Atomization of denatured whey proteins as a novel and simple way to improve oral drug delivery system properties.

In the sphere of drug delivery, denatured whey protein (DWP) has in recent times gained press. However, to date, no scalable and affordable dosage form has been developed. The objective of our study was to evaluate the potential use of spray-dried DWP as a ready to use excipient for oral drug delivery. Therefore, solid state, FTIR spectra and wettability were studied. Dissolution, mucoadhesion and the effect on paracellular permeability were also evaluated. The spray-dried DWP particles were spherical with 4µm mean diameter. Further, relative to native WP, the spray-dried DWP particles bore reduced wettability, and their structure was characterized by the exposure of a high amount of free thiol and by the formation of intermolecular ß-sheets. The DWP powders were mucoadhesive, enzymatic inhibitors, biocompatible and they induced the opening of tight junctions. Our study shows great potential for the use of spray-drying as a technique to modify the dissolution rate of drugs and enhance the oral bioavailability of molecules. That is, the use of spray drying as a single step ready to use DWP excipient.

Denatured Whey Protein Powder as a New Matrix Excipient: Design and Evaluation of Mucoadhesive Tablets for Sustained Drug Release Applications.

PURPOSE: In earlier study, we proposed denatured whey protein (DWP) powder obtained by atomization as a new excipient to promote oral drug delivery. In this work, we evaluate the possibility to formulate tablets based on DWP powders and to characterize their role as a matrix mucoadhesive excipient. METHODS: Tablets containing increased amount of DWP (10 to 30%) were produced by direct compression after mixing with theophylline, microcrystalline cellulose, Aerosil® and magnesium stearate. Dissolution behaviors of obtained tablets were evaluated in different USP buffers (pH 1.2, 4.5 and 6.8) and in simulated gastric and intestinal fluids and mechanisms analyzed by multiple mathematical models. Swelling, erosion and mucoadhesion were also evaluated. Finally, release and absorption were studied in the artificial digestive system (TIM 1). RESULTS: Tablets based on DWP and containing 300 mg of theophylline were obtained by direct compression. These tablets exhibited controlled release driven by diffusion starting from 15% DWP content whatever the pH studied. They also showed a great extent of swelling and water uptake while matrix weight loss was limited. Addition of enzymes accelerated drug release which became governed by erosion according to Peppas model. CONCLUSIONS: The present study shows that DWP powders can be successfully used as a pharmaceutical excipient, and in particular as a matrix mucoadhesive controlled release tablets.

Whey protein mucoadhesive properties for oral drug delivery: Mucin-whey protein interaction and mucoadhesive bond strength.

Whey protein is a natural polymer recently used as an excipient in buccoadhesive tablets but its mucoadhesive properties were barely studied. In this work, we characterize mucoadhesion of whey protein in order to determine the mechanisms and optimal conditions for use as excipient in oral drug delivery. Thus, native and denatured whey protein (NWP and DWP) were investigated and the effect of concentration and pH were also studied. Many methods of characterization were selected to allow the study of chemical and physical interactions with mucin and then the results were bound with an ex vivo experiments. Turbidity of WP-mucin mixture increased at acidic pH 1.2 till 4.5 indicating interaction with mucin but not at pH 6.8. No interaction with mucin was also found by ITC method at pH 6.8 for native and denatured whey protein used at 1% (w/w). Forces of bioadhesion evaluated by viscosity measurements were the best for high concentrated (10.8%) DWP solutions at pH 6.8 and were low at pH 1.2 for NWP and DWP solutions. Addition of chemical blockers indicated that hydrogen bondings and disulfide bridges were the main mechanisms of interactions with mucin. Reticulation of DWP with calcium ions to obtain microparticles (MP) did not influence the ability of interaction with mucin as shown by FTIR analysis. These results correlated with ex vivo study on rat tissue demonstrating important adhesion (75%) of WP MP on the intestine and null on the stomach after 2h of deposit.

Liquid anti-solvent recrystallization to enhance dissolution of CRS 74, a new antiretroviral drug.

This study concerns a new compound named CRS 74 which has the property of inhibiting Human Immunodeficiency Virus (HIV) protease, an essential enzyme involved in HIV replication process. It is proved in this study that the original CRS 74 exhibits poor aqueous solubility and a very low dissolution rate, which can influence its bioavailability and clinical response. In an attempt to improve the dissolution rate, CRS 74 was recrystallized by liquid anti-solvent (LAS) crystallization. Ethanol was chosen as solvent and water as the anti-solvent. Recrystallized solids were compared with the original drug crystals in terms of physical and dissolution properties. Recrystallization without additives did not modify the CRS 74 dissolution profile compared to the original drug. CRS 74 was then recrystallized using different additives to optimize the process and formulate physicochemical properties. Steric stabilizer in organic phase ensured size-controlling effect, whereas electrostatic stabilizer in aqueous phase decreased particle agglomeration. Cationic additives avoided drug adsorption onto stainless steel T-mixer. In general, additive improved drug dissolution rate due to improvement of wetting properties by specific interactions between the drug and the additives, and ensured continuous production of CRS 74 by electrostatic repulsion.

