Enzyme structure and function protection from gastrointestinal degradation using enteric coatings

https://doi.org/10.1016/j.ijbiomac.2018.07.143Get rights and content

Abstract

Bovine Serum Albumin (BSA) and Horseradish Peroxidase (HRP) have been encapsulated within microparticulated matrices composed of Eudragit RS100 by the water-in-oil-in-water double emulsion solvent evaporation method. Good encapsulation efficiencies were achieved for BSA and HRP, 88.4 and 95.8%, respectively. The stability of the loaded proteins was confirmed by using circular dichroism and fluorescence. The gastroresistance of the protein-loaded microparticles was evaluated under simulated gastric conditions demonstrating the preservation of the structural integrity of the proteins loaded inside the particles. The enzymatic activity of HRP after being released from the enteric microparticles was evaluated by using the peroxidase substrate, revealing that the released enzyme preserved its 100% function. The high drug loadings achieved, reduced cytotoxicity and efficient gastric protection point out towards the potential use of those carriers as oral delivery vectors of therapeutic proteins offering a more controlled targeted release in specific sites of the intestine and an enhanced gastrointestinal absorption.

Introduction

In the last decades the development of recombinant DNA technology and gene engineering have facilitated the approval of >239 protein-based therapeutics by the FDA with nearly 380-marketed pharmaceutical products which active principle includes monoclonal antibodies, vaccines, hormones, cytokines or enzymes [1]. Exogenous enzymes are therapeutically used in replacement therapy (i.e., mainly in the treatment of lysosomal storage disorders), in cancer treatment (i.e., for depleting essential aminoacids up-regulated in cancer cells), in the prevention of thrombi (i.e., as plasminogen activators), in hyperuricemia (i.e., to counteract the accumulation of serum uric acid) and in cystic fibrosis (i.e., to hydrolyze extracellular DNA) among several other diseases. Numerous examples of FDA-approved therapeutic enzymes are reviewed elsewhere [2].

Those therapeutic enzymes are generally applied intravenously, intramuscularly or subcutaneously; but in some applications, such as antimicrobial prevention, in the treatment of lactose and sucrose intolerance and in pancreatic insufficiency, therapeutic enzymes are orally administered [3]. Compared to parenteral routes, this route of administration has the great advantage of improving patient compliance and reducing administration costs. In addition, several randomized clinical trials have demonstrated the benefits of using orally administered enzymes in other pathologies. For instance, synergetic effects have been demonstrated by Jayachandran et al. [4] when showing that the combined administration of diclofenac sodium and several enzymes (i.e., bromelain, trypsin, rutoside trihydrate) produced a better outcome in osteoarthritis-associated pain management than the administration of just the enzymes or just the drug, after performing a randomized clinical trial with 30 patients. In another randomized, double-blind, placebo-controlled clinical trial with 150 patients the same enzymatic combination showed similar effects than sodium diclofenac in relieving pain associated to knee osteoarthritis but with fewer side effects than the ones caused by this nonsteroidal anti-inflammatory drug alone [5]. Also, the results of a double-blind, placebo controlled, randomized Phase 1 cross-over study performed on 30 healthy volunteers with hyperoxaluria induced by the ingestion of a high oxalate diet, demonstrated that a low calcium diet treated with oxalate degrading enzymes administered orally produced a significant reduction (>10%) in urinary oxalate excretion compared to placebo in 60% of the volunteers [6].

Enzymes are also consumed orally as dietary supplements by patients with different conditions and by consumers that simply want to improve their general health despite the absence of robust clinical trial data that corroborate their demonstrated benefits [7].When orally administered, to prolong the gastrointestinal stability of therapeutic enzymes, those have been: i) covalently coupled to different polymers (e.g., poly ethylene glycol, PEG), ii) genetically modified to avoid unfolding and enzymatic hydrolysis under gastric conditions, iii) protected with gastric proton pump inhibitors or iv) protected within enteric coatings [3, 8]. In some cases, instead of encapsulating the enzyme, bacteria resistant to the gastrointestinal passage that endogenously produce the enzyme of interest have been used [9]; however the microencapsulation with enteric-resistant materials presents clear advantages compared to the use of live bacteria including an easier storage, shelf life and handling and a more controlled targeted release in specific sites of the intestine. The state of ionization of the aminoacids composing the enzyme is pH dependent and consequently under acidic conditions the conformation and functionality of several enzymes can be hindered [10, 11].

Frequent enteric coatings used are of natural origin (e.g., sodium alginate, chitosan, etc.) or synthetic (e.g., cellulose acetate derivatives, methyl acrylate-methacrylic acid copolymers, etc.). The medical outcome improves after protecting the therapeutic enzyme with the enteric coating. For example, Mas et al. [12] reported the results of a multicenter randomized blinded positive controlled trial for canine exocrine pancreatic insufficiency showing that dogs receiving a methacrylate-based enteric-coating on pellets of pancreatic enzymes responded better to therapy (i.e., higher body condition score and lower clinical disease severity score) than those given an otherwise identical uncoated product.

