-Cys) was synthesized by covalent immobilization of thiol groups on poly(acrylic acid) (PAA) with a molecular mass of 250 kDa followed by immobilization of 2-mercaptonicotinic acid (2MNA) to thiol groups via disulfide bond formation resulting in PAA-Cys-2MNA. P-gp inhibitory effect of this preactivated thiomer was evaluated on Caco-2 cells. Transports of rhodamine 123 at 37 °C with and without verapamil and at 4 °C were performed to evaluate P-gp function of cells. In total, 1571.81 ± 156.18 µmol thiol groups were immobilized per gram of polymer that were in the next step by 99.88% preactivated. The enhancement ratios of Papp calculated from the ratio between Papp of rhodamine 123 in the presence of P-gp inhibitors and Papp of rhodamine 123 alone were 2.36, 2.09, and 1.84-fold in the presence of PAA-Cys-2MNA, PAA-Cys, and PAA, respectively. Because of its pronounced P-gp inhibitory effect, PAA-Cys-2MNA could be considered as promising macromolecular P-gp inhibitor for various drug delivery systems.

Evaluation of the impact of multivalent metal ions on the permeation behavior of Dolutegravir sodium.

Interactions between active pharmaceutical ingredients (APIs) and polyvalent cations are an important factor within drug absorption in the gastrointestinal tract. Dolutegravir sodium, as a second-generation integrase stand transfer inhibitor for the treatment of HIV was investigated regarding chelation with Al(3+), Ca(2+), Fe(3+), Mg(2+ )and Zn(2+) ions at three different molar ratios. Furthermore, the influence of drug-ion chelates on the permeability of the drug across two intestinal membrane models was analyzed. For this purpose, Caco-2 monolayer model and Ussing chamber technique utilizing freshly excited rat intestinal mucosa were chosen and a buffer system without additional Mg(2+) and Ca(2+) ions was tested regarding cell detachment. The addition of polyvalent cations in an equal molar ratio to the drug solution decreased the dissolved drug by at least 11%. An increased multivalent cation concentration in a ratio of 1:10 afforded an API drop in the solution of at least 88% with the exception of Mg(2+). In particular, Dolutegravir sodium was chelated with iron ions to nearly 100%. Overall, the higher the amount of metal ions in the solution, the lower was the detected amount of the drug. The permeation experiments across the Caco-2 monolayer and the rat intestinal mucosa pointed out that the addition of AlCl3, CaCl2 and ZnCl2 in a molar ratio of 10:1 to the drug led to significantly decreased drug permeation. According to these results the co-administration of Al(3+), Ca(2+ )or Zn(2+ )as well as of supplementary medications containing these polyvalent ions is in case of oral Dolutegravir delivery not recommended.

Novel in vitro transport method for screening the reversibility of P-glycoprotein inhibitors.

The purpose of this study was to establish a novel in vitro method for screening reversibility of P-glycoprotein (P-gp) inhibitors. Caco-2 cells with 21days of cultivation were used as an in vitro model. Transport of rhodamine 123 in the presence of various inhibitors and after removing of inhibitors was determined. Transport of rhodamine 123 at 4°C and in the secretory direction assured that Caco-2 cells exhibited P-gp function at all time of experiment. The apparent permeability coefficient (Papp) of rhodamine 123 in the presence of verapamil, cyclosporin A, ritonavir, quinidine, N-ethylmaleimide, Cremophor® EL, Tween 80 and poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-cys-2MNA) was 2.3-, 3.8-, 2.3-, 3.1, 7.5-, 2.1-, 2.9- and 2.5-fold higher than Papp of rhodamine 123 alone. After removing of the inhibitors, Papp decreased to the same range of control except in the case of N-ethylmaleimide which was 2.4-fold higher than the control. These results revealed a reversible inhibition of verapamil, cyclosporin A, ritonavir, quinidine, Cremophor® EL, Tween 80 and PAA-cys-2MNA and an irreversible inhibition of N-ethylmaleimide for P-gp. Thus, this novel established that in vitro method might be an effective tool for screening the reversibility of inhibition of P-gp inhibitors.

In vivo evaluation of anionic thiolated polymers as oral delivery systems for efflux pump inhibition.

Recently, the cationic polymer thiolated chitosan has been reported to modulate drug absorption by inhibition of intestinal efflux pumps. The objective of this study was to evaluate in vitro and in vivo whether thiolated anionic biopolymers also show an efflux pump inhibitory effect in order to improve intestinal transcellular drug uptake. Therefore, three thiomers have been synthesized due covalent attachment of cysteine to various polymer backbones: pectin-cysteine (pect-cys), carboxymethylcellulose-cysteine (CMC-cys) and alginate-cysteine (alg-cys). In vitro, the permeation enhancing properties of these thiomers and their corresponding unmodified polymers have been evaluated on rat small intestine in Ussing-type chambers, using sulforhodamine 101 (SR-101) as MRP2 model substrate. In comparison to buffer only, SR-101 transport in presence of pect-cys, CMC-cys and alg-cys was improved 1.5-fold, 1.8-fold and 3.0-fold, respectively. Due to the comparatively best in vitro performance of thiolated alginate, it has been chosen for in vivo studies: a SR-101 solution containing 4% (w/v) alg-cys led to an AUC0 ≥ 12 of SR-101 of 109 ng ml(-1)h in rats representing a 3.8-fold improvement in comparison to a SR-101 buffer solution. Unmodified alginate improved the AUC0 ≥ 12 of SR-101 by a factor of 1.9. These findings suggest thiolated alginate as promising auxiliary agent for drugs being anionic efflux pump substrates, since the oral bioavailability of a MRP2 substrate could be significantly improved.

