Kniga na apstrakti book of abstrcts

Formulation and evaluation of diazepam hydrogel 
for rectal administration
M. Dastevska Mitevska, M. Glavas Dodov, K. Goracinova
Institute of Pharmaceutical Technology, Faculty of Pharmacy, 
University "Ss Cyril and Methodious", Vodnjanska 17, 1000 Skopje, Macedonia
Diazepam (DZP) is a long-acting, benzodiazepine with anticinvulsant, anxiolytic, sedative and muscle relaxant
properties. Incorporated in different pharmaceutical dosage forms it is administered orally, parenterally and rectally.
DZP has become a commonly used drug for treatment of acute repetitive epileptic seizures and febrile con-
vulsions in children. Considering the advantages of rectal administration of DZP, the objective of our study was to
formulate and evaluate rectal hydrogel formulations containing DZP as a drug substance in combination with suit-
able co-solvents (ethanol/propylene glycol 1:3) and preservatives (benzyl alcohol, benzoic acid and sodium ben-
zoate) using a contemporary approach in designing hydrogel preparations.
Prepared HPMC-E5 (hydroxypropyl methylcellulose) hydrogels containing different concentrations of DZP
(2 mg mL
-1
, 4 mg mL
-1
and 6 mg mL
-1
respectively) manifested good quality in respect to physico-chemical param-
eters (pH value, drug content, ingredients content), adequate rheological characteristics, antimicrobial efficiency and
microbiological quality. Under the proposed HPLC conditions (LiChrospher
®
60 RP- selected B, 125 x 4 mm, 5 
µm
colums, using a mixture of methanol and water = 70:30 V/V as a mobile phase; injection volume od 20 
µl; flow rate
of 1ml/min and UV detection at 254 nm)  satisfactory separation of DZP and the preservatives used was achieved.
In vitro release studies have shown that the total amount of the drug substance was released in a period of 3 hours. 
Under the conditions characteristic of the second climate zone, the dosage forms were stable for a period of
4 months. For that reasons, prepared DZP delivery systems should be stored at a room temperature of 15-30
0
C, pro-
tected from light and moisture, and should be used within a period of 4 mounths.
Macedonian pharmaceutical bulletin 53 (1,2) 96-97 (2007)
PP - 37
96
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Formulacija i ispituvawe na diazepam rektalen gel
M. Da{tevska Mitevska, M. Glava{ Dodov, Katerina Gora~inova
Institut za farmacevstka tehnologija, Farmacevtski fakultet, 
Univerzitet Sv. Kiril i Metodij, Vodwanska 17,Skopje,Makedonija
Diazepamot spa|a vo grupata na benzodiazepini so dolgo deluvawe koi poseduvaat antikonvulzivni,
anksioliti~ni, sedativni i miorelaksantni osobini. Inkorporiran vo razli~ni farmacevski dozirani
formi, se aplicira per oralno, parenteralno i rektalno.
Osnovata na urgentnata antikonvulzivna terapija denes vo sovremenata farmakoterapija vo pedi-
jatrijata pretstavuva diazepamot. Koga se aplicira diazepamot intravenski so cel da se stopiraat akut-
nite konvulzii sekoga{ postoi rizik od depresija na centarot za respiracija. Intramuskulnata prime-
na na diazepamot ne dava nagli visoki koncentracii vo krvta, bidej}i diazepamot se vrzuva vo muskulnoto
tkivo, taka {to resorpcijata e odlo`ena i nepotpolna. Rektalnata aplikacija se karakterizira so zadovoli-
telna bioraspolo`ivost i e naj~esto primenuvan na~in na administracija za prevencija na konvulziite.
Celta na ovoj trud pretstavuva formulacija na bezbedna i efikasna rektalna dozirana forma so
ednostavna primena vo preventivnata antikonvulzivna terapija kaj pedijatriska populacija. 
Postavenite rabotni zada~i bea: formulirawe i izrabotka na diazepam rektalen gel, farma-
cevtsko tehnolo{ka i biofarmacevtska karakterizacija na podgotvenite formulacii i pratewe na
nivnata stabilnost.
Spored postavenite celi vo tekot na formulaciskiot pristap bea podgotveni formulacii na
diazepam rektalen gel so razli~ni koncentracii na inkorporiranata lekovita supstancija (2 mg mL
-1
, 4
mg mL
-1
i 6 mg mL-1), koi sodr`ea ist odnos na korastvoruva~i kako i antimikrobni sredstva. Gel matrik-
sot be{e izraboten so primena na 15% rastvor na hidroksipropil metilceluloza (HPMC-E5) vo voda.