Whey protein and alginate hydrogel microparticles for insulin intestinal absorption: evaluation of permeability enhancement properties on Caco-2 cells.

The evaluation of encapsulated insulin intestinal absorption enhancement was investigated by in vitro methods. Insulin-loaded microparticles (INS-MP) made of whey protein (WP) and alginate (ALG) were prepared by a cold gelation technique. Effect of INS encapsulation toward trypsin and chymotrypsin degradation was performed. Permeability studies using in vitro (Caco-2 cells) experiments were conducted. INS was partially protected by encapsulation toward enzymatic degradation. Moreover INS transport experiments showed that WP and, in lesser extent, ALG were able to enhance INS absorption both as MP and as polymeric solutions by opening the tight junctions. These experiments reinforced the interest of encapsulation in WP/ALG hydrogel combination.

Efficacy of mucoadhesive hydrogel microparticles of whey protein and alginate for oral insulin delivery.

PURPOSE: To evaluate the efficacy of mucoadhesive insulin-loaded whey protein (WP) /alginate (ALG) microparticles (MP) for oral insulin administration. METHODS: Insulin-loaded microparticles (ins-MP) made of whey protein and alginate were prepared by a cold gelation technique and an adsorption method, without adjunction of organic solvent in order to develop a biocompatible vehicle for oral administration of insulin. In vitro characterization, evaluations of ins-MP in excised intestinal tissues and hypoglycaemic effects after intestinal administration in healthy rats were performed RESULTS: The release properties and swelling behaviors, investigated in different pH buffers, demonstrated a release based on diffusion mechanism following matrix swelling. Mucoadhesion studies in rabbits and insulin transport experiments with excised intestinal rat tissues revealed that encapsulation in microparticles with mucoadhesive properties promotes insulin absorption across duodenal membranes and bioactivity protection. In vivo experiments reinforced the interest of encapsulation in whey protein/alginate combination. Confocal microscopic observations associated with blood glucose levels bring to light duodenal absorption of insulin biologically active following in vivo administration. CONCLUSIONS: Insulin-loaded WP/ALG MP with high quantities of drug entrapped, in vitro matrix swelling and protective effect as well as excellent mucohadesive properties was developped. Improvement of intestinal delivery of insulin and increased in bioavailability were recorded.

Development and characterization of coated-microparticles based on whey protein/alginate using the Encapsulator device.

The aim of this study is to prepare whey protein (WP)-based microparticles (MP) using the Encapsulator(®) device. The viscosity dependence of the extrusion device required to mix WP with a food-grade and less viscous polymer. Mixed WP/ALG MP were obtained with the optimized WP/alginate (ALG) ratio (62/38). These particles were further coated with WP or ALG using non-traumatic and solvent-free coating process developed in this study. Size and morphology of coated and uncoated MP were determined. Then, swelling and degradation (WP release) of formulations were investigated in pH 1.2 and 7.5 buffers and in simulated gastric and intestinal fluids (SGF, SIF) and compared to pure ALG and pure WP particle behaviours. At pH 1.2, pure ALG shrank and pure WP swelled, whereas the sizes of mixed WP/ALG matrix were stable. In SGF, WP/ALG MP resisted to pepsin degradation compare to pure WP particles due to ALG shrinkage which limited pepsin diffusion within particles. Coating addition with WP or ALG slowed down pepsin degradation. At pH 7.5, WP/ALG particles were rapidly degraded due to ALG sensitivity but the addition of a WP coating limited effectively the swelling and the degradation of MP. In SIF, pancreatin accelerated MP degradation but ALG-coated MP exhibited interesting robustness. These results confirmed the interest and the feasibility to produce coated WP-based MP which could be a potential orally controlled release drug delivery system.

Development and in vitro characterization of insulin loaded whey protein and alginate microparticles.

Insulin was encapsulated into microparticles (MP) made of denaturized whey proteins (WP) and alginate (ALG) using an extrusion/cold gelation process with calcium ions. High encapsulation efficiency of 85% was obtained. Influence of insulin on polymeric viscosity and on microparticle behavior was evaluated. Insulin seemed to interact with WP chains by non covalent binding and steric hindrance. This influence was balanced by ALG addition. Nevertheless, insulin was released rapidly by diffusion at both acidic and intestinal dissolution media. Despite this fast in vitro release, WP/ALG MP showed an important enzymatic inhibition effect on trypsin and alpha-chymotrypsin. Thus, WP/ALG MP contributed to an effective insulin protection towards enzymatic degradation. The aforementioned results suggested that WP based microparticles are a promising carrier for improving oral delivery of insulin.

