RU2583923C1 - Method of producing microencapsulated form of therapeutic protein superoxide dismutase for oral application - Google Patents

Abstract

FIELD: chemistry.

SUBSTANCE: invention relates to high-molecular chemistry and pharmacology, and is intended for use as an oral form of therapeutic protein superoxide dismutase (SOD). Invention consists in adding SOD into porous calcium carbonate (CaCO₃) of core by coprecipitation of solutions Na₂CO₃ and CaCl₂×2H₂O and further formation of alginate microcapsules, containing said core. For suppressing peptidase activity in intestinal medium in system includes peptidase inhibitor - ovomucoid (OM), which, as well as SOD, but independently from it, is included in core. CaCO₃ cores are generated according to following procedure: equal volumes of 0.33 m of solutions Na₂CO₃ and CaCl₂×2H₂O is drained and mixed with 30 s. Suspension matures in 15 minutes, then core is washed with water and acetone, filtered and dried at T = 40 °C. Formation of alginate microcapsules, containing a core with proteins, carried out by ionotropic cross-linking. Dimensions of alginate microcapsules is 800-900 mcm. Inclusion of SOD and OM therein is 10-30 mcg/mg and 5-20 mcg/mg, respectively. Such a configuration of oral delivery system maintains activity of SOD in presence of digestive enzyme trypsin. Release of SOD in medium simulating gastric (0.05 m HCl pH = 1.2) is minimum.

EFFECT: in a medium simulating intestinal (phosphate buffer, pH 8), SOD is released on 60 % for 24 hours that provides prolonged action of preparation.

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Description

The proposal relates to the field of high molecular chemistry and pharmacology and is intended for use as a microencapsulated form of the therapeutic protein superoxide dismutase (SOD) for oral use.

The literature provides various methods for producing encapsulated forms of SOD in biodegradable microcapsules (MK). Preparations based on chitosan MK obtained by coprecipitation [Ö. Çelik, J. Akbuğa. Preparation of superoxide dismutase loaded chitosan microspheres: Characterization and release studies. European Journal of Pharmaceutics and Biopharmaceutics, 66 (2007) 42-47], or based on MKs formed by an emulsion method using poly (D, L-lactide-co-glycolide) [S. Giovagnoli, P. Blasi, M. Ricci, and C. Rossi. Biodegradable Microspheres as Carriers for Native Superoxide Dismutase and Catalase Delivery, AAPS Pharm Sci Tech, 5 (2004) 1-9], have the ability to prolong the SOD release process with partial retention of enzymatic activity. A significant drawback of these methods is the preparation of injection forms of drugs, while the use of oral preparations is much more convenient for patients and mitigates the requirements for ensuring the sterility of the manufacturing process of dosage forms. A significant disadvantage of the aerosol preparation SOD obtained by the double emulsion method "solid / oil / water" based on polyketal [V.F. Fiore, M.C. Lofton, S. Roser-Page, S.C. Yang, J. Roman, N. Murthy, T.H. Barker. Polyketal microparticles for therapeutic delivery to the lung, Biomaterials, 31 (2010) 810-817], is the use of dichloromethane, a harmful organic solvent.

At the same time, SOD preparations for oral administration are not currently known. For oral dosage forms, it is necessary to ensure stability in the acidic environment of the stomach and in the alkaline environment of the intestine. For protein dosage forms, in addition, proteolytic intestinal enzymes trypsin, chymotrypsin, elastase, etc.

The closest in essence is a method for producing oral encapsulated insulin, which consists in the formation of microparticles, which were obtained by stepwise adsorption of oppositely charged polyelectrolytes on a matrix, which is an insoluble complex of proteins - insulin and intestinal peptidase inhibitors with polyanion (M. dextransulfate and chitosan) [Pechenkin M. , Balabushevich N.G., Zorov I.N., Izumrudov V.A., Klyachko N.L., Kabanov A.V., Larionova N.I. The use of protease inhibitors in the composition of polyelectrolyte microparticles to increase the bioavailability of encapsulated proteins when administered orally. CFC 47 (2013) 49-56].

