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.

Thiomers: Impact of in situ cross-linkers on mucoadhesive properties.

The aim of this study was to evaluate the impact of in situ cross-linkers on the gelling and mucoadhesive properties of thiomers. Polycarbophil-cysteine conjugate (PCP-cys) was synthesized by covalent attachment of l-cysteine to polycarbophil via amide bond formation mediated by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDAC) and N-hydroxysuccinimide (NHS) whereas in situ cross-linkers (PAA-cys-MNA) were synthesized by the same bond formation between poly(acrylic acid) (PAA) of 2.1-, 6-, and 15kDa and 2-((2-amino-2-carboxyethyl)disulfanyl)nicotinic acid (cys-MNA) used as ligand. The in situ cross-linking properties were studied via rheological measurements of dynamic viscosity of mixtures of PCP-cys and PAA-cys-MNA with purified porcine intestinal mucus and via rotating cylinder method. The diffusion of polymers in purified porcine intestinal mucus was studied via rotating tube technique. The results showed that in situ cross-linkers (PAA 2.1-, 6-, 15kDa) increase the dynamic viscosity of PCP-cys/mucus mixtures by 5.1-, 5.6-, and 3.5-fold. Combinations of 10% of in situ cross-linkers PAA 2.1-, 6- or 15kDa and 90% PCP-cys increased the adhesion time 1.1-, 2.0- and 4.9-fold, respectively, compared to PCP-cys alone. Diffusion study showed that low molecular mass PAAs highly penetrate into the mucus gel layer due to their high polymer chain mobility compared to PCP-cys. The results provide evidence for the potential of in situ cross-linking agents as gelling and mucoadhesion enhancers.

Improved mucoadhesive properties of self-nanoemulsifying drug delivery systems (SNEDDS) by introducing acyl chitosan.

This study was aimed to improve the mucoadhesive properties of SNEDDS by the incorporation of acyl chitosan including octanoyl chitosan (OC), lauroyl chitosan (LC) and palmitoyl chitosan (PC). SNEDDS and acyl chitosan SNEDDS were characterized regarding droplet size and zeta potential. Their mucoadhesivity on porcine intestinal mucosa was evaluated by falling liquid film technique using Sudan Red G as marker. Degree of substitution of chitosan was determined to be 52.8%, 64.8 and 48.5% for OC, LC and PC, respectively. SNEDDS and acyl chitosan SNEDDS displayed a droplet size less than 50nm and 80-300nm as well as a zeta potential of -0.2 to -1.6 and 0.05 to 0.99mV, respectively. Introducing 2% acyl chitosan into SNEDDS increased the residence time of SNEDDS on intestinal mucosa 2-fold. It is concluded that due to the incorporation of acyl chitosan into SNEDDS, their mucoadhesive properties can be increased.

Self-emulsifying drug delivery systems (SEDDS): Proof-of-concept how to make them mucoadhesive.

AIM: The objective of this study was to provide a proof-of-concept that self-emulsifying drug delivery systems can be made mucoadhesive by the incorporation of hydrophobic mucoadhesive polymers. METHODS: In order to obtain such a hydrophobic mucoadhesive polymer, Eudragit® S100 was thiolated by covalent attachment of cysteamine. After determination of the thiol group content, in vitro mucoadhesion studies (rotating cylinder and rheological measurements) were performed. Then, synthesized conjugate was incorporated into self-emulsifying drug delivery systems (SEDDS) and their toxic potential as well as that of unmodified and thiolated Eudragit® S100 was examined on Caco-2 cell line. Lastly, the mucoadhesiveness of developed SEDDS on porcine intestinal mucosa was determined. RESULTS: Generated thiolated Eudragit® S100 displaying 235±14µmol of free thiol groups and 878±101µmol of disulfide bonds per gram polymer showed a great improvement in both: dynamic viscosity with mucus and adhesion time on mucosal tissue compared to the unmodified polymer. Resazurin assay revealed that unmodified and thiolated polymers and also SEDDS dispersions were non-toxic over Caco-2 cells. Furthermore, the incorporation of 1.5% (w/w) of such thiomer into SEDDS led to remarkably improved mucoadhesiveness. Blank SEDDS were completely removed from the mucosa within 15min, whereas >60% of SEDDS containing thiolated Eudragit® S100 were still attached to it. CONCLUSION: These results provide evidence that SEDDS can be made mucoadhesive by the incorporation of hydrophobic mucoadhesive polymers.

