RU2522846C2 - Drug preparation and method for improving rheological properties of sputum and pulmonary administration of this preparation - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: group of inventions refers to medicine and may be used for improving rheological properties of sputum and inhibiting the bacterial biofilm formation in bronchi in treating cystic fibrosis. That is ensured by using recombinant human deoxyribonuclease-1 covalently bond to homopolymeric polysaccharide containing 80 links of alpha-2,8 sialic acid as an inhalation agent. There are also presented a method and administration of a drug preparation for improving the rheological properties of sputum.

EFFECT: group of inventions enables maintaining a higher level of DNA-hydrolytic activity of the administered drug preparation in bronchial mucus.

3 cl, 3 dwg, 4 ex, 2 tbl

Description

The invention relates to medicine, and specifically to drugs that improve the rheological properties of sputum in the treatment of cystic fibrosis and prevent the development of bacterial biofilms in the airways of patients.

Cystic fibrosis is a systemic hereditary disease caused by a mutation in the gene for the transmembrane regulator of cystic fibrosis and characterized by damage to the endocrine glands, severe respiratory and gastrointestinal tract dysfunctions. Cystic fibrosis is the most common hereditary disease leading to death. Currently, cystic fibrosis in the Russian Federation affects approximately 8,000 people, most of whom are under the age of 30.

The accumulation of viscous purulent secretion (sputum) in the airways of patients with cystic fibrosis plays a role in reducing the functional ability of the lungs and in exacerbations of the infectious process. Sputum of patients with cystic fibrosis has a number of characteristic features. The viscosity of bronchial secretions is due to the content of two macromolecules: mucoid glycoproteins and DNA. The main source of DNA is decaying polymorphonuclear neutrophils, which accumulate in the airways in response to a chronic bacterial infection, as well as biofilms of microbial agents, in particular P. Aeruginosa. A characteristic feature of the chronic inflammatory process in the lungs of patients with cystic fibrosis is persistent infiltration of lung tissue with massive neutrophilia in the airways .. Derivatives of dying neutrophils - elastase, cathepsin G, protease, collagenase, gelatinase, plasminogen activation factor, free radicals, myeloperoxidase, oxidase, oxidase oxidase, endotoxin - contribute to the development of a "respiratory explosion" and can directly influence and inactivate enzyme preparations that realize their pharmacologists esky effect in such aggressive microenvironment. (E. Gembitskaya, 2008)

Pulmozyme is used to improve respiratory function in patients with cystic fibrosis over the age of 5 years.

Pulmozyme (Dornase alpha) is a phosphorylated glycosylated recombinant deoxyribonuclease I, identical to human deoxyribonuclease isolated from urine, and consists of 260 amino acids having a molecular weight of 37 KDa. and is produced by fermentation of a recombinant strain of Chinese hamster ovary cells. The therapeutic efficacy of the drug is based on its ability to hydrolyze the high molecular weight DNA of the mucoalveolar secretion of patients, thereby improving the rheological properties of the secretion.

The main disadvantage of pulmozyme is its limited effectiveness, which is a consequence of the following reasons:

A rapid decrease in sputum concentration resulting from systemic absorption - with inhalation of 2.5 mg pulmozyme, the average concentration of the drug in sputum decreases from 3 μg / ml to 0.6 μg / ml within 2 hours after inhalation. (Pulmozyme; Instructions for medical use)

The suppression of the enzymatic activity of pulmozyme by the degradation products of cell cytoskeleton (actin), contained in large quantities in the mucoalveolar secretion of patients with cystic fibrosis. The G-form of actin binds to dornase alpha mainly in the Tyr65-Val66-Val 67 region and inhibits the hydrolytic activity of the enzyme (Fujihara, 2006). Moreover, the profile of N-glycosylation characteristic for pulmozyme does not prevent actin inactivation and proteolysis mediated by bacterial and leukocyte peptidases.

The objective of the invention is to provide a drug for the hydrolysis of high molecular weight extracellular DNA of sputum in patients with cystic fibrosis, which has the ability to maintain a high level of DNA hydrolytic activity in bronchial mucus for a long time and has a reduced sensitivity to the action of natural inhibitors and proteases contained in sputum.

