Fabian Hintzen

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 evidence of oral vaccination with PLGA nanoparticles containing the immunostimulant monophosphoryl lipid A.

Although oral vaccination has numerous advantages over the commonly used parenteral route, degradation of vaccine and its low uptake in the lymphoid tissue of the gastrointestinal (GI) tract still impede their development. In this study, the model antigen ovalbumin (OVA) and the immunostimulant monophosphoryl lipid A (MPLA) were incorporated in polymeric nanoparticles based on poly(D,L-lactide-co-glycolide) (PLGA). These polymeric carriers were orally administered to BALB/c mice (Bagg albino, inbred strain of mouse) and the resulting time-dependent systemic and mucosal immune responses towards OVA were assessed by measuring the OVA-specific IgG and IgA titers using an enzyme-linked immunosorbent assay (ELISA). PLGA nanoparticles were spherical in shape, around 320 nm in size, negatively charged (around -20 mV) and had an OVA and MPLA payload of 9.6% and 0.86%, respectively. A single immunization with formulation containing (OVA + MPLA) incorporated in PLGA nanoparticles induced a stronger IgG immune response than that induced by OVA in PBS solution or OVA incorporated into PLGA nanoparticles. Moreover, significantly higher IgA titers were generated by administration of (OVA + MPLA)/PLGA nanoparticles compared to IgA stimulated by control formulations, proving the capability of inducing a mucosal immunity. These findings demonstrate that co-delivery of OVA and MPLA in PLGA nanoparticles promotes both systemic and mucosal immune responses and represents therefore a suitable strategy for oral vaccination.

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 Evaluation of a Novel Mucus Diffusion Test System Approved by Self-Nanoemulsifying Drug Delivery Systems

The aim of this study was the development of a novel mucus diffusion model and the approval thereof by self-nanoemulsifying drug delivery systems (SNEDDSs). For diffusion experiments, various SNEDD formulations were developed, spiked with fluorescein diacetate, and evaluated for their mucus diffusion behavior through an intestinal mucus layer within the novel setup. In brief, SNEDD formulations resulting in particle sizes of 12.0 nm produced 70.3% of diffused model drug through the mucus layer. In comparison, SNEDDSs with particle sizes of 455.5 nm led to a permeation of 8.3% only. Apart from this size dependence, two SNEDDS excipients namely Cremophor RH 40 and triacetin were identified to strongly affect the permeation through mucus. Hence, it could be demonstrated that particle size and single excipients can positively influence mucus diffusion of SNEDDSs. Furthermore, it could be shown that the developed mucus diffusion model is a promising tool for pharmaceutical research in comparison with already established systems as it allows an easy handling coupled with the possibility to test different kinds of mucus in parallel within one setup.

Development and in vitro evaluation of slippery nanoparticles for enhanced diffusion through native mucus

AIM The aim of this study was to investigate the mucus-penetrating properties of neutral nanoparticles comprising poly(acrylic acid) (PAA) and poly(allylamine) (PAM). MATERIALS & METHODS PAA and PAM nanoparticles were prepared on the basis of ionic interactions between the two polymers. Nanoparticles were characterized by particle size as well as surface charge. The cytotoxicity was examined via resazurin and lactate dehydrogenase assays. Using a modified Ussing chamber with mucus, the diffusion properties of obtained neutral nanoparticles were compared with control particles. RESULTS The obtained PAA-PAM nanoparticles demonstrated no significant cytotoxicity and displayed smooth and spherical surfaces, a particle size range of 200 nm and ζ-potential of 0.9 mV. The diffusion efficiency of neutral nanoparticles was 2.5- and 1.8-fold higher than PAM and PAA nanoparticles, respectively. CONCLUSION Taking enhanced mucus-penetrating properties into account, neutral nanoparticles were shown to be very promising in drug delivery via mucus membranes of different cavities.

Synthesis and in vitro characterization of entirely S-protected thiolated pectin for drug delivery.

The study was aimed to synthesize a thiolated polymer (thiomer) that is resistant to oxidation in solutions above pH 5. In order to protect a pectin-cysteine conjugate against premature oxidation, the thiomer was S-protected by a disulfide connected leaving group. Therefore, 2-mercaptonicotinic acid was first coupled to L-cysteine by a disulfide exchange reaction and the purified product was subsequently attached to pectin by a carbodiimide mediated amid bond formation. The obtained fully S-protected thiolated pectin was in vitro characterized with respect to co- and mucoadhesive properties and stability toward oxidation. The results indicated a 1.8-fold and 2.3-fold enhanced disintegration time at pH 6.8 of the S-protected thiolated pectin (Pec-Cys-MNA) compared to thiolated pectin (Pec-Cys) and unmodified pectin (Pec). Moreover, rheological measurements of polymer/mucus mixtures showed a 1.6-fold (compared to Pec-Cys) and 6.7-fold (compared to Pec) increased dynamic viscosity of Pec-Cys-MNA. On the other hand, in the presence of a strong oxidizing agent such as H2O2 (0.3% v/v), no increase in viscosity of Pec-Cys-MNA could be observed. A 6-month experiment also demonstrated the long-term stability of a liquid formulation based on Pec-Cys-MNA. Further investigations proved that the first time all thiol groups on a thiolated polymer could be protected owing to the novel synthesis. Accordingly, these features may help to develop thiomer based liquid or gel formulations targeting mucosal surfaces such as nasal, ocular or vaginal drug delivery systems.

