82 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drug Research Oral Presentation 
Senior Scientists’ Forum 
 
  
  83
The binding properties of some novel ruthenium (III) complexes 
with human serum transferrin 
 
1Arsene Andreea L., 2Uivarosi Valentina, 1Mitrea Niculina, 1Dragoi Cristina M.,  
1Nicolae Alina  
 
University of Medicine and Pharmacy ”Carol Davila”, Faculty of Pharmacy, Bucharest, Romania 
1Department of Biochemistry 
2Department of Inorganic Chemistry 
 andreeanitulescu@hotmail.com 
 
Background. The transferrin cycle has gained increased interest in recent years and it 
holds promise as an attractive system for strategies of drug targeting to tumor tissues. 
Indeed, tumor cells exhibit a large demand of iron for their growth and therefore express 
the transferrin receptor at a high rate. As a consequence, transferrin conjugates that retain 
a good affinity for the transferrin receptor can preferentially interact with cancer cells. 
This strategy is exploited nowadays for targeting novel anti-cancer drugs. 
Following the success of cisplatin, several metal complexes other than platinum have 
been considered over the years as possible alternatives to cisplatin, particularly it was 
found that ruthenium (III) compounds possess antitumor and antimetastatic activities. 
The literature studies showed the affinity of these complexes for crucial biomolecules 
(like transferrin) and provide evidence for formation of stable adducts between them. 
Objectives. The paper presents the transferrin-binding properties of some ruthenium (III) 
complexes with quinolones and dimethylsulfoxide, having general formula 
RuL2(DMSO) mCl3·nH2O ((Ru-nf) L: norfloxacin (nf), m = 1, n = 1; (Ru-cpx) L: 
ciprofloxacin (cpx), m = 2, n = 2; (Ru-of) L: ofloxacin (of), m = 1, n = 1; (Ru-levo) L: 
levofloxacin (Levo), m = 2, n = 8; (Ru-pip) L: pipemidic acid (pip), m = 1, n = 2, DMSO: 
dimethylsulfoxide). In this regard we investigated, in vitro, the interactions of these 
ligands with human transferrin through spectroscopic techniques, with the ultimate goal 
of preparing adducts with good selectivity for cancer cells. 
Results and Conclusions. By analyzing the obtained experimental results a series of 
conclusions could be drawn: all studied complexes interact with human serum transferrin; 
the molar ratio [complex]/[transferrin] strongly influences the binding affinity and our 
research established that the best interaction between the complexes studied and 
transferrin is achieved for a molar ratio of 8; the best interaction was registered for 
ruthenium (III) complex with pipemidic acid (Ru-pip), followed by ruthenium (III) 
complex with norfloxacin (Ru-nf) 
 
