journal of the faculty of pharmacy

Transkript

ISSN : 1300-0608
HACETTEPE UNIVERSITY
JOURNAL OF THE
FACULTY OF PHARMACY
HACETTEPE ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ
DERGİSİ
VOLUME 32 NUMBER 1 JANUARY 2012
Owner and Publisher
L. Ömür Demirezer
(Dean, Hacettepe University, Faculty of Pharmacy Ankara, Turkey)
Executive Place
Hacettepe University, Faculty of Pharmacy, 06100, Sıhhiye, Ankara.
www.eczfakder.hacettepe.edu.tr e-mail : [email protected],
Tel: (0312) 305 1088, Fax: (0312) 311 4777
Executive Editor
Sacide ALTINÖZ
(Hacettepe University, Ankara, Turkey), [email protected]
Associate Editors
Nesrin GÖKHAN KELEKÇİ (Hacettepe University, Ankara, Turkey),
Selma ŞAHİN (Hacettepe University, Ankara, Turkey)
Technical Associate Editor
Mustafa ÇELEBİER (Hacettepe University, Ankara, Turkey)
Type of Publication: Scientific Journal
Linguistic of Publication: English, Turkish
Manner of Publication: Semiannual
Publishing Place: Ankara
Date of Publication: 08.04.2013
Printing: Hacettepe University Hospitals Printing House,
06100 Sıhhiye, Ankara.
Tel: (0312) 310 9790 e-mail: [email protected]
Editorial Board
Prof.Dr. Nurşen Başaran (Hacettepe University, Ankara, Turkey).
Prof.Dr. İnci Erdemli (Hacettepe University, Ankara, Turkey).
Prof.Dr. Süeda Hekimoğlu (Hacettepe University, Ankara, Turkey).
Prof.Dr. İclal Saraçoğlu (Hacettepe University, Ankara, Turkey).
Assoc.Prof.Dr. Meral Özalp (Hacettepe University, Ankara, Turkey).
Advisory Board
Prof.Dr. Okan Atay (Gazi University, Ankara, Turkey)
Prof.Dr. Metin Balcı (METU, Ankara, Turkey)
Assoc.Prof.Dr. Dumitru Baleanu (Çankaya University, Ankara, Turkey)
Prof.Dr. Sema Çalış (Hacettepe University, Ankara, Turkey)
Prof.Dr. İsmet Çok (Gazi University, Ankara, Turkey)
Prof.Dr. Turgay Dalkara (Hacettepe University, Ankara, Turkey)
Prof.Dr. Sedef Kır (Hacettepe University, Ankara, Turkey)
Prof.Dr. Erhan Pişkin (Hacettepe University, Ankara, Turkey)
Prof.Dr. Filiz Öner (Hacettepe University, Ankara, Turkey)
Prof.Dr. Tanju Özçelikay (Ankara University, Ankara, Turkey)
Prof.Dr. Sibel Özkan (Ankara University, Ankara, Turkey)
Prof.Dr. Selma Saraç (Hacettepe University, Ankara, Turkey)
Assoc.Prof.Dr. Ahmet Aydın (Gulhane Military Medical Academy, Ankara, Turkey)
Prof.Dr. Tuncer Değim (Gazi University, Ankara, Turkey)
Prof.Dr. Murat Kartal (Ankara University, Ankara, Turkey)
Subscription Price
Turkey: 2,5 TL. per year (postage included), 1,5 TL. per single copy (postage
included) Foreign Countries: $80.00 per year (postage included)
This journal is indexed in Chemical Abstracts, Analytical Abstracts and
International Pharmaceutical Abstract
(5187 numaralı Basın Yasası mucibince gösterilmesi zaruri bilgiler)
Sahibi: Prof.Dr. L. Ömür Demirezer
Sorumlu Yazı İşleri Müdürü: Prof.Dr. Sacide Altınöz
Yönetim Yeri: Hacettepe Üniversitesi, Eczacılık Fakültesi,
06100 Sıhhiye, Ankara.
Telefon: 305 1088 Faks: 311 4777
Yayın Türü: Yerel Süreli Yayın
Yayın Dili: İngilizce, Türkçe Yayınlanma Biçimi: Altı ayda bir
Basım Yeri: Hacettepe Üniversitesi Hastaneleri Basımevi,
06100 Sıhhiye, Ankara. Telefon: 310 9790
Basım Tarihi: 08.04.2013
CİLT 32 / SAYI 1 / OCAK 2012
VOLUME 32/ NUMBER 1 / JANUARY 2012
CİLT 30 / SAYI 1 / OCAK 2010
VOLUME 30 / NUMBER 1 / JANUARY 2010
HACETTEPE ÜN‹VERS‹TES‹
HACETTEPE UNIVERSITY
ECZACILIK FAKÜLTES‹ DERG‹S‹
JOURNAL OF THE FACULTY OF PHARMACY
İÇİNDEKİLER / CONTENTS
Araştırma Makaleleri
Research Articles
İÇİNDEKİLER
/ /CONTENTS
1
Microbiological Investigation of Used Cosmetic Samples
Kullanılmış Kozmetik Örneklerinin Mikrobiyolojik Yönden Araştırılması
FATMA KAYNAK ONURDAĞ, SELDA ÖZGEN,DUYGU ABBASOĞLU
17
Antimicrobial and Cytotoxic Activities of the Extracts Obtained From the
Araştırma Makaleleri / Research Articles
1
Flowers
of Alcea Rosea L.
Investigation
of Interaction
of Simvastatin with Human Genomic DNA
İnsan Genomik
DNA
ile Simvastatinin Etkileşmesinin İncelenmesi
Sitotoksik
Aktiviteleri
TUBA MERT,
TUĞÇE
FAFAL,Özaltın
BİjEN KıvÇAK, H. TANSEL ÖZTüRK
Mehmet Lütfi
YOLA,
Nuran
17
Paeoniflorin
Diminishes
Maximal
and
PTZ -A Induced
Event
Reporting and
the Effect Electroshockof Pharmacovigilance
Training:
Hospital
Experience
Convulsions
in Mice
PaeoniflorinBildirimi
Farede
Maksimal
ve PTZ
Nöbetlerini
Azaltır
Hakkındaki
BilgiElektroşok
ve Tutumları üzerine
Etkisi:
Bir Özel Hastane
Eczanesi Deneyimi
Semra Koyunoğlu,
Okan Arıhan, Yıldırım Sara, Rüştü Onur,
NAZLı ŞENcAN, MERYEM ALTıNKAYNAK, ıRMAK FERAH,
Sedef Kır,ASLı
İhsan
ÇalışEMEL MASHAKı cEYLAN, PHıLıP MARTıN cLARK
ÖZYıLDıRıM,
31
41 Attitudes,
In vitro Cytotoxic
Activity
Sternbergia
S. lutea andAbout
Pancratium
Patients’
Beliefs
andofDesire
forsicula,
Information
maritimum Extracts
Methotrexate
Therapy
in
Arthritis
Disease
Management:
of
Sternbergia sicula, S. lutea ve Pancratium maritimum EkstrelerininInfluences
ın vitro
Sitotoksik Aktiviteleri
Social Deprivation
and Involvement of a Pharmacist
GüLEN İREM KAYA, BUKET SARıKAYA, DERYA ÇİÇEK,
Artrit Tedavisinde
Hastaların
NEHİR üNvER
SOMER* Oral Metotreksat Tedavisi Hakkında Bilgi
İstekleri, İnançları ve Yaklaşımları: Eczacının Katılımı ve Sosyal Deprivasyonun
Etkisi
Derlemeler / Review Articles
Aygin BAYRAKTAR Ekincioglu
Alcea rosea L. Çiçeklerinden Hazırlanan Ekstrelerin Antimikrobiyal ve
25
The Knowledge and Attitudes of Physicians and Nurses Towards Adverse
Farmakovijilans Eğitiminin Hekim ve Hemşirelerin Advers İlaç Reaksiyonu
49
Etnobotanik ve Türkiye’de Yapılmış Etnobotanik Çalışmalara Genel Bir
53
Adherence Bakış
of Drug Advertisements to the International Marketing Codes
Ethnobotany and a General view of Ethnobotanical Studies in Turkey
GüLSEN KENDİR,
AYŞEGüLPazarlama
GüvENÇ
İlaç Reklamlarının
Uluslararası
Kurallarına Uygunluğu
81
Antiinflamatuvar Tedavide Yeni Bir Yaklaşım: Siklooksijenaz ve
Bilge SÖZEN
ŞAHNE,
Selen
YEĞENOĞLU,
M. Mithat ÜNER,
5-Lipooksijenazın Dual İnhibitörleri
A New Avenue in Anti-İnflammatory Therapy: Dual ınhibitors of
Albert I. WERTHEIMER
cyclooxygenase and 5-Lipoxygenase.