Enteric polymers as acidifiers for the pH-independent sustained delivery of a weakly basic drug salt from coated pellets.

Weakly basic drugs and their salts exhibit a decrease in aqueous solubility at higher pH, which can result in pH-dependent or even incomplete release of these drugs from extended release formulations. The objective of this study was to evaluate strategies to set-off the very strong pH-dependent solubility (solubility: 80 mg/ml at pH 2 and 0.02 mg/ml at pH 7.5, factor 4000) of a mesylate salt of weakly basic model drug (pK(a) 6.5), in order to obtain pH-independent extended drug release. Three approaches for pH-independent release were investigated: (1) organic acid addition in the core, (2) enteric polymer addition to the extended release coating and (3) an enteric polymer subcoating below the extended release coating. The layering of aspartic acid onto drug cores as well as the coating of drug cores with an ethylcellulose/Eudragit L (enteric polymer) blend were not effective to avoid the formation of the free base at pH 7.5 and thus failed to significantly improve the completeness of the release compared to standard ethylcellulose/hydroxypropyl cellulose (EC/HPC)-coated drug pellets. Interestingly, the incorporation of an enteric polymer layer underneath the EC/HPC coating decreased the free base formation at pH 7.5 and thus resulted in a more complete release of up to 90% of the drug loading over 18 h. The release enhancing effect was attributed to an extended acidification through the enteric polymer layer. Flexible release patterns with approximately pH-independent characteristics were successfully achieved.

Selection of the most suitable dissolution method for an extended release formulation based on IVIVC level A obtained on cynomolgus monkey.

OBJECTIVE: The purpose of this study is primarily to identify the most suitable in vitro dissolution method(s) for their ability to predict the in vivo performance of extended release prototype tablet formulations designed for a new chemical entity, Biopharmaceutic Classification System class II drug, weak base, based on the data collected in cynomolgus monkey. MATERIALS AND METHODS: Different types of buffer at different pH were selected as dissolution medium resulting in a broad variety of release patterns (slow to fast). The in vivo and in vitro data were put in relation. RESULTS: As a consequence of the discrimination between both tested formulations, the in vitro-in vivo correlation (IVIVC) qualities and shapes changed significantly. The obtained level A showed that the simple HCl medium was superior to biorelevant media and media containing surfactant when investigating IVIVCs in cynomolgus monkey. In addition, the results of dissolution in HCl suggested rather a diffusion mechanism of the extended release matrix formulation as the main factor of the release. CONCLUSION: Adjusting dissolution testing conditions to match the behavior of the formulations in vitro with that in vivo by taking into account the properties of the drug and the formulation is a straightforward and useful approach in identifying a predictive method in the development of the IVIVC. These investigations will definitely help by derisking of new formulations as well as by rating changes in existing formulations with regard to their impact on bioavailability before entry into human.

Coated whey protein/alginate microparticles as oral controlled delivery systems for probiotic yeast.

Viable Saccharomyces boulardii, used as a biotherapeutic agent, was encapsulated in food-grade whey protein isolate (WP) and alginate (ALG) microparticles, in order to protect and vehicle them in gastrointestinal environment. Yeast-loaded microparticles with a WP/ALG ratio of 62/38 were produced with high encapsulation efficiency (95%) using an extrusion/cold gelation method and coated with ALG or WP by a simple immersion method. Swelling, yeast survival, WP loss and yeast release in simulated gastric and intestinal fluids (SGF and SIF, pH 1.2 and 7.5) with and without their respective digestive enzymes (pepsin and pancreatin) were investigated. In SGF, ALG network shrinkage limited enzyme diffusion into the WP/ALG matrix. Coated and uncoated WP/ALG microparticles were resistant in SGF even with pepsin. Survival of yeast cells in microparticles was 40% compared to 10% for free yeast cells and was improved to 60% by coating. In SIF, yeast cell release followed coated microparticle swelling with a desirable delay. Coated WP/ALG microparticles appear to have potential as oral delivery systems for Saccharomyces boulardii or as encapsulation means for probiotic cells in pharmaceutical or food processing applications.

Investigation of coated whey protein/alginate beads as sustained release dosage form in simulated gastrointestinal environment.

