The 4th International Conference
on Pharmaceutical Sciences and Pharmacy Practice
dedicated to the 75th anniversary of
Lithuanian Pharmacopoeia
Book of abstracts
The International Conference on Pharmaceutical Sciences and Pharmacy practice is organized by Faculty of Pharmacy of Lithuanian Health Sciences University in collaboration with Lithuanian Pharmaceutical Association and LSMU FF Alumni Association.
Conference Organizers: Vitalis Briedis (Kaunas, Lithuania)
Vilma Petrikaitė (Kaunas, Lithuania) Antanas Tauras Mekas (Kaunas, Lithuania) Eduardas Tarasevičius (Vilnius, Lithuania) Jerzy Lazowski (Warszaw, Poland) Ilze Barene (Riga, Latvia)
ISBN 978-9955-15-308-5
© LSMU, 2013
Language of abstracts was not corrected.
CONFERENCE PROGRAMME
Zigmas Januškevičius Hall of Lithuanian University of Health Sciences (Scientific-laboratory building), Eivenių street 4, LT-50004 Kaunas, Lithuania
10.30-11.00 Registration
11.00-13.00 PLENARY SESSION, Chair: Prof. Vitalis Briedis
11.00-11.10 Opening and wellcome speech Prof. Vitalis Briedis 11.10-11.40 75th anniversary of the Lithuanian
Pharmacopoeia
Dr. Tauras Antanas Mekas
11.40-12.10 International, regional and national
Pharmacopoeias: importance and challenges of their harmonization
Prof. Eduardas Tarasevičius 12.10-12.40 Latvian Pharmacopoeia Dr. Sabine Lauze
Riga Stradins University 12.40-13.00 European Pharmacopoeia and Quality of
Medicinal Products Roma Mockutė Lithuanian State Medicines Control Agency 13.00-14.00 Lunch
14.00-15.00 ORAL PRESENTATIONS (part I), Chair: Prof. Eduardas Tarasevičius
14.00-14.15 Biological and Biosimilar Drugs. Present State of Art
Dr. Jerzy Łazowski Polish Oncology Pharmacists Association 14.15-14.30 Microscopic and macroscopic identification
of common spruce species raw material from Latvian forests
Sanita Siksna
Riga Stradins University 14.30-14.45 Assessment of digoxin pharmacokinetic
measurements in tertiary level hospital
Rima Minkutė 14.45-15.00 Pharmacokinetic properties of Ibogaine and
Noribogaine in plasma of mice
Audrius Sveikata
15.00-15.30 Coffee break
15.30-16.30 ORAL PRESENTATIONS (part II), Chair: Prof. Vitalis Briedis
15.30-15.45 Evaluation of antioxidant activity of Ginkgo biloba flavonoids using postcolumn HPLC method
Prof. Nijolė Savickienė
15.45-16.00 Counseling of pregnant and breastfeeding women in Lithuanian pharmacies: structural equitation modeling approach
Švitrigailė Grincevičienė
16.00-16.15 Results of effect of hypochlorous acid on platelet aggregation in healthy people and in patients with heart failure in vitro
Aušra Mongirdienė
16.15-16.30 Anticancer activity and structure-activity relationship of new quinoxaline derivatives
Agnė Vegytė
CONTENT
WELLCOME SPEACH 8
ORAL TALKS 9
75th anniversary of the Lithuanian Pharmacopoeia
T. A. Mekas, V. Briedis
9
International, regional and national Pharmacopoeias: importance and challenges of their harmonization
E. Tarasevičius, H. Rodovičius, T. A. Mekas
10
Latvian Pharmacopoeia
S. Lauze, B. Maurina, V. Sidlovska
11
European Pharmacopoeia and Quality of Medicinal Products
R. Mockutė
13
SHORT TALKS 15
Biological and Biosimilar Drugs. Present State of Art
Łazowski Jerzy
15 Microscopic and macroscopic identification of common spruce species raw material from Latvian forests
S. Siksna, I. Barene, I. Daberte
16
Assessment of digoxin pharmacokinetic measurements in tertiary level hospital
R. Minkutė, V. Briedis, R. Steponavičiūtė, A. Vitkauskienė, R. Mačiulaitis
18
Pharmacokinetic properties of Ibogaine and Noribogaine in plasma of mice
A. Kubilienė, A. Sveikata, I. Sadauskienė, L. Ivanov
21
Evaluation of antioxidant activity of Ginkgo biloba flavonoids using postcolumn HPLC method
A. Špadienė, N. Savickienė, L. Rimkienė, V. Jakštas, H. Rodovičius
24
Counseling of pregnant and breastfeeding women in Lithuanian pharmacies: structural equitation modeling approach
S. Grinceviciene, J. Grincevicius, L. Kubiliene, A. Baranauskas
26
Results of effect of hypochlorous acid on platelet aggregation in healthy people and in patients with heart failure in vitro
A. Mongirdienė
28
Anticancer activity and structure-activity relationship of new quinoxaline derivatives
A. Vegytė, J. Šarlauskas, A. Pavilonis, V. Petrikaitė
ABSTRACTS OF POSTERS 32 Effect of ethanol on energy metabolism and potency of extracellular matrix synthesis of pancreatic stellate cells
R. Baniene, S. Trumbeckaite, A. Maziukiene, A. Jakstaite, L. Venclauskas
32
Comparison of hemagglutinating and antibacterial activity in lectin enriched fractions from Urtica dioica L.
G. Balčiūnaitė, N. Savickienė, A. Pavilonis,D. Baniulis
33
Pharmacognostical studies of introducated plant in Lithuania Monarda
didyma L.