Za opredeluvawe na sodr`inata na diazepamot vo podgotvenite gel formulacii be{e primenet HPLC
metod, posle ekstrakcija na diazepamot od hidrogelot so metanol. So primena na utvrdenite hromato-
grafski uslovi, postignato be{e dobro razdeluvawe na diazepamot i pomo{nite supstancii benzil al-
kohol i benzoeva kiselina/natrium benzoat. 
Podgotvenite formulacii ispituvani vedna{ posle izrabotkata poka`aa zadovolitelen kvalitet
vo soglasnost so propisite na Ph. Eur. IV (pH 7.2; istisnata masa od ambala`ata ne pomala od deklariranata,
2.5 ml; sodr`ina na diazepam vo formulaciite 2.23%, 4.27% i 6.23%, soodvetno; soodveten mikrobiolo{ki
kvalitet; viskoznost od 2 Pas i psevdoplasti~ni osobini na te~ewe.
Brzinata na osloboduvawe na lekovitata supstancija zavise{e od nejzinata inicijalna koncen-
tracija vo formulaciite (50% premin za vreme od 1h ili 0.5h). Ispituvawata na stabilnost poka`aa deka
vo tek na ~etiri meseci ~uvawe na sobna temeratura od (15 
0
C do 25 
0
C) ne bea zabele`ani nikakvi prome-
ni na ispituvanite parametri na gelovite (organolepti~kite osobini, istisnata masa, sodr`ina na dia-
zepam, brzina na osloboduvawe, viskoznost, mikrobiolo{ki kvalitet). 
Kratkotrajnite izlo`uvawa na zgolemena temperatura poka`aa deka nastanuva promena vo organo-
lepti~kite osobini i sodr`inata na aktivnata supstancija posle eden mesec  na poka~ena temperatura od
37 
0
C.Kratkotrajnite izlo`uvawa na poniska temperatura poka`aa deka nastanuva precipitacija vo gelot
posle ~uvawe vo fri`ider vo tek na 7 dena na temperatura od 4-8 
0
C.Rezultatite uka`uvaat na potreba od
preporaka za ~uvawe na rektalniot gel na temperatura od 15 
0
C - 25 
0
C (sobna temperatura karakteristi~na
za 2-ta klimatska zona) vo predvideniot rok na upotreba.
Macedonian pharmaceutical bulletin 53 (1,2) 96-97 (2007)
PP - 37
97
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98

KLINI^KA FARMACIJA 
I BIOMOLEKULARNI NAUKI 
Sekciski vovedni predavawa  SPL 5-8
Posterski prezentcii  PP 38-77
CLINICAL PHARMACY
AND BIOMOLECULAR SCIENCES
Section plenary lectures  SPL 5-8
Poster presentation PP 38-77

Strategies for vaccine improvement. 
The use of microbial Heat Shock Proteins 
as adjuvants or carrier molecules
Carmen Fernandez
Department of Immunology, WGI, Stockholm University, Stockholm, Sweden
There is widespread recognition of the need for improved vaccines for control of infectious diseases, and
scientists are searching for appropriate combinations of antigens and adjuvants or suitable carrier molecules for inclu-
sion in subunit vaccines. Heat shock proteins (HSPs) are some of the most conserved proteins present in all prokary-
otes and eukaryotes. They undertake crucial functions in maintaining cell homeostasis. From an immunological point
of view, HSPs have attracted increasing interest, since they serve as carriers of antigens and effectively induce anti-
gen-specific B- and T-cell responses without the need of adjuvant. These immunomodulatory functions of HSPs are
based on various properties: (i) HSPs stimulate the production of chemokines which attract immunological cells; (ii)
HSPs possess the ability to activate dendritic cells, thus initiating innate immune responses; and (iii) HSPs are capa-
ble of delivering peptides to major histocompatibility complex molecules for the priming of adaptive immunity.
Moreover, the use of high-MW HSPs as carriers is particularly interesting for the development of vaccine strategies,
since most humans have been in contact with microbial HSPs. BCG is widely used as a vaccine against tuberculo-
sis, and a large number of people are sensitized to mycobacteria or other parasites through natural contacts. We dis-
cuss the utility of HSPs in vaccine design. 