Preactivated thiolated pullulan as a versatile excipient for mucosal drug targeting.

AIM: The purpose of the present study was to generate a novel mucoadhesive thiolated pullulan with protected thiol moieties and to evaluate its suitability as mucosal drug delivery system. METHODS: Two different synthetic pathways: bromination-nucleophilic substitution and reductive amination including periodate cleavage were utilized to synthesize such thiolated pullulans. The thiomer (pullulan-cysteamine) with the highest amount of free thiol groups was further enrolled in a reaction with 6-mercaptonicotinamide and its presence in pullulan structure was confirmed via NMR analysis. Furthermore, unmodified, thiolated and preactivated thiolated pullulan were investigated in terms of mucoadhesion via rotating cylinder studies and rheological synergism method as well as their toxicity potential over Caco-2 cells. RESULTS: Comparing both methods the reductive amination seems to be the method of choice resulting in comparatively higher coupling rates. Using this procedure pullulan-cysteamine conjugate displayed 1522±158µmol immobilized thiol groups and 280±70µmol free thiol groups per gram polymer. Furthermore, 82% of free thiol groups on this conjugate were linked with 6-mercaptonicotinamide (6-MNA). The adhesion time on the rotating cylinder was up to 46-fold prolonged in case of the thiolated polymer and up to 75-fold in case of the preactivated polymer. Rheological measurements of modified pullulan samples showed 98-fold and 160-fold increase in dynamic viscosity upon the addition of mucus within 60min, whereas unmodified pullulan did not show an increase in viscosity at all. Both conjugates had a minor effect on Caco-2 cell viability. CONCLUSION: Because of these features preactivated thiolated pullulan seems to represent a promising type of mucoadhesive polymers for the development of various mucosal drug delivery systems.

Impact of lipases on the protective effect of SEDDS for incorporated peptide drugs towards intestinal peptidases.

AIM: The aim of this study is the development of self-emulsifying drug delivery systems (SEDDS) differing in amounts of ester substructures and to evaluate their stability in presence of pancreatic lipase and protective effect against luminal enzymatic metabolism using leuprorelin as model peptide drug. METHODS: Hydrophobic leuprolide oleate was incorporated into three different SEDDS formulations and their stability towards pancreatic lipases was investigated utilizing a dynamic in vitro digestion model. Protective effect of SEDDS in respect to peptide drug stability against proteolytic enzymes, trypsin and α-chymotrypsin, was determined via HPLC. RESULTS: Results of in vitro digestion demonstrated that 80% of SEDDS containing the highest amount of ester linkages was degraded within 60min. In comparison to that, SEDDS without ester bonds showed no degradation. With increasing oil droplets hydrolysis the remaining amount of peptide encapsulated into formulation decreased. Furthermore, after 180min incubation with trypsin up to 33.5% and with α-chymotrypsin up to 60.5% of leuprolide oleate was intact while leuprorelin acetate aqueous solution was completely metabolized by trypsin within 120min and by α-chymotrypsin within 5min. Protective effect in environment containing lipases was lower due to oil phase degradation, however, the amount of peptide in ester-free SEDDS was remarkably higher compared to SEDDS susceptible to lipases. CONCLUSION: The present study revealed that SEDDS stable towards hydrolysis is able to exhibit a protective effect for oral peptide delivery.

S-protected thiolated hydroxyethyl cellulose (HEC): Novel mucoadhesive excipient with improved stability.

The aim of this study was the design of novel S-protected thiolated hydroxyethyl cellulose (HEC) and the assessment of its mucoadhesive properties and biodegradability compared to the corresponding unmodified polymer. Thiolated HEC was S-protected via disulfide bond formation between 6-mercaptonicotinamide (6-MNA) and the thiol substructures of the polymer. In vitro screening of mucoadhesive properties was accomplished using two different methods: rotating cylinder studies and viscosity measurements. Moreover, biodegradability of these polymers by cellulase, xylanase and lysozyme was evaluated. MTT and LDH assays were performed on Caco-2 cells to determine the cytotoxicity of S-protected thiolated HEC. Thiolated HEC displayed 280.09±1.70µmol of free thiol groups per gram polymer. S-protected thiolated HEC exhibiting 270.8±21.11µmol immobilized 6-MNA ligands per gram of polymer was shown being 2.4-fold more mucoadhesive compared to thiolated HEC. No mucoadhesion was observed in case of unmodified HEC. Results were in a good agreement with rheological studies. The presence of free thiol moieties likely caused lower degree of hydrolysis by xylanase, whereas the degradation by both enzymes cellulase and xylanase was more hampered when 6-MNA was introduced as ligand for thiol group's protection. Findings in cell viability revealed that all three conjugates were non-toxic. S-protection of thiolated hydroxyethyl cellulose improved mucoadhesive properties and provided pronounced stability towards enzymatic attack, that makes this excipient superior for non-invasive drug administration over thiolated and unmodified forms.

