A mucoadhesive nanoparticulate system for the simultaneous delivery of macromolecules and permeation enhancers to the intestinal mucosa.

The feasibility of combining safe permeation enhancers in a mucoadhesive particulate system for the oral delivery of peptide drugs was investigated in this study. Polyelectrolyte complex nanoparticles (NPs) were prepared by ionic interaction of spermine (SPM) with polyacrylic acid (PAA) polymer. Cytotoxicity studies in Caco-2 monolayers revealed the safety of the delivery system in the concentration range used for permeation enhancement. The cellular transport of fluorescein isothiocyanate dextran (FD4) showed higher permeation enhancing profiles of SPM-PAA NPs, as compared to SPM solution or PAA NPs prepared by ionic gelation with MgCl(2) (Mg-PAA NPs). These permeation enhancing effects were associated with a reversible decrease in TEER values, suggesting a paracellular permeation pathway by reversible opening of the tight junctions. Furthermore, confocal microscopy results revealed strong association of the NPs prepared using fluorescence labeled PAA to Caco-2 cells. The permeation enhancing properties of SPM-PAA NPs were further evaluated in vivo after oral administration to rats, using FD4 and calcitonin as models of poorly permeating drugs. Confocal microscopy images of rats' small intestine confirmed previous findings in Caco-2 cells and revealed a strong and prolonged penetration of FD4 from the mucosal to the basolateral side of the intestinal wall. In addition, the proposed NPs were efficient in improving the oral absorption of calcitonin, as evidenced by the significant and prolonged reduction of the blood calcemia in rats.

Nanoparticles of glycol chitosan and its thiolated derivative significantly improved the pulmonary delivery of calcitonin.

A novel thiomer derivative of glycol chitosan (GCS) was synthesized by coupling with thioglycolic acid (TGA) and evaluated for the pulmonary delivery of peptides. Nanoparticles (NPs) based on GCS and GCS-TGA were obtained by the ionic gelation method and demonstrated a particle size in the range of 0.23-0.33 microm with positive surface charge and high calcitonin entrapment. Fluorescent GCS-TGA NPs resulted in a 2-fold increase in mucoadhesion to lung tissue after intra-tracheal administration to rats as compared to non-thiolated NPs. Evaluation of pulmonary toxicity revealed the biocompatibility of the two nanoparticulate formulations with lung tissue. The efficacy of the prepared NPs to enhance the pulmonary absorption of peptides was evaluated after pulmonary administration to rats using a liquid micro-sprayer technique. Calcitonin-loaded GCS and GCS-TGA NPs resulted in a pronounced hypocalcemic effect for at least 12 and 24 h, and a corresponding pharmacological availability of 27 and 40%, respectively. These findings suggest that both GCS and its thiomer derivative are promising and safe carriers for pulmonary peptide delivery.

Carbopol-lectin conjugate coated liposomes for oral peptide delivery.

Within the current study, a delivery system based on a novel polymer-lectin conjugate (carbopol-lectin) was evaluated for the oral delivery of therapeutic peptides and proteins. It was demonstrated that covalent attachment of lectin to carbopol does neither decrease nor abolish the specific binding properties of lectin. Bioadhesion studies revealed that liposomes coated with carbopol lectin are more bioadhesive than liposomes coated with unmodified carbopol. Finally, the in vivo data suggest that carbopol-lectin conjugate coated liposomes are effective oral peptide delivery systems which are capable of increasing the pharmacological effect of orally administered calcitonin.

Oral protein delivery: a patent review of academic and industrial approaches.

Protein therapeutics are used in the treatment of a broad variety of diseases, however, usually they are not available as peroral formulations. Oral delivery systems of proteins including insulin, glucagon like peptide, calcitonin or parathyroid hormone are highly demanded by patients suffering from chronic diseases such as diabetes or osteoporosis. The need for oral protein formulations has been recognized by researchers of various scientific disciplines and a number of patents have been filed that deal with technologies capable of facilitating oral protein delivery. Within the current review, patents based on approaches such as particulate delivery systems, multifunctional polymers, enzyme inhibitors, permeation enhancers and ligand-specific binding and uptake are discussed. In addition, the technology platforms of several innovative drug delivery companies are highlighted.

Development and in vitro characterization of liposomes coated with thiolated poly(acrylic acid) for oral drug delivery.