 84 
Comparison of dual acting and conventional NSAIDs towards 
parameters of NO-synthase system and oxidative stress in mucosal 
membrane of large intestine of rats with experimental ulcerative 
colitis  
1Sklyarov A. Ya., 2Lesyk R. B., 1Panasyuk N. B., 1Fomenko I. S., 2Havryluk D. Ya. 
Danylo Halytsky Lviv National Medical University, 
69, Pekarska St., Lviv, Ukraine, 79010 
1 Department of Biochemistry  
2 Department of Pharmaсeutical, Organic and Bioorganic Chemistry  
sklyarov@meduniv.lviv.ua; dr_r_lesyk@org.lviv.net 
Background. Although the etiology of ulcerative colitis remains unknown, mounting 
evidence implicates increased eicosanoids production in the inflammatory process of this 
disease. In the past few decades several compounds have been developed to block both 
COX and 5-LOX. Dual COX/LOX inhibitors constitute a valuable alternative to classical 
NSAIDs and selective COX-2 inhibitors for treatment of inflammatory diseases. 
Moreover, they appear to be almost exempt from gastrointestinal toxicity. The role of 
COX/PGs and LOX/LTs systems in the pathogenesis of ulcerative so far remains 
disputable and leaves much to be elucidated.  
Aim of this research was to compare the action of nonspecific COX inhibitor 
indomethacine, COX-2 selective inhibitor celecoxib, 5-LOX blocker AA-861 and 2-
amino-5-(3,5-ditertbutyl-4-hydroxybenzylidene)-thiazol-4-one (2A5DHT), which is the 
active substance of dual COX-2/5-LOX inhibitor darbufelone on indexes of NO-synthase 
system and intensity of oxidative stress in the mucous membrane of the large intestine 
(MMLI) under condition of experimental ulcerative colitis.  
Results. In an experimental model of inflammatory bowel disease, nonspecific COX 
inhibition with indomethacin resulted in a significantly worse clinical condition, as 
compared with acetic acid colitis alone. Under COX-2 inhibition with celecoxib 
cytoprotective processes in MMLI were enhanced and oxidative stress levels were 
reduced. 5-LOX inhibition with AA-861 revealed no significant effect on structure-
hemorrhagic lesions in MMLI caused by the administration of 4% acetic acid. 
Concomitantly, iNOS activity was reduced, MDA content displayed a tendency to the 
decrease. COX-2/5-LOX inhibition by 2A5DHT compound did not cause considerable 
destructive changes of the MMLI of rats. The activity of inducible nitric oxide synthase 
(iNOS) declined more than 2 fold as compared to their activity in colitis. The intensity of 
lipoperoxidation processes was found to be much lower than under the separate effect of 
celecoxib or indomethcine.  
Conclusions. Dual COX-2/5-LOX inhibition with 2A5DHT compound displayed 
significant cytoprotective effect, manifested by the decreased area of the MMLI lesions, 
and the decline of NO-synthases activities and the intensity of lipoperoxidation 
processes. The substance 2А5DHT significantly over exceeds the cytoprotective effects 
of both selective and non-selective COX/LOX inhibitors and can be used in the treatment 
of inflammatory bowel disease. 
  85
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drug Research Oral Presentation  
Yong Scientists’ Forum  
 
 86 
Design, synthesis and antitumor activity screening of novel 
heterocyclic derivatives of 4-thiazolidinones based on the hybrid 
pharmacophore approach 
 
Havrylyuk Dmytro, Zimenkovsky Borys, Lesyk Roman 
 
Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National 
Medical University,  
69, Pekarska St., Lviv, Ukraine, 79010 
d‐havrylyuk@ukr.net; dr_r_lesyk@org.lviv.net 
 
4-Thiazolidinone and 1H-indole-2,3-dione are the privileged scaffolds in modern 
medicinal chemistry having broad spectrum of biological activity and wide possibility to 
their chemical modification. The evaluation of antitumor activity is actual and promising 
for these compounds. The systematic study of 4-thiazolidinones derivatives with 
heterocyclic fragments in the molecules allowed us to identify a number of high-active 
compounds as potential antitumor agents [1,2]. The mechanisms of antitumor 4-
thiazolidinones can be associated to their affinity to JNK-stimulating phosphatase-1 (JSP-
1), tumor necrosis factor TNF, anti-apoptotic biocomplex Bcl-XL-BH3, integrin αvβ3 
receptor, nonmembranic protein tyrosine phosphatase (SHP-2), etc [2]. Besides that, the 
1H-indole-2,3-dione derivatives possesses a potent anticancer activity as tyrosine kinase 
inhibitors and inhibitors of cyclin-dependent kinases (CDKs). From another hand various 
diazole derivatives (pyrazoles and pyrazolines) as inhibitors of cyclin-dependent kinase, 
heat shock proteins, vascular endothelium growth factors and P-glycoprotein were 
identified.  
It is known that combination of different bioactive fragments with complementary 
pharmacophoric functions or with different mechanisms of action often showed 
synergistic effects. Therefore, we synthesized non-condensed heterocyclic compounds by 
linking the main structural unit of the 4-thiazolidinone, 1,3-dihydroindol-2-one and 
pharmacological attractive pyrazoline moieties and carried out the evaluation of their 
antitumor activity in vitro. 
Antitumor activity screening is conducted within the framework of DTP scientific 
program of National Institutes of Health (Bethesda, USA). Anticancer activity assays of 
18 compounds allowed us to determine the high activity for 16 derivatives on leukemia, 
melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancers cell lines in 
concentration 10-5 M. Subsequently, these compounds were selected in advanced assay 
against a panel of approximately sixty tumor cell lines at 10-fold dilutions of five 
concentrations (100µM, 10µM, 1µM, 0.1µM and 0.01µM). Finally, the tested 
compounds showed a broad spectrum of growth inhibition activity against all human 
tumor cells in micromolar concentrations at the GI50 level (average logGI50 values <-
6.00). Among the tested compounds, Les-3833 was found to be the most active candidate 
with average logGI50 and logTGI values -7.46 and -6.81 respectively. Currently, the 3 
  87
lead-compounds from this group are under in-depth in vivo studies according to the 
decision of NCI Biological Evaluation Committee. One should note, that mentioned 
compounds showed no toxicity in Nontumored Animal Toxicity Assays.  
 