Contributing to this work: KR Qazi, MR Qazi, E Julián, M Singh, M Abedi-Valugerdi
Macedonian pharmaceutical bulletin 53 (1,2) 100 (2007)
SPL - 5
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Therapeutic significance of recurrent gene fusions 
in hematological and epithelial tumors
J. Gogusev
Hopital Necker, 161, Rue de Sevres 75015-Paris, France
Chromosomal aberrations that accompany carcinogenesis have been documented for almost half of centu-
ry, with gene fusion being the most prevalent type of alteration. Gene fusions leading to generation of aberrant fusion
proteins or aberrant expression of normal proteins are a potent route to carcinogenesis and have recently emerged as
attractive therapeutic targets. In hematological malignancies (leukemia’s and lymphomas) and in soft tissue tumors
(sarcomas) that together represent only 10% of all human cancers, recurrent structural aberrations and gene fusions
that typify most cancer genes have been observed. Intriguingly, gene fusions have been far less frequently described
in the more common epithelial carcinomas. In fact, recurrent structural aberrations are rare in the epithelial carcino-
mas perhaps because of some fundamental difference from hematological malignancies. Conversely, this discrepan-
cy may simply indicate that epithelial carcinomas are not amendable to the same analytical techniques that are suc-
cessfully used for detecting chromosomal aberrations in liquid or soft-tissue tumors. 
Two types of structural aberrations of “cancer genes” have been described. One type involves the regulato-
ry elements of a gene (promoter and /or enhancer) becoming aberrantly apposed to a proto-oncogene, thus driving
deregulated expression of the oncogene (e.g. immunoglobulin and T-cell receptor regulatory regions drive aberrant
expression of the c-MYC oncogene in B and T cell malignancies, respectively). The other type of structural aberra-
tion occurs when the coding regions of two genes are juxtaposed, resulting in a chimeric transcript that produces a
fusion protein with a new or altered activity, e.g. the t(9;22) result in the breakpoint cluster region (BCR)-v-abl
Abelson murine leukemia viral oncogene homolog 1(ABL1) gene fusion in chronic myelogeneous leukemia (CML).
Recurrent chromosomal aberrations have causal roles in oncogenesis; therefore, it is not surprising that they provide
specific therapeutic targets, including for exemple Trastuzumab for breast carcinoma with ERBB2/HER2 amplifi-
cation and Imatinib for CML.
Mechanistically, it has been recently proposed that technical issues, rather than any fundamental dichotomy
between hematological and solid cancers, account for the under-representation of gene fusions in epithelial cancers.
Remarkably, recent reports support this contention and provide evidence of widespread recurrent gene fusions in
prostate cancer using a novel analysis of gene-expression profiles. Here, we provide an appraisal of the state of the
knowledge of gene fusions in some epithelial cancers. 
Future implications of gene fusions in common epithelial cancers are likely to become of tremendous impor-
tance in understanding the fine chromosomal aberrations in carcinogenesis and to serve as a target for effective ther-
apeutic interventions. 
Macedonian pharmaceutical bulletin 53 (1,2) 101 (2007)
SPL - 6 
101
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Structure-based design of subtype-selective AMPA
receptor antagonists
Tommy N. Johansen,
1
Ewa Szymanska,
1
Darryl Pickering,
2
Zorica Serafimoska,
1
Elena Frola
1
1
Departments of Medicinal Chemistry and  
2
Pharmacology and Pharmacotherapy, 
Faculty of Pharmaceutical Sciences, University of Copenhagen, Denmark.
Binding of the excitatory (stimulating) neurotransmitter glutamate to ionotropic glutamate receptors (iGluRs)
is a key step in the mechanism of fast excitatory synaptic transmission among nerve cells within the brain. Present
in more than 80% of all excitatory synapses, iGluRs are critically important for normal brain function. Therefore,
understanding the structure, function and regulation of this receptor family is a prerequisite for explaining the cel-
lular as well as molecular basis for the mechanisms that underlie several of the essential brain functions including
thought, perception and encoding of information. Furthermore, dysfunction in glutamatergic transmission has severe
consequences on normal brain function and is implicated in several brain diseases including schizophrenia, Alzheimer’s
disease, brain damage following stroke and epilepsy. iGluRs are involved in the development of these diseases and/or
provide attractive targets for therapeutic intervention. Therefore, detailed understanding of iGluR structure and func-
tion is essential for providing a rational basis for design of therapeutic strategies for treating such disorders.
iGluRs function as ligand-gated ion channels by coupling the energy of glutamate binding to the opening of
a transmembrane ion pore that allows flow of Na
+
, K
+
and Ca2
+
ions, leading to depolarization and ultimately to the
generation of an action potential. On basis of their ability to assemble with each other, these subunits are divided
into three different classes: the (S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl)propionic acid (AMPA)-, kainate-
and N-methyl-D-aspartic acid (NMDA)-type. Structurally, AMPA-receptors are tetrameric assemblies of highly
homologous GluR1–4 subunits.