UMUT SALGıN GÖKŞEN, NESRİN GÖKHAN KELEKÇİ
Derlemeler / Review Articles
67
Kanser Tedavisinde Lenfatik Hedeflendirme
Lymphatic targeting in Cancer Therapy
Özge Çevik, Uğur Aydın, R. Neslihan Gürsoy
91
Arnebia Forssk. Türlerinin Kullanışları ve Biyolojik Aktiviteleri
Usage and Biological Activities of Arnebia Forssk. species
Merve Yüzbaşıoğlu, Ayşe Kuruüzüm-Uz
Hacettepe University Journal of the Faculty of Pharmacy
Volume 32 / Number 1 / January 2012 / pp. 1-16
Investigation of Interaction of Simvastatin
with Human Genomic DNA
Received:14.12.2011
Revised:
19.02.2012
Accepted:20.03.2012
Mehmet Lütfi Yola*0, Nuran Özaltın*
Introduction
Simvastatin (SIM) (Figure 1) reduces levels of circulating atherogenic
lipoproteins by competitive inhibition of the microsomal enzyme 3-hydroxy-3-methylglutaryl-co-enzyme A(HMG-CoA) reductase, which catalyzes the conversion of HMG-CoA to mevalonate, a critical intermediary
in the biosynthesis pathway of cholesterol1. Also SIM inhibits oxidation
of native and modified low-density and high-density lipoproteins2. So, it
is used in the treatment of hypercholesterolemia3,4.
Deoxyribonucleic acid (DNA) plays a central role in life process since
it contains all of the genetic information required for cellular function.
However, DNA molecules are prone to be damaged under various conditions, especially by interaction with some molecules and this damage
may lead to various pathological changes in living organisms. Studies
on the binding interactions of small molecules with DNA are of interest
for both therapeutic and construction of new and more efficient drugs
targeted to DNA as well as in understanding how proteins recognize and
bind to specific DNA sequences5-7.
* Hacettepe University, Faculty of Pharmacy, Department of Analytical Chemistry,
Ankara, Turkey
o
Corresponding author: E-mail: [email protected]
2
HACETTEPE UNIVERSITY JOURNAL OF THE FACULTY OF PHARMACY
Figure 1
Chemical structure of SIM
A variety of small molecules interact reversibly with double-stranded
DNA, primarily through three modes: electrostatic interactions, with the
negative charged nucleic sugar-phosphate structure, which are along
the external DNA double helix and do not possess selectivity; binding
interactions with two grooves of DNA double helix; and intercalation between the stacked base pairs of native DNA8.
In recent years, electrochemical methods have gained growing interests in the investigation of DNA – drug interaction9-13. In this study,
detailed investigations on the electrochemical behavior of the interaction
between SIM and DNA were carried out using cyclic voltammetry (CV)
and differential pulse voltammetry (DPV) at hanging mercury dropping
electrode (HMDE). Moreover, UV–vis spectra were studied to characterize
the interaction mode and the interaction mechanism. It is suggestive for
further fruitful research to quantify the therapeutic efficacy of SIM.
Material and Methods
Genomic DNA isolation from whole blood
Human genomic DNA was isolated from whole blood using ammonium sulfate precipitation method14. To confirm the purity of the genomic
DNA extract we used A260/A280 ratio and solutions of the genomic DNA
gave rations of UV absorbance at 260 and 280 nm (A260 / A280) of approxi-
INVESTIGATION OF INTERACTION OF SIMVASTATIN WITH HUMAN GENOMIC DNA
3
mately 2.1, indicating that the genomic DNA was sufficiently proteinfree15. The purified genomic DNA was run on 1.2 % agarose gel at 10 V/
cm for 40 minutes. The gel was stained with Ethidium Bromide and the
gel image was acquired using Kodak Molecular Imaging System.
Human genomic DNA (400 µg mL-1) was dissolved in water and
stored at 4 0C. Solutions were incubated at 37 0C for 2 hours. SIM stock
solution (1.0 x 10-4 M) were prepared in methanol and kept away from
light to avoid photochemical decomposition. Working standard solutions
were prepared daily by appropriate dilution of the stock standard solutions. A series of supporting electrolytes [borate, acetate, phosphate and
Britton-Robinson] were tested in the presence of 1.64 µg mL-1. The results showed that SIM in Britton-Robinson buffer gave the highest signal
response. If not specially stated, the supporting electrolyte was BrittonRobinson (BR) buffer. BR buffers (pH 7.1), ionic strength 0.2 mol dm-3,
were prepared (0.04 M of each of acetic, o-phosphoric and boric acids,
adjusted to the required pH with 0.2 M sodium hydroxide) and used
as supporting electrolytes. All reagents were of analytical grade quality.
Deionized water was used throughout to prepare solutions. Proteinase K,
agarose, ethidium bromide, FX 174 DNA marker and cyan-orange DNA
stain were purchased from Invitrogen (UK). Ammonium Sulfate, SDS,
Ethanol, Tris were purchased from Sigma Aldrich (MO,USA).
Instrumentation
BAS 100 B/W model electrochemical workstation was used. The reference electrode was Ag/AgCl and a platinum wire was used as the auxiliary electrode and HMDE was used as the working electrode. Shimadzu
Spectrophotometer UV-1800 was used for spectrophotometric analysis.
All the pH measurements were made with Metler Toledo MA 235.
Electroanalytical procedure
Prior to the experiments, high purity nitrogen was used to deaeration
of 900 µL supporting electrolyte for at least 10 min. Then 100 µL of 1.0
x 10-4 M stock solution of SIM was placed into the cell to make up 1000
4
µL mixture solution at a SIM concentration of 1.0 x 10-5 M and N2 was
passed through in 2 seconds. The voltammograms were recorded with
cyclic potential scan between -1.0 V and -1.6 V. During the experiments,
nitrogen atmosphere was maintained over the solutions to prevent the
reentry of atmospheric oxygen. All experiments were typically carried out
at room temperature.
Results and Discussion
Interaction of SIM with DNA
The electrochemical behaviour of SIM at HMDE was investigated employing CV and DPV. To prevent DNA from acidic or basic denaturing, BR
buffer of pH 7.1 was chosen as supporting electrolyte. The CV behaviour
of SIM showed one reduction peak at -1.404 V in BR buffer of pH 7.1 with
a scan rate of 100 mV s-1 (Figure 2). No peak was observed in the reverse
scan suggesting that reduction of SIM at HMDE is irreversible.
The effect of scan rate (v) on the peak current (Ip) of SIM have been
studied. For this, we recorded CV of 1.0 x 10-5 M SIM at HMDE in the
scan rate range of 25 – 300 mV s-1 (Figure 3-A). The plots of log Ip versus
logv in the scan rate range of 25 – 300 mV s-1 yielded a straight line with
Figure 2
Cyclic voltammograms of 1.0 x 10-5 molL-1 SIM in the (1) absence and (2) presence of 1.73
µg mL-1 DNA. Supporting electrolyte: Britton–Robinson buffer, pH 7.1, scan rate 100 mVs-1
5
Figure 3
(A) Cyclic voltammogram of 1.0 x 10-5 molL-1 SIM at HMDE Supporting electrolyte:
Britton–Robinson buffer (pH 7.1) ; (a) 25 mV s-1 ; (b) 50 mV s-1; (c) 100 mV s-1 ; (d) 200
mV s-1; and (e) 300 mV s-1
(B) Relationship between the peak currents of the reduction wave of SIM in the absence ()
and presence of () 2.15 µg mL-1 DNA and the square root of scan rates
slope of 0.96 for SIM. These values are close to the therotical value of
1.00, which is expected for an ideal reaction condition for adsorption –
controlled electrode process 16,17. Furthermore, the smaller linear slopes
of DNA complex demonstrated (Figure 3-B) that SIM could interact with
DNA in solution, forming SIM – DNA adducts with large molecular weight,
resulting in a considerable decrease in the apparent diffusion coefficient.