NH
NAr1
Ar2
S
NH
O
S NR2
O
O
R 1
N
N
Ar1
Ar2
S
N O
NR2O
R 1
A design for synthesis of 4-thiazolidinones 
     based on the molecular hybridization
Tested 18 compounds
Selected 16 "hits" in vitro
Current status of antitumor activity screening
                by NCI DTP Program 
I Phase
Prescreening at 60 cancer 
cell lines in concentration 10-5M
II Phase
60 cell lines assay at 10-fold 
dilutions of five concentrations  
III Phase
Consideration by 
Biological Committee NCI
IV Phase
Advanced biological studies 
in vivo 
Tested 16 compounds
Selected 5 compounds for
consideration by 
Biological Committee NCI
Selected 3 compounds for
in-depth study as
drug-candidates 
In-depth study of 3 compounds
are in progress   
 
In conclusion, these preliminary results allowed us: to identify the high group potency of 
synthesized compounds on antitumor activity assays in vitro; to select 3 lead-compounds 
for in vivo studies; to determine no toxicity of mentioned compounds in vivo. 
Consequently, the hybridization of 4-thiazolidinone template with 1,3-dihydroindol-2-
one and pyrazoline moieties in one molecule is promising approach in drug-like 
molecules design. 
 
[1] R. B. Lesyk, and B. S. Zimenkovsky. 2004. 4-Thiazolidones: Centenarian History, Current Status and 
Perspectives for Modern Organic and Medicinal Chemistry. Current Organic Chemistry, 8: 1547-1577. 
[2] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko et al. 2009. Synthesis of novel thiazolone-based 
compounds containing pyrazoline moiety and evaluation of their anticancer activity. European Journal of 
Medicinal Chemistry, 44(4): 1396-1404. 
 88 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Drug Research Young Scietists’ 
Poster Presentation 
 
  89
Synthesis, transformations and anti-inflammatory activity study of  
3H-thiazolо[4,5-b] pyridine derivatives 
 
Chaban T. I., Ogurtsov V. V. 
Danylo Halytsky Lviv National Medical University, Lviv, Ukraine 
69, Pekarska St., Lviv, Ukraine, 79010 
 
chemdep@meduniv.lviv.ua 
The intensive researches have been focused on five-membered heterocycles condensed with the 
pyridine ring in recent year as the potential anti-inflammatory drugs. Thiazolо[4,5-b]pyridines 
are ones of the least accessible and in turn the least-studied organic compounds. Their biological 
activity screening data are also not provided extensively. Studies on biochemical properties of 
these structures reveals they are possessing antifungal activity, can act as H3-histamine and 
metabotropic glutamate 5 (mGluR5) receptors agonists and also been found to provide high 
inhibitory activity against epidermal growth factor receptor and a number of other enzymes. 
In this work we are reporting the convenient 3H-thiazolo[4,5-b]pyridine-2-one derivatives 
synthetic route development. 4-Iminothiazolidone-2 was used as the starting compound, which 
was treated with fenylazoacetylacetone to form 5,7-dimethyl-6-fenylazo-3H-thiazolo[4,5-
b]pyridine-2-one. The compound properties allowed to detect the acidic character of the proton 
in the thiazole ring third position, so its treatment with potassium hydroxide yielded the 
corresponding potassium salts. The nucleophilic properties of 5,7-dimethyl-6-fenylazo-3H-
thiazolo[4,5-b]pyridine-2-one were exhibited in its reaction with electrophilic agents to form N-
alkylated products. A series of appropriate alkyl halides, monochloroacetic acid and its 
derivatives were used as alkylating agents. 
 