This problem is solved in that a synthetic glycoprotein is used as a medicine for the hydrolysis of high molecular weight extracellular DNA of sputum in patients with cystic fibrosis, which is a recombinant human deoxyribonuclease-1 obtained in the E. coli microorganism and covalently linked by its N-terminus to a polysaccharide containing 80 polysaccharide containing alpha-2.8 sialic acid in the polysaccharide chain. Thus, the present invention provides a medicament for improving the rheological properties of sputum and suppressing the formation of bacterial biofilms in the bronchi, in a patient with cystic fibrosis, containing as an active substance an effective amount of recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha 2.8 sialic acid.

The invention further provides a method for improving the rheological properties of sputum and suppressing the formation of bacterial biofilms in the bronchi of a patient with cystic fibrosis, comprising administering to a patient in need thereof an effective amount of a drug containing, as an active substance, a recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid.

Further, according to the invention, there is provided the inhalation use of a medicament containing, as an active substance, a recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2,8 sialic acid to improve the rheological properties of sputum and suppress the formation of bacterial biofilms in the bronchi when treatment of cystic fibrosis.

Polysialic acids (PSA) are naturally occurring unbranched polymers of sialic acid produced by certain strains of bacteria and in certain mammalian cells (Roth., 1993). They can be obtained with different degrees of polymerization, from n = about 80 or more sialic acid residues to n = 2 by acid hydrolysis or by cleavage with neuraminidases, or by fractionation of the natural polymer species produced by bacteria. The composition of various polysialic acids also changes so that homopolymer forms exist, i.e. alpha-2.8 - linked polysialic acid, including the capsular polysaccharide of E. coli strain K1 and the B-group of meningococci, which is also found in the embryonic form of a cell neuron adhesion molecule (N-CAM). Heteropolymer forms also exist, such as alternating alpha-2.8 alpha-2.9 polysialic acid of E. coli strain K92 and C group polysaccharides of N. meningitidis. Sialic acid can also be found in alternating copolymers with monomers other than sialic acid, such as group W135 or group Y N. meningitidis.

Polysialic acids have important biological effects, including evading pathogenic bacteria from the immune system and complement system. Alpha-2.8 - linked polysialic acid of E. coli strain K1 is also known as "colominic acid" and it (of various lengths) is used in the present invention.

Among the bacterial polysaccharides alpha-2.8, the bound form of polysialic acid is the only non-immunogenic one (that does not cause either T-cell response or antibody formation in mammals, even when conjugated with immunogenic protein carriers). Shorter forms of the polymer (up to n = 4) are found in gangliosides of the cell surface, which are widespread in the body and are considered effective for communicating and maintaining immunological tolerance to polysialic acid.

In recent years, the biological properties of polysialic acids, in particular the biological properties of alpha-2.8 linked homopolymer polysialic acid, have been used to modify the pharmacokinetic properties of protein or low molecular weight drug molecules [Gregoriadis, 2001; Jain et al., 2003; US-A-5846951, WO-A-0187922].

Alpha-2,8 - linked polysialic acid (PSA) offers an attractive alternative for protein modification, being an immunologically invisible biodegradable polymer that is a natural part of the human body and that is broken down by tissue neuraminidases to sialic acid, a non-toxic saccharide

The choice is based on the results of a study conducted by the authors of the present invention, which found the ability of recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2,8 sialic acid in the polysaccharide chain to significantly increase the duration of the specific pharmacological action of recombinant human deoxyribonuclease-1 in patients with cystic fibrosis. In the process of working on the invention, the authors unexpectedly discovered that the proposed synthetic glycoprotein has the ability to inhibit the growth of bacterial biofilms in the sputum of patients with cystic fibrosis.

The invention is illustrated by two examples of obtaining the claimed medicinal product is a table with the results of comparative tests, and figures, where it is given:

Table 1 presents the effect of the claimed drug on the sputum fluidity of patients with cystic fibrosis compared with pulmozyme.

Pulmozyme and the claimed drug were added to sputum samples to a concentration of 10 μg / ml protein (BCA method). 15 minutes after the administration of the test drugs, each sputum sample was diluted with an equal volume of fresh sputum from the same patient.

Sputum fluidity was determined on a Keal scale:

Figure 1 shows the effect of the claimed drug and pulmozyme on the degree of polymerization of DNA isolated from the sputum of patients with cystic fibrosis.

Electrophoresis of a pooled sputum DNA sample 30 minutes after dilution of the test aliquot with an equal volume of fresh sputum (10 and 50 μg / ml). DNA size is indicated in the database standards.