Mucoadhesive drug delivery systems offer promising opportunities for oral drug delivery. The aim of this study was to investigate the feasibility of preparing liposomes that are coated with the multifunctional polymer poly(acrylic acid)-cysteine (PAA-Cys). Cationic multilamellar vesicles (MLV) as well as cationic submicron-sized liposomes (ssLip) were prepared and coated with PAA-Cys. Size, zeta potential, amount of free thiol groups, aggregation behavior, drug-loading, and drug release of these novel carriers were evaluated. A switch of the initial positive zeta potential to a negative value after coating indicated the successful coating procedure. In both size ranges, MLV and ssLip, the amount of free thiol groups was comparable to that in a PAA-Cys solution of the same concentration. Drug loading of the hydrophilic marker fluorescence-isothiocyanate 4 kDa (FD4) was higher in PAA-Cys liposomes in comparison to noncoated liposomes, but lower in comparison to liposomes coated with unmodified poly(acrylic acid) (PAA). Only a minor ssLip or no increase MLV of the drug-loading was observed when using carboxyfluorescein (CF). These effects were attributed to interactions between the markers and the poly(acrylates). Coating of liposomes with PAA-Cys and PAA did not influence the release profile of FD4 and CF, whereas the release profile was affected by the molecular mass of the marker and the liposome size. In conclusion, the feasibility of coating liposomes with PAA-Cys was demonstrated, and it could be shown that this novel carrier system fulfills the basic requirements for an intended use in oral drug delivery.

Modified chitosans for oral drug delivery.

The cationic polysaccharide chitosan has been extensively studied for oral drug delivery. In recent years, chemically modified chitosans developed in order to improve the properties of chitosan for oral drug delivery have gained increasing attention. Representatives of these novel polymers are trimethyl-chitosans, thiolated chitosans, carboxymethyl chitosan and derivatives, hydrophobic chitosans, chitosan succinate and phthalate, PEGylated chitosans and chitosan-enzyme inhibitor conjugates. Besides their use for oral delivery of therapeutic peptides and proteins, they have recently been evaluated regarding their potential for the delivery of other substance classes, including genes and efflux pump substrates. Within the current review, various modified chitosan derivatives, their properties and synthesis are discussed.

Chitosan-aprotinin coated liposomes for oral peptide delivery: Development, characterisation and in vivo evaluation.

In order to improve the systemic uptake of therapeutic peptides/proteins after oral administration, the polymer-protease inhibitor conjugate chitosan-aprotinin was synthesised and polyelectrolyte complexes between negatively charged multilamellar vesicles (MLV) and positively charged chitosan-aprotinin conjugate were prepared. It could be demonstrated that chitosan-aprotinin was capable of significantly inhibiting Trypsin in vitro in concentrations of 0.05% and 0.1%, whereas no inhibition was observed in the presence of 0.1% chitosan. The size range of the prepared MLV was between 3 and 4.5microm and the initially negative zeta potential (ca. -90mV) of the core liposomes switched to a positive value after polymer coating (ca. +40mV). Confocal laser microscopy studies showed comparable mucoadhesive properties of chitosan-aprotinin coated MLV and chitosan coated MLV. In comparison to calcitonin in solution, the area above the blood calcium concentration-time curve (AAC) after oral administration of calcitonin loaded chitosan coated MLV to rats increased around 11-fold, and around 15-fold in the case of calcitonin loaded chitosan-aprotinin coated MLV. Data gained in the current study are believed to contribute to the development of novel polymer-protease inhibitor based delivery systems.

Thiolated chitosans: useful excipients for oral drug delivery.

To improve the bioavailability of orally administered drugs, formulations based on polymers are of great interest for pharmaceutical technologists. Thiolated chitosans are multifunctional polymers that exhibit improved mucoadhesive, cohesive and permeation-enhancing as well as efflux-pump-inhibitory properties. They can be synthesized by derivatization of the primary amino groups of chitosan with coupling reagents bearing thiol functions. Various data gained in-vitro as well as in-vivo studies clearly demonstrate the potential of thiolated chitosans for oral drug delivery. Within the current review, the synthesis and characterization of thiolated chitosans so far developed is summarized. Features of thiolated chitosans important for oral drug delivery are discussed as well. Moreover, different formulation approaches, such as matrix tablets and micro-/nanoparticles, as well as the applicability of thiolated chitosans for the oral delivery of various substance classes including peptides and efflux pump substrates, are highlighted.

Effect of a thiolated polymer on oral paclitaxel absorption and tumor growth in rats.