 
The structures of 5,7-dimethyl-6-
fenylazo-3H-thiazolo[4,5-b]pyridine-2-
one and its N-alkyl derivatives were 
established on the basis of H-NMR 
spectra.  
The anti-inflammatory activity of the 
synthesized compounds was examined. 
They were evaluated for anti-exudative 
effect in vivo using carrageenin-induced 
paw oedema test in white rats. Ibuprofen, 
butadion and diclofenac sodium in their effective therapeutic dose were used as standards. The 
screening results demonstrate the significant anti-inflammatory action of 3-substituted 5,7-
dimethyl-6-phenylazo-3H-thiazolo[4,5-b]pyridine-2-ones while the nature of the substituent in 
thiazole ring third position is the defining criteria for the anti-inflammatory activity enhancing. 
The current research provides a new lead for developing potential anti-inflammatory drugs based 
on 3H-thiazolo[4,5-b]pyridine-2-one scaffold.
S
NH
NH
O
CH3 OH
N
CH3
ONPh
S
NH
O
NCH3
CH3
N
NPh
S
NK
O
NCH3
CH3
N
NPh
S O
NCH3
CH3
N
NPh N
Alk
KOH
HalAlk
 90 
Design of new anticancer agents – thiopyranothiazole derivatives 
 
Kaminskyy Danylo, Kryshchyshyn Anna, Lesyk Roman 
 
Department of Pharmaceutical, Organic and Bioorganic Chemistry,  
Danylo Halytsky Lviv National Medical University, 
69, Pekarska St., Lviv, Ukraine, 79010 
 
dankaminskyy@gmail.com; dr_r_lesyk@org.lviv.net 
 
Discovery of 4-thiazolidone derivatives anticancer potential provides wide opportunities 
for new anticancer agents design using this scaffold. Fused thiopyranothiazoles show 
special interest as potential source of antitumor agents. The background for our research 
was determining of the crucial role of substituent existence in the C5 position of basic 
heterocycle for antitumor activity appearance and the hypothesis that condensed 
heterocyclic systems possibly imitate the biological activity of their synthetic precursors, 
namely 5-ylidene-4-thiazolidinones. Previous research held in the department of 
Pharmaceutical, Organic and Bioorganic Chemistry and SAR data allowed us to outline 
the directions of rational design of novel thiopyranothiazole derivatives. 
S
N
S
R1
X
R
R2
S
NS
R
X
R2 R1
S
H
H
S
N
R
X
SN
S S
N
XX
RR S
O
S
N
X
H
H
R
X = O, S 
Thiopyrano[2,3-d][1,3]thiazoles 
sites for modification 
Structure modification directions
Target classes of thiazolidinone(thione) derivatives
I II III
Increase of activity level  N-3 Unsubstituted compounds (I-III) were obtained using methods developed by us and 
lie in iso- and thiorhodanines usage as starting materials in the Knoevenagel, Diels-Alder 
and domino-Knoevenagel-hetero-Diels-Alder reactions as well. It is worth to mention 
that compound I belongs to the new class of 4-thiazolidinone derivatives and is obtained 
for the first time.  
 
  91
S
S
N
O
NH
O
X
S
S
N
O
O
NH
R
S
S
N
O
O
S
S
N
O
O
O R
S
S
N
O O
N N
NOS
S
N
O
O
O
O
H
H
S
O
Me
-(CH2)nCO-
Most active derivatives Not effective modification of side chainHal or NO2
 