K. Rastenytė, N. Savickienė, O. Ragažinskienė, A. Maruška, M. Stankevičius, E. Šeinauskienė
36
Phytochemical analysis of Chamerion angustifolium L.by means of gas chromatography-mass spectrometry
M. Stankevičius, T. Drevinskas, I. Akuneca, A. Maruška, O. Ragažinskienė, V. Briedis, K. Ramanauskienė, A. Stelmakienė, R. Ugenskienė
38
Antimicrobial properties of Rosmarinus officinalis L. leaf extracts
I. Petrauskaitė, U. Čižauskaitė, J. Bernatonienė, A. Pavilonis, V. Petrikaitė
39 Antimicrobial properties of thiophene derivatives
S. Jankevičiūtė, J. Šarlauskas, A. Pavilonis, V. Petrikaitė
41 Chromatographic analysis of phenolic acids in honey of Kazakh and
Lithuanian origin
M. Žilius, B.G. Machatova, Z.B. Sakipova, K. Ramanauskienė, U.M. Dathaev, V. Briedis
42
Evaluation and comparison of quality indicators and antioxidant activity of natural fresh grape juices from the different Vitis vinifera species
S. Kazlauskaitė, G. Kasparavičienė, S. Velžienė, M. Žilius
44
The influence of solvent on the quantity and antioxidant activity of
Sambucus nigra L. extracts
L. Pudžiuvelytė, S. Velžienė, G. Kasparavičienė
47
Ethanolic extracts of Rosmarinus officinalis leaves, Matricaria recutita flowers and Foeniculum vulgare fruit production and evaluation
S. Velžienė, G. Kasparavičienė, M. Žilius, L. Jašmontaitė
49
Comparative analysis of line of ‘Bronchos’ teas
S. Velžienė, G. Kasparavičienė, M. Žilius, I. Piepolytė
50 Total protein and metallothioneins synthesis in mice liver under exposure of lead and nickel ions for two weeks
I. Šveikauskaitė, J. Šulinskienė, I. Sadauskienė, L. Ivanov
52
Oncology patients experience the additional measures used in addition to a physician prescribed treatment study
L. Nekrasaite, T.A. Mekas
ABSTRACTS OF POSTERS
The research of five antidepresants mixture using thin layer chromatography
D. Kazlauskienė, I. Matukaitytė
55
Immune-chromatographic system for detection of Amanita phalloides toxins in body fluids
Y. Bidnychenko, Z. Barsteigienė, V. Skyrius
58
Extraction of paroxetine from human plasma on H-klinoptilolite columns for GC-MS detection
I. Halkevych, L. Ivanauskas, Z. Barsteigiene
60
Pharmacognostic properties of Satureja hortensis L. introduced in Lithuania
S. Šuštauskaitė, O. Ragažinskienė, A. Maruška, M. Stankevičius, Z. Barsteigienė, E. Šeiniauskienė
61
Total flavonoid and antiradical assay of meadowsweet (Filipendula ulmaria L.) raw material during the phenological cycle
R. Žemaitytė, D. Burdulis
63
Evaluation of flavonoids of Ginkgo biloba L. leaves growing in Lithuania using HPLC method
L. Rimkienė, L. Ivanauskas,V. Jakštas
65
Modification of postcolumn HPLC method for evaluation of antioxidant activity on Solidago canadensis herbal extracts
M. Marksa, L. Ivanauskas, V. Jakštas, J. Radušienė, R. Marksienė
67
Penetration of resveratrol into the human skin ex vivo
V. Juškaitė, K. Ramanauskienė, V. Briedis
68 Synthesis of new 4-thiazolidinone sulfanilamides derivates
A. Mazurkevičiūtė, L. Šlepikas, E. Tarasevičius, J. Salys, H. Rodovičius
70 Synthesis of new rhodanine derivatives
V. Petreikis, L. Šlepikas, E. Tarasevičius, J. Salys, H. Rodovičius
72 Rhodanine derivates targeting Staphylococcus aureus
L. Zubavičius, L.Šlepikas, E. Tarasevičius, J. Salys, H. Rodovičius
73 Structure – activity evaluation of 3-Aminorhodanine derivatives with potential antifungal activity
J. Dambrauskaitė, H. Rodovičius
75
Methods of detecting genetically modified organisms and products and their ingredients in Ukraine
U. Lototska-Dudyk, N. Krupka
76
Mercazolyl biomodel of secondary iodine deficiency for toxicological researches
S.T. Zub, M.O. Kovaliv
ABSTRACTS OF POSTERS
The prevalence of self-reported underuse of medications due costs in elderly: results from seven European urban communities
A. Stankuniene, J. Lindert, R. Radziunas, A. Baranauskas, G. Kuncaite, J.F.J. Soares
80
Rapid Test for Clomipramine Detection in Blood
L. P. Kostyshyn, I. Y. Halkevych
83
Phenolic compound identification, quantification and antioxidant activity in Malus domestica Borkh. leaf ethanolic extracts
V. Žvikas, M. Liaudanskas, J. Viškelis
84
Total impact of non-ionic surfactants to stability of an (o/w) emulsion
A. Kapočiūtė, J. Bernatonienė
86
The antioxidant effects of green tea extract (GTE) in mice liver
K. Čijauskaitė, A. Špadienė, R. Bernotienė, N. Savickienė, V. Jakštas, L. Ivanauskas, L. Ivanov
88
Influence of ammonium salts of N-(3-methyl-7-acetylmethyl xanthine-8) piperazine on excitability of visceral nociceptors
V.I. Kornienko, B.A. Samura
90
New therapeutic perspectives in prevention of cardiovascular events in patients with chronic lymphocytic leukemia
B.B. Samura
91
Training of specialists in the treatment of drugs in the Republic of Belarus.
N.S. Gurina
92
Searching for the optimal European mistletoe (Viscum album L.) drug forms in oncology
E. Kazlauskis
94
Metabolites of protein nature in European wild and cultivated plants: prospects for use of lectin family glycoproteins of Urtica dioica L. and Solanum tuberosum L. in Veterinary and Medicine
O. Kandelinskaya, H. Grischenko, P. Krasochko, N. Shukanova, T. Shman, H. Elyashevich, V. Golubkov, P. Beliy, N. Savickiene, J. Bernatoniene, A. Savickas, G. Draksiene
96
Search for Promising Sources of Hyperoside
R. Lysiuk, R. Darmahray
99
Trends in the usage of hormonal preparations in Lithuania, the comparative overview of estrogenic-like active compounds of herbal origin
K. Jačunskaitė, A. Dagilytė
102
WELLCOME SPEACH
Dear participants of the conference,
It is a pleasure to welcome you all at the 4th international conference of pharmacy and allied sciences which is dedicated to the 75th anniversary of Lithuanian Pharmacopoeia. It is already a tradition to meet you at the annual conference, jointly organized by Lithuanian University of Health Sciences, Alumni organization of Faculty of Pharmacy, and Lithuanian Pharmaceutical Society. This conference is dedicated to remind essential milestones in the development of pharmacy science and practice in Lithuania. National Pharmacopoeia established a clear reference for regulation of pharmacy sphere, including scientific research and industrial manufacture of pharmaceuticals. It was a strong impetus for implementing international level quality standards, and motivating national pharmaceutical industry to orient to international markets.
Pharmacy sciences and practices are becoming more integrated with related areas of biomedical research. The development of pharmaceuticals has become increasingly challenging and needs multilateral approach today. It presents major challenges to research, development and manufacturing, to all aspects related to the safety and efficacy of products from quality control to regulatory affairs. Thus you are invited to present original approaches to existing unsolved issues in pharmacy theory and practice, to discuss the risks and opportunities. You are welcomed to enjoy the oral presentations and poster session on pharmacy history, pharmaceutical chemistry, analytical chemistry, clinical aspects of pharmacy, pharmacognosy, pharmaceutical technology, biopharmaceutics, and social pharmacy. The program of the conference contains a lot of interesting reports and presentations, and we strongly believe that established and young scientists will find new opportunities for developing scientific networking, resulting in initiation of fruitful discussions and mutually beneficial collaborations. Let me wish you all the very best for the most successful conference.
75
thanniversary of the Lithuanian Pharmacopoeia
Tauras Antanas Mekas*, Vitalis BriedisLithuanian University of Health Sciences
*Corresponding author. E-mail address: tauras.mekas@gmail.com
Seventy-five years ago – in late 1938 – the first edition of the Lithuanian Pharmacopoeia was published. This book – although being a necessary and the most important book at a pharmacy – had to travel a long way until it reached pharmacists. Before its publication, the Russian 6th Pharmacopeia published before World War I (in 1910) was mandatory in Lithuania. With time - and an increasing number of pharmacies in Lithuania - the supplies of this Pharmacopeia ran out. On the other hand, a new generation of pharmacists emerged in Lithuania – and they did not speak Russian. In Lithuania Minor, the German Pharmacopoeia was used. This resulted in different drug formulations – e.g. ung. camphorae in Klaipėda district was produced from camphor and petroleum jelly (petrolatum), and in Lithuania Major – from camphor, wax, and pork fat.
Pharmacies, pharmaceutical companies, and the Department of Health needed the Lithuanian Pharmacopeia, which had to standardize the production of medications throughout the country, and to provide regulations and methods for the control of medicines.
The first steps towards the Lithuanian Pharmacopoeia were taken in 1929. The Lithuanian Council of Ministers allocated 50000 litas for this purpose. The same year, the Department of Health made an agreement with Prof. Petras Raudonikis (1869 – 1950) that the Pharmacopoeia would be prepared and published within the period of two years. Due to unfavorable circumstances, this aim was not achieved, and the allocated funds were not used. The pharmacist society was embittered by this, and sarcastic articles discussing the fate of the Pharmacopoeia appeared in professional press.
