Is hyaluronic acid the perfect excipient for the pharmaceutical need?

Hyaluronic acid has become an interesting and important polymer as an excipient for pharmaceutical products due to its beneficial properties, like solubility, biocompatibility and biodegradation. To improve the properties of hyaluronic acid, different possibilities for chemical modifications are presented, and the opportunities as novel systems for drug delivery are discussed. This review gives an overview over the production of hyaluronic acid, the possibilities of its chemical modification and the current state of in vitro and in vivo research. Furthermore, market approved and commercially available products are reviewed and derivatives undergoing clinical trials and applying for market approval are shown. In particular, hyaluronic acid has been studied for different administrations in rheumatology, ophthalmology, local anesthetics, cancer treatment and bioengineering of tissues. The present work concludes with perspectives for future administration of pharmaceuticals based on hyaluronic acid.

A gellan gum derivative as in-situ gelling cationic polymer for nasal drug delivery.

The aim of the present study was the development of a novel gellan gum derivative exhibiting mucoadhesive properties for nasal application. Accomplishing this, amino groups have been introduced to the polymeric backbone. The resulting synthesis products were characterized in terms of the amount of attached amino groups, regarding hydration, zeta potential and gel characteristics. Mucoadhesiveness was assessed studying rheological synergism, by rotating cylinder and regarding tensile studies. Next to erythrocyte-/cytotoxicity evaluation, the impact on ciliary beat frequency of nasal epithelial cells was investigated. Results revealed coupling rates up to 1259.50 ± 75.98 µmol/g polymer as well as accelerated hydration of the derivatives. Comparing aminated with unmodified gellan, enhanced mucoadhesion was verified by a 32-fold increase in viscosity of polymer/mucus mixtures and by a 14-fold extended mucosal adhesion time. Tensile studies demonstrated a 9-fold higher total work of adhesion and a 3.75-fold elevated maximum detachment force. Cellular membrane was not seriously impaired. CBF studies proved a reversible inhibition due to the application of the novel derivative. According to the outlined findings, aminated gellan gum can be considered as a promising excipient for nasal dosage forms improving drug bioavailability by superior adhesive features.

Strategies for improved hair binding: Keratin fractions and the impact of cationic substructures.

Keratin extracts and hydrolysates from varying sources, their chemical modifications and compositions thereof have shown potential in the restoration of hair properties. Within this study on reactivity of thiol groups and the shielding effect of anionic charges the binding of keratin-associated proteins (KAP) and α-keratins (Ker) extracted from human hair to natural and permed hair fibers was evaluated. Selectively extracted KAP and Ker were preactivated with 6-mercaptonicotinamide in a quantity of 194 ± 21 µmol/g for KAP and 169 ± 27 µmol/g for Ker resulting in 1.9- and 1.4-fold enhanced binding to natural hair, respectively. The amount of accumulated Ker on hair fibers was furthermore increased by 1.7-fold in presence of 25 mM L-arginine. Perming of hair impaired binding characteristics of Ker with negligible effects for preactivation, whereas unmodified and preactivated KAP showed results comparable to natural hair. Strongly enhanced penetrability after perming was reflected by the mean penetration depth for fluorescein of 25 µm compared to 5 µm for natural fibers.

Development and In Vitro Evaluation of Stearic Acid Phosphotyrosine Amide as New Excipient for Zeta Potential Changing Self-Emulsifying Drug Delivery Systems.

PURPOSE: Development of zeta potential changing SEDDS containing newly synthesized derivative stearic acid phosphotyrosine amide. METHODS: Stearoyl chloride was conjugated with phosphotyrosine, which is substrate for the brush border enzyme intestinal alkaline phosphate. The synthesized derivative was implemented in different SEDDS formulations and the zeta potential changing properties and the concluding mucus diffusion abilities were evaluated. RESULTS: Stearic acid phosphotyrosine amide was successfully synthesized and incorporated into SEDDS. A SEDDS formulation containing the new derivative showed a zeta potential of -14 mV before, and + 2 mV after enzymatic cleavage by intestinal alkaline phosphatase. Experiments on a Caco-2 monolayer demonstrated that the phosphate cannot only be cleaved by isolated enzyme, but also by enzyme, which was expressed by cells. The mucus diffusion abilities of the untreated, negatively charged SEDDS were significantly higher compared to the enzymatically cleaved, positively charged SEDDS. CONCLUSION: The developed stearic acid phosphotyrosine represents a promising excipient for zeta potential changing SEDDS. Graphical Abstract.

