Christiane Müller

Preactivated thiomers as mucoadhesive polymers for drug delivery

This study was aimed to synthesize polymeric excipients with improved mucoadhesive, cohesive and in situ-gelling properties to assure a prolonged retention time of dosage forms at a given target site, thereby achieving an increased uptake and improved oral bioavailability of certain challenging therapeutic agents such as peptides and proteins. Accordingly, poly(acrylic acid)-cysteine-2-mercaptonicotinic acid (PAA-cys-2MNA) conjugates were synthesized by the oxidative S–S coupling of PAA-cys (100-, 250- and 450 kDa) with 2-mercaptonicotinic acid (2MNA). Unmodified PAAs, PAAs-cys (thiomers) and PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates were compressed into tablets to perform disintegration tests, mucoadhesion studies and rheological measurements. Moreover, cytotoxicty of the polymers was determined using Caco-2 cells. The resulting PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 113.5 ± 12.7, 122.7 ± 12.2 and 117.3 ± 4.6 μmol/g of 2-mercaptonicotinic acid, respectively. Due to the immobilization of 2MNA, the PAA-cys-2MNA (pre-activated thiomers) conjugates exhibit comparatively higher swelling properties and disintegration time to the corresponding unmodified and thiolated polymers. On the rotating cylinder, tablets based on PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates displayed 5.0-, 5.4- and 960-fold improved mucoadhesion time in comparison to the corresponding unmodified PAAs. Results achieved from tensile studies were found in good agreement with the results obtained by rotating cylinder method. The apparent viscosity of PAA-cys-2MNA (100-, 250- and 450 kDa) conjugates was improved 1.6-, 2.5- and 206.2-fold, respectively, in comparison to the corresponding unmodified PAAs. Moreover, pre-activated thiomers/mucin mixtures showed a time dependent increase in viscosity up to 24 h, leading to 7.0-, 18.9- and 2678-fold increased viscosity in comparison to unmodified PAAs (100-, 250- and 450 kDa), respectively. All polymers were found non-toxic over Caco-2 cells. Thus, on the basis of achieved results the pre-activated thiomers seem to represent a promising generation of mucoadhesive polymers which are safe to use for prolonged residence time of drug delivery systems to target various mucosa.

In vivo evaluation of an oral self-microemulsifying drug delivery system (SMEDDS) for leuprorelin

The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) for the model peptide drug leuprorelin to prove a protective effect against luminal enzymatic metabolism. In order to incorporate leuprorelin into microemulsion droplets (o/w), the commercially available hydrophilic leuprolide acetate was modified by hydrophobic ion paring with sodium oleate. The obtained hydrophobic leuprolide oleate was dissolved in the SMEDDS formulation (30% (m/m) Cremophor EL, 30% (m/m) Capmul MCM, 10% (m/m) propylene glycol and 30% (m/m) Captex 355) in a concentration of 4 mg/g showing a mean droplet size of 50.1 nm when dispersed in a concentration of 1% (m/v) in phosphate buffer pH 6.8. The microemulsion was able to shield leuprolide oleate from enzymatic degradation by trypsin and α-chymotrypsin, so that after 120 min 52.9% and 58.4%, respectively, of leuprolide oleate were still intact. Leuprolide acetate dissolved in an aqueous control solution was completely metabolized by trypsin within 60 min and by α-chymotrypsin within 5 min. Moreover, an in vivo study in rats showed a 17.2-fold improved oral bioavailability of leuprolide oleate SMEDDS compared to a leuprolide acetate control solution. This is the first time, to our knowledge, that hydrophobic ion pairing is utilized in order to incorporate a peptide drug in SMEDDS and evidence of a protective effect of oil-in-water (o/w) microemulsion droplets against enzymatic degradation of a peptide drug was provided. According to these results, the system could be likely a novel platform technology to improve the oral bioavailability of peptide drugs.