The anticancer agent paclitaxel is currently commercially available only as an infusion due to its low oral bioavailability. An oral formulation would be highly beneficial for patients. Besides the low solubility, the main reason for the limited oral bioavailability of paclitaxel is that it is a substrate of the efflux pump P-glycoprotein (P-gp). Recently, it has been demonstrated that P-gp can be inhibited by thiolated polymers. In this study, an oral paclitaxel formulation based on thiolated polycarbophil was evaluated in vivo in wild-type rats and in mammary cancer-induced rats. The paclitaxel plasma level after a single administration of paclitaxel was observed for 12 h in healthy rats. Moreover, cancer-induced rats were treated weekly for 5 weeks with the novel formulation. It was demonstrated that (1) co-administration of thiolated polycarbophil significantly improved paclitaxel plasma levels, (2) a more constant pharmacokinetic profile could be achieved and (3) the tumor growth was reduced. These effects can most likely be attributed to P-gp inhibition. According to the achieved results, thiolated polymers are believed to be interesting tools for the delivery of P-gp substrates such as paclitaxel.

New-generation efflux pump inhibitors.

The development of novel efflux pump inhibitors is an emerging and challenging research field. Besides the use of such excipients in cancer therapy, efflux pump inhibitors are gaining increasing interest with regards to drug delivery. In particular, inhibition of efflux pumps located in the intestine and the blood-brain barrier offers promising prospects. Nowadays, third-generation inhibitors, such as elacridar, zosuquidar, laniquidar, OC144-093 and tariquidar, have been evaluated in clinical trials. Apart from these small, molecular inhibitors, which will be discussed within the current review, a focus has been set on polymeric or polymer-based inhibitors, including poly(ethylene glycol) and derivatives, poloxamers and thiomers.

Natural and synthetic polymers as inhibitors of drug efflux pumps.

Inhibition of efflux pumps is an emerging approach in cancer therapy and drug delivery. Since it has been discovered that polymeric pharmaceutical excipients such as Tweens or Pluronics can inhibit efflux pumps, various other polymers have been investigated regarding their potential efflux pump inhibitory activity. Among them are polysaccharides, polyethylene glycols and derivatives, amphiphilic block copolymers, dendrimers and thiolated polymers. In the current review article, natural and synthetic polymers that are capable of inhibiting efflux pumps as well as their application in cancer therapy and drug delivery are discussed.

Polymeric and low molecular mass efflux pump inhibitors for oral drug delivery.

Transmembrane located transporter proteins can be responsible for the low bioavailability of orally administered drugs. Drug delivery systems which can overcome this barrier caused by efflux pumps are therefore highly on demand. Within the current review, intestinal located efflux transporters, methods to identify efflux pump substrates and inhibitors as well as strategies to minimize efflux pump mediated transport of drugs are discussed. Methods include in silico screening, transport and accumulation studies and monitoring of the ATPase activity. An emphasis has been placed on efflux pump inhibitors including low molecular mass inhibitors such as cyclosporine, PSC833 or KR30031 and polymeric inhibitors such as myrj, thiomers and cremophor EL. Also formulation approaches to circumvent intestinal segments with high efflux pump expression are briefly addressed.

Oral peptide delivery: in-vitro evaluation of thiolated alginate/poly(acrylic acid) microparticles.

The purpose of this study was to develop an oral thiomer-based microparticulate delivery system for insulin by ionic gelation. The microparticulate matrix consisted of either poly(acrylic acid)-cysteine (PAA-Cys) and alginate-cysteine (Alg-Cys) or the corresponding unmodified polymers (PAA, Alg). Two different viscosities of alginates were provided for the study, low and medium. Three different types of microparticles were prepared via ionic gelation with calcium (Alg, AlgPAA and AlgPAA-Cys) and their different properties evaluated in-vitro (particle size and shape, drug loading and release profile, swelling and stability). The mean particle size of all formulations ranged from 400 to 600 microm, revealing the lowest for thiolated microparticles. SEM micrographs showed different morphological profiles for the three different types of microparticles. Encapsulation efficiency of insulin increased within the following rank order: Alg (15%) < AlgPAA (40%) < AlgPAA-Cys (65%). Alginate and AlgPAA microparticles displayed a burst release after 30 min, whereas the thiolated particles achieved a controlled release of insulin over 3 h. The swelling ratio was pH dependent: in simulated intestinal fluid microparticles exhibited a much higher water uptake compared with simulated gastric fluid. Due to the formation of intraparticulate disulfide bonds during the preparation process, thiolated particles revealed a higher stability. It was also observed that the viscosity of the two alginates used had no influence on the properties of the particles. According to these results AlgPAA-Cys microparticles obtained by ionic gelation and stabilized via disulfide bonds might be an alternative tool for the oral administration of therapeutic peptides.