Intestinal enzyme delivery: Chitosan/tripolyphosphate nanoparticles providing a targeted release behind the mucus gel barrier.

AIM: The aim of this study was to evaluate the potential of chitosan/tripolyphosphate (TPP) nanoparticles to provide a targeted release of ß-galactosidase behind the intestinal mucus gel barrier. METHODS: Nanoparticles were prepared by ionic gelation of chitosan and TPP in the presence of ß-galactosidase. Particles were characterized regarding size, polydispersity index and drug load. Target mediated hydrolysis of the TPP cross-linker followed by particle degradation and release of ß-galactosidase was investigated during incubation with isolated as well as cell and tissue associated intestinal alkaline phosphatase (IAP). Phosphate content in the media was quantified via malachite assay, whereas particle disintegration was monitored in parallel by measuring the decrease in particle size as well as in optical density at 600 nm. The released amount of ß-galactosidase was either determined utilizing bicinchoninic acid (BCA) protein detection or via an enzymatic activity assay with 2-nitrophenyl ß-D-galactopyranoside (ONPG) as substrate. Protection towards tryptic degradation was verified by ONPG assay. RESULTS: The size of nanoparticles was 573 ±â€¯34 nm and a payload of 376 ±â€¯18 µg ß-galactosidase per mg particles was achieved. Degradation studies with isolated IAP revealed a maximum phosphate cleavage of 118 ±â€¯1 µg/mg particles, a size decrease up to 38 ±â€¯7 % and a release of 58 ±â€¯0.5 % ß-galactosidase. Release of 94 ±â€¯6 % of the incorporated initial amount of ß-galactosidase was proven after 3 h incubation on porcine mucosa. Furthermore a protection against tryptic degradation was attained resulting in a 3-fold higher residual enzymatic activity of encapsulated ß-galactosidase compared to a control of free enzyme. CONCLUSION: Chitosan/TPP nanoparticles seem to be qualified as a suitable carrier for a targeted delivery of active ingredients to mucosal tissues expressing alkaline phosphatase.

Cationic starch derivatives as mucoadhesive and soluble excipients in drug delivery.

The aim of this study was to develop a novel mucoadhesive cationic polymer by introducing primary amino groups to the polymeric backbone of starch. This newly synthesized polymer should exhibit superior properties over chitosan regarding solubility, mucoadhesiveness and cytotoxicity. Increasing amounts of sodium periodate were used to cleave and oxidize vicinal diols under aldehyde formation obtaining three different degrees of modification. In a subsequent step, primary amines were introduced via reductive amination with ammonia. Degree of amination was examined with TNBS-assay and zeta potential measurements. Mucoadhesiveness was investigated by rotating cylinder, tensile studies and rheological measurements. Primary amino groups were successfully attached to the polymer, proven by zeta potential measurements and UV-spectroscopy. Depending on the amount of periodate used in the reaction, coupling rates of up to 514 µmol/g polymer were achieved. All synthesized derivatives showed 100% solubility in a pH range of 1-9. Aminated starch with the highest coupling rate of 514 µmol/g showed a 9.5-fold prolonged retention time on intestinal mucosa and a 2.7-fold higher total work of adhesion on the mucosal tissue compared to chitosan. Furthermore, cytotoxic examinations of all tested polymers showed only a low impact on cell viability after 24 h, whereby starch derivatives possessed even less cell toxic effects than chitosan. Summarizing these results, cationic starch derivatives seem to be promising excipients for mucosal drug delivery with superior properties compared to chitosan, the most examined cationic polymer.

In Vitro-in Vivo Correlation of Mucoadhesion Studies on Buccal Mucosa.