Bard and co-workers 18 reported that positive shifts in the peak potential of intercalators were observed in the binding form via hydrophobic
interactions (intercalation) while electrostatic interactions led to negative
shifts. Based on this report, the positive shifts in the peak potential of
SIM upon binding to DNA should be as a result of intercalative interaction to DNA.
The single reduction peak of SIM is attributed to two electron reduction of ester group on the position 1 (Figure 1)19. So a possible mechanism
is proposed to explain the electrochemical reduction of SIM as below:
The electron transfer coefficient ‘α’ is calculated from the difference
between peak potential (Ep) and half wave potential (Ep/2) according to
6
equation given below 20. The value of electron transfer coefficient is 0.5
for irreversible electrochemical reaction whereas the value of electron
transfer coefficient is 1.0 for reversible electrochemical reaction.
ΔEp=Ep– Ep/2 = (47.7 / αn) mV (irreversible reaction, at 298K)
(1)
The value of α is calculated to be 0.51 ± 0.05 for SIM. So it is suggesting that reduction of SIM at HMDE is irreversible19. For an irreversible
cathodic reaction, we may use the following equation to calculate standard rate constant 21,22.
Ep=E0 + (RT / αnF)[ln(RTk0 / αnF)] – lnv]
(2)
Where E0 is the formal potential, R was the universal gas constant
(8.314 J K-1 mol-1), T (K) was the Kelvin temperature, α was the transfer
coefficient, k0 (s-1) was the electrochemical rate constant and F was the
Faraday constant (96487 C mol-1). The value of E0 was obtained from the
intercept of the Ep versus v plot by the extrapolation to the vertical axis at
v = 0. The values of k0 were evaluated from the plot of Ep versus lnv and
found to be 2.31 (± 0.15) x 105 s-1 for SIM. The value of obtained k0 was
high so it was suggesting that no peak was obtained on the reverse scan.
Furthermore, chronocoulometry was performed in the solutions of
5.0 x 10-5 mol cm-3 SIM in Britton–Robinson buffer of pH 7.1 as the electrolyte. The diffusion coefficient (D) can be determined according to the
formula given by Anson23:
Q(C) = 2nFACD1/2t1/2π-1/2 + Qdl + Qads
(3)
where A (cm2) was the surface area of the working electrode, C (mol
cm ) was the concentration of SIM, D (cm2 s-1) was the diffusion coefficient of SIM, t (s) was time, Qdl (C) was double-layer charge and Qads
(C) was the faradaic charge due to the reduction of adsorbed drug. In
our experiment, the effects of double-layer charge Qdl can be eliminated
via subtraction of the background charge and the plots Q (µC) against
t (s) were converted into the plots of Q against t1/2, which was depicted
in Figure 4. It was clear that the charges (Q) have linear relationships
with the square roots of time (t1/2) for the reduction reaction. According
to Eq.(3), the diffusion coefficient of SIM and SIM-DNA complex can be
estimated from the slope of the plot of Q-t1/2. The diffusion coefficients of
SIM and SIM-DNA complex were calculated as 8.07 (± 0.25) x 10-8 cm2 s-1,
1.32 (± 0.25) x 10-9 cm2 s-1, respectively. Diffusion coefficients decrease
because large complexes decrease mass transfer at electrode surface.
-3
7
Figure 4
The linear relationship between the charges (Q) and the square-root of time (t1/2) for (a)
SIM and (b) for SIM – DNA.
The decrease in peak current and diffusion coefficient of SIM upon
addition of DNA may be attributed to several possible reasons. These
are as follows: The major electrochemical kinetic parameters (α and k0)
of SIM in presence and absence of DNA can demonstrate whether DNA
influences the electrochemical kinetics of SIM or not. The calculated values of α and k0 are noticed to be 0.51 (± 0.05) and 2.31 (± 0.15) x 105 s-1
for SIM in absence of DNA and 0.53 (± 0.05) and 2.48 (± 0.20) x 105 s-1
for SIM – DNA in presence of DNA. In this way, appreciable difference in
the values of α and k0 in presence and absence of DNA was not observed
indicating that the DNA did not alter the electrochemical kinetics of SIM
reduction. In order to prove that the decrease in peak current is not
due to increase viscosity of the solution or the blockage of the electrode
surface by DNA adsorption 24. The special CV experiment was designed
in K4[Fe(CN)6] solution in presence and absence of DNA at glassy carbon
electrode by Kalanur et al 25. The Fe(CN)6-3/-4 ions did not interact with
DNA due to coulombic repulsion between their negative charges. The peak
currents of Fe(CN)6-3/-4 are observed to be almost same in presence and
absence of DNA. The CV experiment was designed in Fe(CN)6-3/-4 solution
in presence and absence of DNA at HMDE by us. The voltammograms of
Fe(CN)6-3/-4 in presence of DNA are the same as that in absence of DNA,
So any interaction was not observed at HMDE. In addition we think that
the same behaviour should be valid for the other electrodes in terms of
interaction only. Hence, we conclude that the decrease in peak current is
not due to change in viscosity of the solution or blockage of the electrode
8
surface by DNA adsorption. Therefore, the decrease in peak currents of
SIM upon the addition of DNA is attributed to decrease in equilibrium
concentration of free SIM. Hence, we propose that SIM and DNA formed
an electrochemically inactive SIM–DNA complex. Ester group of SIM on
the position 1 interacted with DNA so inactive SIM-DNA complex occured
with increasing concentration of DNA. This indicates interaction between
DNA and drug.
If it is assumed that the interaction of DNA with drugs produces only
a single complex, DNA – mDRUG, then the equation shown below holds
good26:
DNA + mSIM ↔ DNA - mSIM
where m is the binding ratio. The equilibrium constant, βS can be
deduced from the following equation:
βS=[DNA–mSIM]/{[DNA][mSIM]m}
(4)
According to the Ilkovic equation for an irreversible electrode process,
ΔImax=kCDNA
(5)
ΔI=k[DNA—mSIM]
(6)
[DNA]+ [DNA — mSIM] = CDNA
(7)
ΔImax-ΔI=k(CDNA–[DNA—mSIM]]
(8)
ΔImax-ΔI=k[DNA]
(9)
From Eqs. (4), (5), and (8) we get
(1/ΔI)=(1/ΔImax)+{[1/(βSΔImax)]x(1/[SIMm])}
(10)
or
log[ΔI/(ΔImax–ΔI)]=logβS+mlog[SIM]
(11)
where ΔI is the difference in peak current in presence and absence of
DNA and ΔImax corresponds to the obtained value when the concentration
of SIM is extremely higher than that of DNA. CDNA, [DNA] and [DNA –
mSIM] are the total, free and bound concentrations of DNA in the solution, respectively. If DNA interacts with SIM to form a single complex,
then the plot of log [ΔI /(ΔImax - ΔI)] versus log [SIM] shows linearity. The
values of binding ratio and binding constant are obtained from the slope
and intercept, respectively, and these values are found to be 2.0 and
9
1.66 (± 0.10) x 104 M-1 for SIM-DNA. Thus, the formation of a stable 1:2
complex (DNA:SIM) are proposed.The other method for the calculation of
binding constant is Bard’s method:
Amperometric current titrations were performed by keeping the constant concentration of the drug while varying the concentration of DNA
using both DPV and CV at pH 7.1. The interaction of drug with DNA can
be described using the following equation:
drug + DNA ↔ drug – DNA
An equation for amperometric titration can be deduced according to 9,10,18,27
(12)
Where K is the apparent binding constant, IG and IH-G the peak current of the free guest (G) and the complex (H-G), respectively. Under the
assumption of diffusion – controlled electron transfer and the complex of
drug with DNA (in nucleotide phosphate) is 1:1 association complex, then
the plot of log (1 / [DNA]) versus log (IH-G / (IG - IH-G)) becomes linear with
the intercept of log (K). The binding constant of this complex were evaluated according Eq. (12) and the results are listed in Table I.