Development and in vivo evaluation of papain-functionalized nanoparticles

The aim of the present study was to develop a novel nanoparticulate delivery system being capable of penetrating the intestinal mucus layer by cleaving mucoglycoprotein substructures. Nanoparticles based on papain grafted polyacrylic acid (papain-g-PAA) were prepared via ionic gelation and labeled with fluorescein diacetate. In vitro, the proteolytic potential of papain modified nanoparticles was investigated by rheological measurements and diffusion studies across fresh porcine intestinal mucus. The presence of papain on the surface and inside the particles strongly decreases viscosity of the mucus leading to facilitated particle transition across the mucus layer. Results of the permeation studies revealed that enzyme grafted particles diffuse through mucus layer to a 3.0-fold higher extent than the same particles without enzyme. Furthermore, the penetration behavior of the nanocarriers along the gastrointestinal tract of Sprague Dawley rats was investigated after oral administration of nanoparticles formulated as enteric coated capsules. The majority of the papain functionalized particles was able to traverse across the mucus layer and remained in the duodenum and jejunum of the small intestine where drug absorption primarily occurs. Polymeric nanoparticles combined with mucolytic enzymes that are capable of overcoming intestinal mucus barriers offer an encouraging new attempt for mucosal drug delivery.

RNAi: ancient mechanism with a promising future

RNA interference (RNAi) is a gene silencing mechanism that has been conserved in evolution from yeast to man. Double stranded RNA, which is either expressed by cellular genes for small non-coding RNAs, by parasitic nucleic acids, such as viruses or transposons, or is expressed as an experimental tool, becomes processed into small RNAs, which induce gene silencing by a variety of different means. RNAi-induced gene silencing controls gene expression at all levels, including transcription, mRNA stability and translation. We are only beginning to understand the physiological roles of the RNAi pathway and the function of the many small non-coding RNA species, which are found in eukaryotic genomes. Here we review the known functions of genes in RNAi in various species, the experimental use and design of small RNAs as a genetic tool to dissect the function of mammalian genes and their potential as therapeutic agents to modulate gene expression in patients.

In vitro and ex vivo evaluation of an intestinal permeation enhancing self-microemulsifying drug delivery system (SMEDDS)

The objective of this study was to develop a self-microemulsifying drug delivery system (SMEDDS) that enhances drug absorption through intestinal membranes and to evaluate the mechanism of improvement. The developed formulation was optimized in order to reduce the droplet size and characterized in several environments. Furthermore, a pseudo-ternary phase diagram was mapped in order to identify the o/w microemulsion region. The formulation contains 30 % (m/m) Cremophor EL, 30 % (m/m) Capmul MCM, 10 % (m/m) propylene glycol and 30 % (m/m) Captex 355. Permeation studies were carried out in vitro on Caco-2 cell monolayers and ex vivo on rat intestine in Ussing-type chambers using model compounds rhodamine-123 (Rho-123) and fluorescein isothiocyanate-dextran 4 (FD4). The selected formulation improved in a concentration of 0.5 % (m/v) absorption of FD4 1.85-fold in vitro and 1.65-fold ex vivo via tight junction opening. Furthermore, the system had the ability to inhibit P-glycoprotein and improved the permeation of Rho-123 1.49 fold in vitro and 1.64 fold ex vivo. Stability studies were carried out to assure that the SMEDDS was stable during transport studies and in several media and buffers. According to these results, the developed formulation showed great promise in terms of improving the bioavailability of low-permeability drugs—often the rate-limiting step for oral drug delivery systems.

Thiolated hydroxyethyl cellulose: design and in vitro evaluation of mucoadhesive and permeation enhancing nanoparticles.

Within this study, HEC-cysteamine nanoparticles with free thiol groups in the range of 117-1548 μmol/g were designed and characterized. Nanoparticles were generated via ionic gelation of the cationic polymer with tripolyphosphate (TPP) followed by covalent crosslinking via disulfide bond formation using H2O2 as oxidant. The mean diameter of the particles was in the range of 270-360 nm, and zeta potential was determined to be +4 to +10 mV. Nanoparticles were evaluated in terms of mucoadhesive, permeation enhancing, and biocompatible properties as well as biodegradability. The particles remained attached to porcine intestinal mucosa up to 70% after 3h of incubation. The more nanoparticles were oxidized; however, the less were their mucoadhesive properties. Nanoparticles applied in a concentration of 0.5% (m/v) with the highest content of free thiol groups improved the transport of fluorescein isothiocyanate dextran 4 (FD4) across Caco-2 cell monolayer 3.94-fold in comparison with control (buffer). In addition, the transport of FD4 was even 1.84-fold enhanced in the presence of 0.5% (m/v) nanoparticles with the lowest free thiol group content. The higher the disulfide bond content within nanoparticles was, to a lower degree nanoparticles were hydrolyzed by cellulase. None of these nanoparticles showed pronounced cytotoxicity. Accordingly, HEC-cysteamine could be a promising excipient for nanoparticulate delivery systems for poorly absorbed drugs.