BACKGROUND: For the development of novel buccoadhesive formulations, their physicochemical properties, strength of the interfacial joint, and residence time on the buccal mucosa are considered as a measure for their in vivo mucoadhesive properties. Focusing on these parameters, the predictive power of established in vitro systems was assessed for mucoadhesive properties in humans using discs as the model solid dosage form. METHODS: Compressed into discs, hydroxyethyl cellulose, carboxymethyl cellulose, carbopol, polycarbophil, alginate, and xanthan gum were used as model polymers. Mucosal residence time, maximum detachment force (MDF), and total work of adhesion (TWA) were determined ex vivo on the porcine buccal mucosa and in vivo on healthy volunteers. The impact of detachment velocity, humidification, and experimental set-up employed for tensile studies was examined and correlated to in vivo studies. RESULTS: Ex vivo results for mucosal residence time showed a very high correlation ( r = 0.997) with data obtained in vivo. For tensile studies, a set-up optimized for moistening the interface, speed, and alignment of the tensile force provided ex vivo results with very high correlation to in vivo experiments with r = 0.983 obtained for MDF and r = 0.973 for TWA, respectively. CONCLUSIONS: Experimental set-ups for the determination of mucosal residence time and tensile studies could be identified as valid methods for the development of intraoral solid dosage forms.

Thiolated polymers: Bioinspired polymers utilizing one of the most important bridging structures in nature.

Thiolated polymers designated "thiomers" are obtained by covalent attachment of thiol functionalities on the polymeric backbone of polymers. In 1998 these polymers were first described as mucoadhesive and in situ gelling compounds forming disulfide bonds with cysteine-rich substructures of mucus glycoproteins and crosslinking through inter- and intrachain disulfide bond formation. In the following, it was shown that thiomers are able to form disulfides with keratins and membrane-associated proteins exhibiting also cysteine-rich substructures. Furthermore, permeation enhancing, enzyme inhibiting and efflux pump inhibiting properties were demonstrated. Because of these capabilities thiomers are promising tools for drug delivery guaranteeing a strongly prolonged residence time as well as sustained release on mucosal membranes. Apart from that, thiomers are used as drugs per se. In particular, for treatment of dry eye syndrome various thiolated polymers are in development and a first product has already reached the market. Within this review an overview about the thiomer-technology and its potential for different applications is provided discussing especially the outcome of studies in non-rodent animal models and that of numerous clinical trials. Moreover, an overview on product developments is given.

In vitro evaluation of a self-emulsifying drug delivery system (SEDDS) for nasal administration of dimenhydrinate.

The objective of the study was the development and in vitro characterization of a self-emulsifying drug delivery system (SEDDS) for the nasal application of dimenhydrinate. Final composition of SEDDS was established based on drug solubility and stability studies. Dimenhydrinate was loaded into the SEDDS pre-concentrates to 7.5% (m/v). The droplet size of the final SEDDS formulations was in a range between 60 and 220 nm. Permeability, as well as tissue toxicity, of the formulations was investigated using bovine nasal mucosa. Enhancement in permeation up to 2.8-fold compared to pure dimenhydrinate was confirmed. Furthermore, toxicity studies did not reveal any serious tissue damages related to the SEDDS. Additionally, irritation potential of SEDDS was evaluated in ciliary beat frequency measurements. Incorporation of dimenhydrinate into SEDDS might therefore be considered as a promising approach within the field of nasal delivery of antiemetics by utilizing permeation enhancement strategy.

N-Hydroxysulfosuccinimide Esters versus Thiomers: A Comparative Study Regarding Mucoadhesiveness.