 
Anticancer activity evaluation of synthesized compounds was carried out within the 
Development Therapeutic Program (NCI, NIH, USA). As evidenced by the results of 
research the level of anticancer activity of tested compounds increases when passing 
from compound I to III. The derivatives of isorhodanine (Х=О) have higher activity 
level than thiorhodanine isosteres (Х=S). The best way of molecules optimization is 
introduction of the substituent in the position 3. This approach allows achieving a 
significant increase of the level and/or selectivity of the antitumor effect of the tested 
compounds in comparison with N-unsubstituted analogues. Among mentioned 
derivatives the highest anticancer activity was observed for the compounds with N-
arylacetamide or ester fragments. Complication or simplification of given fragment led to 
activity level decreasing. Introduction of the nitro group or Br atom in the position 10 of 
the compound III is needed for the anticancer activity realization unlike introduction of 
the methyl group in the position 5a. 
 92 
Anti-inflammatory, analgetic and diuretic activity prediction for   
6{[({[phenyl]sulfonyl}amino)-(oxo)acetyl]amino}hexanoic acid 
derivatives by RDF descriptors approach 
1Klenina O. V., 2Tsepelevsky A. A.  
1- Danylo Halytsky Lviv National Medical University, Lviv, Ukraine 
69, Pekarska St., Lviv, Ukraine, 79010 
2- National pharmaceutical university, Kharkiv, Ukraine 
olena_klenina@yahoo.com 
QSAR analysis methodology makes use of the molecular descriptors offering valuable 
information about the structure of molecules, which is used later in the elaboration of the 
predictive models. This allows cost savings by reducing the laboratory resources needed, and the 
time required to create and investigate new drugs with certain desired biological activity. In this 
study, RDF descriptors were used to predict anti-inflammatory, analgetic and diuretic activity for 
a series of 21 compounds 6-{[({[phenyl]sulfonyl} amino)(oxo)acetyl]amino}hexanoic acid 
derivatives, which were synthesized at National pharmaceutical university, Kharkiv, Ukraine. 
They possess significant diuretic activity (the Berlin’s test, %), 17 of them possess analgetic (%) 
against acetic acid induced writhing, and 13–anti-inflammatory (%) against histamine induced 
rats paw oedema activities. Furocemide, hydrochlorothiazide, metamizole sodium and diclofenac 
were used as standards.   
 