AMPA receptor antagonists are considered to have clinical potential as neuroprotective drug candidates in
epilepsy and acute ischaemic stroke. Even though a large number of competitive AMPA receptor antagonists are
known today, none of these compounds are able to discriminate between the individual AMPA receptor subunits.
One reason for this lack of receptor selectivity is a very high degree of homology between amino acid residues form-
ing the ligand binding domain of the individual AMPA receptor subunits. Only one amino acid residue in the cent-
ral part of this region is non-conserved; i.e. residue number 702, which in GluR1 and 2 is a tyrosine residue, whereas
in GluR3 and 4 it is a phenylalanine residue. This particular amino acid residue has been identified to be an impor-
tant molecular determinant for AMPA receptor agonist subunit selectivity.
The main target of the present project is to design and synthesize competitive AMPA receptor antagonists
selective for the individual GluR1–4 subunits. On the basis of published crystal structures of the GluR2 binding core
co-crystallized with competitive AMPA receptor antagonists, series of compounds were designed and synthesized,
with the main aim to achieve strong interactions between the ligand and the Tyr – 702 residue of GluR2 either by
direct interactions or through a water binding network.
Macedonian pharmaceutical bulletin 53 (1,2) 102 (2007)
SPL - 7
102
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Studies on Synthesis and Evaluation of Anticonvulsant Drugs
Unsal Calis
Hacettepe University, Faculty of Pharmacy, Department of Pharmaseutical Chemistry, 
06100 Sihhiye-Ankara / Türkiye.
The treatment of epilepsy is still one of the major problems because of the uncontrolled seizures and med-
ication toxicity. Antiepileptics which are used symptomatically in the treatment of epilepsies, have to be adminis-
tered for a long time even throughout one’s life as they have to be used chronically. Although there are a number of
antiepileptic drugs available in the market, development of new compounds are still popular in anticonvulsant ther-
apy as the present ones possess the risk of tolerance developing, side effects, moreover it is still not possible to get
under control some types of epilepsies. Today most of the drugs used in the treatment of epilepsies contain a ureid
structure as barbiturated, glutarimids, oxazolidinediones and succinimids. In the last decade, new anticonvulsant
compounds with substantially different structures than the classical anticonvulsant drugs have emerged. One of the
recently discovered and structurally distinct anticonvulsant drug group is that of the (arylalkyl)imidazole. Nafimidone
(I) and denzimol (II) are two representatives of this class undergoing clinical evaluation.
Both compounds are composed of a lipophilic aryl group, an imidazole ring and an alkyl bridge between the
two rings. The imidazole ring in nafimidone was substituted with various groups to investigate the significance of
the imidazole ring in anticonvulsant activity. For this purpose, some compounds having various groups instead of
the imidazole ring in the nafimidone molecule, have been synthesized. 
Anticonvulsant activity of all these compounds were determined by phase I tests of Antiepileptic Drug Development
(ADD) Programme. It was determined that during the seizures in ScMet tests, 1-(2-Naphthyl)-2-(N-succinimidyl)-1-
ethanolne (III) in which succinimide was replaced instead of imidazole in structure was effective in 30 mg/kg dose.
In literature, 3-substituted-6-arylhexahydropyrimidine-2,4-diones (IV) which have a ureid structure have
been reported as a new group that have been found to be active in the anticonvulsant therapy. 
In our studies, we synthesized some new 3-alkyl-6-arylhexahydropyrimidine-2,4-dione derivatives having naph-
tacyl, phenacyl and dithiocarbamate functional group at position 3 of the hexahydropyrimidine-2,4-dione ring as below.
Group I
Group II
According to the activity studies, 3-alkyl-6-arylhexahydropyrimidine-2,4-dione derivatives in Group I were
found to be protective against only ScMet. On the other hand , the derivatives in Group II were found protective
against both MES and ScMet. Neurotoxicity was not observed in any of the synthesized compounds in Group I and
II which were administered to mice in the dose range of 30-300 mg/kg.