Linear range of DNA determination
The decrease in peak current of SIM resulted from the addition of
DNA into SIM solution can be employed to determine the concentration
of DNA. The peak current of DPV of SIM at -1.388 V was used as the
detection signal. Under the optimum experimental condition of Figure
5, the decreases in the DPV peak current were linearly related to DNA
concentration in the range of 0 – 10.15 µg mL-1 when SIM concentration
were fixed at 20.0 ng mL-1, detection limit of DNA was found as 0.0025 µg
mL-1. Data of the calibration curves for the proposed method were given
in Table II.
Limit of detection (LOD) was estimated by the equation:
LOD=3.3S/m
(13)
S: Standart deviation of the intercept, m: Slope of the regression line 28
The relative standard deviation (RSD) of six experiments performed
at DNA concentration level of 1.85 µg mL-1 was % 1.4, indicating that the
10
TABLE I
Binding constant of SIM – DNA complex calculated from the results of voltammetry at pH
7.1
Complex
Cyclic voltammetry, K (M-1)
Differential pulse
voltammetry, K (M-1)
SIM – DNA
1.48 (± 0.15) x 104
1.57 (± 0.15) x 104
Figure 5
Differential pulse voltammograms of 20.0 ng mL-1 SIM in the absence (a) and presence
of (b) 1.73 (c) 3.85 (d) 5.15 (e) 7.15 µg mL-1 DNA. Step potential (Es) = 0.004 V and pulse
amplitude 0.05 V
proposed method can provide a reproducible determination. The results
suggested that the proposed method is simple and sensitive hence it can
be applied to the determination of many kinds of DNA.
Effect of the ionic strength on the interaction of SIM with DNA
The effect of the ionic strength on the interaction of SIM with DNA
using cyclic voltammetry was also investigated to acquire possible binding modes. It has been shown that when a charged ligand is added to
a polyelectrolyte solution, its binding constant, K, depends on the total
counter ion concentration, [Na+], as follows29.
dlogK/dlog[Na+]=Δr= -zΨ
(14)
Where z is the charge on the ligand molecule, Ψ is the fraction of
counterions and Δr is the number of counter ions released upon binding
11
TABLE II
Data of the calibration curves for the proposed method (n = 7)
Regression equation
Standart error of slope
Standart error of intercept
Correlation coefficient (r)
Linearity range (DNA concentration)
Number of data points
LOD
y = - 19.189 x + 319.78
1.18
1.74
0.9994
0 – 10.15 µg mL-1
7
0.0025 µg mL-1
y = ax + b; y: peak current (nA), x: DNA concentration (µg mL-1), a: Slope, b: Intercept,
LOD: Limit of Detection
of the ligand with charge z. The theoretical slope of the log K vs. log[Na+]
relation is -0.88 for a singly charged ligand bound to the B form of DNA
in aqueous solutions at 250C 29. The plot of log K vs. log[Na+] was linear
with a slope of -0.44. A comparison of the experimentally determined
value of the slope with that predicted by theory indicates that electrostatic attractions are less important for the interaction of SIM with DNA.
UV/Vis spectra
Figure 6 showed the UV/Vis absorption spectra of SIM in the absence and presence of different concentrations of DNA. The maximum
absorbance of SIM was located around at 230, 238 and 245 nm. It was
observed that on the addition of DNA, SIM showed an decrease in molar absorptivity with a red shift of 1 - 6 nm. This hypochromic effect
is thought to be due to the interaction between the electronic states of
the intercalating chromophore and those of the DNA bases30. The SIM
solution exhibited hypochromic effect and bathochromic shift in UV/Vis
spectra upon binding to DNA, a typical characteristic of an intercalating
mode 31.
Based on the variations in the absorbance spectra of SIM upon binding to DNA, the binding constant (K) was calculated according to the
equation (15) 32,33. The absorbance at 238 nm was used to calculate the
binding constant (K) in UV/Vis spectra.
(15)
12
Figure 6
UV-VIS absorption spectra of 1.0 x 10-5 mol L-1 SIM in the absence (a) and presence of (b)
1.73 (c) 3.85 (d) 5.15 µg mL-1 DNA
where A0 and A are the absorbances of drug in the absence and presence of DNA, εG and εH-G are the absorption coefficients of drug and its
complex with DNA, respectively. According to Eq. (15), the plot of A0 / (A –
A0) versus 1 / [DNA] was constructed using the data from the absorbance
titrations and a linear fitting of the data yielded the binding constant (K)
1.41 x 104 M-1 for SIM - DNA. These results are close to that from voltammetry (Table I).
Conclusions
In this study, the interaction of SIM with DNA was studied by cyclic
voltammetry, differential pulse voltammetry and UV/Vis spectroscopy.
The binding of SIM to DNA resulted in a series of changes in the electrochemical behavior and spectra characteristics. Upon binding of SIM to
DNA, absorption spectra of SIM showed hypochromic effect and bathochromic shift. From these experimental results, it could be affirmed that
the interaction of SIM with DNA through intercalative mode. In addition
that the effect of the ionic strength on the interaction of SIM with DNA
13
was investigated and It was found that electrostatic interactions were
less important for the interaction of SIM with DNA. The results demonstrate that the electrochemical and spectroscopic methods are available,
and offers great promise for study of the mechanism of the interaction
between DNA and targeting compounds. The electrochemical method developed here offers important advantages of low detection limit, low cost,
sensitivity, rapid and easy applicability.
Summary
The interaction of simvastatin (SIM), a hipolipidemic drug, with human genomic DNA has been investigated by cyclic voltammetry (CV), differential pulse voltammetry (DPV) and UV/Vis spectroscopy. As a result
of the interaction of SIM with human genomic DNA, a considerable decrease in the SIM peak currents and a hypochromic and bathochromic
shift in the maximum adsorption bands of SIM was observed. The changes in the electrochemical and spectral characteristics of SIM indicated
SIM bind to DNA by intercalative mode. Binding constants were determined using voltammetric and spectroscopic data. In addition that the
effect of ionic strength on the interaction support the intercalation of SIM
into the DNA double helix. These studies are valuable for a better understanding SIM – DNA interaction, which should be important into the
determination of therapeutic efficacy of the drug and design of new DNA
targeted drug in future.
Key Words: Simvastatin, Human genomic DNA, Electrochemistry,
UV/Vis spectroscopy
Özet
İnsan Genomik DNA ile Simvastatinin etkileşmesinin
incelenmesi
Bir hipolipidemik ilaç olan simvastatinin insan genomik DNA ile
etkileşmesi dönüşümlü voltametri (CV), differansiyel puls voltametrisi
(DPV) ve UV/Vis spektroskopisi yöntemleri kullanılarak incelenmiştir.
14
Simvastatinin insan genomik DNA ile etkileşmesi sonucunda simvastatinin pik akımında önemli bir azalmayla birlikte maksimum absorpsiyon bandlarında hipokromik ve batokromik etki gözlenmiştir.
Simvastatinin elektrokimyasal ve spektral özelliklerindeki değişiklikler
DNA ile interkalasyon bir şekilde etkileştiğini belirtmektedir. Voltametrik
ve spektroskopik veriler kullanılarak bağlanma sabitleri tayin edilmiştir.
Ayrıca etkileşme üzerine iyonik şiddetinin etkisi simvastatinin DNA çift
sarmalına interkalasyonunu desteklemektedir. Bu çalışmalar ilacın tedavi edici etkisinin belirlenmesi açısından önemli olan simvastatin – DNA
etkileşiminin daha iyi anlaşılması ve ilerde yeni DNA hedefli ilaçların
tasarlanması açısından değerlidir.
Anahtar Kelimeler: Simvastatin, İnsan Genomik DNA, Elektrokimya,
UV/Vis spektroskopisi
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Paeoniflorin Diminishes Maximal
Electroshock - and PTZ - induced
Convulsions in Mice
Received:18.02.2012
Revised:
23.03.2012
Accepted:03.04.2012
Semra Koyunoğlu1*, Okan Arıhan2, Yıldırım Sara2, Rüştü Onur2,
Sedef Kır3, İhsan Çalış4
1. Introduction
Paeonia Radix is one of the most commonly used herbal drug in traditional Chinese medicine1 and folkloric medicine of different countries
since ancient times2. A number of chemical and pharmacological studies have been conducted on the roots of Paeonia species. Monoterpen
glycosides are the active constituents, among them; paeoniflorin is the
main compound in genus Paeonia3. In traditional medicine aqueous decoctions of Paeonia have been used against several types of seizures1,4,5.