The first edition of the Lithuanian Pharmacopoeia had 528 pages with 616 articles describing reagents and solutes, indicators, titrated solutions, and sterilization. The Pharmacopoeia contained tables on single and daily dosage of toxic and potent substances, tables on the highest single doses of toxic and potent substances for veterinary use, an inventory of devices and glassware used at a pharmacy, a table on the atomic mass of the more important elements, and indices.
The terminology coined for the Lithuanian Pharmacopoeia had a long-standing importance for Lithuania’s pharmacy, and its traces are still found today. The life of the Lithuanian Pharmacopoeia did not end with the occupation of the country. Because it was compiled following the 7th edition of the USSR Pharmacopoeia, and the majority of Lithuanian pharmacists did not speak Russian, the Lithuanian Pharmacopoeia was used in Soviet Lithuania’s pharmacies until the 8th edition of the USSR Pharmacopoeia was published – and in many pharmacies it survived as a silent memorial to the pharmacy of Independent Lithuania.
International, regional and national Pharmacopoeias: importance
and challenges of their harmonization
Eduardas Tarasevičius1*, Hiliaras Rodovičius2, Tauras Antanas Mekas2 1
Lithuanian Pharmaceutical Association 2Lithuanian University of Health Sciences
*Corresponding author. E-mail address: taredas@gmail.com
According official description, Pharmacopoeia is a book containing directions for the identification and quantitative analysis of drug samples, determination of possible impurities and the compounding procedures of different pharmaceutical formulations. This drug‘s quality standard book is published by the authority of a government or a medical or pharmaceutical society. By now existing pharmacopoeias is possible to classify by geografic area. Large majority are National Pharmacopoeias, few – Regional and one only is International Pharmacopoeia.
In European region most countries issued National Pharmacopoeias. The British Pharmacopoeia (BP) will be celebrating its 150th Anniversary in 2014. The first edition of the BP was published in 1864. A lot has changed since the first edition was published and the anniversary will be used as an opportunity to reflect on the past and to look forward to the future. First edition of the German Pharmacopoeia „Deutsches Arzneibuch“ (DAB) was published in 1872. „Pharmacopoeia Hungarica“ was published in 1871. First edition of the Russian Pharmacopoeia was published in 1778. Oldest was first edition of the Polish Pharmacopoeia „Farmakopea Polska“ published in 1817.
In Asian region the first edition of the Japanese Pharmacopoeia (JP) was published in 1886. Australia and Canada are just two of the countries that have adopted the BP as their national standard alongside the UK. Very important was for countries of the European region to develop Regional Pharmacopoeia. European Directorate for the Quality of Medicines and HealthCare under Council of Europe in 1967 issued first edition of regional „European Pharmacopoeia“. In Africa Committee on Scientific and Technical Research of the African Union in 1985 issued African Pharmacopoeia.
The need for International Harmonisation was recognized by WHO Expert Committee on Specifications for Pharmaceutical Preparations and in 1951 was published first edition of the International Pharmacopoeia. For the first time in 10 years, representatives from pharmacopoeias from 23 countries came together in 2012 at WHO headquarters in Geneva and committed to working further towards harmonization and strengthening WHO's role when developing global standards for the production and testing of drugs.
Globalisation and expansion in international trade present a growing need to develop global quality standards for medicines. Harmonisation among the world's three major pharmacopoeias, the European Pharmacopoeia (Ph. Eur.), the Japanese Pharmacopoeia (JP) and the United States Pharmacopoeia (USP), is an important and challenging task. In 1990, the International Conference on Harmonisation (ICH), for the harmonisation of testing of medicines among the European Union, the United States and Japan was set up. This programme aims to reduce the overall cost of pharmaceutical research worldwide by avoiding duplication of work such as the preparation of dossiers and studies, thus reducing the time required for innovative medicines to become available.
Latvian Pharmacopoeia
Lauze Sabine*, Maurina Baiba, Sidlovska VentaLatvian pharmacopoeia started composing in year 1923 and was written almost 20 years; finally published in year 1940. This publishing of pharmacopoeia is mentioned as one of the biggest achievements in field of Latvian pharmacy, especially in the first era of independence in Latvia. Before that Latvian pharmacists were using Russian pharmacopoeia VI edition, which was released in 1910 and was very out of date. And as press released news that there in Latvia the chemical and pharmaceutical producing in different places were using different methods, so it appears that it was also used German pharmacopoeia and maybe pharmacopoeia from other countries.
The initiator and also the chief editor of Latvian pharmacopoeia was pharmacy professor from University of Latvia Jānis Maizīte (1883 – 1950). Thanks to him, in year 1923 Latvian Pharmacy Administration established a pharmacopoeia commission, which in year 1926 was approved by Cabinet. It was created pharmacognosy, chemical, galenical, pharmacology, and manual versions of the panel, composed of leading university professors, health department and public organizations.
It was also decided to issue a Pharmacy Manual, which included a variety of recipes of the medicines which were popular in pharmacies. Pharmacy Manual was prepared already in year 1927, but work with pharmacopoeia was slow. During the year the committee had met 10 times and created a list of treatments that should be included in the pharmacopoeia. Pharmacopoeia included all the medications that were often prescribed. From rarely used products included toxic substances and products which have been forged. When the commission's list was made and published; after that the commission was waiting pharmacists and medical staff reviews and comments. With the establishment of a list real work only started. To establish the Latvian pharmacopoeia, the authors noted and studied other pharmacopeia of European countries, including Russia and the United States Pharmacopoeia. But were mainly used German and Russian pharmacopoeia methods. Jānis Maizīte wrote about the work on pharmacopoeia in Latvian Pharmacist journal, this article was published with title "On the pharmacopoeia of medicinal substances investigative techniques" in year 1924. It is noted that in order to verify and compare the already developed medicinal preparation and analytical methods were developed dozens of graduation. Students compared the number of pharmacopoeia methods and gave his opinion, which of the methods of a particular case considered optimal.
developed under professor’s Maizīte management concluded that the professor monitored the students the studies and the conclusions drawn.
Pharmacopoeia was on 968 pages, the general part of it involved international decisions about poisons and drugs, methods of analysis (essential oils determination of drugs, density determination, melting point determination, etc.), the rules for sterilization. The special part found in more than 658 pharmacopoeia articles that contain characteristics of formulations (capsules, slurry, powder, ointment, etc.), as well as herbal drugs, chemicals, animal products.
References:
1. The Pharmacopeia of Latvia. Riga: Ministry of the Wealth of Nations 1940, 968. S.
2. Government regulations. The Pharmacopoeia of Latvia. Journal of pharmacists in Latvia, 1924, 2(1): 30–32.
3. Maizīte J. On the pharmacopeia of medicinal substances investigative techniques. Journal of pharmacists in Latvia, 1924, 2 (6/7). 167–171.
4. The science for homeland in 20 years from 1918 to 1938. Riga: University of Latvia, 1938. 47–61. 5. The project of the Pharmacopeia of Latvia. Journal of pharmacists in Latvia, 1934, 12 (3):
110-114, 12(4): 145-148, 12(5): 187-192, 12(6): 237-242.
European Pharmacopoeia and Quality of Medicinal Products
Roma MockutėHead of Medicines Control Laboratory of State Medicines Control Agency under the Ministry of Health of the Republic of Lithuania, the member of Lthuanian delegation in the European Pharmacopoeia Commission
E-mail address: romamockute@vvkt.lt Aim of Pharmacopoeia:
- Harmonisation of requirements in the field of medicinal products within a region.