A significant disadvantage of this method of obtaining an oral microencapsulated form of the protein is that the method is designed to encapsulate insulin. In the case of using the method for encapsulating a protein with other characteristics (primarily with a different value of the isoelectric point), the joint administration of the target protein and peptidase inhibitors during their coprecipitation with the polymer does not exclude the interaction of the components of the system and a decrease in their activity. Aggregates of proteins with a polymer are extremely heterogeneous in size and morphology. The stage of microcapsule formation by the method of polyelectrolyte adsorption is extremely laborious and leads to the loss of the target product.

The technical task and the positive result of the proposed method is the development of a method for obtaining a microencapsulated form of a therapeutic protein superoxide dismutase for oral use in the form of a two-level microcapsule, which is resistant to the acidic environment of the stomach and with prolonged excretion into the alkaline environment of the intestine, and is introduced into the drug to protect SOD from intestinal peptidases peptidase inhibitor - ovomukoid (OM).

The specified task and technical result is achieved in a method for producing a microencapsulated oral form of a therapeutic protein of superoxide dismutase, which includes the separate introduction by the method of coprecipitation of the target enzyme — SOD and the peptidase inhibitor — OM in primary carriers — porous calcium carbonate nuclei with their subsequent inclusion in the secondary membrane — alginate MK, formed by ionotropic crosslinking. Namely: the target protein superoxide dismutase, like ovomucoid, an inhibitor of peptidase activity in the intestinal medium, is introduced independently into porous calcium carbonate (CaCO ₃ ) nuclei by the method of coprecipitation of equal volumes of 0.33 M solutions of Na ₂ CO ₃ and CaCl ₂ × 2H ₂ O (the latter contains SOD or OM proteins at a concentration of 2-3 mg / ml) with stirring for 30 s, after the maturation of the suspension for 15 min, the cores are washed on a No. 16 Schott filter, filtered, then washed with acetone and dried in an oven with T = 40-50 ° C; hereinafter, alginate microcapsules (AMA), which serve as the outer shell for both types of nuclei, are formed by ionotropic crosslinking, according to the following procedure: the dispersion of nuclei in a 3% solution of sodium alginate (not more than 200 mg of nuclei in 1 ml of solution) is introduced dropwise into the precipitation bath containing a 1% solution of CaCl ₂ in a 0.2-0.5% solution of chitosan in 1% acetic acid, then filtered on a Buchner funnel, washed with water and dried at room temperature; the sizes of the AMA obtained in this way are 800-900 μm, the inclusion of SOD and OM in the proposed systems is 10-30 μg / mg and 5-20 μg / mg, respectively.

The method is described more fully in the examples given, where the following reagents were used in the experiments: OM (Reakhim, Russia), trypsin (Sigma-Aldrich), Na ₂ CO ₃ , CaCl ₂ × 2 H ₂ O and anhydrous CaCl ₂ - all puriss pa ( Sigma-Aldrich), Na ₂ HPO ₄ , NaH ₂ PO ₄ - chda (Reakhim Russia), acetic acid, chda (Vecton Russia), deionized water extra pure reagent grade III (Acros Organics), low-viscosity medical alginate Na (Arkhangelsk plant) , acid-soluble chitosan, TU 15-01482-88 with MM = 560 kDa and MM = 35 kDa, Ransod reagent kit (SD125) for determining the activity of SOD (Randox).

