Development of target delivery system based on actinomycin class drugs and recombinant alpha-fetoprotein.

A recombinant alpha-fetoprotein (rAFP) was obtained in the yeast P. pastoris system, and its functional activity was confirmed. A method for producing polymer particles loaded with dactinomycin was developed, and a conjugate of these nanoparticles with rAFP was synthesized. The efficiency of the obtained conjugate on the HeLa, SKOV3, and MG-63 tumor cells and the absence of toxicity on the normal cells was shown. Experiments in vivo demonstrated a significant increase in the antitumor efficacy of the conjugate at a lower general toxicity as compared to the commercially available dactinomycin.

[Accumulation of doxorubicin conjugates with dendritic polymer and vector protein in normal and tumor cells in vitro].

Accumulation of doxorubicin (Dox), its conjugates with the second generation dendritic polymer (G2-Dox) and vector pro- tein (recombinant third domain of alpha-fetoprotein - 3D-G2- Dox) in normal and tumor cells was studied in vitro within the framework of the development of selective transport system of anticancer drugs to the target cells. The objects of the study were cells of peripheral blood mononuclear fraction of healthy donors and cells of breast adenocarcinoma lines MCF-7 and MCF-7/MDR1, differing in chemosensitivity. G2-Dox and 3D-G2-Dox accumulated in tumor cells of the both lines better than free Dox (p<0,05). However removal of these drugs out of cells MCF-7 and MCF-7/MDR1 was significantly different: in the latter case all free Dox was excluded from the cells for 24 hours while Dox, accumulated in composition with dendrimers, still remained in the cells. It was important that 3D-G2-Dox (unlike the G2-Dox) accumulated in normal cells worse than free Dox (p<0.01). Thus, the results indicate that the use of 3D-G2-Dox is the most promising because it accumulates in tumor cells better and in normal cells worse than free Dox. Furthermore it can be assumed that the use of 3D-G2-Dox would be especially useful in cases of multi-drug resistance associated with the high expression of P-glycoprotein.

Increasing the Efficiency of Parkinson's Disease Treatment Using a poly(lactic-co-glycolic acid) (PLGA) Based L-DOPA Delivery System.

To compare the efficacy of L-DOPA administered intranasally in the form of nanoparticles (nano-DOPA) and in standard drug forms using a rat Parkinson's Disease (PD) model. L-DOPA-containing nanoparticles (250±50 nm) were synthesized using the double emulsion method. The efficacy of nano-DOPA therapy was studied in Wistar rats with 6-OHDA-induced PD. Drugs were administered daily, 0.35 mg/kg (by L-DOPA). Animals' motor coordination and behavior were analyzed using the forelimb placing task and several other tests. Thirty minutes after the first administration, animals treated with L-DOPA, L-DOPA+benserazide, and nano-DOPA showed equally significant (p<0.05) improvements in coordination performance in comparison to the non-treated group. After 4 weeks of treatment, coordination performance in the nano-DOPA group (89±13% of the intact control level) was twice as high as in the L-DOPA and L-DOPA+benserazide groups, which did not differ from non-treated animals. The effect of nano-DOPA was significantly higher and more long-lasting (90±13% at 24 h after administration); moreover, it was still significant one week after the treatment was discontinued. Intranasal nano-DOPA was found to provide a lasting motor function recovery in the 6-OHDA-induced rat PD model with the effect sustained for one week after discontinuation, while the same doses of standard drugs provided significant effect only after the first administration. L-DOPA administered in the form of PLGA-based nanoparticles had a higher effective half-life, bioavailability, and efficacy; it was also efficiently delivered to the brain by intranasal administration.

Telomerase as a tumor marker in diagnosis of prostatic intraepithelial neoplasia and prostate cancer.

BACKGROUND: Early diagnosis of prostate cancer (CaP) can be addressed by studying prostatic intraepithelial neoplasia (PIN) as precancer (high-grade PIN or HGPIN). This article attempts to analyze the diagnostic role of telomerase as an early marker of carcinogenesis. METHODS: Complex urological patient evaluation and assessment of telomerase activity. RESULTS: Out of 92 patients 44% were diagnosed with CaP, 49% with low-grade PIN (LGPIN) in association with benign prostatic hyperplasia (BPH), and 7% with HGPIN in association with BPH. Active telomerase (AT) in prostate biopsy specimens was detected in 98% of patients with CaP, in 33% of patients with HGPIN, and in 20% of patients with LGPIN. In the event of simultaneous detection of AT and PIN in initial prostate biopsy specimens, further monitoring for 0.5-4.0 years revealed CaP development in 50-56% of cases. Further follow-up of patients with PIN and absent telomerase activity in initial biopsy specimens did not demonstrate the development of CaP. The PSA level was significantly higher in patients with active telomerase in the prostate tissue than in telomerase negative patients. CONCLUSIONS: Telomerase activity in the prostate tissue increases the risk of CaP development in patients with PIN. Detection of telomerase activity in prostate biopsy specimens from patients with PIN enables selection of a group of patients with high risk of CaP development and reduction of the number of prostate biopsies performed in other patients.