Historically:
- 1872 Pharmacopoeia Germanica, edition 1;
- 1963 First discussion EC about free circulation of medicinal products; - 1964 European Pharmacopoeia;
- 1948 Internation Pharmacopoeia, WHO;
- 1990 International Conference on harmonisation of technical requirements (ICH) – Pharmacopoeial Discussion Group (PDG) process;
European Pharmacopoeia set the mandatory requirements (standards), described in:
- General monographs; - General chapters;
- Specific monographs (substances for pharmaceutical use, vaccines). Implementation the European Pharmacopoeia in the Lithuania:
- Regulators:
• important tool during assessment;
• Medicines control laboratories (OMCL) carries out marketing control. - Industry:
• collaboration Regulators – Industry; • predictable requirements.
- Community and hospital pharmacies:
• relevant and significant quality standards for pharmacist;
• quality standards for extemporaneous preparations, i.e. pharmaceutical preparations individually prepared for a specific patient or patient group, supplied after preparation and for stock preparations, i.e. pharmaceutical preparations prepared in advance and stored until a request for a supply is received.
Prepared for the future:
Biological and Biosimilar Drugs. Present State of Art
Jerzy ŁazowskiPolish Oncology Pharmacists Association, Warsaw, Poland E-mail address: jlazowski@psfo.org
Biopharmaceuticals are medicines whose active substance is made by living cells. They are large molecules and every biologics has various sections in its structure that perform specific functions and enable the biologic to do its job. These structural features attributes of molecule play a pivotal role in the biologic medicine ability to treat the disease.
Little known but important feature of biologics is that these structural attributes and corresponding functions varies slightly from batch to batch over time and more pronounced shifts are seen after manufacture changes. In reality no two batches of the same biologic can be considered identical.
Manufacturing a biologic is a complex process and even small changes in the process can affect the final product and how it could react in human body. Over past 30 years biological medicines provided treatments for some of the most serious medical conditions. The outcomes of patients have been remarkable. Further development of new biologic medicines may be the best hope for producing effective treatments for many diseases that currently have no cure. Some major biotechnology derived medicines have lost or will soon lose patent protection and will become open to development and manufacture by other companies.
Because of their intrinsic complexity such copies are similar, but not identical to the original biologic drug (reference drug) and therefore are called “biosimilars”. EMEA defines biosimilars as follows: A biosimilar medicine is a medicine which is similar to a biological medicine that has already been authorized (the “biological reference medicine”. The active substance of a biosimilar medicine is similar to the one of the biological reference medicine. Biosimilar and biological reference medicine are used in general at the same dose to treat the same disease. Since biosimilar and biological reference medicine are similar, but not identical, the decision to treat patient with reference opr a biosimilar medicine should be taken following the opinion of qualified healthcare professional. The named “Appearance and packing of a biosimilar drug should differ to those of biological reference medicine.”
Therefore, switching between similar biopharmaceuticals in patients should be prevented.
Although biosimilars do not target new illnesses, they may expand access to biologic therapy, provided that they are sufficiently safe, effective, and used in an appropriate and well-monitoring setting. They may also significantly decrease costs of disease treatments.
Microscopic and macroscopic identification of common spruce
species raw material from Latvian forests
Sanita Siksna*, Ilze Barene, Irena Daberte Riga Stradins University, Riga, Latvia
*Corresponding author. E-mail address: sanita.siksna@rsu.lv
Introduction. It has long been known that green plants contain many necessary substances for human survival and health providing – different valuable natural substances and products for practical use are obtained. An important source of biologically active substances is spruce greenery, and most of the substances are obtained from Picea abies L. – Norway spruce, that is the only spruce species into the wild in Latvia. However, there are more spruce species introduced here and some of them can be moved into the wild and be as possible impurities in mono-source material for natural substances obtaining. Spruce branches are crushed in 1-3mm small pieces after collection therefore certain criteria of individual distinguishing feature of various spruce species in Latvia forest must be known to identify this source.
Aims. The aim of this research was to investigate characteristics of five different Picea genus species plants branches and their needles microscopic and macroscopic properties for these species identification and differentiation from Picea abies L. needles raw material mono-source.
Results. Results from the branches and needles macroscopic analysis are listed in the table.
If we talk about results from needles raw material microscopic analysis, than most of needles cross-section shapes are rhomboid with approximate length relation of the diagonals 1:3, which is observed not only in P. abies case, but also P. engelmannii, P. pungens and P. orientalis species. Expressed square shape needles has P. glauca, but very wide equilateral triangle shaped needles with inconspicuous apical edge has P. omorika.
Cuticle layer thickness differences are observed – it is particularly thick in P. engelmannii and P. glauca species, but P. orientalis and P. pungens has very thin layer. Wavy cuticle layer thickness is distinctive for P. omorika needles.
Epidermis is very similar for all species, significant differences were not observed. However, hypodermis has differences – most of the species have cells in one layer there, P. orientalis and P. engelmannii passim has thicker spots with 2 or 3 layers of cells, but P. pungens has hypodermis from 2 or 3 layers of cells, rarely only in one cell thickness.
Table 1. Results from the six various spruce species branches and needles
macroscopic analysis
Differences are observed in mesophyll tissue structure – P. glauca and P. engelmannii has regular folded parenchyma, but P. omorika mesophyll is formed from very different size particularly folded cells, but layer is still uniform. P. pungens needles mesophyll has identical, symmetrically round shaped cells with minimal folds. In case of P. abies and P. orientalis, along hypodermis palisade Picea species features Picea abies L. Picea engelmannii (Parry ex Engelm.) Picea glauca (Moench) Picea pungens (Engelm.) Picea omorika (Pančić) Picea orientalis L. Hairs of
shoots very rare few, russet - - few many, dense Colour of shoots brown-yellow yellow-brown bright gray-yellow orange-brown gray-brown bright brown – bright yellow Resin on buds - lightly - - - -
Colour of
needles dark green
dark blue-gray green blue-green blue-white gray-green very dark green Length of needles, cm 1.0-2.5 1.5-2.5 0.6-1.8 1.8-3.0 0.5-2.0 0.4-0.8 Apex shape of needles acute, medium axial very acute
and axial truncate
acute, very axial
short and
axial truncate Deflection of
needles erect bent
parenchyma in one layer is visible, but closer to the central part is spongy parenchyma in several layers.
Endodermis is without significant differences, as well as transfusion tissue – merely P. orientalis needles parenchyma is formed from relatively smaller cells, which are arranged only in 2 or 3 layers. Vascular bundle structure was without significant differences.
Conclusions. Morphological and anatomical structure features of branches with needles can be used in Norway spruce (Picea abies L.) needles raw material quality assessment, possible impurities detection and identification, because macroscopic analysis showed that needle samples have variety of colours and length, but microscopic analysis showed remarkable differences in needles cross-section shape, cuticle layer thickness, hypodermis and mesophyll tissue structure. References:
1. Mauriņš A., Zvirgzds A. Dendroloģija. – Rīga, LU Akadēmiskais apgāds, 2006. –Pp. 94–101. 2. Hannick F., Waterkeyn L., Weissen F., Van Praag J. H. Vascular tissue anatomy of Norway spruce
needles and twigs in relation to magnesium deficiency // Tree Physiology, 1993; 13:337-349. 3. Puech L., Mehne-Jakobs B. Histology of magnesium-deficient Norway spruce needles influenced
by nitrogen source // Tree Physiology, 1997; 17:301-310.
Assessment of digoxin pharmacokinetic measurements in tertiary
level hospital
Rima Minkutė1*, Vitalis Briedis1, Rasa Steponavičiūtė2, Astra Vitkauskienė2, Romaldas Mačiulaitis3,4
1
Department of Clinical Pharmacy, Lithuanian University of Health Sciences 2
Clinic of Laboratory Medicine, Hospital of Lithuanian University of Health Sciences Kauno Klinikos
3
Institute of Physiology and Pharmacology, Lithuanian University of Health Sciences
4
Clinic of Nephrology, Hospital of Lithuanian University of Health Sciences Kauno Klinikos *Corresponding author. E-mail address: rima.minkute@lsmuni.lt
Methods: In this retrospective study, measurements of DSC in three years were analysed and compared with recommended therapeutic level. Concentrations below 1.0 nmol/ml were considered as “too low concentration”, in the range 1.0 – 2.6 nmol/ml – “therapeutic” and above 2.6 nmol/ml – “too high concentration”. Influence of patients’ gender and age on DSC was evaluated. Consumption of digoxin in hospital departments was calculated to evaluate frequency of DSC measurements.