Development and in vitro evaluation of an oral SEDDS for desmopressin.

CONTEXT: Self-emulsifying drug delivery systems (SEDDS) are among most promising tools for improving oral peptide bioavailability. OBJECTIVE: In this study, in vitro protective effect of SEDDS containing desmopressin against presystemic inactivation by glutathione and α-chymotrypsin was evaluated. MATERIALS AND METHODS: The partitioning coefficient (log P) of desmopressin was increased via hydrophobic ion pairing using anionic surfactants. Solubility studies were performed to select the appropriate solvents for SEDDS preparation. Subsequently, droplet size and emulsification properties of 22 SEDDS formulations were evaluated. Moreover, the peptide-surfactant complex was dissolved in two chosen SEDDS formulations. Finally, SEDDS containing desmopressin were characterized regarding lipase stability, toxicity, and in vitro protective effect toward glutathione and α-chymotrypsin. RESULTS: Desmopressin log P was increased from initial -6.13 to 0.33 using sodium docusate. The resulting desmopressin docusate complex (DES/AOT) was incorporated in two different SEDDS formulations, containing Capmul 907 P as main solvent. DES/AOT-SEDDS-F4 (containing 0.07% w/w DES/AOT) was composed of 50% Capmul 907P, 40% Cremophor RH40, and 10% Transcutol. The comparatively more hydrophilic formulation DES/AOT-SEDDS-F15 (containing 0.25% w/w DES/AOT) consisted of 20% Capmul 907P, 40% Acconon MC8-2, and 40% Tween 20. Both formulations were stable toward digestion by lipase and protected desmopressin toward α-chymotrypsin degradation. Moreover, DES/AOT-SEDDS-F4 also protected the peptide from thiol/disulfide exchange reactions with glutathione and was not cytotoxic at a concentration of 0.375% (w/w). CONCLUSION: DES/AOT-SEDDS-F4 protected desmopressin from in vitro glutathione and α-chymotrypsin degradation. DES/AOT-SEDDS-F4 was metabolically stable and nontoxic. Therefore, it could be considered as a potential delivery system for oral desmopressin administration.

Evaluation of thiolated silicone oil as advanced mucoadhesive antifoaming agent.

CONTEXT: Silicone oils, such as dimethicone, are commonly administered against gastrointestinal gas accumulation and are attributed with mucoprotective features. OBJECTIVE: Evaluation of thiolated silicone oil as advanced antiflatulence with a prolonged retention on small intestinal mucosa as an intended site of action. MATERIALS AND METHODS: 3-Mercaptopropionic acid (MPA) as a thiol ligand was covalently attached to silicone oil. This thiomer was assessed with regard to foam inhibiting action, droplet size of a suitable self-emulsifying system, mucoadhesion and cytotoxicity. RESULTS: Antifoaming activity of silicone-MPA was complying with United States Pharmacopeia (USP) requirements for simethicone as standard antiflatulence. Another antifoaming test performed on porcine mucosa supported silicone-MPA's outstanding foam destruction, as this thiomer was superior in comparison to non-thiolated silicone oil and dimethicone with 14.7 ± 2.1 versus 73.3 ± 9.1 and 66.3 ± 7.5 s, respectively. A significantly enhanced mucoadhesiveness (p < 0.02) with 26.2 ± 7.1% silicone formulation remaining on small intestinal mucosa after 8 h was evident for the thiomer without any toxic effect. CONCLUSION: Thiolated silicone oil was found to exhibit excellent antifoaming and superior mucoadhesive features. The prolonged residence time of thiolated silicone oil promises to be beneficial in the treatment of flatulence, aerophagy and inflammation throughout the whole gastrointestinal tract.

Self-emulsifying drug delivery systems in oral (poly)peptide drug delivery.