Epilepsy is a common chronic neurological disorder characterized
by recurrent seizures, resulting from excessive electrical activity of the
brain. Medical treatment of epilepsy mainly consists of preventing seizure
activity by anti-epileptic drugs. Anti-epileptic drugs prevent seizures in
about two third of the patients6,7. Most of the anti-epileptic drugs are not
individually effective in inhibiting all types of seizures and they mostly
Department of Basic Pharmaceutical Sciences, Faculty of Pharmacy, Hacettepe
University
2
Department of Pharmacology, Faculty of Medicine, Hacettepe University
3
Department of Analytical Chemistry, Faculty of Pharmacy, Hacettepe University
4
Department of Pharmacognosy, Faculty of Pharmacy, Hacettepe University, Ankara,
Turkey
o
Corresponding author: e-mail: [email protected]
1
18
can suppress only one type of seizure and even sometimes they worsen
the other epileptic disorder types. For instance, fenitoin, a first line drug
for partial and tonic-clonic epilepsies, aggravates absence type of epilepsy. Some of the epileptic patients are resistant to currently used drugs.
Therefore, research for new drugs with different mechanisms of action
and broader spectrum of activity is still required.
Screening plant-originated molecules is a promising approach to develop new antiepileptic agents. Various compounds derived from Paeonia
were shown to have antiseizure effects in several studies8,9. Although
Paeonia species have been used to treat all types of epilepsies for centuries in traditional medicine, antiseizure effects of paeoniflorin in generalized tonic-clonic and/or absence type of epilespy have not been evaluated
yet. Sugaya et al10 extracted several compounds from Paeonia species
and found that they alter PTZ-induced EEG power spectrum changes
in anesthezied rats and they reported that paeoniflorin was one of the
active compound among the other molecules tested in this study10. Although, the exact mechanism of antiseizure action of paeoniflorin is not
clear, previous studies demonstrated that it exerts inhibitory effect on
L-type Ca2+ channels11, iberiotoxin-sensitive large conductance calciumactivated potassium channels12 and voltage-gated sodium channels13,14.
In this study, we aimed to evaluate the efficacy of paeoniflorin in
generalized seizures in mice. In order to test the antiseizure activity of
paeoniflorin, we employed MES and PTZ which are both standard procedures for testing the effectiveness of antiepileptic agents in generalized
tonic-clonic and absence type of epilepsies, respectively15,16.
2. Material and Methods
2.1. Plant material
Paeonia mascula subsp. arietina was collected from Sikirin Tepe, Bitlis, Turkey, in June, 2004. A voucher specimen (ADD-11999) has been
deposited at the Herbarium of the Department of Biology, Faculty of Sciences, Hacettepe University, Beytepe, Ankara, Turkey.
PAEONIFLORIN DIMINISHES MAXIMAL ELECTROSHOCK - AND PTZ - INDUCED CONVULSIONS IN MICE
19
2.2. Reagents
All chemicals were analytical reagent grade. Deionized water was prepared using a Barnstead Nanopure Diamond Analytical (USA) ultrapure
water system.
2.3. Extraction and isolation of the compound
The air-dried powdered roots of Paeonia mascula subsp. arietina
(480g) were extracted with MeOH under reflux (40oC). The solvent was
removed by rotary evaporation yielding 30.191 g methanolic extract. The
methanolic extract was subjected to vacuum liquid chromatography using reversed-phase material (Sepralyt 40 mm, 100 g), as eluents, employing H2O (400 mL), H2O- MeOH (75:25, 100 mL, 50:50 mL, 25:75
mL) and MeOH (400 mL ); to give 27 fractions (100 ml/fractions), which
were combined to nine main groups (I-IX ) based on their TLC profiles:
Fr. II (1.103 g) was subjected to normal-phase silica-gel (100 g) CC with
CH2Cl2-MeOH-H2O mixtures with increasing polarity (80:20:1®80:20:2)
to give compound paeoniflorin (477 mg).
Paeoniflorin is an amorphous white powder; [a]D: -12.90 (c = 1.0,MeOH);
The 1H-, 13C-NMR spectra and physical constants were similar to the previous report3,17.
2.4. Animals
All experimental procedures involving animals were conducted in accordance with the guidelines for animal experiments of Hacettepe University Ethical Committee (Approval number: 2008/16-2) together with
the recommendations from the Declarations of Helsinki. Male mice, 2-4
months old, weighing 25-40 g were used in the experiments. Animals
were maintained in a room where temperature (24±2°C) and relative humidity (55±15%) were kept within constant limits with a light-dark cycle
of 12 h/12 h, (lights were on at 07:00). Water and food were available ad
libitum. Groups of 12 to 18 mice were used for each dose of the drugs and
during the experiments mice were housed in individual cages. Experiments were carried out between 12:00 and 18:00 hrs.
2.5. Drugs
The following drugs were used; phenytoin (Sigma, USA) 15 mg/kg,
PTZ (Sigma) 80mg/kg and paeoniflorin (obtained from Paeonia species
20
from Turkey) at 100, 250 and 500 mg/kg. The drugs were dissolved in
saline and were administered intraperitoneally in a volume of 10 ml/kg
of body weight. Control mice received saline and standard drug (positive
control) group received phenytoin alone.
2.6. Statistical analysis
All data are expressed as the mean±SEM. Data were analyzed with
Student’s unpaired t-test and accepted as significant when P<0.05.
3. Experimental Procedure for Antiepileptic Activity
3.1. Maximal electroshock
Convulsions were induced with rectangular current pulses delivered
by a Grass S44 stimulator (Grass, USA) and a constant current unit
(Grass CCU1A) using corneal electrodes. Electrodes were soaked with
saline containing 1% lidocaine was applied to the eyes in order to ensure
sufficient electrical conduction and to avoid pain18. MES was applied
at current intensities between 32.5-42 mA with 3 ms duration and 60
Hz for 1 sec. Maximal seizure was defined by the tonic extension of the
hindlimbs to an angle close to 180° to the plane of the body axis.
A maximal electroshock seizure threshold test was also performed.
For the assessment of threshold for convulsions, four groups of mice consisting of at least 12 animals were used. Groups were tested with various
electrical stimuli in different current intensities. The shocks were aimed
to yield 10-30%, 30-50%, 50-70% and 70-90% of animals with fully developed seizures. Following this, a current intensity-effect curve was constructed according to a log-probit method by Litchfield and Wilcoxon19.
Using this curve, a median current strength (CS50 in mA) and its 95%
confidence limits with SEM were calculated20. The CS50 value obtained
from the control mice was used to evaluate the efficacy of paeoniflorin on
MES-induced convulsions at different doses and time intervals following
drug administration. MES was applied 40, 90, and 120 minutes after
drug administration. In the evaluation of anticonvulsive effect, following
parameters were compared: 1) Number of animals having convulsions. 2)
Duration of hindlimb extension. 3) Duration of post-convulsive recovery
period, including recovery from righting position and from stupor state.
21
3.2. PTZ test
Convulsions were induced with 80 mg/kg pentylenetetrazolium (PTZ)
given intraperitoneally. Maximal seizure was defined by the tonic extension of the hindlimbs to an angle close to 180° to the plane of the body
axis. Paeoniflorin was given at 100 and 250 mg/kg doses 90 minutes
prior to PTZ injection. The time period before the initiation of the first
generalized convulsion and the duration of the convulsion were accepted
as a measure of anticonvulsive effect.
3.3. Rotarod test
Animals treated with paeoniflorin were also tested with a rotarod device 4.5 cm in diameter with a rotation speed of 12 r.p.m. in order to determine the possible effects of paeoniflorin on motor performance.
4. Results
4.1. Effects of paeoniflorin on MES-induced seizures
In order to assess the effect of paeoniflorin on the seizure threshold
for MES, we first determined the CS50 value. Control mice had CS50 value
of 35.3± 1.0 mA (n=12 animals for different current intensities). After
250 mg/kg paeoniflorin treatment, the seizure threshold increased to
39.4±1.4 mA (Table I, n=12 animals, p<0.05).