Firstly geometry 
optimization of 
compound using semi-
empirical PM3 method 
included in HyperChem 
7.5 was carried out. The 
DRAGON computer 
software web version 3.0 was used to calculate 150 RDF descriptors. The atomic masses, van der 
Waals volumes, Sanderson electronegativities and polarizabilities were used as bond weightings. 
The mathematical models were obtained by means of the Multiple linear Regression Analysis  
implemented in BuildQSAR software. The GA was used as the variable selection strategy. 
Regression model’s predictive power was validated by calculating Q2 values. The statistical 
significance was determined by the regression correlation coefficient, the Fischer ratio and the 
standard deviations. In order to avoid collinearity, Randic orthogonalization procedure was 
carried out.  
Obtained three-variables modes for diuretic activity contain RDF075m, RDF115m descriptors 
both making positive contribution and RDF145(u,e,p) descriptors making negative contributions 
(r=0.714-0.802, F=5.90-9.62, Q2=0.27-0.37). This corresponds to a radius of 7.5 to 14.5 Å. Two-
variables QSAR models involving anti-inflammatory activity contain positively contributing 
RDF130m and negatively contributing RDF060(m,e,p) descriptors (r=0.853-0.867, F=12.03-
13.63, Q2=0.402-0.664). This corresponds to a radius of 6.0-13.0 Å. One- and two-variables 
models involving analgetic activity contain positively contributing RDF040(u,e,p), RDF140(p,v) 
and negatively contributing RDF060(u,e,v) descriptors (r=0.842-0.88, F=17.09-24.03, Q2=0.628-
0.709), which corresponds to a radius of 4.0 to 6.0 and 6.0 to 14.0 Å. In this sense a spherical 
molecular volume could have certain restrictions to the addition of bulky substituents. 
SO N
O
O
O
N O
O
R1
R2
R3
R4
X
X = CH2 or none
R1 = H, Br, NO2          R2 = H, NO2, COOH
R3 = H, CH3, COOH, NH2, NO2, Cl, 
         Br, OCH3, COOCH3, COOC2H5,
         CH2NH2, NHCOOCH3, 
         CH2NHCOCH3, NHCONHC6H11(cyclo)
R4 = H, Cl, Br 
  93
Expression of regulatory genes in streptomycetes and 
overproduction of nogalamycin, doxorubicin and aranciamycin 
1Klymyshin D., 1Honchar M., 2Nimets O., 3Chaikovska A., 3Kychabska M., 
1Kolysnyk H., 2Fedorenko V. 
1Institute of Animal Biology, Stoosa str. 38, Lviv, Ukraine, 79034; 
2Ivan Franko National University of L’viv, Hrushevskuy str. 4, Lviv, Ukraine, 79005; 
3Danylo Halytsky Lviv National Medical University, Pekarska str. 69, Lviv, Ukraine, 79010 
dedima@rambler.ru 
Streptomycetes are an abundant source of many commercially valuable natural products, 
including clinically important antibiotic, anticancer, and antiviral compounds along with agents 
utilized in agricultural, veterinary, and food industries. Doxorubicin, nogalamycin and 
arancyamycin are the anthracycline-type antitumor drugs produced by Streptomyces peucetius 
ATCC 27952, S. nogalater Lv65 and S. echinatus DSM40730. They act against the cancer cells 
of some leukemia; breast, stomach, lung, ovary, and thyroid cancers; soft tissue sarcoma; 
multiple myeloma and other cancer types. Bacterial strains isolated from nature usually produce 
only discrete amounts of particular secondary metabolites; production of a given metabolite of 
interest must therefore be improved in order to meet commercial requirements for its isolation. 
Most secondary metabolite biosynthetic pathways involve specific activator genes and global 
regulators. These regulators are considered important targets for metabolic engineering efforts. 
In this work, regulatory studies for anthracycline producing strains improvement including 
investigations of regulatory mechanisms that act in the production of antibiotics during their 
biosynthesis in the parental strains are discussed.  
Expression of global regulators in heterological hosts is an effective approach in overproduction 
of clinically important drugs. That is why we used different global and site-specific regulators to 
improve production of doxorubicin, nogalamycin and arancyamycin. AfsS, RelA, AbsA2 
pleiotropically regulate antibiotic biosynthesis in S. coelicolor. Overexpression of these genes in 
S. coelicolor and related species causes antibiotic stimulatory effects in the host organism. In 
order to express these regulators in S. peucetius, S. nogalater and S. echinatus srains a set of 
plasmid constructions were obtained. Regulatory genes were subcloned in pKC1218E plasmid 
under the control of the erythromycin promoter and transferred into streptomycetes by 
conjugation from Escherichia coli ET12567 (pUB307). Our data shows that expression of afsS 
and absA2 in S. peucetius, S. nogalater and S. echinatus had no significantly enhancing on 
antibiotics production. Amplification of relA on a high-copy-number plasmid conferred 
overproduction of doxorubicin, nogalamycin and arancyamycin in these streptomycetes. We 
observed previously that multiple copies of site-specific regulator snorA coused to 
overproduction of nogalamycin in S. nogalater. We overexpressed this gene in S. peucetius and 