Targeted delivery of doxorubicin: drug delivery system based on PAMAM dendrimers.

Polyamidoamine (PAMAM) dendrimers of the second generation (G2) are branched polymers containing 16 surface amino groups that allow them to be used as universal carriers on creating systems for drug delivery. G2 labeled with fluorescein isothiocyanate (FITC) efficiently bound with the surface of tumor cells at 4°C and was absorbed by the cells at 37°C. The covalent binding to G2-FITC of a vector protein, a recombinant fragment of the human alpha-fetoprotein receptor-binding domain (rAFP3D), increased the binding and endocytosis efficiency more than threefold. Covalent conjugates of G2 with doxorubicin (Dox) obtained by acid-labile linking of cis-aconitic anhydride (CAA) without the vector protein (G2-Dox) and with the vector protein rAFP3D (rAFP3D-G2-Dox) were accumulated by the tumor cells with high efficiency. However, a selective effect was observed only in rAFP3D-G2-Dox, which also demonstrated high cytotoxic activity against the human ovarian adenocarcinoma SKOV3 cells and low cytotoxicity against human peripheral blood lymphocytes. Based on these results, rAFP3D-G2 conjugate is promising for selective delivery of antitumor drugs.

A new system for targeted delivery of doxorubicin into tumor cells.

The use of vector molecules for the targeted delivery of antitumor drugs provides their selectivity for cancer cells. The recombinant receptor-binding fragment of alpha-fetoprotein (rAFP3D) was used as a vector molecule. The specific receptor of alpha-fetoprotein is a universal tumor marker, being expressed on the surface of many tumor cells, but not in normal human tissues. And rAFP3D includes the receptor binding cite of AFP. A three-component delivery system including vector protein rAFP3D, PAMAM G2 dendrimer and antitumor antibiotic doxorubicin (Dox) was synthesized. The attachment of two dendrimer molecules to the vector protein did not affect the effectiveness of rAFP3D binding to AFP receptor on the surface of tumor cells nor the effectiveness of receptor-mediated endocytosis. Dox was conjugated with G2 via cis-aconitic anhydride (acid labile linker). The in vitro Dox release study showed that the conjugate was stable at neutral pH but was labile at pH<6. The Dox release was correlated with the intracellular distribution of conjugate in tumor cells. The rAFP3D-G2-Dox conjugate demonstrated a high cytotoxic activity against human ovarian adenocarcinoma cell lines: Dox-sensitive SKOV3 cells and Dox-resistant SKVLB cells and was low-toxic against human peripheral blood lymphocytes. Based on our findings, we may conclude that it is possible to significantly increase the effectiveness of Dox delivery to tumor cells by using the targeted delivery system comprising the recombinant third domain rAFP3D as a vector molecule.

[The distribution of iodine-125 labeled alpha-fetoprotein in the animal organism and its accumulation in the tumor].

The distribution of iodine-125 labeled human alpha-fetoprotein in mice was studied after its intravenous injection. The maximal accumulation of alpha-fetoprotein in different tissues and organs of animals was observed mainly 5 hours after injection. Then the protein was gradually eliminated from the body. In the liver, intestine and blood of intact animals 125I-alpha-fetoprotein persists for at least three days. Accumulation of alpha-fetoprotein in various tissues and organs may determine the different biological effects of this protein. In the mice with transplanted lymphatic leukemia cells P388 the high level of alpha-fetoprotein accumulation was detected in the tumor tissue, reaching 6% of the injected amount per 1 g of tissue. This allows considering the radionuclide-labeled alpha-fetoprotein as a promising medical radionuclide marker for the radiological detection of malignant tumors.

New protein vector ApE1 for targeted delivery of anticancer drugs.