Immediately after the electroshock administration in the control animals, a tonic flexion of all limbs (with a slight tremor lasting approximately 2 s) was observed (n=18, Table II). Following this phase, tonic extension
of the hindlimbs occurred and lasted for 15±0.2 s. After the cessation of
seizure activity, animals displayed a period of stupor for 38±2 s and then
they recovered. Paeoniflorin at 100 mg/kg was ineffective at preventing
convulsions. However, the number of animals displaying convulsions after 250 and 500 mg/kg paeoniflorin administration was significantly lower
after 90 min of injection. Convulsions were observed only in 25 % of mice,
90 min after 250 mg/kg of paeoniflorin injection. This anticonvulsant
effect persisted after 120 minutes. Greater inhibitory effect was observed
at 500 mg/kg. Seventeen percent of the mice displayed convulsions 120
22
TABLE I
The effect of paeoniflorin on electroshock threshold in mice
Treatment
(mg/kg)
CS50
N
S.E.M.
Control
Paeoniflorin
(Saline)
250
35.3 (33.3-37.4)
39.4 (36.1-41.2)*
12
12
1.0
1.4
Results are presented as median current intensities (CS50 values with 95% confidence
limits) necessary to produce tonic hindlimb extension in 50% of animals tested in
the electroshock-induced seizure threshold test. Paeoniflorin was administered
intraperitoneally 90 min before the electroshock. Data was statistically evaluated by
unpaired Student’s t-test. * P<0.05 vs. control group.
TABLE II
Effects of Paeoniflorin on the Electroshock-induced Convulsions in Mice
Treatment (mg/kg)
Convulsive
Control
Paeoniflorin
Paeoniflorin
Paeoniflorin
Phenytoin
Saline
100
250
500
15
Time after
injection
(min)
N of
mice having
convulsions
Duration of
hindleg
extension (s)
Postrecovery
period (s)
40
9/18
15.0 ± 0.2
38.6 ± 2.7
90
9/18
15.7 ± 0.3
41.0 ± 3.5
120
9/18
14.1 ± 0.5
45.7 ± 3.7
40
6/12
14.0 ± 0.5
41.2 ± 2.7
90
5/12
14.4 ± 0.5
44.4 ± 8.4
120
6/12
14.5 ± 0.5
49.3 ± 8.7
40
6/12
13.7 ± 0.6
40.6 ± 3.6
90
3/12
12.3 ± 1.2
50.3 ± 12.8
120
3/12
15.0 ± 1.5
48.7 ± 9.6
40
6/12
14.3 ± 0.3
43.0 ± 4.7
90
3/12
14.0 ± 0.9
120
2/12
15.2 ± 0.7
54.0 ± 7.3
40
0/12
0
0
90
1/12
13.0
43.2
120
0/12
0
0
72.0 ± 8.9
Table presents results of control, paeoniflorin and phenytoin groups in MES test.
Paeoniflorin was given at increasing doses to the groups. Times of intraperitoneal
injections for paeoniflorin were 40, 90 and 120 minutes before MES. All values presented
as mean±SEM.
23
TABLE III
Effects of Paeoniflorin on the PTZ-induced Convulsions in Mice
Time after
injection
(min)
N of
mice having
convulsions
Latency for
the
1st generalized
convulsion (s)
Duration of
convulsion
(s)
Saline
90
8/8
245.5 ± 50.3
11.1 ± 1.1
Paeoniflorin
100
90
8/8
346.0 ± 45*
12.0 ± 1.1*
Paeoniflorin
250
90
6/8
684.8 ± 105.8*
16.3 ± 0.8*
Treatment (mg/kg)
Control
Table presents results of control and paeoniflorin groups in PTZ test. Paeoniflorin was
given at two different doses to different groups. Time of intraperitoneal injection for
paeoniflorin was, 90 minutes before PTZ injection. All values presented as mean±SEM. *
P<0.05 vs. control group.
minutes after paeoniflorin injection. The duration of hindlimb extension
in paeoniflorin-treated animals was not different from the controls at all
doses tested. The recovery period was prolonged in all animals treated
with paeoniflorin. This effect was significant at 250 and 500 mg/kg.
Paeoniflorin administration caused a mild sedation and decreased locomotor activity. This sedative effect was detectable at 100 mg/kg and it
increased in a dose-dependent fashion, but these animals still responded
to the external stimuli. Paeoniflorin-induced sedative effect was not evaluated quantitatively. However, all of the animals injected with 500 mg/
kg paeoniflorin maintained to stay on the rotarod longer than 1 min at 12
r.p.m showing that motor control was not impaired.
Phenytoin was tested as a standard drug (positive control) to compare the antiseizure activity of paeoniflorin. As it is well documented20-22,
phenytoin (15 mg/kg) prevented the convulsions induced by MES in 11
animals out of 12 (n=12, Table II) and these animals did not display stupor or behavioral alterations after electroshock administration.
4.2. Effects of paeoniflorin on PTZ-induced seizures
Following PTZ administration all animals in control (saline) and 100
mg/kg paeoniflorin groups had generalized tonic-clonic seizures (Table
III). On the other hand, only 6 out of 8 mice had convulsions after PTZ
injection in 250 mg/kg paeoniflorin group. However, the latencies for the
initiation of these seizures significantly increased in paeoniflorin groups
24
in a dose dependent manner (p<0.05). Latencies were 245 ± 5 s, 346 ± 45
s, and 684 ± 105 s in the control group, 100 mg/kg paeoniflorin group
and 250 mg/kg paeoniflorin group, respectively.
Duration of seizures also showed a similar dose-dependent prolongation. In the control group, duration was 11.1±1 s and after 100 mg/kg
paeoniflorin administration duration was increased to 12±1 s and it was
16.3±0.8 s in the 250 mg/kg paeoniflorin group. These prolongation of
seizure durations following 100 and 250 mg/kg paeoniflorin administrations were statistically significant when compared to the control values
(P<0.05 for both 100 and 250 mg/kg paeoniflorin groups).
4.3. Rotarod test and other effects of paeoniflorin
In order to assess the possible toxic effects of paeoniflorin at the
maximum dose, we observed the animals for 15 days and all animals
survived without any noticeable signs of toxicity or complications. Animals did not display any abnormality in their gait and posture within
this period. Additionally, we assesed the possible effects of paeoniflorin
on the motor coordination and/or fatigue resistance on mice by using
Rotarod test. Animals from both paeoniflorin and control groups performed similarly in Rotarod test showing paeoniflorin did not alter motor
performance of the mice.
5. Discussion
The outcome of this study indicates that paeoniflorin, the active constituent of Paeonia plant, displays an antiseizure activity in both tonicclonic and absence type of generalized seizure tests in mice. Paeoniflorin
effectively reduced the number of animals having convulsions subsequent to the electroshock administration and these animals displayed
significantly exceeding seizure thresholds for MES. This antiseizure activity of paeoniflorin was observed at 250 mg/kg and higher doses for
MES whereas it was apparent at 100 and 250 mg/kg doses in PTZ test.
Paeoniflorin-induced antiseizure activity appears to be dose-dependent.
Only two out of twelve mice displayed convulsion at the maximum dose
(500mg/kg) of paeoniflorin in MES experiments at 120 minutes. Although
paeoniflorin effectively increased the seizure threshold for MES, it did
25
not alter the duration of the hindlimb extension in animals having convulsions. When the antiseizure activity of paeoniflorin is compared with
that of phenytoin, paeoniflorin exerted antiseizure activity with a lower
potency. Phenytoin and paeoniflorin seems to have different antiseizure
activity spectrums23.
In order to evaluate the activity of paeoniflorin as an absence type of
epilepsy drug we employed a commonly used screening test for the drugs
effective against absence type of epilepsy24. In this study, paeoniflorin
did not alter the number of animals having convulsions after PTZ injection. However, paeoniflorin significantly prolonged latency period for the
appearance of the first convulsion as well as the seizure duration in the
PTZ tests. These findings indicate that paeoniflorin may also have some
activity against absence type of epilepsies.
Contrary to phenytoin25, paeoniflorin seems to be effective on both
absence and tonic-clonic types of generalized seizures. These findings
may imply that paeoniflorin utilizes a different mechanism of action than
phenytoin. Furthermore, it may be proposed that combination of phenytoin with paeoniflorin may help to reduce phenytoin dose and consequently, its side effects as well. Since there is scarce information about the
mechanism of paeoniflorin, similar plant based pharmacological studies
may provide additional information about its mode of action. A recent
ethnopharmacological study demonstrates that brain monoamines such
as serotonin can behave as an inhibitory molecule in seizure activity26.