The objective of the study was to compare poly(acrylic acid)- N-hydroxysulfosuccinimide reactive esters (PAA-Sulfo-NHS) and poly(acrylic acid)-cysteine conjugates (PAA-Cys) regarding their mucoadhesiveness. Polymer conjugates were synthesized in a water free environment and characterized by UV-vis spectroscopy and FTIR. Water uptake studies were performed, and the polymers were further examined for their mucoadhesive properties and cohesiveness using the rotating cylinder method. Tensile force measurements were conducted to define the strength of adhesion to porcine intestinal mucosa. Additionally, polymer-mucus mixtures were assessed for rheological synergism by measuring the increase in dynamic viscosity. Both modifications led to a prolonged adhesion time compared to unmodified PAA. Fast dissolution of PAA-Sulfo-NHS derivatives was monitored, whereas PAA-Cys tended to extensively swell while exhibiting high cohesive properties. Measurements of tensile force revealed up to 2.7-fold (PAA-Sulfo-NHS) and 2.3-fold (PAA-Cys) enhancement of the maximum detachment force and 7.6-fold (PAA-Sulfo-NHS) and 3.6-fold (PAA-Cys) increase in the total work of adhesion. Formation of a gel network between polymer and mucus was confirmed by a 10.8-fold (PAA-Sulfo-NHS) and 20.8-fold (PAA-Cys) increase in viscosity. Both types of polymers show high mucoadhesive properties due to the formation of covalent bonds with the mucus. As thiolated polymers are capable of forming stabilizing disulfide bonds within their polymeric network, they are advantageous over PAA-Sulfo-NHS.

Reactive keratin derivatives: A promising strategy for covalent binding to hair.

HYPOTHESIS: Restoration of damaged hair structure by replacing lost keratin is still of paramount interest. On account of the fact that native keratin is a highly cross-linked protein with numerous disulfide bonds but just a few nucleophilic thiol groups, binding affinity to hair is comparatively low. Hence, the design of reactive keratin derivatives bearing free sulfhydryl groups that are optionally S-protected and preactivated should enhance permanent binding to hair fibers. EXPERIMENTS: Keratin was extracted from human Caucasian hair and reduced with NaBH4 to obtain free sulfhydryl groups (keratin-SH). These thiol groups were S-protected via disulfide linkage to 2-mercaptonicotinic acid (keratin-MNA). Hair fibers were either utilized in their natural form or chemically damaged by bleaching. Amount of keratin derivatives being bound to hair fibers was quantified by fluorescence detection of fluorescein isothiocyanate labeled keratins. FINDINGS: Both modifications induced higher affinity of keratin to hair fibers, resulting in up to 1.7-fold (keratin-MNA) improved binding to natural hair and up to 3.6-fold (keratin-MNA) improved binding to bleached hair. Confocal laser microscopy confirmed the accumulation of keratin derivatives in distinct regions of the cuticle layer. Thiol functionalization seems therefore to be a promising strategy for efficient durable binding of keratin to hair.

Surface phosphorylation of nanoparticles by hexokinase: A powerful tool for cellular uptake improvement.

The aim of the study was to develop zeta potential changing nanoparticles (NPs) via surface phosphorylation in order to improve their uptake by epithelial cells. Polymeric NPs were formed via in situ gelation between chitosan (CS) and chondroitin sulphate (ChS). Phosphorylation of these NPs was carried out by using hexokinase. The resulting phosphorylated NPs (p-NPs) were characterized in respect of their size and zeta potential. Phosphate release was quantified by incubating the particles with isolated as well as cell-associated intestinal alkaline phosphatase (AP). In parallel, resulting change in zeta potential was monitored. In-vitro mucus permeation of these particles was evaluated on porcine intestinal mucus. Furthermore, toxicity and cellular uptake studies were performed on Caco-2 cells. p-NPs exhibited a mean size of 412 ±â€¯3.24 nm and a zeta potential of -12.4 mV. When these p-NPs were incubated with isolated as well as cell-associated AP, a significant amount of phosphate was released within 4 h and zeta potential raised up to -1.2 mV. p-NPs showed improved mucus permeation in comparison to unmodified NPs. Due to de-phosphorylation by AP, cellular uptake of p-NPs increased almost 2-fold. Moreover, particles displayed no toxicity. Findings of this study show that zeta potential changing p-NPs provide effective mucus permeation and enhanced cellular uptake.

Development and in vitro characterization of a papain loaded mucolytic self-emulsifying drug delivery system (SEDDS).