From left to right:

lane 1 - Pulmozyme 50 μg / ml

lane 2 - Pulmozyme 10 μg / ml

lane 3 - The inventive drug 50 mcg / ml

lane 4 - The inventive drug 10 mcg / ml

Figure 2 shows the results of a Western blot analysis of the covalent conjugate of human recombinant deoxyribonuclease-1 with an alpha-2.8 sialic acid homopolymer weighing 22 kDa.

The analysis of conjugates of recombinant human deoxyribonuclease-1 and homopolymer polysaccharide alpha-2,8 sialic acid by immunoblotting

Figure 3 shows micrographs of bacterial biofilms developing in the sputum of patients in the presence of pulmozyme and the inventive synthetic glycoprotein.

The effect of recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid at a concentration of 5.0 μg / ml (A) and pulmozyme (B) at a concentration of 5.0 μg / ml on the growth of bacterial biofilms in sputum of a patient with cystic fibrosis 24 hours after the addition of drugs.

The inventive drug is obtained, for example, as follows.

Example 1. Obtaining recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid

The synthetic gene encoding human deoxyribonuclease and expression strain were constructed according to the method described by Worrall (Worrall AF, Connolly BA. The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. J Biol Chem. 1990 Dec 15 ; 265 (35): 21889-95). Protein refolding and purification was performed according to the method described by Linardou (Linardou H, Epenetos AA, Deonarain MP. A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I. Int J Cancer. 2000 May 15; 86 (4): 561-9.). A non-glycosylated protein was obtained with a molecular weight of 29 KDa and an amino acid sequence that fully corresponds to the amino acid sequence of deoxyribonuclease I from human urine but does not have an N-linked oligosaccharide group. The specific activity of the obtained protein, measured by the Kunitz method, corresponded to the published specific activity of N-glycosylated recombinant deoxyribonuclease 1 person

The resulting human recombinant deoxyribonuclease-1 was dissolved in 0.15 M phosphate buffered saline (PBS) (pH 7.4) and covalently bound to oxidized polysialic acid. Polysialic acid with a molecular weight of 22 kDa was used, adding it to human recombinant deoxyribonuclease-1 (2 mg) in molar ratios of polysialic acid: deoxyribonuclease-1 (12.5: 1). Sodium cyanoborohydride was added to a final concentration of 4 mg / ml. The reaction mixture was sealed and stirred using a magnetic stirrer for 24 hours at 35 ± 2 ° C. The mixture was then precipitated with ammonium sulfate ((NH ₄ ) ₂ SO ₄ ) by slowly adding salt with continuous stirring to achieve 70% w / v. saturation, stirred for 1 hour at 4 ° C, then centrifuged (5000 · g) for 15 minutes, and the granules were re-suspended in a saturated solution of (NH ₄ ) ₂ SO ₄ and again centrifuged for 15 minutes (5000 · g). The precipitates isolated were redissolved in 1 ml PBS pH 7.4 and subjected to active dialysis (24 hours) at 4 ° C against the same buffer. Controls included conjugation of the native protein in the presence of unoxidized polysialic acid or in the absence of polysialic acid. Shaking was minimized to avoid concomitant protein denaturation. The conjugate was isolated sequentially by ion exchange and reverse phase chromatography. Purified covalent conjugate of human recombinant deoxyribonuclease-1 with an alpha-2.8 sialic acid homopolymer containing 80 sialic acid units was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot analysis. The results are shown in Figure 2.

Example 2. Obtaining recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid.

At the first stage, thiol groups were introduced into the sequence of human recombinant deoxyribonuclease-1 by thiolation of the amine lysine. The human recombinant deoxyribonuclease-1.4 mg obtained as described previously in Example 1 was dissolved in 0.25 ml PBS + 10 mM EDTA and 0.498 mg 2-iminothiolane (2-IT or Trout reagent 50 mol equivalents 3.6 was added) × 10 ^-6 mol) in 0.25 ml of the same buffer. The tube was closed with foil and left to incubate with stirring with a flexible stirrer for 1 h at 37 ° C. The thiolated human recombinant deoxyribonuclease-1 was purified from free 2-IT (Trout reagent) by gel filtration (PD-10) and a 0.5 ml fraction was analyzed for the presence of protein (BCA analysis) or thiol (Elman analysis). Oxidized polysialic acid with a molecular weight of 22 kDa was reacted with 5 molar equivalents of N- [b-maleimidepropionic acid hydrazide] in 0.1 M sodium acetate for 2 hours at 37 ° C. The resulting hydrazone (CAM) was precipitated in ethanol, re-suspended in sodium acetate and re-precipitated in ethanol, re-dissolved in water and subjected to dry freezing. To thiolated human recombinant deoxyribonuclease-1 (3.6 mg) in 2 ml of PBS / EDTA was added 22.5 mg of CAM (9 × 10 ^-7 mol, 15 mol equivalents). The tube was sealed and left to incubate at 37 ° C for 1 h with gentle stirring. The resulting conjugate was then isolated sequentially by ion exchange and reverse phase chromatography. Purified covalent conjugate of human recombinant deoxyribonuclease-1 with an alpha-2.8 sialic acid homopolymer containing 80 sialic acid units was analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate and Western blot analysis.