Example 1. Two-level AMA containing CaCO ₃ nuclei with the antioxidant enzyme SOD and peptidase inhibitor OM included in them were prepared as follows. Superoxide dismutase (SOD) is a key enzyme in the body's antioxidant defense. The physiological effect is associated with the detoxification of superoxide radicals formed during the development of various pathological conditions. The main limitation in the therapeutic use of SOD is a short plasma half-life of only 6 minutes. In this work, we used the Rexod drug (VNII OBChP), the main active ingredient of which is recombinant human SOD obtained from the yeast Saccharomyces cerevisiae, strain Y2134. Rexod is an original drug, not described in foreign pharmacopoeias. Registration certificate LSR-007164/09 from 09/10/2009. OM is an inhibitor of peptidase enzymes in the intestines - trypsin, chymotrypsin and elastase. The first level of encapsulation, the deposition of SOD in the porous CaCO ₃ vaterite was carried out as follows: equal volumes of 0.33 M solutions of Na ₂ CO ₃ and CaCl ₂ × 2H ₂ O (the last contains SOD or OM proteins at a concentration of 2-3 mg / ml) were drained and stirred for 30 s. The suspension matured for 15 min, then the cores on a 16 Schott filter were washed with a 6-fold volume of water, filtered, then washed with acetone and dried in an oven at T = 40-50 ° C. The temperature regime affects the morphology of CaCO ₃ crystals. Drying of the CaCO ₃ gel at T below 30 ° C leads to the formation of non-porous calcites, at T above 50 ° C a mixture of porous spherical vaterites and non-porous aragonites forms, and the fraction of aragonites increases with temperature. The introduction of an OM peptidase inhibitor at the first level of encapsulation was carried out similarly to the introduction of SOD. The sizes of carbonate nuclei containing SOD or OM proteins are 4–6 μm. The second level of encapsulation - the formation of AMA containing nuclei with proteins, was carried out by ionotropic crosslinking. A dispersion of nuclei (not more than 200 mg per 1 ml of solution) in a 3% sodium alginate solution was introduced dropwise with stirring into a precipitation bath containing a 1% aqueous CaCl ₂ solution. To improve the surface quality of AMA, chitosan additives are introduced; for this, the CaCl ₂ precipitation bath contained a 0.2-0.5% solution of chitosan in 1% acetic acid. When sodium alginate in the droplet enters the bath, it is spatially crosslinked with divalent calcium ions, forming a strong elastic AMA, inside which carbonate nuclei are enclosed with protein. Then the AMA was filtered, washed with water and dried at room temperature. Dimensions AMK are 800-900 microns. The obtained AMA are the main component of the long-acting oral SOD preparation. The inclusion of SOD and OM in the proposed systems is 10-30 μg / mg and 5-20 μg / mg, respectively. The release of SOD in a simulated gastric medium (0.05 M Hcl, pH = 1.2) is minimal. On Wednesday, simulating intestinal (Na phosphate buffer, pH = 8.0), SOD is released by 60% in 24 hours, which ensures a prolonged effect of the drug. The curves for the release of SOD into the model intestinal environment from a two-level system, as well as from AMA without carbonate nuclei and only from CaCO ₃ - systems of the first level are shown in FIG. 1. It is seen that carbonate nuclei practically do not retain SOD in the intestinal environment. Comparison of SOD release profiles from the claimed two-level system and AMC on a more significant prolongation of the process in the first case.

Example 2. It differs from Example 1 in that, at the first level of encapsulation, co-precipitation of SOD and OM was performed.

This example is provided to compare the effectiveness of delivery systems of various configurations in the presence of a proteolytic enzyme inhibitor.