Preactivated thiomers: evaluation of gastroretentive minitablets.

The object of this study was to evaluate the potential of a recently developed preactivated thiolated pectin derivative as mucoadhesive excipient in drug delivery to the gastric cavity. Pectin (Pec) was chemically modified with L-cysteine (Cys). The free thiol groups of resulting thiomer were activated with 2-mercaptonicotinic acid (MNA) in order to improve stability and reactivity of attached thiol groups over a broad pH range. Multiunit dosage form properties of the resulting conjugate (Pec-Cys-MNA) were compared to unmodified pectin and the intermediate thiolated using rosuvastatin calcium as a model drug in loaded minitablets. Obtained results were compared with unmodified pectin and the intermediate thiolated pectin. Approximately half of attached thiol groups (507 µmol/g polymer) have been preactivated. Minitablets were evaluated regarding mucoadhesive properties, hardness, disintegration behavior, swelling characteristics and release of rosuvastatin calcium. Mediated by covalent bonds between the polymer and cysteine-rich subdomains in mucus, total work of adhesion increased more than 5-fold. The modification had no impact on hardness of compressed tablets but implementation of the aromatic ligand went along with reduction in hydrophilic properties. Disintegration time was prolonged more than 2-fold while water uptake capacity increased. Weight gain for Pec-Cys-MNA was at least 16-fold. Further, a sustained release of rosuvastatin calcium over 36 h was determined. Neither biodegradability nor CaCo-2 cell viability was affected. The study shows that Pec-Cys-MNA is a promising excipient for the development of mucoadhesive gastric dosage form.

Development and in vivo evaluation of an oral vitamin B12 delivery system.

Recently, poly(acrylic acid)-cysteine (PAA-cys) based formulations have shown to modulate vitamin B12 absorption across Caco-2 cells monolayers and rat intestinal mucosa. The aim of the present study was to provide a proof-of-principle for a delivery system based on PAA-cys in vivo by administering vitamin B12 to Sprague Dawley rats. In vitro, the permeation enhancing effect of unmodified and thiolated PAA was evaluated using rat intestinal mucosa mounted on Ussing type chambers and was compared to that of verapamil and reduced glutathione (GSH). Vitamin B12 transport in the presence of 0.5% (m/v) PAA-cys was 3.96-fold improved compared to buffer, while 91.5% and 56.5% increased compared to verapamil and GSH, respectively. In vivo, the oral administration of minitablets based on 0.5mg vitamin B12 with 4.5mg PAA or PAA-cys resulted in a significant improvement of vitamin B12 absolute bioavailability. The area under the serum concentration-time curve (AUC0₋8) of vitamin B12 after administration of PAA and PAA-cys minitablets was 1.74-fold and 2.92-fold higher in comparison with oral solution, respectively. Thiolated formulations provided an absolute bioavailability of 0.89%. According to the achieved results, PAA-cys can be considered a valuable tool for improving the oral bioavailability of vitamin B12.

Thiomers: influence of molar mass on in situ gelling properties.

The aim of this study was to investigate the influence of molar mass of thiolated polymers (thiomers) on their in situ gelling properties. Chitosan-thioglycolic acid (chitosan-TGA) and pectin-cysteine (pectin-Cys) of increasing molar mass were chosen to produce in situ gels in combination with carbamide peroxide. Low molar mass chitosan (~2 kDa) was prepared by oxidative degradation with NaNO(2), whereas pectin was depolymerized by heat treatment. Thiomers, displaying 1271-1616 µmol (chitosan-TGA) and 305-403 µmol (pectin-Cys) free thiol groups per gram polymer, were synthesized via amide bond formation mediated by a carbodiimide. The results showed that a reduction of molar mass combined with increased concentrations of both cationic chitosan-TGA and anionic pectin-Cys leads to higher final viscosities and to a higher relative increase in viscosity within 60 min and 180 min, respectively. Using this method, the dynamic viscosity of a very low molar mass chitosan-TGA (~2 kDa) could be increased 100,000-fold within 60 min and 390,000-fold within 180 min. In view of these in situ gelling properties carbohydrate thiomers might be useful for various pharmaceutical applications such as vehicle for drug delivery or as wound dressing material.

In situ gelling properties of anionic thiomers.

The aim of this study was to investigate in situ crosslinking systems of anionic thiolated polymers. In order to accelerate the increase in dynamic viscosity of thiolated polymers (thiomers), they were combined with hydrogen peroxide, carbamide peroxide and ammonium persulfate. Thiomers (pectin-cysteine (Pec-Cys), sodium carboxymethylcellulose-cysteine (NaCMC-Cys) and poly(acrylic acid)-cysteine (PAA-Cys)) were synthesized via amide bond formation between the carboxylic acid group of polymers and the primary amino group of l-cysteine. The rheological properties of 1% (m/v) thiomer solutions with oxidizing agents were compared by oscillatory measurements over time (120 min). Pec-Cys and NaCMC-Cys with hydrogen and carbamide peroxide showed a sol-gel phase transition within a few minutes and scored up to 13,000-fold increase in dynamic viscosity. Furthermore, only thiomers exhibiting a polysaccharide backbone (Pec-Cys and NaCMC-Cys) showed a significant increase in viscosity (p < 0.05). In contrast, measurements of carbohydrate thiomers in combination with ammonium persulfate showed an initial increase in viscosity. Afterwards, a decrease in viscosity was observed likely caused by chain scission. According to these results, carbohydrate thiomer/oxidizing agent systems might be useful for various pharmaceutical applications such as for in situ gelling liquid/semisolid formulations or in tissue engineering.