A new chimeric gene ApE1 encoding the receptor-binding domain of the human alpha-fetoprotein fused to a sequence of 22 glutamic acid residues was constructed. A new bacterial producer strain E. coli SHExT7 ApE1 was selected for ApE1 production in a soluble state. A simplified method was developed to purify ApE1 from bacterial biomass. It was shown that the new vector protein selectively interacts with AFP receptors on the tumor cell surface and can be efficiently accumulated in tumor cells. In addition, ApE1 was shown to be stable in storage and during its chemical modification. An increased number of carboxyl groups in the molecule allows the production of cytotoxic compound conjugates with higher drug-loading capacity and enhanced tumor targeting potential.

[Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters.

Molecular and physiological mechanisms of membrane receptor systems functioning.

Molecular physiology is a new interdisciplinary field of knowledge that looks into how complicated biological systems function. The living cell is a relatively simple, but at the same time very sophisticated biological system. After the sequencing of the human genome, molecular physiology has endeavored to investigate the systems of cellular interactions at a completely new level based on knowledge of the spatial organization and functions of receptors, their ligands, and protein-protein interactions. In recent years, the achievements in molecular physiology have centered on the study of sensor reception mechanisms and intercellular data transfer, as well as the immune system physiology, amongst other processes.

[High-efficient renaturation of immobilized recombinant C-terminal fragment of human alpha-fetoprotein].

C-terminal fragment of a human oncofetal alpha-fetoprotein (AFP) may be used in targeted cytostatics delivery to malignant cells of many tumors. AFP fragment (from 404 to 595 amino acids residues of a full-sized protein) was cloned and produced in Escherichia coli cells, BL21 strain (DE3) in the form of inclusion bodies. To obtain a functionally active protein, is it necessary to renature the protein. The renaturation procedure of the AFP third domain (rAFP3D) is considerably complicated by the fact that the protein is hydrophobic and contains a large number of S-S bonds. A renaturation technique of rAFP3D immobilized on silicic metal chelate resin has been developed. The yield of renatured C-terminal fragment was no less than 60% with purity on the order of 98%. The developed technique has been applied for the first time for hydrophobic protein with a large number of S-S bonds. The approach can be applied for efficient renaturation of other hydrophobic proteins with a large number of disulfide bonds for scientific and practical purposes.

Targeted delivery of anti-cancer growth inhibitory peptides derived from human alpha-fetoprotein: review of an International Multi-Center Collaborative Study.

The alpha-fetoprotein derived growth inhibitory peptide (GIP) is a 34-amino acid peptide composed of three biologically active subfragments. GIP-34 and its three constituent segments have been synthesized, purified, and studied for biological activity. The GIP-34 and GIP-8 have been characterized as anticancer therapeutic peptides. An multicenter study was initiated to elucidate the means by which these peptide drugs could be targeted to tumor cells. The study first established which cancer types were specifically targeted by the GIP peptides in both in vitro and in vivo investigations. It was next demonstrated that radiolabeled peptide ((125)I GIP-34) is specifically localized to rodent breast tumors at 24 h post-injection. The radionuclide studies also provided evidence for a proposed cell surface receptor; this was confirmed in a further study using fluorescent-labeled GIP-nanobeads which localized at the plasma membrane of MCF-7 breast cancer cells. Finally, it was readily demonstrated that GIP conjugated to either fluorescein or doxorubicin (DOX) underwent tumor cell uptake; subsequently, DOX-GIP conjugates induced cytotoxic cell destruction indicating the utility of GIP segments as cancer therapeutic agents. Following a discussion of the preceding results, a candidate cell surface receptor family was proposed which correlated with previous published reports for a putative AFP/GIP receptor.

[Purification and characterization of recombinant human alpha-fetoprotein fragment, corresponding to the C-terminal structural domain].

Human alpha-fetoprotein (hAFP) is the main human oncofetal protein. Receptor of hAFP is expressed on the surface of different types of cancer cells, but not produced by normal cells of the adult organism. The hAFP interacts with the receptor via its third domain. The conjugates of native hAFP with a variety of natural cytostatic agents inhibit growth of cancer cells in vivo and in vitro. The C-terminal hAFP fragment comprising amino acids from 404 to 595 of the native hAFP was expressed in E. coli BL21 (DE3) strain. The level of the protein expression was no less than 150 mg/l. Highly efficient purification and refolding procedures were developed. The final protein yield was no less than 50% with purity of about 95%. Refolded rAFP3D bound specifically with cancer cells carrying hAFP receptor and was accumulated by them. This rAFP3D can be used as a carrier for the targeted drug delivery to cancer cells.

[Telomeric oligonucleotide complexes with PGEk protein vector: internalization by target cells and antiproliferative properties].