Tests of the activity of AMA containing SOD obtained according to the claimed invention (Example 1), and by the methods described in Example 2, were carried out in vitro. SOD activity was determined using a Randox Ransod (SD125) kit. SOD activity was calculated by the inhibition of the reaction of the formation of formazan as a result of oxidation of the chromogen (2- (4-iodophenyl) -3- (4-nitrophenol) -5-phenyltetrazolium chloride) with oxygen radicals generated by xanthine oxidase during the oxidation of xanthine. The optical density of the solutions was measured using a FLx800 flat panel photometer (Biotek). Samples were diluted 3,000 times with 10 mM sodium phosphate buffer. 6 μl of diluted sample or standards and 200 μl of reagent 1 (xanthine) were added to a 96-well plate and mixed. Then, 30 μl of xanthine oxidase-containing reagent 2 was added, mixed, and the initial optical density An was measured at a wavelength of 505 nm. After 8 minutes of incubation, the final absorbance Ak was measured. All measured values for standards and samples (the average of two measurements) were converted to percent inhibition: Ing = 100- [dAH / dAK] · 100%. As a calibration curve, we used the curve of the inhibition of the reaction of the formation of formazan on the logarithm (Log ₁₀ ) of SOD activity in the standard, expressed in units / ml. Temporal profiles of changes in the activity of SOD during the release of an enzyme from two-level AMA of various compositions, corresponding to Examples 1-2, into a model intestinal environment, namely: in 0.07 M Phosphate buffer, pH = 7.8, containing trypsin at a concentration of 0.125 mg / ml, are given in FIG. 2. An attempt to introduce a peptidase OM inhibitor in CaCO ₃ nuclei together with SOD (Example 2) did not lead to the expected result. The interaction of proteins during the procedure for the formation of the encapsulation system is not excluded.

Preferred is the microencapsulated form of SOD obtained by the method described in Example 1, when OM is included in the CaCO ₃ nucleus regardless of SOD. This configuration of the oral delivery system effectively retains SOD activity in vitro in the presence of the intestinal trypsin enzyme. The release of SOD in a simulated gastric medium (pH = 1.2) is minimal. On Wednesday, simulating the intestinal (phosphate buffer, pH = 8.0), SOD is released by 60% in 24 hours, which ensures a prolonged effect of the drug.

In the inventive method, the entire process is carried out using aqueous solutions, environmentally friendly, fireproof. This allows you to get a drug with the following positive characteristics: the oral form of taking the drug SOD is much more convenient than parenteral for both the patient and staff. In the production of oral forms, the requirements for sterility of production are less stringent than in the production of parenteral forms. When passing through the gastrointestinal tract, the oral microencapsulated form of SOD protects the therapeutic protein in the gastric environment and ensures its gradual release into the intestinal environment. The prolonged action of the drug reduces the number of doses of the drug. Due to the optimal variant of inclusion of an intestinal peptidase inhibitor, the proposed preparation retains the activity of the therapeutic protein SOD in the presence of the intestinal trypsin enzyme.

Claims (1)

A method for producing a microencapsulated form of a therapeutic superoxide dismutase protein for oral administration, comprising the formation of a two-level microcapsule containing the target protein and a peptidase inhibitor inside, characterized in that the target superoxide dismutase protein, like the ovomucoid, an inhibitor of peptidase activity in the intestinal medium, is introduced independently into porous calcium carbonate (CaCO ₃₎ of the core by the coprecipitation of equal volumes of solutions of 0.33 M Na ₂ CO ₃ and CaCl ₂ × 2H ₂ O, in the latter contained SOD proteins or OM in conc ntratsii 2-3 mg / ml with stirring for 30 seconds, after maturation in the suspension for 15 min nucleus washed on the sinter №16 water, filtered off, then washed with acetone and dried in oven at T = 40-50 ° C; subsequently, alginate microcapsules serving as the outer shell for both types of nuclei are formed by ionotropic crosslinking, according to the following procedure: the dispersion of nuclei in a 3% sodium alginate solution of not more than 200 mg of nuclei in 1 ml of a solution is introduced dropwise into the precipitation bath containing 1 % solution of CaCl ₂ in a 0.2-0.5% solution of chitosan in 1% acetic acid, then filtered on a Buchner funnel, washed with water and dried at room temperature; the sizes of alginate microcapsules obtained in this way are 800-900 μm, the inclusion of superoxide dismutase and ovomukoid in them is 10-30 μg / mg and 5-20 μg / mg, respectively.

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