Numerous therapeutic enzymes have been encapsulated within those synthetic enteric polymers. β-Galactosidase (lactase) has been protected against gastric degradation preserving its hydrolytic activity against lactose by its encapsulation within polylactic acid nanoparticles (100–200 nm) which were subsequently filled within hard gelatin capsules coated with hydroxypropyl methylcellulose phthalate used as a pH-sensitive enteric polymer [13]. Fibrinolytic enzymes have been protected in the gastrointestinal track in vivo using methacrylic acid copolymers [14]. In that case, by using radiolabelling, those authors reported a long biological half-life of the proteases after oral administration and large intestinal absorption. The same methacrylic copolymers have been used to coat hard gelatin capsules containing prolyl endopeptidase for the treatment of celiac disease [15].The resulting capsules preserved the activity of the enzyme from gastric degradation in an in vitro gluten digestion model. Serratiopeptidase has also been encapsulated within methacrylated based polymers (Eudragit S100) forming microparticles by the double emulsion solvent evaporation method [16].

In general, those methacrylic polymers remain insoluble in gastric conditions thanks to their protonation and dissolve or erode releasing their content in the intestine. Some of them are pH responsive and dissolve when their carboxylic groups ionize in aqueous media at a specific pH (i.e., Eudragit L100-55); others such as Eudragit RS100 are pH independent and being mucoadhesive they release their content following a time-controlled sustained release, thanks to their chemical modification (i.e., with quaternary ammonium groups). Eudragit copolymers are widely used as enteric coatings in the protection of active pharmaceutical ingredients whereas for the protection of dietary supplement ingredients Eudraguard copolymers are mainly used in targeted colonic delivery. The enteric polymer can be applied as a coating on capsules or forming microparticles in a core-shell structure (i.e., also known as microcapsules) or as a matrix homogeneously blended with the active principle ingredient or nutraceutical. Usually spray processes including span coaters or fluidized bed coaters are used to form those gastrointestinal resistant coatings on hard and soft capsules. Microparticles, on the other hand, can be prepared by following different techniques including solvent casting and grinding, emulsification-solvent evaporation, extrusion, polymer phase separation (coacervation), electrospraying, and so on [17]. The selection of the proper microencapsulation technique is of paramount importance in order to assure an efficient entrapment of active pharmaceutical ingredients (APIs). In this work, emulsification-solvent-evaporation was selected because the double emulsion (w/o/w) method seems to be the most suitable procedure to encapsulate hydrophilic water-soluble APIs. Additional aspects such as reproducibility, high throughput, solvent economy and toxicity, as well as protein stability, evidence that the selected microencapsulation technique overpasses the rest of aforementioned available techniques.

Compared to the use of enteric coatings on large soft or hard capsules, microencapsulation allows a more controlled targeted release in specific sites of the intestine and an enhanced gastrointestinal absorption [18]. Compared to single-unit doses produced by capsules or tablets, microparticles provide an improved bioavailability with a longer sustained release of the active principle reducing high local concentration on the intestinal lining and minimizing potentially adverse side effects [19].

Herein, we have demonstrated that two different enzymes can be encapsulated within methacrylate-based polymers forming microparticulated mixed matrices and their biocatalytic functionality preserved after simulated gastric conditions demonstrating the potential applicability of this micro-encapsulation process for preserving therapeutic enzymes.

Section snippets

Synthesis of unloaded and protein-loaded enteric microparticles

Eudragit RS100 was gently donated by Evonik Industries AG. Dichloromethane, poly(vinyl alcohol) (PVA, MW: 85,000–124,000 Da), triethyl citrate (TEC), Bovine Serum Albumin-fluorescein 5-isothiocyanate (BSA-FITC), Bovine Serum Albumin (BSA), Peroxidase from Horseradish (HRP), acetic acid and sodium hydroxide were purchased from Sigma-Aldrich and used as received.

Simulated gastric (HCl (0.1 N), NaCl and H2O, pH = 1.1) and intestinal fluids (NaCl + Na2HPO4 + KH2PO4 + H2O, pH 6.8) without enzymes

Results and discussion

Fig. 1(A1–D1) showed the spherical morphology of the empty Eudragit RS100-based microparticles having an average particle size of 143.1 ± 36 μm. High magnification SEM image showed the absence of pores or cracks on the surface of the microparticles (Fig. 1; B1 and C1). BSA-loaded microparticles showed an average particle size of 172.9 ± 73 μm and 88.4% of encapsulation efficiency reaching a protein loading of 5.1 ± 0.3 w/w% (Fig. 1; A2–D2). These BSA loading achieved was superior to those

Conclusions

In this study, BSA- or HRP-loaded Eudragit RS100 microparticles were prepared by a double emulsion solvent evaporation method. Interestingly, excellent encapsulation efficiencies were achieved, 88.4% for BSA with a protein loading of 5.1 ± 0.3 w/w% and 95.8 ± 2.3% for HRP reaching an enzyme loading of 4.6 ± 0.4 w/w%. The structural integrity of the encapsulated proteins was preserved after the encapsulation process. BSA or HRP in vitro release patterns from loaded microparticles were different

Acknowledgements

The authors gratefully acknowledge the financial support of the ERC Consolidator Grant program (ERC-2013-CoG-614715, NANOHEDONISM). CIBER-BBN is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions and financed by the Instituto de Salud Carlos III (Spain) with assistance from the European Regional Development Fund. Instituto de Salud Carlos III and co-funded by European Union (ERDF/ESF, “Investing in your future”) FIS project

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