[Nonimmunogenic staphylokinase in the treatment of acute ischemic stroke (FRIDA trial results)]. / Neimmunogennaya stafilokinaza ­ novyi tromboliticheskii preparat v lechenii ishemicheskogo insul'ta (rezul'taty issledovaniya FRIDA).

AIM OF THE STUDY: To investigate the efficacy and safety of non-immunogenic staphylokinase (NS) compared with alteplase (A) in patients with acute ischemic stroke (AIS) within 4.5 h after symptom onset. MATERIAL AND METHODS: 336 patients with IS within 4.5 h after symptom onset were included in a randomized, open-label, multicenter, parallel-group, non-inferiority comparative trial of NS vs A (168 patients in each group). NS was administered as an intravenous bolus in a dose of 10 mg, regardless of body weight, over 10 s, A was administered as a bolus infusion in a dose of 0.9 mg/kg, maximum 90 mg over 1 hour. The primary efficacy endpoint was a favorable outcome, defined as a modified Rankin scale (mRS) score of 0-1 on day 90. Safety endpoints included all-cause mortality on day 90, symptomatic intracranial haemorrhage, and other serious adverse events (SAEs). RESULTS: At day 90, 84 (50%) patients reached the primary endpoint (mRS 0-1) in the NS group, 68 (41%) patients - in the A group (p=0.10, OR=1.47, 95% CI=0.93-2.32). The difference between groups NS and A was 9.5% (95% CI= -1.7-20.7) and the lower limit of the 95% CI did not cross the margin of non-inferiority (pnon-inferiority<0.0001). There were no significant differences in the frequency of deaths between the groups: on day 90, 17 (10%) patients in the NS group and 24 (14%) in the A group had died (p=0.32). There was a trend towards significant differences in the frequency of symptomatic intracranial haemorrhage: NS group - 5 (3%) patients, A group - 13 (8%) patients (p=0.087, OR=0.37, 95% CI=0.1-1.13). There were significant differences in the number of patients with SAEs: in the NS group - 22 (13%) patients, in the A group - 37 (22%) patients (p=0.044, OR=0.53, 95% CI=0.28-0.98). CONCLUSION: The presented results of the FRIDA trial are the first in the world to use a drug based on NS in patients with IS. It has been shown that a single bolus (within 10 s) administration of NS at a standard dose of 10 mg, regardless of body weight, allows to conduct fast, effective and safe thrombolytic therapy in patients with IS within 4.5 h after symptom onset. In further clinical tials of NS, it is planned to expand the therapeutic window beyond 4.5 h after symptom onset in patients with IS.

[Production of mycobacterial antigenes merged with cellulose binding protein domain in order to produce subunit vaccines against tuberculosis].

Protein genes Ag85A, Esat-6, and Cfp10 of Mycobacterium tuberculosis were sequenced using the database GenBank to implement selection and synthesis of primer pairs of given genes. PCR was used to obtain target amplicons of the genes. Chromosome DNA of M. tuberculosis H37Rv was used as the DNA amplification matrix. The PCR products were obtained using the plasmid pQE6, cloned, and amplified in the Escherichia coli M15 strain. Chimere products containing mycobacterial genes and cellulose binding protein domain (CBD), were obtained using the plasmid treated with restriction endonucleases. CBD fragment obtained using similar treatment of the ptt10 plasmid. The plasmids containing merged sequences of mycobacterial genes-antigenes and CBD were selected. The 3 mycobacterial genes were expressed in the E. coli M15 cells resulting in biosynthesis of corresponding recombinant proteins of expected molecular weight. Concentration of CBD, Cfp10-CBD, Ag85A-CBD, and ESAT6-CBD was 20%, 15%, and 15% total protein, respectively. The resulting chimere proteins provide high affinity for cellulose and high stability. Immobilization of CBD-containing recombinant proteins proceeds as one-stage process providing target protein purification and adsorption on cellulose. The vaccines produced using this technology are inexpensive because of low cost of cellulose sorbents as well as simultaneous use of cellulose for purification and immobilization of protein. Many cellulose preparations are not toxic, biocompatible, and widely used in medicine.

[Production of the recombinant human bone morphogenetic protein-2 in Escherichia coli and testing of its biological activity in vitro and in vivo].

Bone morphogenetic protein-2 (rhBMP-2) represents the osteoinductive protein factor which plays a dominant role in growth and regeneration of a bone tissue. In clinical practice the bone grafting materials on the basis of rhBMP-2 are widely applied; the Russian analogues of similar materials are not produced. The fragment of the bmp2gene coding for a mature protein was cloned in Escherichia coli. The effective overproducing strain of rhBMP-2 was created on a basis of the E. coli BL21 (DE3). The rhBMP-2 production was about 25% of total cell protein. The biologically active dimeric form of rhBMP-2 was obtained by isolation and purification of protein from inclusion bodies with subsequent refolding. The rhBMP-2 sample with more than 80% of the dimeric form was obtained, which is able to interact with specific antibodies to BMP-2. Biological activity of the received rhBMP-2 samples was shown in the in vitro experiments by induction of alkaline phosphatase synthesis in C2C12 and C3H10T1/2 cell cultures. On model of the ectopic osteogenesis it was shown that received rhBMP-2 possesses biological activity in vivo, causing tissue calcification in the place of an injection. The protein activity in vivo depends on way of protein introduction and characteristics of protein sample: rhBMP-2 may be introduced in an acid or basic buffer solution, with or without the carrier. The offered method of rhBMP-2 isolation and purification results in increasing common protein yield as well as the maintenance of biologically active dimeric form in comparison with the analogues described in the literature.

[Human recombinant interferon-B constructed on the basis of affinity-binding domain technology].

Fusion gene consisting of dextran-binding domain from Leuconostoc mesenteroides subsp. Mesenteroides (DBD) and human recombinant interferon-beta (IFN-beta) incorporated between the nucleotide sequence encoding for the recognition site of human enteropeptidase (DDDDK) was installed and constructed in Escherichia coli. The overproducing strain of the chimeric protein DBD-IFN-beta consisting of the IFN-beta, spacer including 10 GS-repeats, human enteropeptidase recognition site, and dextran-binding domain from Leuconostoc mesenteroides was constructed. Free human recombinant interferon-beta was obtained as a result of treatment of the chimeric protein DBD-IFN-beta immobilized on sephadex G-25 with human enteropeptidase. The ability of free and immobilized protein to protect human cells from viral infection was demonstrated. The developed approach can be used for purification of the recombinant proteins with different biological activity and possible construction of new immunostimulating and antiviral drugs, growth factors, anti-cancer drugs, etc.

[Isolation and characterization of collagen-binding domains from human von Willebrand factor].

DNA fragments encoding for two collagen binding decapeptides from the human von Willebrand factor (vWF-H1 and vWF-H2) were cloned in the Escherichia coil culture. Overproducing strains of the chimeric proteins vWF(H1)-CBD and vWF(H2)-CBD consisting of the corresponding decapeptide, Gly-Ser spacer and a cellulose binding domain (CBD) from Anaerocellum thermophilum were constructed. Using one-stage purification on cellulose, the highly purified samples of vWF(H1)-CBD and vWF(H2)-CBD proteins were obtained and the ability of these proteins to bind collagen was studied. These constructions are planned to be used for development of the recombinant collagen binding proteins with different biological activities, which, in its turn, will be used for development of the new generation products and materials for medicine, such as different kinds of implants, the coats, etc.