Macedonian pharmaceutical bulletin 53 (1,2) 103 (2007)
SPL - 8
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C
CH
2
O
N
O
O
(III)
 
NH
N
O
O
1
2
3
4
5
6
(IV)
Ar
R
    
NH
N
O
O
CH
2
-CO-R
2
R
1
R
1
: -H, -Cl
R
2
 :  Naphtyl, Phenyl, 4-Chlorophenyl, 4-Bromophenyl, 
        4-Methylphenyl
    
NH
N
O
O
CH
2
-CH
2
-S-CS-R
3
R
1
R
1
: -H, -Cl
R
3
 :  Morpholine, Pyrrolidine, Piperidine, 4-Phenylpiperazine
R
2
R
2
: -H, -Cl
 

Introduction of 
99m
Tc-EDDA/HYNIC-TOC, 
a novel radiophamaceutical in nuclear oncology
S. Kuzmanovska
1
, O. Vaskova
1
, T. Tripunoski
1
, M. Kocovska - Zdraveska
1
, C. Decristoforo
2
1
Institut za patofiziologija i nuklearna medicina, Medicinski fakultet, Skopje, R Macedonia
2
Universitatsklinik fur Nuklearmedizin, Innsbruk, Austria
Aim: The use of radiolabelled peptide ligands as diagnostics and therapeutics in nuclear oncology has increased
recently. One of the most frequently used radiopharmaceutical is
99m
Tc-EDDA/HYNIC-TOC, a somatostatine ana-
logue with an affinity for certain types of somatostatine receptors, expressed on some neuroendocrine tumors (lympho-
mas, pheochromocytoma, pituitary adenomas, insulinomas). The radiopharmaceutical (RF) is not comercialy avail-
able, therefore our goal was to introduce its “in house” preparation in our lab, within project activities supported by
IAEA (International Atomic Energy Agency). We introduced a labelling protokol, performed quality control studies
and initial imaging on patients.
Materials and methods: The ligand used for labelling was Tyr
3
octreotide, conjugated with hydrazinonicoti-
namide (HYNIC), supplied by Polatom, Poland. The radiopharmaceutical was procuced “ex tempore” by “wet
labelling” procedure, with addition of co-ligands ethylene diamine diacetic acid (EDDA) and tricine. A fresh eluat-
ed
99m
Tc-pertechnetate, with an activity of 1 GBq was introdiced to the mixture, together with stannous chloride and
the vial was incubated at 70 ºC for 30 min. Than after, the radiopharmaceutical was purified using SepPak mini catr-
tridge (Waters, Milford, USA) and sterilized by filtration. The radiochemical purity was assessed by ITLC on Silica
gel (Merck, 5333) with 3 different solvents: methylethylketone, Na-citrate and acetonytrile. The biodistribution stud-
ies were performed on normal Wistar rats, using static imaging under Gamma camera (Siemens e.cam) after 15 min
and 4h of i.v. injection of the RF. The normal organ distribution was performed by measuring the radioactivity of the
organs of interest (blood, liver, kidneys, spleen, pancreas, adrenals gut and muscle) and was expressed as %ID/g
organ. The first three scintigraphy studies were performed on patients suspecting of pheochromocytoma.
Results: The radiochemical purity of the labeled, conjugated peptide, as assessed by ITCL-SG, was always
> 95%. The free 
99m
Tc-pertechnetate impurities (in MEK) were 0.4% ± 0.26, the non peptide bound 
99m
Tc co-lig-
and impurities were 2.8% ± 1.06 (in Na-citrate) and for the 
99m
Tc-colloid impurities, we obtained 1.23% ± 0.37. The
rat organ biodistribution after 4h of the injection showed the highest accumulation in kidneys (3.2%%ID/g), in the
liver 1.3%ID/g and the lowest percentage was found in muscles 0.15%ID/g. These findings were confirmed by the
static animal scintigraphs. On the first patient images after 15 min and 4h of injection we did not observe any pathol-
ogycal accumulation in the adrenal area. The highest retention was found in kidneys, the liver and spline.
Conclusion: Following the proscribed labeling procedure, we produced a radiopharmaceutical which fulfil-
led the quality control criteria for radiochemical purity and normal biodistribution, as compared with the data from
literature. Our future activities will be focused on the clinical application and evaluation on a group of properly select-
ed patients.
Macedonian pharmaceutical bulletin 53 (1,2) 104-105 (2007)
PP - 38
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