S. echinatus. Multicopies of snorA caused S. peucetius and S. echinatus to overproduce 
doxorubicin and aranciamycin, indicating that this regulator stimulates antibiotic production in 
the heterological hosts. 
Overexpression of global and site-specific regulators is a good strategy for generating of 
industrial strains at the molecular genetic level. This in turn led us to study regulatory genes 
critical for secondary metabolite overproduction. Such experiments followed by detailed 
investigation of regulatory networks would contribute to strain improvement in forthcoming 
research. 
This work was partly supported by WUBMRC Grant. 
 94 
Metabolic engineering by interspecies protoplast fusion of 
angucycline producing streptomycetes 
1Kobylyanskyy A., 2Luzhetskyy A., 2Bechthold A., 3Fedorenko V.  
1Institute of Cell Biology,  
14/16, Drahomanov St., Lviv, Ukraine79005  2Albert-Ludwigs-Universität, Institut für Pharmazie,  
19, Stefan-Meier-Straße,  79104 Freiburg, Germany 3Ivan Franko National University, Department of Genetics and Biotechnology, 
4, Hrushevskyy St. Lviv, Ukraine 79005 
kob.anton@googlemail.com 
Angucyclic antibiotics are quinone natural polyketide products bearing a characteristic 
four-ring frame of aglycon moiety, which is assembled in an angular manner. There are 
few hundreds of them characterised, all possessing antitumor activity. The majority of 
them are actinomycetes’ secondary metabolites. For three last decades protoplast fusion 
has been successfully used to modify the phenotypic traits of streptomycetes. It allows 
achieving of high frequency gene transfer and recombination. Recently this process was 
optimized towards a recursive multiparental fusion and combined with high-throughput 
screening, which greatly accelerated the process of strain development. 
Our approach involves genome shuffling of Streptomyces strains by means of protoplast 
fusion. These parental strains carry antibiotic resistance marker insertions in angucycline 
biosynthetic pathway genes. It stipulates recombination between them, leading to the 
formation of chimerical clusters, consistent of two parental ones. By applying this 
technology we aimed to provide the production of novel biologically active secondary 
metabolites and extend our knowledge about recombination events and enzyme substrate 
specificity. Fused mutants should be able to restore the missing glycosylation or 
oxygenation steps owing to complementation of metabolic pathways by the enzymes of 
both parental metabolic pools. Flexibility of polyketide modifying enzymes allows 
introduction of new functional groups or additional sugars in unusual for both parents’ 
products positions. 
Obtained recombinants displayed different morphology and secondary metabolites 
spectra in comparison with parental strains. The most remarkable was recombinant of S. 
fradiae ∆urdQ/R (olivose-biosynthesis deficient mutant of S. fradiae Tü2717) and S. 
globisporus M12 (oxygenase gene mutant of landomycin E producer S. globisporus 
1912) called Rec5. It accumulates 40 angucyclic compounds, among which four are 
novel, i.e. glycosilated tetrangomycin and tetrangulols. 
Metabolic engineering by means of interspecies protoplast fusion with consequent 
screening of antibiotic resistant recombinants is fruitful in case of angucycline producing 
mutants with damaged terminal polyketosynthetic steps. As a result of one fusion we 
obtained a recombinant producing a whole collection of compounds. It is a promising 
method to proceed in strain modification when inductive mutagenesis reaches deadlock. 
Beside pure combinatorial profit, a great deal of data on gene functions and nature of 
enzymes’ interaction had been won. 
  95
The cytoprotective effect of hexapeptide arginyl-alfa-aspartyl-lysyl-
valyl-tyrosyl-arginine in experimental gastric lesions in rats 
Nasadyuk Christina, Sklyarov Alexandr 
Danylo Halytsky Lviv National Medical University 
69, Pekarska St., Lviv, Ukraine, 79010 
sklyarov@meduniv.lviv.ua 
 Background. Recent research showed the high gastroprotective activity of some 
olygopeptides in particular glyprolines, BPC 160 and hexapeptide arginyl-alfa-aspartyl-
lysyl-valyl-tyrosyl-arginine (AALVTA). The positive effects of olygopeptides on gastric 
mucosa (GM) could be explained by their multidirectional influence on homeostasis, 
intercell communication, receptors of γ-aminobutyric acid cooperating with the systems 
of dopamine, NO and prostaglandins [Ilic S. et al. 2009; Samonina G. et al. 2008; O. 
Orlovsky, 2007].  
Objective and methods: Aim of research was to study the cytoprotective action of 
AALVTA in interconnection with the systems of NO-synthases (NOS), cyclooxygenases 
(COX) and lipooxygenases (LOX) in experimental gastric lesions (GL) in rats. GL in rats 
were induced by intraperitoneal application of epinephrine (2 mg/kg). Experimental 
animals were pretreated with AALVTA (1µg/100g) solely and in combination with 
COX/LOX blocker darbufelon (20 mg/100g) and iNOS blocker aminoguanidine (20 
mg/kg).  