The aim of the study was to create a self-emulsifying drug delivery system (SEDDS) with mucolytic properties based on incorporated papain for improved mucus permeation. In order to increase the lipophilicity of the enzyme and to dissolve it in SEDDS, hydrophobic ion pairing with sodium deoxycholate in a molar ratio of 20:1 (surfactant: enzyme) was performed. The yield of precipitated papain was 86.8±2.7% and the ion pair was loaded into the formulations to 1% (m/m). Suitable formulations were chosen according to their properties to dissolve the ion pair and characterized regarding droplet size and polydispersity index. Prepared emulsions were in a droplet size range between 50 and 120nm. Enzyme activity assay of complex and loaded SEDDS was conducted to ensure proteolytic qualities for following permeation and diffusion studies. SEDDS loaded with the ion pair showed an almost 2-fold increase in mucus permeation compared to the control without complex. Furthermore, 3-fold enhanced mucus diffusion could be confirmed in a second assay and an increase of mucosal residence on porcine intestinal mucosa up to 3- and 5-fold was observed as against the blank formulations. Consequently the incorporation of enzymes exhibiting proteolytic properties in self-emulsifying drug delivery systems may be considered as a promising strategy to enhance mucus permeation and overcome intestinal barriers.

Totally S-protected hyaluronic acid: Evaluation of stability and mucoadhesive properties as liquid dosage form.

AIM: It is the aim of this study to synthesize hyaluronic acid (HA) derivatives bearing mucoadhesive properties and showing prolonged stability at pH 7.4 and under oxidative condition as liquid dosage form. METHODS: HA was modified by thiolation with l-cysteine (HA-SH) and by conjugation with 2-mercaptonicotinic acid-l-cysteine ligand to obtain an S-protected derivative (HA-MNA). The polymers were characterized by determination of thiol group content and mercaptonicotinic acid content. Cytotoxicity, stability and mucoadhesive properties (rheological evaluation and tensile test) of the polymers were evaluated. RESULTS: HA-SH and HA-MNA could be successfully synthesized with a degree of modification of 5% and 9% of the total moles of carboxylic acid groups, respectively. MTT assay revealed no toxicity for the polymers. HA-SH resulted to be unstable both at pH 7.4 and under oxidative conditions, whereas HA-MNA was stable under both conditions. Rheological assessment showed a 52-fold and a 3-fold increase in viscosity for HA-MNA incubated with mucus compared to unmodified HA and HA-SH, respectively. Tensile evaluation carried out with intestinal and conjunctival mucosa confirmed the higher mucoadhesive properties of HA-MNA compared to HA-SH. CONCLUSIONS: According to the presented results, HA-MNA appears to be a potent excipient for the formulation of stable liquid dosage forms showing comparatively high mucodhesive properties.

2,2'Dithiodinicotinyl ligands: Key to more reactive thiomers.

The aim of this study was to establish a novel type of preactivated thiomers exhibiting a comparatively higher reactivity with mucus and consequently improved mucoadhesive properties. In order to achieve this goal, the dimeric form of 2-mercaptonicotinic acid (MNA-MNA) was directly attached to the polymeric backbone of chitosan (CHI) via amide bond formation mediated by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) used as a coupling reagent. The remaining free amino groups were in the following reacted with succinic anhydride (Succ) in order to obtain a uniformly anionically charged polymer (CHI-Succ-MNA-MNA). Within this study, different coupling rates of up to 170 µmol MNA-MNA per gram polymer were achieved. The attachment of the dimeric ligand resulted in a preactivated thiomer with a comparatively more reactive disulfide substructure due to the additional nitrogen atom in conjugation over the aromatic moieties. Furthermore, the obtained polymer is entirely preactivated and thus prevented against undesired oxidation reactions. Kinetic studies of disulfide exchange reactions showed a 3.8-fold higher reactivity of CHI-Succ-MNA-MNA in comparison to a state-of-the-art preactivated thiomer. Within rheological measurements, CHI-Succ-MNA-MNA with a coupling rate of 170 µmol (CHI-Succ-MNA-MNA 170) led to a 5.7-fold higher mucus viscosity than the non-thiolated control polymer (CHI-Succ) indicating a rheological synergism due to mucoadhesive properties. These results were confirmed by a second mucoadhesion study, which showed a significantly prolonged retention time of CHI-Succ-MNA-MNA on the small intestinal mucosa compared to CHI-Succ (P<0.02). Accordingly, the double preactivation seems to be a promising strategy in order to obtain entirely preactivated polymers with enhanced mucoadhesive properties.