Table 1 shows the effect of using the claimed method of a drug - a covalent conjugate of human recombinant deoxyribonuclease-1 with a homopolymer of alpha-2.8 sialic acid weighing 22 kDa containing 80 units of sialic acid on the sputum fluidity of patients with cystic fibrosis in comparison with pharmaco-pharmacodynamic pharmacodynamics . From table 1 it can be seen that the claimed medicinal product is superior to pulmozyme in the ability to improve the rheological properties of individual samples of sputum of patients in all studied samples.

Figure 1 shows the effect of using the claimed method of the drug - a covalent conjugate of human recombinant deoxyribonuclease-1 with a homopolymer of alpha-2.8 sialic acid weighing 22 kDa containing 80 units of sialic acid on the degree of polymerization of DNA isolated from the sputum of patients with cystic fibrosis after processing the claimed drug and pulmozyme. Figure 1 shows that the claimed drug is superior to pulmozyme in its ability to enhance the defragmentation of high molecular weight extracellular DNA of sputum in patients with cystic fibrosis.

Figure 2 shows the results of a Western blot analysis of the covalent conjugate of human recombinant deoxyribonuclease-1 with an alpha-2.8 sialic acid homopolymer weighing 22 kDa containing 80 units of sialic acid, indicating the formation of a true conjugate.

Figure 3 shows the effect of using the claimed method of a drug - a covalent conjugate of human recombinant deoxyribonuclease-1 with a homopolymer of alpha-2.8 sialic acid weighing 22 kDa, containing 80 units of sialic acid on the growth of bacterial biofilms in the sputum of a patient with cystic fibrosis compared with pulmonary. Figure 3 shows that the claimed drug is superior to pulmozyme in its ability to inhibit the growth of bacterial biofilms in the sputum of a patient with cystic fibrosis.

Example 3. The inventive drug - recombinant human deoxyribonuclease-1, covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid was prepared as described in Example 2.

Additionally, a comparative preparation was prepared — recombinant human deoxyribonuclease-1, covalently linked to a homopolymer polysaccharide containing 40 units of alpha-2.8 sialic acid, as described in Example 2, except that oxidized polysialic acid was used to create the conjugate molecular mass of 11 kDa, not 22 kDa, and containing, respectively, not 80, but 40 units of sialic acid.

Pulmozyme, the claimed drug (recombinant human deoxyribonuclease-1, covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid), as well as a comparison drug (recombinant human deoxyribonuclease-1, covalently linked to a homopolymer of 40 polysaccharide alpha-2.8 sialic acid) was added to the sputum samples of patients with cystic fibrosis to a concentration of 10 μg / ml protein (ICA method). 15 minutes after the test preparations were introduced into the medium, each sputum sample was diluted with an equal volume of fresh sputum from the same patient.

Sputum fluidity was determined immediately, then after 15 and 45 minutes on the Keal scale:

The results of the study are presented in table 2.

Table 2 shows that deoxyribonculease conjugated to a shorter polysaccharide containing only 40 alpha-2.8 sialic acid units has a significantly lower ability to increase sputum fluidity in patients with cystic fibrosis, and in some patients it did not significantly affect sputum fluidity, which said that such conjugation led to a significant loss of the enzymatic activity of the enzyme without increasing the duration of its action.

Example 4. Improving the rheological properties of sputum and suppressing the formation of bacterial biofilms in the bronchi with cystic fibrosis with inhalation use of the inventive drug.