Results: The rats introduced to epinephrine developed severe GL, accompanied by 
increase of the activity of total NOS (by 133%) in GM in particular due to the increase of 
iNOS activity (6 times), increase of NO (by 50%) and decrease of the level of L-arginine 
in plasma (by 37%).  Pretreatment with AALVTA led to 50% decrease of the area of GL, 
56% decrease of the activity of total NOS and 62% decrease of іNOS, 31% decrease of 
the content of NO in GM as well as tendency to increase of the level of L-arginine in 
plasma. Pretreatment with AALVTA under conditions of iNOS blockage by 
aminoguanidine in rats introduced to epinephrine provided decrease of the damaged area 
of the stomach by 61% compared to mono action of AALVTA, enhanced blockage of 
iNOS, decrease of NO in GM and increase of the level of L-arginine in plasma. The 
administration of COX/LOX blocker darbufelon on the background of AALVTA in 
animals introduced to epinephrine didn’t provide significant cytoprotection and showed 
tendency to increase of the activity of eNOS.  
Conclusions: AALVTA provides cytoprotection in experimental GL in rats in particular 
through the modulation of the activity of NOS, which is more expressed under the 
conditions of iNOS blockage by aminoguanidine and is not enhanced by the dual 
COX/LOX blocker darbufelon. So the mechanisms of the cytoprotective effect of 
AALVTA in interconnection with the systems of COX/LOX need further profound 
investigations. 
 96 
Сhanges in PUFAs due to amaranth oil introduced at the 
background of experimental colitis  
Panasyuk N.B., Sklyarov O.Ya. 
Danylo Halytsky Lviv National Medical University  
69, Pekarska St. Lviv, Ukraine, 79010 
sklyarov@meduniv.lviv.ua 
Introduction: Ulcerative colitis is the most common pathology of large intestine. 
Development of ulcerative colitis is accompanied by structure-hemorrhagic changes and 
increase in proanti inflammatory cytokins, proapoptic proteins and prooncogens. In 
ulcerogenic colitis, balance between pro-oxidant and antioxidant systems becomes impaired 
and activity of inducible NO-synthase enhanced. Role of the short-chain and long-chain fatty 
acids in cytoprotective mechanisms in the mucous membrane of digestive organs is essential 
because these acids are the source for synthesis of prostaglandins and leukotrients 
Material and methods: Experimental colitis was modeled by 1ml of 4 % acetic acid 
injected into rats’ large intestine. Amaranth oil were given per os in the dose of 0.2 ml/100 g 
of body weight Then were determined: area and degree of destructive changes in the mucous 
membrane of large intestine, alterations in lipoperoxidation processes (MDA), activity of 
SOD, catalase, NOS, iNOS, cNOS, NO in MMLI and L-arginine contents in blood. Fatty 
acids were determined by the method of norm setting, peak areas of methyl derivatives of the 
fatty acids were measured and their content calculated in percentage. Obtained results were 
processed by the variation statistics method at Student’s t-criterium determined. 
Results: Injection of acetic acid caused ulcerative defects, erosions, hemorrhages in the 
MMLI. There were observed: 2.5-fold enhancement of general NOS activity (P<0.05), сNOS 
– by 21%, iNOS – 6.9-fold (P<0.001), NO content increased by 64%. Blood L-arginine 
decreased by 50% (P<0.05), MDA content increased 2.2-fold (P<0.001), SOD activity – by 
71% and catalase – by 54%. Content of polyunsaturated acids (linoleic, arachidonic) in the 
blood decreased by 15-34 %, content of unsaturated acids (oleic, linoleic, arachidonic) 
decreased up to 13 % versus the contents in intact animals. On introduction of amaranth oil 
at the background of ulcerogenic action of acetic acid, lesion area in MMLI diminished – 
necrotic changes, ulcers, erosions were absent; and only solitary punctuate hemorrhages were 
found at the background of hyperemized mucous membrane, macroscopic signs of colitis 
were absent at all, content of MDA products decreaced by 34 % (p<0.05), SOD activity – 
1.4-fold (p<0.01), catalase by 20%, and iNOS (by 48%, P<0.05), сNOS activity changed 
slightly, NO content in the MMLI decreased by 44% (P<0.05), concentration of L-arginine 
in blood plasma increased. Content of polyunsaturated acids in the blood increased by 26 %, 
content of unsaturated acids increased up to 16 % versus the contents in colitis animals. Most 
pronounced was the increase of linoleic acid whose content in amaranth oil is 52 %. Content 
of saturated fatty acids in the blood increased by 24 % in the animals with colitis and 
decreased up to 19 % versus the contents in colitis animals. Protective effect of amaranth oil 
is likely to be associated with the contents of unsaturated fatty acids in this oil (palmitic – 23 
%, palmitoleinoic – 0.18 %, stearic – 1.3 %, oleinoic– 22.6 %, linoleic – 52.4 %, linolenoic – 
0.39 %). Summary: Thus, obtained results of our investigation prove that amaranth oil 
displays cytoprotectiveeffect associated with the participation of w-3 and w-6 fatty acid