The recombinant protein PGEk, containing residual of the human epidermal factor (hEGF) bearing DNA binding sequence, retains ability of hEGF to bind with hEGF receptor and to induce cell proliferation was shown. On an example of PGEk complexes with telomeric mimic-oligodeoxyribonucleotide d(TTAGGG)4 and with its thio-analogue we had found such systems can be effectively and selectively internalized by hEGF receptors super expressing cells. The association of this process with a protein/oligonucleotide ratio in complexes was investigated. The intracellular localization of oligonucleotides was explored. We had shown that PGEk not only promotes intensive delivery of oligonucleotides, but also protects them from degradation by nucleases. The oligonucleotides in composition of complexes have considerably more expressed cytotoxic activity in comparison with free oligonucleotides.

[Prostate-specific antigen and telomerase in prostatic cancers].

A correlation between the blood level of prostate-specific antigen (PSA) and the prostatic tissue activity of telomerase was analyzed in prostate cancer (PC), low- and high-grade prostatic intraepithelial neoplasia (LG PIN, HG PIN) and in benign prostatic hyperplasia (BPH). The study was based on the results of a comprehensive examination of 92 patients from the Clinic of Urology, I. M. Sechenov Moscow Medical Academy. 44% of the patients were diagnosed as having PC; 49% had LG PIN and BPH; 7% had HG PIN and BPH. Active telomerase in the prostate biopsy specimens was found in 98% of the patients with PC, in 33% of those with HG PIN, and in 20% of those with LG PIN. When active telomerase was detected in the prostate biopsy specimens of patients with urological cancer (PC, PIN, BPH, the blood content of total PSA) was statistically significantly higher than that in the absence of this enzyme.

[Anatomical pathology and molecular diagnosis].

150 years ago, R. Virchow proposed a "cellular pathology" theory that forced one to revise many concepts of the mechanisms responsible for the development of disease and marked the beginning and further development of anatomical pathology as an independent discipline. Rapid progress in immunology, genetics, biotechnology, and cellular and molecular biology in the late 1980s to the early 1990s gave rise to a new field, namely molecular medicine. Damage changes the profile of expression some genes, activates various signal systems, and, due to of intercellular and cellular-matrix interactions, then spreads first at the level of organs, then at that of the whole body if a pathological process cannot localize. By involving some cells, the pathological process cannot cause characteristic morphological changes and therefore traditional studies yield a negative result. Molecular pathology became a necessary additional tool in the work of a pathologist, by allowing him to obtain the information that had been earlier beyond the reach, which increased the validity of diagnosis. Some points of the Virchow "cellular pathology" theory are supported by molecular pathology.

[Inactivation and sensitization of the tumor cells by the gene Bax transfection].

After the transfection of the gene Bax into the cultured tumor cells of human ovary adenocarcinoma SKOV3 and uterus carcinoma HeLa in vitro the high sensitivity of the cells SKOV3 to the protein Bax produced after the gene Bax transfection was found. The sensitivity of the cells HeLa to the gene Bax transfection was much smaller. The hyperexpression of gene Bax and hypersensitivity to doxorubicin were seen in HeLa cells received as a result of the gene Bax transfection and subsequent selection. All cells of the line SKOV3 with the increased expression of the transfected gene Bax died. In the cell line SKOV3 the mutation in a gene Bax was found which has a genotype G7/G9 against a native type of a gene Bax--G8/G8. It was concluded that the found in the exone 3 of the gene Bax mutation G7/G9 in cells SKOV3 results in an inactivation of proapoptotic activity of the protein Bax.

[PCR system for the detection of the RNA of hepatitis A virus]. / Razrabotka i ispytanie PTSR test-sistemy dlia detektsii RNK virusa gepatita A.

The detection of the causative agent of hepatitis A in the patient's body is the necessary element of the diagnostics of this disease. In this work the PCR system for the analysis of the RNA of hepatitis A virus in the patient's blood is presented and characterized. The method of the detection of the RNA of hepatitis A virus is based on the "nest" principle and consists of two consecutive reactions. In the first reaction the reverse transcription and amplification with the external pair of primers are carried out. The product thus obtained is used as material for the second reaction with the internal pair of primers. This method was used for the study of 44 blood samples from hepatitis A patients and 23 blood samples from healthy donors. The detection rate of the RNA of hepatitis A virus in blood samples from the patients was 82%. Viral RNA could be detected in the serum in 72% of cases, both in the serum and in mononuclear blood cells in 20% of cases, in mononuclear blood cells only in 8% of cases.