Patient, 14 years old, Diagnosis: Cystic fibrosis, moderate, mixed form. In sputum culture, growth of Pseudomonas aeruginosa in association with staphylococcus, alpha - hemolytic streptococcus, and fungi of the genus Candida is noted.

A 2.5 ml solution (at a concentration of 1 mg / ml) of the covalent conjugate of human recombinant deoxyribonuclease-1 with an alpha-2.8 sialic acid homopolymer containing 80 units of sialic acid, obtained as described in Example 1 in physiological saline (0 , 9% sodium chloride solution) was inhaled for 30 days once a day, at a dose of 2.5 mg through a nebulizer, at the same time, in the first half of the day.

The cough became wet (liquefaction of sputum) on the 4th day, while, in previous hospitalizations (treatment without the use of the claimed drug), a wet cough appeared on average only on the 10-12th days. Also on day 5, separation of purulent sputum began. In previous hospitalizations (treatment without the use of the claimed drug), purulent sputum separation began not earlier than 11-12 days. Staphylococcus aureus and fungi of the Candida genus disappeared in sputum cultures, the number of Pseudomonas aeruginosa decreased.

Thus, the claimed drug effectively improves the rheological properties of sputum and inhibits the formation of bacterial biofilms in the bronchi with cystic fibrosis with inhalation use.

Thus, the above materials suggest that the claimed use of recombinant human deoxyribonuclease-1, covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid to improve the rheological properties of sputum and inhibit the formation of bacterial biofilms in the bronchi in the treatment of cystic fibrosis new. Applicants are not aware of the use of recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 alpha-2.8 sialic acid units for the treatment of cystic fibrosis from published sources of information.

The inventive drug is industrially applicable, as evidenced by examples of its manufacture and use.

The claimed application is not obvious. The biological activity of recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid is significantly different from that of natural human deoxyribonuclease. The use of a homopolymer polysaccharide containing from 80 units of alpha-2.8 sialic acid to alter the improvement in pharmacological properties of recombinant deoxyribonuclease during inhalation is new.

The mechanisms of this phenomenon are currently being investigated by the authors.

From the foregoing, it follows that the claimed application is new, non-obvious and industrially applicable, i.e. meets all the requirements for the invention.

Junko Fujihara et. al., Actin-inhibition and folding of vertebrate deoxyribonuclease I are affected by mutations at residues 67 and 114. Comparative Biochemistry and Physiology, Part B 143 (2006) 70-75

E. Gembitskaya, A.G. Chermensky, Pulmonary Damage in Cystic Fibrosis: Current Aspects, Consilium Medicum, No. 3, 2008

Roth, J., Rutishauser, U., Troy, F.A. (Eds.), Polysialic acid: from microbes to man, Birkhauser Verlag, Basel, Advances in Life Sciences, 1993.

Gregoriadis, G., Drug and vaccine delivery systems, in: PharmaTech, World Markets Research Center Limited, London (2001) 172-176.

Jain, S., Hirst, DH, Laing, P., Gregoriadis, G., Polysialylation: The natural way to improve the stability and pharmacokinetics of protein and peptide drugs, Drug Delivery Systems and Sciences, 4 (2) (2004) 3 -9

Worrall AF, Connolly BA. The chemical synthesis of a gene coding for bovine pancreatic DNase I and its cloning and expression in Escherichia coli. J Biol Chem. 1990 Dec 15; 265 (35): 21889-95.

Linardou H, Epenetos AA, Deonarain MP. A recombinant cytotoxic chimera based on mammalian deoxyribonuclease-I. Int J Cancer. 2000 May 15; 86 (4): 561-9.

Claims (3)

1. A drug for improving the rheological properties of sputum and suppressing the formation of bacterial biofilms in the bronchi, in a patient with cystic fibrosis, containing as an active substance an effective amount of recombinant human deoxyribonuclease-1 covalently bound to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid . 2. A method for improving the rheological properties of sputum and suppressing the formation of bacterial biofilms in the bronchi during cystic fibrosis, comprising administering to a patient in need of this patient an effective amount of a drug containing, as an active substance, a recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 alpha units 2.8 sialic acid. 3. The use of a medicinal product containing, as an active substance, a recombinant human deoxyribonuclease-1 covalently linked to a homopolymer polysaccharide containing 80 units of alpha-2.8 sialic acid, as an inhalation agent for improving the rheological properties of sputum and inhibiting the formation of bacterial biofilms in the bronchi in the treatment of cystic fibrosis.

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