Design and evaluation of a chitosan-aprotinin conjugate for the peroral delivery of therapeutic peptides and proteins susceptible to enzymatic degradation.

One main barrier for the peroral administration of therapeutic peptides and proteins is the enzymatic barrier, that is mediated by luminally secreted and membrane bound proteolytic enzymes. It was the aim of the study to synthesise, characterise and evaluate a novel polymer-inhibitor conjugate in order to improve the bioavailability of orally-administered peptides and proteins. The trypsin/chymotrypsin inhibitor aprotinin was covalently bound to chitosan. The percentage of the inhibitor in the polymer-inhibitor conjugate (m/m) was determined to be between 1.11 +/- 0.36 and 1.92 +/- 0.05%. In vitro enzyme assays clearly demonstrated the potential of the novel conjugate to inhibit trypsin and chymotrypsin. Moreover, studies in rats were performed to evaluate the efficacy of the conjugate in vivo. Eight hours after oral administration of tablets containing insulin and the novel chitosan-aprotinin conjugate, the mean blood glucose level decreased to 84 +/- 6%. In contrast, the mean blood glucose level in the control group increased to 121 +/- 8% of the initial measured blood glucose level. In conclusion it was demonstrated that chitosan-aprotinin conjugate represents a novel and promising tool for the oral administration of therapeutic peptides and proteins susceptible to enzymatic degradation caused by trypsin and chymotrypsin.

Synthesis and in vitro evaluation of thiolated hyaluronic acid for mucoadhesive drug delivery.

It was the aim of this study to synthesize and characterize a novel hyaluronic acid-cysteine ethyl ester (HA-Cys) conjugate providing improved mucoadhesive properties and a significantly lowered biodegradation rate. Mediated by carbodiimide and N-hydroxysuccinimide, L-cysteine ethyl ester hydrochloride was covalently attached to hyaluronic acid (HA, hyaluronan) via the formation of an amide bond. The adhesive properties of HA-Cys conjugates were evaluated in vitro on a freshly excised porcine mucosa via the rotating cylinder method. The cohesive properties of the resulting conjugates were evaluated by oxidation experiments. Biodegradability studies were carried out by viscosity measurements and spectrophotometric assays. Release studies were performed with fluorescein isothiocyanate-dextrans (FD) as model compounds. The obtained conjugate displayed 201.3+/-18.7 micromol immobilized free thiol groups and 85.7+/-22.3 micromol disulfide bonds per gram polymer. Results from the rotating cylinder method showed more than 6.5-fold increase in the adhesion time of HA-Cys versus unmodified HA. In aqueous solutions, the obtained conjugate demonstrated improved cohesive properties. The hydrolysis degree of HA-Cys was lower compared with the corresponding unmodified HA in the framework of viscosity experiments. In addition, the cross-linking process via disulfide bonds additionally reduced the rate of degradation of the new derivative. Cumulative release studies out of matrix tablets comprising HA-Cys and the model compound FD demonstrated a sustained drug release for more than 12h due to in situ formation of inter- and intramolecular disulfide bonds in the thiomer matrix. According to the results of the present study, this novel thiolated polymer seems to represent a promising multifunctional excipient for the development of various drug delivery systems.

Evaluation of in vitro enzymatic degradation of various thiomers and cross-linked thiomers.

The aim of this study was to examine the biodegradability of thiomers and cross-linked thiomers in comparison with unmodified polymers. Disulfide-cross-linked conjugates were prepared by air oxidation at room temperature. Thiomers were investigated by viscosity measurements and spectrophotometric assays. The influence of different factors on the hydrolysis rate, such as the degree of modification of thiomers, structure of the conjugates, pH value of the reaction medium, and the impact of the process of cross-linking were evaluated. Due to the modification, thiolated chitosans degraded 12.9-24.7% less than unmodified chitosan in the framework of viscosity measurements. In addition, the hydrolysis degree of thiolated alginates and modified carboxymethylcelluloses was 25.6-32.4% and 18.4-27.0% lower, respectively, in comparison to the corresponding unmodified polymers. Conjugates with higher coupling rate of thiol groups were degraded even more slowly. Moreover, the cross-linking process via disulfide bonds additionally reduced the rate of thiomer degradation. The range of degradation rates achieved in vitro could be modified by alterations of the contents of thiol and disulfide groups, as well as by suitable design of the polymer structure and ligands used. These results represent helpful basic information for the development of mucoadhesive drug delivery systems, implantable delivery systems and tissue engineering constructs.