Results: There were analysed 123, 86 and 78 DSC in 2010, 2011 and 2012, respectively. A half of DSC was measured in patients who used digoxin at home, were hospitalized and medication was not prescribed further. Consumption of digoxin in hospital departments is presented in fig. 1. The majority of digoxin pharmacokinetic measurements were done in department T6: 43.5 % of all DSC in 2010 (1 DSC for 0.32 DDD/100 OBD), 58.3 % - in 2011 (1 DSC for 0.36 DDD/100 OBD) and 53.1 % - in 2012 (1 DSC for 0.42 DDD/100 OBD).
0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 R5 R2 R3 T6 T15 R 1 T3 C2 T16 T17 C3 T18 R7 R6 C 6 T7 C1 T9 T2 Other
Hos pital de partm e nts
D D D /1 0 0 O B D 2010 2011 2012
Fig.1. Digoxin consumption in DDD/100 OBD in hospital departments in 2010 -
2012.
DDD/100 OBD – defined daily doses in 100 occupied bed days; other – 13 departments in 2010, 14 departments in 2011, 17 departments in 2012.
45.5 (10) 58.3 (14) 35.5 (11) 72.2 (13) 58.6 (17) 60.0 (15) 36.4 (8) 33.3 (8) 38.7 (12) 22.2 (4) 20.7 (6) 24.0 (6) 18.2(4) 8.3 (2) 25.8 (8) 5.6 (1) 20.7 (6) 16.0 (4) 0 10 20 30 40 50 60 70 80 1 2 1 2 1 2 2010 2011 2012 Ye ars %
Too low Therapeutic Too high
Fig. 2. Appropriateness of digoxin serum concentration to recommended interval
in patients treated with digoxin at home, % (n). 2010, n = 54; 2011, n = 49; 2012, n = 46. 1 – male, 2 – female. 23.5 (4) 40.0 (6) 33.3 (6) 31.6 (6) 31.0 (9) 50.0 (20) 47.1 (8) 40.0 (6) 38.9 (7) 42.1 (8) 37.9 (11) 40.0 (16) 29.4 (5) 20.0 (3) 27.8 (5) 26.3 (5) 31.0 (9) 10.0 (4) 0 10 20 30 40 50 60 70 80 1 2 1 2 1 2 2010 2011 2012 Ye ars %
Too low Therapeutic Too high
Fig. 3. Appropriateness of digoxin serum concentration to recommended interval
in patients in hospital, % (n). 2010, n = 69; 2011, n = 37; 2012, n = 32. 1 – male, 2 – female.
References:
1. Yang EH, Shah S, Criley JM. Digitalis toxicity: A fading but crucial complication to recognize. Am J Med. 2012 Apr;125(4):337-43.
2. Cheng JW, Rybak I. Use of digoxin for heart failure and atrial fibrillation in elderly patients. Am J Geriatr Pharmacother. 2010 Oct;8(5):419-27.
3. Haynes K, Hennessy S, Localio AR, Cohen A, Leonard CE, Kimmel SE, et al. Increased risk of digoxin toxicity following hospitalization. Pharmacoepidemiol Drug Saf. 2009 Jan;18(1):28-35. 4. Winter M. Digoxin. In: Basic Clinical Pharmacokinetics. 4th ed. Baltimore, MD: Lippincott
Williams&Wilkins; 2004. p. 183-227.
Pharmacokinetic properties of Ibogaine and Noribogaine in plasma
of mice
Asta Kubilienė1*, Audrius Sveikata2, Ilona Sadauskienė3, Leonid Ivanov3 1
Department of Analytical and Toxicological Chemistry, Lithuanian University of Health Sciences
2
Department of Theoretical and Clinical Pharmacology, Lithuanian University of Health Sciences
3
NeuroscienceInstitute, Lithuanian University of Health Sciences *Corresponding author. E-mail address: astakubiliene@gmail.com
Ibogaine is an indole alkaloid found in African plant Tabernanthe iboga Baill. (Apocynaceae). This substance facilitates the symptoms of abstinence [1]. In the late 20th century many scientific researches started to be conducted with an aim to find out the mechanism of action of ibogaine. The pharmacology of ibogaine is quite complex, affecting many different neurotransmitter systems simultaneously. However, the pharmacological targets underlying the physiological and psychological actions of ibogaine are not completely understood [2]. Ibogaine is metabolized into active metabolite noribogaine which can condition the long-term effect of ibogaine [3,4]. According to the results of researches, noribogaine is deemed to be more safe and less toxic and causing less undesirable reactions as compared with its predecessor.
Objective. To evaluate pharmacokinetic constants of ibogaine and noribogaine in plasma of mice.
Pharmacokinetic constants were estimated by program „Kinetica“ using non-compartment model. Calculated pharmacokinetic parameters include the following: volume of distribution (Vd), area under curve (AUC), elimination half-life (t1/2), maximum concentration (Cmax), clearance (CL), elimination rate constant (ke).
Results. The variation of ibogaine, noribogaine and metabolite N1 concentrations in laboratory mice blood plasma within the period of 24 hours after the administration of substances is illustrated in Fig.1.
Fig.1. Time-concentration profiles for ibogaine, metabolite N1 and noribogaine in plasma of mice after intragastric administration of ibogaine (26.3 mg/kg) or noribogaine (31.5 mg/kg)(n=3)
The pharmacokinetic parameters in blood plasma of mice determined during our research after single dose of ibogaine (26.3 mg/kg intragastrically) and noribogaine (31.5 mg/kg intragastrically) are presented in Table 1.
Following intragastric administration circulating levels of ibogaine and metabolite N1 peaked (Cmax) at 30 min, whereas levels of noribogaine increased slowly to plateau at 4 h. Metabolite N1 Cmax (185±0.02 ng/ml) was much less than that of ibogaine (475±0.05 ng/ml), giving a noribogaine-to-ibogaine Cmax ratio of 0.39. These data show, a much smaller fraction of ibogaine is converted to metabolite N1 (noribogaine) when ibogaine is administered intragastrically. Noribogaine Cmax was three-fold less than that of ibogaine.
Table 1. Pharmacokinetic properties of ibogaine, metabolite N1 and noribogaine in plasma of mice (mean±SD)(n=3)
Pharmacokinetic properties Ibogaine Metabolite N1 Noribogaine
tmax, (h) 0.5 0.5 4 Cmax (ng/mg) 475±0.05 185±0.02 150±0.02 AUClast (ng×h/mg) 1753.31±1.37 823.44±1.06 730.22±0.91 AUCtot (ng×h/mg) 1759.38±1.39 841.59±1.21 753.88±1.02 t1/2 (h) 1.95±0.11 4.42±0.09 6.14±0.21 Vd (ml/mg) 0.04±0.001 0.20±0.01 0.37±0.05 CL (ml/h/mg) 0.015±0.00 0.03±0.001 0.04±0.001 ke (h–1) 0.4±0.01 0.16±0.01 0.11±0.02
Ibogaine half-life in mice blood plasma was 1.95±0.11 h, i.e., more than twice shorter than that of metabolite N1 (t1/2 4.42±0.09 h) and three times shorter than that of noribogaine (6.14±0.21 h) (Table 1). Accordingly, ibogaine elimination constant (0.4±0.01 h–1) is by 2.5 times higher than that of metabolite N1 (0.16±0.01 h–1) and more than three times higher than that of noribogaine (0.11±0.02 h–1).