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.

Poly(acrylic acid)-cysteine for oral vitamin B12 delivery.

The aim of this study was to investigate the potential of poly(acrylic acid)-cysteine (PAA-cys) solution and microparticles to enhance the transport of vitamin B12 (VB 12) across Caco-2 cell monolayer and rat intestinal mucosa. Thiolated PAA was synthesized by covalent attachment of L-cysteine. Microparticles were prepared by spray-drying and characterized regarding their size, morphology, thiol group content, VB 12 payload and release, swelling behavior, mucoadhesion, permeation-enhancing effect, and cytotoxicity. Particles with a mean diameter of 2.452±2.26 μm, a payload of 1.11±0.72%, and 190.2±8.85 μmol of free thiol groups per gram were prepared. Swelling behavior studies revealed that the stability of thiolated particles was improved compared with unmodified ones. Of the total VB 12 loaded, 95±0.12% was released within 3 h from thiolated particles. PAA-cys particles exhibited 2.24-fold higher mucoadhesive properties compared with unmodified particles. Permeation experiments with Caco-2 cells proved that permeability of VB 12 with PAA-cys solution and particles was 3.8- and 3.6-fold higher than control, respectively, and with rat intestinal mucosa it was 4.8- and 4.4-fold higher than control, respectively. Negligible cytotoxicity was assessed. PAA-cys is a promising excipient for oral delivery of VB 12 as a solution and as microparticles.

Thiopyrazole preactivated chitosan: combining mucoadhesion and drug delivery.

The objective of this study was to develop a preactivated chitosan derivative by the introduction of thioglycolic acid followed by 3-methyl-1-phenylpyrazole-5-thiol (MPPT) coupling via disulfide bond formation. The newly synthesized conjugate was characterized in terms of water-absorbing capacity, cohesive properties, mucoadhesion and drug release kinetics. Further in vitro characterization was conducted regarding permeation enhancement of the model compound fluorescein isothiocyanate dextran (FD4) and cytotoxic effects on Caco-2 cells. Based on the attachment of the hydrophobic residue, chitosan-S-S-MPPT test discs showed increased stability of the polymer matrix as well as improved water uptake and liberation of fluorescein isothiocyanate dextran (FD4) compared to chitosan only. The mucoadhesive qualities on porcine intestinal mucosa could be improved 38-fold based on the enhanced bonding between chitosan-S-S-MPPT and mucus through the thiol/disulfide exchange reaction of polymer and mucosal cysteine-rich domains supported by MPPT as the leaving group. This novel biomaterial presents a disulfide conjugation-based delivery system that releases the antibacterial thiopyrazole when the polymer comes into contact with the intestinal mucosa. These properties, together with the safe toxicological profile, make chitosan-S-S-MPPT a valuable carrier for mucoadhesive drug delivery systems and a promising matrix for the development of antimicrobial excipients.

Development and in vitro evaluation of a buccal drug delivery system based on preactivated thiolated pectin.

Abstract The aim of this study was to evaluate the potential of preactivated thiolated pectin (Pec-Cys-MNA) for buccal drug delivery. Therefore, a gel formulation containing this novel polymer and the model drug lidocaine was prepared and investigated in vitro in terms of rheology, mucoadhesion, swelling behavior and drug release in comparison to formulations based on pectin (Pec) and thiolated pectin (Pec-Cys). Both pectin derivatives showed gel formation without addition of any other excipient due to self-crosslinking thiol groups. Under same conditions, pectin did not show gel formation. Viscosity of Pec-Cys-based formulation increased 92-fold and viscosity of Pec-Cys-MNA-based formulations by 4958-fold compared to pectin-based formulation. Gels did not dissolve in aqueous environment during several hours and were able to take up water. Mucoadhesion of pectin on buccal tissue could be improved significantly, value of total work of adhesion increased in the following rank order: Pec-Cys-MNA > Pec-Cys > Pec. The retention time of a model drug incorporated in gel formulations on buccal mucosa under continuous rinsing with phosphate-buffered saline was prolonged, after 1.5 h 3-fold higher amount of a model drug was to be found on tissue after application of Pec-Cys-MNA-based formulation compared to pectin-based and 2-fold compared to Pec-Cys-based formulation. The Pec-Cys-MNA-based gel showed a more sustained release of lidocaine than Pec-Cys-based gel, whereas pectin solution revealed an immediate release. According to these results, the self-crosslinking pectin-derivative is a promising tool for buccal application.