Oral gene delivery: Strategies to improve stability of pDNA towards intestinal digestion.

PURPOSE: Gastrointestinal (GI) nucleases are responsible for a rapid presystemic degradation of orally administered transgenes. Within the current study, the activity of these degrading enzymes as well as the effect of various nuclease inhibitors on the degradation process were evaluated in order to assess their potential as auxiliary agents in oral gene delivery. METHODS: Digestion assays of pDNA with DNaseI and in GI juices were performed in absence and presence of inhibitors. Consequently, a chitosan conjugate with covalently bound ethylendiaminetetraacetic acid disodium salt dihydrat (EDTA) was synthesized and its nuclease inhibitory properties were evaluated. RESULTS: Small intestinal juice was shown to possess a nuclease activity per millilitre corresponding to 0.02 Kunitz units of DNaseI. Inhibition studies revealed that inhibitory activity followed the ranking: EDTA > sodium dodecyl sulfate (SDS) > aurintricarboxylic acid (ATA) > poly (acrylic acid) > cysteine. The chitosan-EDTA conjugate offered good nuclease inhibiting properties. CONCLUSION: This study determined the nuclease activity of native porcine small intestinal juice as well as enterocytes homogenate. Moreover, several promising strategies to overcome this enzymatic barrier were identified.

Degradation of teriparatide by gastro-intestinal proteolytic enzymes.

Teriparatide, a recombinant parathyroid hormone (1-34) is the first approved agent for the treatment of osteoporosis that stimulates new bone formation. Currently, the drug is administered daily by s.c. injection. Because of the obvious advantages of oral teriparatide administration, the development of such a delivery system would be of great benefit. Besides other barriers, the enzymatic barrier caused by gastro-intestinal (GI) proteolytic enzymes is believed to be responsible for negligible teriparatide oral bioavailability. It was therefore the aim of the study to evaluate the stability of teriparatide towards a variety of GI proteases under physiological conditions. Results indicate that teriparatide is entirely degraded by trypsin, chymotrypsin and pepsin within 5 min. In contrast, even after 3 h of incubation with elastase about 85% of undegraded teriparatide could still be detected. Within an incubation period of 3 h in the presence of rat small intestinal mucosa, approximately half of the teriparatide was degraded. Experiments with isolated aminopeptidase N demonstrated that this membrane bound peptidase is primarily involved in the degradation process. Results gained from and recorded in this study provide a precise characterisation of the enzymatic barrier for oral teriparatide administration and represents a prerequisite for the development of oral teriparatide delivery systems.

The potential of cystine-knot microproteins as novel pharmacophoric scaffolds in oral peptide drug delivery.

Within this study, the potential of three clinically relevant microproteins (SE-AG-AZ, SE-EM and SE-EP) with cystine-knot architecture as pharmacophoric scaffolds for oral peptide delivery was investigated. Cystine-knot microproteins (CKM) were analysed regarding their stability towards the most important gastrointestinal secreted and membrane bound proteases in physiological concentrations. In addition, their permeation behaviour through freshly excised rat intestinal mucosa as well as important parameters such as aggregation behaviour, stability in rat plasma and isoelectric point were evaluated and compared to the properties of the model peptide drugs bacitracin and insulin. Aggregation studies indicate that under physiological conditions between 25 and 70% of the CKMs occur as monomers, whereas the rest forms di- and trimers. Pepsin and elastase cause no or only minor degradation to CKMs, whereas trypsin and chymotrypsin degrade CKMs extensively. Removing the theoretical chymotrypsin cleavage site from a CKM, however, led to stabilization towards this protease. Two of the three evaluated CKMs are stable against membrane bound proteases. P(app) values were determined to be 5.96 +/- 0.98 x 10(-6) and 6.63 +/- 0.47 x 10(-6) cm/s. In conclusion, this study indicates that CKM are promising novel pharmacophoric scaffolds for oral peptide delivery.