INTRODUCTION: Oral administration of most therapeutic peptides and proteins is mainly restricted due to the enzymatic and absorption membrane barrier of the GI tract. In order to overcome these barriers, various technologies have been explored. Among them, self-emulsifying drug delivery systems (SEDDS) received considerable attention as potential carriers to facilitate oral peptide and protein delivery in recent years. AREAS COVERED: This review article intends to summarize physiological barriers which limit the bioavailability of orally administrated peptide and protein drugs. Furthermore, the potential of SEDDS to protect incorporated peptides and proteins towards peptidases and proteases and to penetrate the mucus layer is reviewed. Their permeation-enhancing properties and their ability to release the drug in a controlled way are described. Moreover, this review covers the results of in vivo studies providing evidence for this promising approach. EXPERT OPINION: As SEDDS can: i) provide a protective effect towards a presystemic metabolism; ii) efficiently permeate the intestinal mucus gel layer in order to reach the absorption membrane; and iii) be produced in a very simple and cost-effective manner, they are a promising tool for oral peptide and protein drug delivery.

An in-vitro exploration of permeation enhancement by novel polysulfonate thiomers.

The study was aimed to synthesize preactivated polysulfonate thiomers using poly(4-styrenesulfonic acid-co-maleic acid) (PSSA-MA) and to evaluate their permeation enhancing properties. PSSA-MA-cysteamine (PC) conjugates with 2-mercaptonicotinic acid (2MNA) having different degree of preactivation (PC1608-2MNA, PC2300-2MNA, PC3100-2MNA) were synthesized from the subsequent PSSA-MA-cysteamine thiomers (PC1608, PC2300 and PC3100). The permeation-enhancing features were evaluated by in-vitro models using low-molecular size marker sodium fluorescein (Na-Flu) and with high-molecular size marker fluorescein isothiocyanate-dextran (FD4). Associating the influence of degree of preactivation on permeation enhancement, following rank order PC3100-2MNA>PC3100>PC2300-2MNA>PC2300>PC1608-2MNA>PC1608>PSSA-MA>control was observed on Caco-2 cell monolayers and with little change in sequence on freshly excised rat intestine. The apparent permeability (Papp) was improved 3.16-fold for Na-Flu and 3.51-fold for FD4 on Caco-2 cell monolayers. Similarly, 4.17- and 3.60-fold improved Papp values were observed on freshly excised rat intestine for Na-Flu and FD4, respectively. More pronounced permeation effects on rat intestine compared to Caco-2 cell monolayer by the thiomer/preactivated conjugates indicated their mucus-interpenetration capability in addition to mucoadhesion. Thus relatively low molecular weight (LMW) preactivated polysulfonate thiomers could to a higher extent enhance permeation on membranes covered by mucus layer compared to high molecular weight thiomers/preactivated conjugates that usually exhibit higher permeation enhancing effects on Caco-2 cell monolayer.

Thiolated alkyl-modified carbomers: Novel excipients for mucoadhesive emulsions.

The aim of this study was the design and evaluation of mucoadhesive emulsifying polymeric excipients. Three thiol bearing ligands with increasing pKa values of their sulfhydryl group, namely 4-aminothiophenol (pKa=6.86), l-cysteine (pKa=8.4) and d/l-homocysteine (pKa=10.0) were coupled to the polymeric backbone of alkyl-modified carbomer (PA1030). Resulting conjugates displayed 818.5µmol 4-aminothiophenol, 698.5µmol cysteine and 651.5µmol homocysteine per gram polymer and were evaluated regarding the reactivity of thiol groups, emulsifying and mucoadhesive properties. In general, the synthesized conjugates showed a pH dependent reactivity, whereby the fastest oxidation occurred in PA1030-cysteine, as almost no free thiol groups could be detected after 120min. Emulsification of medium chain triglycerides was feasible with all synthesized conjugates leading to oil-in-water-emulsions. Emulsions with PA1030-cysteine displayed the highest stability and the smallest droplet size among the tested formulations. Oxidation and consequently cross-linking of the thiomers prior to the emulsification process led to an overall decreased emulsion stability. Evaluating mucosal residence time of thiomer emulsions on porcine buccal mucosa, a 9.2-fold higher amount of formulation based on PA1030-cysteine remained on the mucosal tissue within 3h compared to the unmodified polymer. According to these results, the highest reactive ligand l-cysteine seems to be most promising in order to obtain thiolated polymers for the preparation of mucoadhesive o/w-emulsions.