Interaction of paeoniflorin with such inhibitory neurotransmission during antiseizure activity needs clarification. According to our findings, anticonvulsant activity of paeoniflorin became prominent 90 min after the
injection and this effect persisted over 120 min. These findings suggest
that paeoniflorin is a slow-onset drug in terms of its antiseizure activity and most likely this is a consequence of its pharmacokinetic profile.
Moreover, further research on oral bioavailability and evaluation of possible synergic interactions with standard antiepileptic drugs are required
for the assessment of paeoniflorin in this context to conclude the value
of paeoniflorin as a candidate molecule in the treatment of epilepsy. Nevertheless, this study strongly implies that ethnopharmacological use of
paeoniflorin has a realization in the treatment of epilepsy in folkloric
medicine.
The major limitation of this study is the finding that the effective
doses of paeoniflorin on seizures were higher than 250mg/kg. In another
26
study focusing on medicinal plants, effective doses were found to be as
200 and 400 mg/kg and our results show similar potency in this aspect26.
These high doses seem to be irrelevant in clinical settings (human use)27.
However, it should be taken into account that this acute dose may be
much lower in its chronical use. Additionally, in our study, this molecule
was tested in mice and pharmacological effective doses of paeoniflorin
may be different in humans because of interspecies differences. In support of this view, anecdotal evidence from herbal medicine also implies
that lower doses seem to be effective in patients with epilepsy.
Paeoniflorin induced sedation and inhibited locomotor activity at
doses greater than 250 mg/kg. These effects do not seem to be related
to a neuromuscular blocking action, since animals maintained their gait,
and posture, and their motor performance on the rotarod test was within
the control limits indicating that paeoniflorin does not cause incapacitance of the animals at these doses. In addition, paeoniflorin-treated animals were responsive to external stimuli. Therefore, paeoniflorin-induced
inhibition of locomotor activity seems to be related to its actions on the
central nervous system. Prolonged post-convulsive recovery period may
also be explained with peoniflorin’s inhibitory effect in the central nervous system.
This is the first study demonstrating the antiseizure activity of paeoniflorin against generalized seizures induced either by MES or PTZ in
the experimental animals. Although the mechanism of action of antiseizure activity of paeoniflorin is not elucidated in this study, it seems to be
different from that of phenytoin. Since the effectiveness of paeoniflorin
in PTZ-induced absence type seizure model is also demonstrated in this
study, this property of the glycoside may broaden its antiseizure spectrum. Although extracts of peony have been cited from western medicine since 17th century, only recently the responsible molecules could be
found28. This molecule can be a candidate for a new anti-seizure drug or
as an add-on therapy with other antiepileptic drugs. Further pharmacological studies and molecular modifications on paeoniflorin are essential
for the assessment of this compound.
Research focusing on plant originated chemicals based on ethnopharmacological relevance is a fruitful area and current research on
this subject continues to yield new molecules29. Evaluation of anticonvulsive properties of other monoterpene glycosides, tannins30 and
27
Paeonimetabolin-I31,32 which exert anti-convulsant action from paeony
species may stimulate a research field for the appraisal of new anti-seizure compounds and further modifications of these molecules may provide new options for the treatment of seizures.
Acknowledgements
The authors thank to Ali A. Donmez PhD, for collecting plant material and to Mert Ertunc MD PhD for his scientific contribution to the article. Authors declare that there are no financial or commercial conflicts
of interest concerning this article.
Summary
Paeonia species have been used to treat epilepsia in traditional medicine, however the effects of paeoniflorin, the main constituent of Paeonia
species have not been studied scientifically. We studied the effects of
paeoniflorin with generalized tonic-clonic convulsion model induced by
Maximal Electroshock (MES) and pentylenetetrazolium (PTZ) in mice. In
MES test paeoniflorin was given intraperitoneally at doses of 100, 250
and 500 mg/kg. The current strength which yielded convulsions in 50 %
of the animals was calculated by MES threshold test. The effects of paeoniflorin on convulsions were evaluated firstly via MES test. Paeoniflorin
exerted anticonvulsive effect at 250 and 500 mg/kg and decreased the
number of animals having convulsion. Anticonvulsive effect of paeoniflorin was observed 90 min after injection. In PTZ test we observed that 100
and 250 mg/kg paeoniflorin prolongs both the time period required for
generalized convulsions and the duration in a dose dependent fashion.
Based on these findings, we conclude that paeoniflorin inhibited generalized tonic-clonic convulsions induced by MES, and partially attenuated
seizures in absence seizure model. Paeoniflorin and other monoterpenic
glycosides of Paeonia species may be considered as a candidate for the
development of a new anticonvulsive drug.
Keywords: Paeony, Paeonia, paeoniflorin, maximal electroshock, PTZ,
antiseizure.
28
Özet
Paeoniflorin Farede Maksimal Elektroşok ve PTZ Nöbetlerini
Azaltır
Paeonia türleri halk tıbbında epilepsinin tedavisi amacıyla kullanılan
türlerdir, ancak Paeonia türlerinin ana etken maddesi olan paeoniflorinin etkileri bilimsel olarak incelenmemiştir. Bu çalışmada paeoniflorinin Maksimal Elektroşok (MES) ve pentilentetrazol (PTZ) ile oluşurulan
jeneralize tonik-klonik konvülsiyon modelindeki etkileri araştırılmıştır.
MES testinde paeoniflorin 100, 250 ve 500 mg/kg dozlarında intraperitoneal yolla verilmiştir. MES eşik testi ile hayvanların yüzde ellisinde konvülsiyona neden olan akım ölçülmüştür. Paeoniflorinin konvülsiyonlar
üzerine etkileri once MES testi ile değerlendirilmiştir. Paeoniflorin 250 ve
500 mg/kg dozlarında antikonvülsif etki göstermiş ve konvülsiyona giren
hayvan sayısını azaltmıştır. Paeoniflorinin antikonvülsif etkileri enjeksiyondan 90 dakika sonra gözlenmiştir. PTZ testinde paeoniflorinin 100 ve
250 mg/kg dozlarında jeneralize konvülsiyonlar için gereken süreyi ve
konvülsiyon süresini doza bağımlı olarak uzatmıştır. Bu bulgular ışığında,
paeoniflorinin MES’le indüklenen jeneralize tonik-klonik konvülsiyonları
inhibe ettiğini değerlendirdik. Paeoniflorin ve paeonia türlerinde bulunan
diğer monoterpenik glikozitler yeni antikonvülsif ilaçların geliştirilmesi
için aday olarak değerlendirilebilir.
Anahtar Kelimeler: Şakayık,
elektroşok, PTZ, antikonvülsif.
Paeonia,
paeoniflorin,
maksimal
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Patients’ Attitudes, Beliefs and Desire for
Information About Methotrexate Therapy
in Arthritis Disease Management:
Influences of Social Deprivation and
Involvement of a Pharmacist
Received:03.07.2012
Revised:
19.09.2012
Accepted:27.09.2012
Aygin Bayraktar Ekincioglu1,2
Introduction
Arthritis and its associated conditions are the most common causes
of physical disability and long-standing illness in the United Kingdom
(UK) among the group of musculoskeletal conditions, which leads to social isolation and chronic pain1,2.
Almost all patients with rheumatoid arthritis (RA) receive diseasemodifying anti-rheumatic drugs (DMARDs) at some stage of their disease
and methotrexate is usually the first choice treatment. Although new
drugs have been introduced in the treatment of RA, methotrexate (MTX)
preserves its value and become established in the literature as a ‘standard’ comparative therapy in the assessment of new therapeutic agents3-9.
Many studies have focused on the effects of environmental factors
and lifestyle variations on occurrence, progression and disease outcomes
of RA. Socio-economic environment has also an impact on health status
as well as health care utilisation. It appeared that socially deprived areas do not particularly have an increased risk of developing RA, however
Hacettepe University, Faculty of Pharmacy, Ankara / TURKIYE
University of Strathclyde, Strathclyde Institute for Biomedical Sciences (SIBS), Glasgow
/ Scotland- UK ( at the time of the study)
o
1
2
32
there are possibilities that those who have the lowest socio-economic status may seek more medical attention during their treatment10. Moreover,
the patients domiciled in affluent areas showed better health status and
perceived self-efficacy for pain management and dealing with other arthritic symptoms11. It was also pointed out that the patients in more
deprived areas were more likely to be put on complex second line drug
treatments and also require more inpatient medical care12. Although
these trends lost significance after adjustment of age, sex and treatment
centre variables, it was identified that the patients domiciled in more
deprived areas presented more severe diseases and disease progression
over the first three years.