Conclusions
1. Largest Vd value in plasma calculated for noribogaine. Whereas Vd for ibogaine is much smaller.
2. Total systemic exposure to ibogaine in plasma is much greater than to noribogaine and metabolite. But elimination of this substance is four-fold faster.
3. Longer elimination of noribogaine from plasma indicates that long-lasting effect of ibogaine attributable to metabolite, in agreement with previous reports.
References:
1. Alper KR, Lotsof HS, Frenken GM, Luciano DJ, Bastiaans J. Am J Addict 1999; 8:234-42.
2. Mačiulaitis R, Kontrimavičiūtė V, Bressolle FMM, Briedis V. Human & Exp Toxicol 2008; 27:181-94.
3. Mash DC, Staley JK, Baumann MH, Rothman RB and Hearn WL. Life Sci 1995; 57:45-50. 4. Hearn WL, Pablo J, Hime GW and Mash DC. J Anal Toxicol 1995; 19:427–34.
Evaluation of antioxidant activity of Ginkgo biloba flavonoids using
postcolumn HPLC method
Asta Špadienė1*, Nijolė Savickienė2, Laura Rimkienė3, Valdas Jakštas2, Hiliaras Rodovičius1
1
Department of Drug Chemistry, Lithuanian University of Health Sciences 2
Department of Pharmacognosy, Lithuanian University of Health Sciences 3Department of Analytical and Toxicological Chemistry, Lithuanian University of Health Sciences
*Corresponding author. E-mail address: astaspadiene@gmail.com
Ginkgo biloba leaves extract contain several active constituents including 20-27% flavonoids, 5-7% terpenoids, and 5-10% organic acids. The major flavonoids are quercetin, isorhamnetin, kaempferol derivatives and proanthocyanidins. The primary terpenoids are ginkgolides A, B, C, M, and J, and bilobalide [1]. Although the mechanism of action of ginkgo leaf is only partially understood, one of the theories is that ginkgo leaf might work by protecting tissues from oxidative damage. Ginkgo leaf flavonoids have antioxidant and free radical scavenging properties [2,3]. Protecting tissues from oxidative damage might prevent progression of tissue degeneration in patients with DM.
The aim of this investigation was to evaluate the antioxidant activity of flavonoids (quercetin, isorhamnetin, kaempferol) in capsules of Ginkgo biloba dry extract (JSC ”Aconitum”) using postcolumn HPLC method as described by Raudonis et al. [4]. Chromatographic separation was based on the European Pharmacopoeia [5,6] slightly modified as follows: compounds were separated on ACE C-18 column (150 × 4.0 mm, 5 μm) and concentration of phosphoric acid (eluent A) was reduce up to 0.1 mg/l in order to maintain the required pH in the postcolumn loop. The DPPH radical scavenging activity of Ginkgo biloba flavonoids were calculated from the standard curve of Trolox at concentrations of 0.02, 0.04, 0.06, 0.08, 0.1 and 0.12 mg/ml. Trolox Equivalent Antioxidant Capacity was calculated for individual flavonoids and expressed as micromoles of Trolox equivalent (TE) per 100 gram of dry extract (μmol TE/100g).
Considering that other compounds of Ginkgo biloba extract have no significant influence on total Ginkgo biloba antioxidant activity it can be stated that 100 gram of dry Ginkgo biloba extract has the same antioxidant activity as well as 4.08 µmol of standard antioxidant trolox.
Figure 1. HPLC (a) and UV-DPPH (b) chromatograms of the Ginkgo biloba extract.
Peaks: 1.Quercetin, 2. Kaempferol, 3. Isorhamnetin.
Evaluation of antioxidant activity of Ginkgo biloba (Ginkgo Biloba L.) flavonoids is a part of international „Eureka“ project „Creation of the methodology for effects of natural antioxidants on the development of the Diabetes mellitus complications“.
References:
1. E/S/C/O/P Monographs, The Scientific Foundation for Herbal Medicinal Products, 2003, pp.178-210 2. Rahman K., Studies on free radicals, antioxidants, and co-factors, Clin. Interv. Aging., 2007, 2(2),
219-236
3. Stefek M., Natural flavonoids as potential multifunctional agents in prevention of diabetic cataract, Interdiscip. Toxicol., 2011, 4(2), 69-77
4. Raudonis R, Jakstas V, Burdulis D, Benetis R, Janulis V. Investigation of contribution of individual constituents to antioxidant activity in herbal drugs using postcolumn HPLC method. Medicina. 2009, 45(5):382-394
Counseling of pregnant and breastfeeding women in Lithuanian
pharmacies: structural equitation modeling approach
Grinceviciene S*, Grincevicius J, Kubiliene L, Baranauskas A.Lithuanian University of Health Sciences
*Corresponding author. E-mail address: svitrigaile@gmail.com
Background. According to Lithuanian legislation and FIP recommendations, pharmacy specialists must counsel the Lithuanian people how to promote health, including the aspects of the rational use of medications and healthy lifestyle. However, it is not known what factors encourage them to counsel pregnant and breastfeeding women on these issues.
Methods. A questionnaire containing the newly developed ‚Maternity Counseling in Public Health for Pharmacists‘ (MCPH-P), a previously validated behavioral measure ‚Physician Pharmacist Collaboration Instrument’ (PPCI-P) [1] and social demographic characteristics of pharmacy specialists and pharmacies was administered to a sample of 440 Lithuanian pharmacy specialists. The MCPH-P was developed based on existing literature and FIP reference paper on the effective utilization of pharmacists in improving maternal, new-born and child health (MNCH) [2]. Factor analysis was used to assess the structure of the MCPH-P and MCPH-PMCPH-PCI-MCPH-P and the Cronbach’s alpha coefficient was used to assess the internal consistency of the instrument. Structural equation modeling was used to determine how the PPCI-P and social demographic characteristics of pharmacy specialists and pharmacies influence frequency of counseling for pregnant and breastfeeding women in pharmacy.
Results. Factor analysis revealed the MCPH-P consisted of three factors: ‘frequency of counseling about rational use of medications’ (RUS), ‚frequency of counseling about healthy lifestyle during pregnancy‘(HLP) and ‘frequency of counseling about healthy lifestyle during breastfeeding’ (HLB), with good internal consistency (Cronbach’s alpha = 0.89; 0.86 and 0.84 respectively). In the model HLP and HLB were included to create latent variables – frequency of counseling about healthy lifestyle (HL). The model demonstrated adequate fit (χ2/df = 1.04, CFI = 0.999, RMSEA =0.011, 90% CI [0.00-0.040]).
The model illuminate that HL was directly increased by RUS and more frequent interaction between pharmacy specialists and physicians. HL was indirectly increased in the pharmacies where pharmacy specialists were younger, recommended to conduct more physiological tests and worked in the pharmacies located further from the physician’s office.
Conclusions. The frequent physician and pharmacy specialists’ interaction was the strongest predictor of frequent counseling of pregnant and breastfeeding women. Age of pharmacy specialists, distance of pharmacy from physician specialists’ office and number of physiological tests had indirect effect to the frequency of counseling.
References
1. Zillich AJ, Doucette WR, Carter BL, Kreiter CD. Development and initial validation of an instrument to measure physician-pharmacist collaboration from the physician perspective. Value Health 2005;8:59–66.
2. International Pharmaceutical Federation (FIP) FIP reference paper on the effective utilization of pharmacists in improving maternal, newborn and child health (MNCH). 2012. Available at: www.fip.org.