AIM: The biopharmaceutical behavior of new formulations based on both food-grade polymers, whey protein (WP) and alginate (ALG) was studied using different in vitro methods. The Biopharmaceutical Classification System (BCS) class I drug Theophylline was chosen as drug model. METHOD: Drug release was studied (i) at pH 1.2 (2 hours) followed by pH 7.5, and in simulated gastric fluid (SGF; 2 hours) followed by simulated intestinal fluid (SIF) using the paddle method and (ii) in an artificial digestive system. RESULTS: Freeze-dried mixed WP/ALG (62/38) beads were coated with WP or ALG with encapsulation efficiency 34.9% and 18.3%, respectively. At pH 1.2, coated beads exhibited gastroresistant properties (< 10% of drug released after 2 hours) followed at pH 7.5 by a sustained release behavior (< 60% of drug released at 24 hours) controlled by an erosion mechanism. In SGF, despite enzyme hydrolysis, drug release was still controlled due to ALG shrinkage. After transfer in SIF, formulations were completely degraded in less than 2 h with total drug release. In an artificial digestive system, coated beads appeared gastroresistant, intestinal part sustained drug release was controlled by erosion. CONCLUSION: Combination of in vitro methods allowed prediction of the in vivo potentialities of WP- and ALG- coated WP/ALG beads as oral sustained release systems.

Immunoadsorption of alloantibodies onto erythroid membrane antigens encapsulated into polymeric microparticles.

PURPOSE: Classical immunoadsorbents used for the removal of deleterious molecules in blood such as auto-antibodies are prepared by covalent coupling of antigens onto previously chemically activated supports. Such a chemical treatment may induce a potential toxicity which can be reduced if new immunoadsorbents are prepared by encapsulating erythrocytes-ghosts carrying antigens inside polymeric and porous microparticles. MATERIALS AND METHODS: Erythrocyte-ghosts obtained by hemolysis in hypotonic buffer were encapsulated into ethylcellulose microparticles by w/o/w emulsification. The porosity of microparticles was evaluated by mercury porosimetry. The adsorption ability of encapsulated antigens was evaluated by hemagglutination after contact in tube or elution in column with polyclonal antibody solutions or human blood-plasma. RESULTS: The encapsulation process did not significantly alter the evaluated antigens since a significant decrease in anti-A (from 256 to 4) as well as anti-Kell (from 64 to 2) antibody titer has been observed in column after eight chromatographic runs (2 h). The higher the ghost concentration (total protein content of 6 mg/ml), the higher the adsorption capacity. CONCLUSION: Encapsulation, currently used for drug delivery purposes, may consequently also be applied to the design of new immunoadsorbents as biomaterials.

Oral bioavailability of a low molecular weight heparin using a polymeric delivery system.

Low molecular weight heparins (LMWHs) are the standards of anticoagulant for the prevention of deep vein thrombosis (DVT) in patients undergoing arthroplasty and abdominal surgery. However, LMWHs are so far only administered by parenteral route. Thus, they are usually replaced by oral warfarin for outpatient therapy. Since warfarin has a slow onset and high incidence of drug-drug interaction, there is a great need for the development of an oral LMWH formulation. LMWH (tinzaparin)-loaded nanoparticles prepared with a blend of a polyester and a polycationic polymethacrylate by the double emulsion method were administered orally in fasted rabbits. The plasma tinzaparin concentration was measured by a chromogenic anti-factor Xa assay. After oral administration of two doses of tinzaparin-loaded nanoparticles (200 and 600 anti-Xa U/kg), the oral absorption was observed between 4 and 10 or 12 h, with a delayed onset of action ranging from 3 to 4 h. Mean absolute bioavailabilities were 51% and 59% for the two tested doses. We now report that the encapsulation of tinzaparin into nanoparticles is likely to contribute to its oral efficacy with an anticoagulant effect prolonged up to 8 h.

Anticoagulant activity of heparin following oral administration of heparin-loaded microparticles in rabbits.

Heparin-loaded microparticles, prepared according to the double emulsion method with biodegradable (PCL and PLGA) and nonbiodegradable (Eudragit RS and RL) polymers used alone or in combination, with or without gelatin, were characterized in vitro and in vivo after oral administration in rabbits. The entrapment efficiency and the release of heparin were determined by a colorimetric method with Azure II. The antifactor Xa activity of heparin released in vitro after 24 h was assessed. After oral administration of heparin-loaded microparticles in rabbits, the time course of modification of the clotting time estimated by the activated partial thromboplastin time (APTT) was followed over 24 h. Microparticles with a size ranging from 80 to 280 microm were obtained. Heparin entrapment efficiency as well as heparin release depended on both the nature of the polymers and the presence of gelatin. The Eudragit polymers increased the drug loading but slowed down the heparin release, whereas gelatin accelerated the release. No change in clotting time was observed after oral administration of gelatin microparticles. Heparin-loaded microparticles prepared with blends of PLGA and Eudragit displayed a prolonged duration of action characterized by a twofold increase in APTT and a enhancement of absorption. This study demonstrated the feasibility of encapsulating heparin within polymeric particles, and the significant increase in APTT confirmed the oral absorption of heparin released from polymeric microparticles.