Thiolated chitosan: Development and in vivo evaluation of an oral delivery system for leuprolide

The aim of the present study was to develop an oral delivery system for the peptide drug leuprolide. Gel formulations based on unmodified chitosan/reduced glutathione (GSH) and chitosan-thioglycolic acid (chitosan-TGA)/GSH were prepared, and their effect on the absorption of leuprolide was evaluated in vitro and in vivo in male Sprague Dawley rats. Transport studies were performed with freshly excised rat intestinal mucosa mounted in Ussing-type chambers. Due to the addition of gel formulations comprising 0.5% (m/v) unmodified chitosan/0.5% (m/v) GSH and 0.5% (m/v) chitosan-TGA/0.5% (m/v) GSH, the transport of leuprolide across excised mucosa was improved up to 2.06-fold and 3.79-fold, respectively, in comparison with leuprolide applied in buffer (P(app)=2.87 ± 0.77 × 10⁻⁶ cm/s). In vivo, the addition of oral gel formulation comprising 8 mg of unmodified chitosan, 1mg of GSH and 1mg of leuprolide increased the area under the plasma concentration-time curve (AUC₀₋₈) of leuprolide 1.39-fold in comparison with leuprolide having been administered just in saline. Moreover, the administration of oral gel formulation comprising 8 mg of chitosan-TGA, 1mg of GSH and 1mg of leuprolide resulted in a further enhanced leuprolide plasma concentration, and the area under the plasma concentration-time curve (AUC₀₋₈) of leuprolide was increased 3.72-fold in comparison with the control. With the oral gel formulation comprising 8 mg of chitosan-TGA, a relative bioavailability (versus s.c. injection) of 4.5% was achieved in contrast to the control displaying a relative bioavailability of 1.2%. Thus, according to the achieved results, it is suggested that chitosan-TGA in combination with GSH is a valuable tool for improving the oral bioavailability of the peptide drug leuprolide.

Enzymatic degradation of thiolated chitosan

The objective of this study was to evaluate the biodegradability of thiolated chitosans in comparison to unmodified chitosan. Mediated by carbodiimide, thioglycolic acid (TGA) and mercaptonicotinic acid (MNA) were covalently attached to chitosan via formation an amide bond. Applying two different concentrations of carbodiimide 50 and 100 mM, two chitosan TGA conjugates (TGA A and TGA B) were obtained. According to chitosan solution (3% m/v) thiomer solutions were prepared and chitosanolytic enzyme solutions were added. Lysozyme, pectinase and cellulase were examined in chitosan degrading activity. The enzymatic degradability of these thiomers was investigated by viscosity measurements with a plate–plate viscometer. The obtained chitosan TGA conjugate A displayed 267.7 µmol and conjugate B displayed 116.3 µmol of immobilized thiol groups. With 325.4 µmol immobilized thiol groups, chitosan MNA conjugate displayed the most content of thiol groups. In rheological studies subsequently the modification proved that chitosan TGA conjugates with a higher coupling rate of thiol groups were not only degraded to a lesser extent by 20.9–26.4% but also more slowly. Chitosan mercaptonicotinic acid was degraded by 31.4–50.1% depending the investigated enzyme and even faster than unmodified chitosan. According to these results the biodegradability can be influenced by various modifications of the polymer which showed in particular that the rate of biodegradation is increased when MNA is the ligand, whereas the degradation is hampered when TGA is used as ligand for chitosan.

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.

Uptake of phenothiazines by the harvested chylomicrons ex vivo model: Influence of self-nanoemulsifying formulation design

The aim of this study was to examine the potential of self-nanoemulsifying drug delivery systems (SNEDDS) on the uptake of the lipophilic and poorly water soluble phenothiazines thioridazine and chlorpromazine with the isolated plasma derived chylomicron (CM) ex vivo model. The multi-component delivery systems were optimized by evaluating their ability to self-emulsify when introduced to an aqueous medium under gentle agitation. The uptake of phenothiazines by isolated plasma derived chylomicrons was investigated with short chain triglyceride (SCT) SNEDDS, medium chain triglyceride (MCT) SNEDDS, and long chain triglyceride (LCT) SNEDDS. SNEDDS were also evaluated for their stabilities, dispersibilities, percentage transmittances and by particle size analyses. For thioridazine a 5.6-fold and for chlorpromazine a 3.7-fold higher CM uptake could be observed using a LCT-SNEDDS formulation compared to the drugs without formulation. In contrast, ex vivo uptake by isolated CM was not significantly increased by SNEDDS formulations based on MCT and SCT. Compared with isolated CM, the CM sizes were increased 2.5-fold in LCT-SNEDDS, whereas in MCT-SNEDDS or SCT-SNEDDS only a small, non-significant (P<0.05) increase in CM size was observed. These results show that distinct SNEDDS formulations containing phenothiazines are efficiently uptaken by plasma derived chylomicrons ex vivo.