Results of effect of hypochlorous acid on platelet aggregation in
healthy people and in patients with heart failure in vitro
Aušra Mongirdienė*Laboratory of Molecular Cardiology, Institute of Cardiology, Lithuanian University of Health Sciences
*Corresponding author. E-mail address: ausra.mongirdiene@mail.com
Background. Thrombotic complications appear in 11-44% of patients with CHF (1). It is thought, that one of the causes of these complications can be generated reactive O2 forms. Inflammatory factors are important contributors to pathogenesis of cardiovascular diseases (2). Activated monocytes and neutrophils excrete enzyme myeloperoxidase, which catalyses production of hypochlorous acid (HOCl) from H2O2 and Cl
-. HOCl is unstable compound and strong oxidizing agent affecting plasma lipoproteins, fibrinogen and other proteins. Their alterations have effect on development of atherosclerosis and thrombosis complications. Platelets actively participate in these processes (3). The published data about HOCl effects on platelet aggregation are inconsistent. It is important to explore the impact of the oxidant to the platelets aggregation, because this might be the niche for treatment: to inhibit platelets aggregation with antioxidants. In that case bleeding complications could be avoided too.
The aim of this study was to determine the dependence of effects of hypoclorous acid on the intensity of platelet aggregation in vitro in healthy people and heart failure patients on HOCl concentration.
relative optical density in aggregating platelet-rich plasma against platelet poor plasma was used. The aggregation was initiated with ADP (3,8 mmol/L) solution. We used 6 samples of the same person plasma. To each sample 10 µL HOCl of different concentrations were added: 2.12 mM/L, 7.06 mM/L, 10.59 mM/L, 21.18 mM/L, and 43.4 mM/L as final concentration. To the control sample the same volume of physiological solution was added. The samples were incubated for 30 min at 370C, and the intensity of platelet aggregation using ADP was determined (%).
Results. It was determined that high concentrations of HOCl (from 2.12 to 43.4 mM/L) significantly decreased platelet aggregation in both groups. We observed that the increasing concentration of HOCl was reversely proportional to the intensity of platelet aggregation. The decrease of platelet aggregation intensity in plasma of heart failure patients affected with HOCl in vitro was more expressed than in healthy people (42.6% and 36.1% respectively).
Conclusions. The intensity of platelet aggregation significantly decreased dependently on HOCl concentration in plasma in vitro. Heart failure patients were more affected than healthy people.
References:
1. Palka I, Nessler J, Nessler B, Piwowarska W, Tracz W, Undas A. Altered fibrin clot properties in patients with chronic heart failure and sinus rhythm: a novel prothrombotic mechanism Heart. 2010 Jul;96(14):1114-8. doi: 10.1136/hrt.2010.192740/
2. Louise N. Patona, Tessa J. Mocattaa, A. Mark Richardsb, Christine C. Winterbourna. Increased thrombin-induced polymerization of fibrinogen associated with high protein carbonyl levels in plasma from patients post myocardial infarction. Free radical biology and medicine. Volume 48, Issue 2, 15 January 2010, Pages 223–229.
3. Schildknecht S, van der Loo B, Weber K, Tiefenthaler K, Daiber A, Bachschmid MM. Endogenous peroxynitrite modulates PGHS-1-dependent thromboxane A2 formation and aggregation in human platelets. Free Radic Biol Med. 2008 Aug 15;45(4):512-20. doi:
10.1016/j.freeradbiomed.2008.04.042. Epub 2008 May 15.
Anticancer activity and structure-activity relationship of new
quinoxaline derivatives
Agnė Vegytė1, Jonas Šarlauskas2, Alvydas Pavilonis3, Vilma Petrikaitė1,4* 1
Department of Drug chemistry, Lithuanian University of Health Sciences 2Department of Xenobiotics Biochemistry, Institute of Biochemistry, Vilnius University
3
Institute of Microbiology and Virusology, Lithuanian University of Health Sciences 4
Laboratory of Biothermodynamics and Drug Design, Institute of Biotechnology, Vilnius University
Quinoxalines are perspective anticancer derivatives. They have shown activity against liver carcinoma, brain tumor [1], leukemia [2], melanoma [3], non-small lung cancer, [4,5] glioma [6] and other types of cancer. Moreover, these compounds have antibacterial and antifungal activity [7]. The aim of this research was to test anticancer activity of new quinoxaline derivatives and find the relationship between their structure and activity.
Sixteen new quinoxaline derivatives were studied in this research. Anticancer activity was tested on two selected cancer cell lines: lung carcinoma (A549) and glioblastoma (U87) using tetrazolium/formazan assay (Fig. 1). Cells were plated in 96-well plates in triplicate (2500 cells/well) and incubated at 37°C for 24 hours. Then serial dilutions of different compounds were made in microplate. After 72 hours incubating at 37°C, 10 μl of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was added in each well. After 3 hours the liquid was aspirated from the wells and discarded. Crystals were dissolved in 100 μl of dimethylsulfoxide, and absorption was measured at a wavelength of 570 nm using a multidetection microplate reader.
Fig. 1. Normalized A549 cancer cell line survival as a function of compound
(AJV37, QDO-01) concentration.
Physicochemical parameters of compounds were estimated by calculation of molecular descriptors like LogP, molecular volume and LUMO. Molecular lipophilicity potential (MLP) was calculated from atomic hydrophobicity contributions by miLogP method.
Table 1. GI50 of the most active quinoxaline derivatives. GI50 (μM) Compound A549 U87 AJV1 5.9 5.0 AJV3 5.0 10.1 AJV4 1.7 3.8 AJV15 5.5 2.0 AJV35 2.5 4.0 AJV37 1.7 0.9
The most active compounds have phenyl ring in the second position with para-nitro group or halogen (Br, I) (Fig. 2). In the sixth position they have halogen (Cl, Br, F). Our findings confirm data reported in literature [1,5].
N N O O R2 R1
Fig. 2. General structure of most active quinoxaline derivatives agaist A549 and
U87 cell lines. R1 – nitro group or halogen, R2 – halogen.
Acknowledgment. This research was partialy funded by a grant No. MIP-11248 “Heterocyclic N-Oxides (HNO): synthesis, cytotoxicity and interaction with enzyme-targets” from the Research Council of Lithuania.
References:
1. Ismail MF, Amin KM, Noaman E, Soliman DH, Ammar Y. New quinoxaline 1, 4-di-N-oxides: anticancer and hypoxia-selective therapeutic agents. Eur J Med Chem 2010, 45, 2733-8. 2. Ingle R, Marathe R, Magar D, Patel HM, Surana S. Sulphonamido-quinoxalines: search for
anticancer agent. Eur J Med Chem 2013, 65, 168-86.
3. Khier S, Deleuze-Masquéfa C, Moarbess G, Gattacceca F, Margout D et al. Pharmacology of EAPB0203, a novel imidazo[1,2-a]quinoxaline derivative with anti-tumoral activity on melanoma. Eur J Pharm Sci 2010, 39, 23-9.
4. Noolvi MN, Patel HM, Bhardwaj V, Chauhan A. Synthesis and in vitro antitumor activity of substituted quinazoline and quinoxaline derivatives: search for anticancer agent. Eur J Med Chem 2011, 46, 2327-46.
5. Lee SB, Park YI, Dong MS, Gong YD. Identification of 2,3,6-trisubstituted quinoxaline derivatives as a Wnt2/β-catenin pathway inhibitor in non-small-cell lung cancer cell lines. Bioorg Med Chem Lett 2010, 20, 5900-4.
6. Mielcke TR, Mascarello A, Filippi-Chiela E, Zanin RF, Lenz G et al. Activity of novel quinoxaline-derived chalcones on in vitro glioma cell proliferation. Eur J Med Chem 2012, 48, 255-64. 7. Vieira M, Pinheiro C, Fernandes R, Noronha JP, Prudêncio C. Antimicrobial activity of quinoxaline
Effect of ethanol on energy metabolism and potency of
extracellular matrix synthesis of pancreatic stellate cells
R.Baniene1, S.Trumbeckaite*1, A.Maziukiene2, A.Jakstaite2, L.Venclauskas2 1Institute of Neurosciences, Medical Academy, Lithuanian University of Health Sciences
2
Institute for Digestive Research, Medical Academy, Lithuanian University of Health Sciences
*Corresponding author. E-mail address: sonatai@centras.lt
Pancreatic stellate cells, one of several resident cells in the exocrine pancreas, play the key role in the pathophysiology of the major disorders of the exocrine pancreas, such as pancreatic cancer and chronic pancreatitis [1]. Alcohol consumption is responsible for about 70%–90% of cases of chronic pancreatitis [1, 2]. It is also known, that chronic pancreatitis is one of the predisposing factors in pathogenesis of pancreatic cancer [1]. Our hypothesis is, that activated pancreatic stellate cells produce the synthesis of extracellular proteins that leads to pancreatic fibrosis as a determinant of pancreatic cancer development.
Thus, the aim of our study was to investigate the potency of matrix synthesis (collagen type I and c-fibronectin protein) and energy metabolism of pancreatic stellate cells exposed to different ethanol concentrations.
Collagen-I an fibronectin synthesis was evaluated by Western blot analysis of Stellate cells lysates and ELISA was applied to measure extracellular proteins in supernatants of Stellate cells growth media. Oxygen consumption of pancreatic Stellate cells was measured oxygraphically in cells growths media by using Oroboros-2K oxygraph (respiratory subtrates glutamate+malate).
In conclusion, our results show the increase in pancreatic stellate cell energy metabolism and extracellular matrix proteins synthesis under pretreatment with 0.5% ethanol. The activation of pancreatic stellate cells under alcohol intoxication could lead to progression of pancreatic fibrosis. Further studies are necessary to find out biologically active compounds in order to reduce activation of pancreatic stellate cells.
References:
1. M. Bishr Omary, Aurelia Lugea, Anson W. Lowe, and Stephen J. Pandol. The pancreatic stellate cell: a star on the rise in pancreatic diseases. J Clin Invest. 2007 January 2; 117(1): 50–59. 2. Marco Siech , Zhengfei Zhou , Shaoxia Zhou , Bernd Bair , Andreas Alt , Stefan Hamm , Hans Gross
, Jens Mayer , Hans G. Beger , Xiaodong Tian , Marko Kornmann , Max G. Bachem. Stimulation of stellate cells by injured acinar cells: a model of acute pancreatitis induced by alcohol and fat (VLDL). American Journal of Physiology - Gastrointestinal and Liver Physiology, 2009, 297(6):G1163-71.
Comparison of hemagglutinating and antibacterial activity in lectin
enriched fractions from Urtica dioica L.
Gabrielė Balčiūnaitė1*, Nijolė Savickienė1, Alvydas Pavilonis2,Danas Baniulis3 1
Department of Pharmacognosy, Lithuanian University of Health Sciences 2
Institute of Microbiology and Virusology, Lithuanian University of Health Sciences 3
Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry *Corresponding author. E-mail address: gabriele.balc@gmail.com
Plant lectins are a class of carbohydrate-binding nonimmune origin proteins. In past few decades a lot of lectins were purified and identified (Goldstein et al., 1980; Sharon, 1989; Peumans et al, 2001; de Meija et al., 2003). They have attracted great interest because of their various biological activities, such as cell agglutination, antiproliferative, antitumor, immunomodulatory, antifungal and antiviral (Broekaert et al., 1989; Wang et al., 1996; Does et al., 1999; Ye et al., 2001; Wong et al., 2003; Singh et al., 2004).
There is a theoretical possibility for lectins to be applied in diagnostic microbiology. There are a lot of structures on bacteria surface (peptidoglycans, teichoic acids, lipopolysaccharides, capsule) that could be specifically binded by lectins. There are a lot of reports of the uses of lectins in diagnostic microbiology. However, this method is still very expensive (Sifkin et al, 1990; Santi-Gadelha et al, 2006).
There are the earlier reports about Urtica dioica L. rhizome lectins antiviral (Kumakia et al, 2011), antifungal (Broekaert et al, 1989) and immunomodulatory (Le Moal et al, 1992) properties. However, Urtica dioica L. herb lectins are still unspecified.
Aim of experiment: To compare the antibacterial activity with hemagglutinating activity in lectin enriched fractions from Urtica dioica L. dry herb and dry extract. Experiment tasks: 1. To evaluate antibacterial activity of lectin fractions from Urtica dioica L. dry herb and dry extract to these bacteria species: Proteus, Bacillus subtilis, Staphylococcus aureus. 2. To compare the results with the hemagglutinating activity.
Materials and methods: 1. Lectin-enriched protein fractions were obtained from the dry herb and dry extract of Urtica dioica L. by the extraction and fractonation with ammonium sulphate. 2. The measurement of the hemaggutinating activity: lectin enriched protein fractions were poured on trypsin treated rabbit erthrocytes and incubated for 30 min. in room temperature. 3. Bacteria suspensions were standartised with McFarland standard indicator, mixed with Mueler-Hinton agar and poured to Petri dishes. 4. Extracts were poured into Peni cylinders, standing on Petri dishes with solid Mueler-Hinton agar mixed with bacteria suspensions. 5. Bacteria were cultivated for 24 hours in 37 °C temperature. 6. Antibacterial activity was evaluated by measuring diameter of bacteria growth inhibition area.
Abbrevations: 1st fraction – Precipitate of 40 % saturated ammonium sulphate; 2nd fraction – Precipitate of 80 % saturated ammonium sulphate; 3rd fraction – Supernatant of 80 % saturated ammonium sulphate.
1
Specific hemagglutinating activity was defined as the ratio of the titer/ml and protein concentration (mg/ml); Titer per ml was defined as the reciprocal of the highest dilution giving visible agglutination of the rabbit erythrocytes.
2
Lectin content is based on cell agglutinating activity as compared to specific hemagglutinating activity of lectin from Solanum tuberosum. Tables include representative results from at least two separate experiments.
Results: The strongest hemagglutinating activity detected in 2nd lectin enriched protein fraction from Urtica dioica L. dry extract and in 3rd lectin enriched protein fraction from Urtica dioica L. dry herb. The biggest inhibition area of bacteria growth diameter (14 mm) detected to B. subtilis bacteria affected with 1st and 2nd lectin enriched protein fractions from Urtica dioica L. dry herb and dry extract.
Conclusions: Nevertheless high hemagglutinating activity was detected, there isn‘t correlation with antibacterial activity.
References:
1. Broekaert WF, Parijis JV, Leyns F, Joos H, Peumans WJ. Science. 1989; 245 1100–1102.
2. Goldstein IJ, Hughes RC, Monsigny M, Osawa T, Sharon N, What should be called a lectin? Nature, 1980; 285: 66;
3. Ye XY, Ng TB, Tsang PWK, Wang J. Purification of chrysancorin, a novel antifungal protein with mitogenic activity from garland chrysanthemum seeds. Biol Chem. 2001 Jun;382(6):947-51. 4. Kumakia Y, Wanderseea M. K., Smitha A. J., Zhoub Y, Simmonsb G, Nelsona N. M., Baileya K. W.,
Vesta Z. G., Lid J. K.-K., Chane P. K. S., Smeea D. F., Barnarda D. L. Inhibition of severe acute respiratory syndrome coronavirus replication in a lethal SARS-CoV BALB/c mouse model by stinging nettle lectin, Urtica dioica agglutinin; Elsevier, Antiviral Research. April 2011; 90(1): 22– 32;
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