Introduction of national policies has highlighted the importance of
patient self-management strategies explaining patients’ behaviour in
their disease management13. It is believed that health behaviour has an
impact on patient’s quality of life, therefore modifications in health behaviour and respond to expectancies might yield positive outcomes in
disease management in order to achieve delayed onset of chronic disease or extended active lifetime14,15. Perception of illness threat (vulnerability) and evaluation of behaviours to overcome this threat determines
the likelihood of an individual taking health-related actions. Individuals
are more likely to respond when they perceive an action is beneficial to
overcome the threat that they face. Patients’ willingness to seek help for
disease management originates from a need for better understanding of
their symptoms, and builds on patient’s self-enablement, their relationships with health care professionals and availability of resources in the
community16,17.
Relatively modest amount of research have been undertaken about
involvement of community pharmacist in arthritis disease management18-24; however there is little known about the impact of pharmacist’s
contributions into the multidisciplinary team in the management of arthritis. Therefore, the aim of the study was to evaluate patient’s beliefs
about their medicines and self-care attitudes in the management of rheumatoid arthritis and to assess the influences of social deprivation and
involvement of a pharmacist on these aspects.
PATIENTS’ ATTITUDES, BELIEFS AND DESIRE FOR INFORMATION ABOUT METHOTREXATE THERAPY IN
ARTHRITIS DISEASE MANAGEMENT: INFLUENCES OF SOCIAL DEPRIVATION AND INVOLVEMENT OF A
PHARMACIST
33
Methods
This study was a part of a research which was designed to implement
and evaluate patient monitoring processes in order to ensure continuity
of care by involving community pharmacists among patients diagnosed
with arthritic conditions who receive oral methotrexate therapy25. The
ethics approvals of the study were granted from the Ethics Committee of
the North and the South Glasgow University Hospitals NHS Trusts, the
Greater Glasgow Community/Primary Care Local Research Ethics Committee in September/October 2001.
The study population was selected within previously identified four
Local Health Care Co-operatives (LHCCs), of those served by two rheumatology clinics in Glasgow, Scotland/UK. Approximately 55 community
pharmacists were identified, contacted by the researchers and their consent forms of participation were obtained afterwards. The pharmacies
were considered eligible if they were later nominated by a participated
patient who agreed to take part in the study. The unit of randomisation
was the community pharmacists, which was undertaken by a researcher.
Randomisation was based on the pharmacists in order to avoid bias in
the process of provision of care. Sampling was stratified to ensure similar
numbers of community pharmacies in postcode sectors identified as ‘deprived’ (Carstairs Category 4, 5, 6 and 7) compared with sectors identified
as ‘non-deprived’ (Carstairs Category 1, 2 and 3)26.
The patients were recruited both in the community pharmacies and
in the hospital outpatient clinics during the period of December 2001May 2002. The patient’s demographics including age, sex, duration of
disease, duration of methotrexate therapy, number of previous DMARDs
therapies, number of co-morbid conditions and socio-economic status
of their place of residence were recorded and they were categorised as
residing in a ‘non-deprived’ or ‘deprived’ area in terms of the Carstairs
Category.
Inclusion criteria for patients were being over 16 years of age, able to
read and understand the English language, diagnosed with RA or psoriatic arthritis, currently receiving oral methotrexate therapy and residing
in the identified LHCC environments. The patients were excluded if they
nominate a community pharmacy that has not been agreed to participate,
do not reside in the designated LHCC environments, have other types of
34
arthritis, are pregnant or part of a couple planning a pregnancy, have
learning difficulties, mental illnesses or life threatening disorders and are
identified by the rheumatologist as a patient who is otherwise unsuitable
for inclusion.
During the study period, patients were asked to attend the same
community pharmacy and to bring their methotrexate monitoring cards
to their pharmacist at each visit. The nominated community pharmacists
were asked to counsel the patients and identify their problems about
methotrexate therapy if there is any. At the initial visit, the patients were
given a copy of the questionnaire to complete after they agreed to participate and have been informed about the study, which involved completing
a questionnaire once every 6 months at the clinic and attending the same
community pharmacy during the study period, and their informed consent was sought only if their nominated pharmacist had already agreed
to participate. The patients were then allocated in the control or the study
group according to the results of the randomisation of their community
pharmacy that they had nominated. The patients in the study group
were received a pharmaceutical care plan initiated and issued by rheumatologist and hospital pharmacist at the clinic, followed by their community pharmacists more closely and drug therapy problems, changes
in the laboratory values, drug treatment and compliance issues were
assessed where necessary and the patients were also referred to their
general practitioners (GP) if required. Patients in the control group were
received routine care from their rheumatologist and pharmacists and no
pharmaceutical care plans were issued.
The overall questionnaire was adapted from the previously validated
questionnaires27-30. The administration of the questionnaire was piloted
in order to ensure patient’s acceptability on a sample of patients (n=20) at
the rheumatology clinic not otherwise eligible for inclusion in the study.
The final questionnaire for this study consisted of 35 questions under
three sections including patient’s intrinsic desire for information (IDI- 12
questions); patient’s beliefs about their medicines (BMQ-11 questions)
and patient’s satisfaction about service that they receive at the community pharmacy (8 questions). The answers for each question were categorised on a 5-point Likert scale from ‘strongly disagree’ (1) to ‘strongly
agree’ (5) options. The further four questions were related with any prob-
PHARMACIST
35
lems that patients may have experienced with their medication in the
past. The questionnaire took approximately 15-20 minutes to complete.
The Extent of Information Desired (EID) scale is derived from the
IDI scale on the basis of factor analysis. The original authors extracted
two factors; factor 1 indicates the extent of information desired (EID) by
patients and was considered useful to identify receptive (high scorers) or
refractory (low scorers) patients. The high scorers were defined as those
patients who had the factor score above the mean score of the population.
The factor 2 indicates the inhibited desire for knowledge about illness/
drugs rather than the amount of information patients are concerned28.
The specific Beliefs about Medicines questionnaire consists of the
‘necessity’ and ‘concerns’ scales and total score for each scale range from
5 to 25. The original authors suggested that scores above the mid-point
on the scales indicate strong beliefs in the ‘necessity’ and the ‘concerns’
regarding medicines29. Furthermore, the authors suggested including
another item in the ‘concerns’ scale and studied the internal consistency
of the 6-item scale. As a result, they concluded that if Cronbach’s alpha
value was >0.65, the new item can be included in the analysis which
makes the total score for the ‘concerns’ scale range from 6-30 (Personal
communication with R.Horne in March 2002). The analysis for internal
consistency, which was undertaken in the sampled patient population
for this study, revealed that the Cronbach’s alpha value for the five-item
concerns scale was 0.42 and 0.47 for the six-item scale. It is indicated
that the additional item increased the internal consistency of the concerns scale, therefore the 6th item was included in the further analysis
of the ‘concerns’ scale. However, the results of the 5-item concerns scale
were also reported in order to allow comparison with the results from the
original authors.
The authors of the BMQ also suggested to calculate the ‘necessity’
and ‘concerns’ scales (five items) differential in order to indicate the relative importance of attitudes for individual patients. This is calculated as
the difference between the ‘necessity’ and the ‘concerns’ scores and has
a range of -20 to 20, where the positive value indicates that the patients
perceive that the benefits of medication outweigh the risks [Personal
communication with R.Horne in March 2002].
36
A sample size calculation was based on the questionnaire results
that have been previously reported by the original authors of two previously validated questionnaire instruments for discriminating differences
in patients’ attitudes to their medicines28,29. A total sample size of 88-112
was calculated as the total number of patients required according to the
post-test questionnaire results with 80% power.
All data were collected by the researcher, anonymised and stored
in a computer; the results were analyzed by the Statistical Package for
the Social Sciences (SPSS) for Windows version-9. The differences in responses between the time periods were analyzed by using the appropriate statistical tests for unpaired and paired data and the distribution of
the scores for questionnaire scales for normality was assessed by Kolmogorov Smirnov test where appropriate.
In comparison of the study and the control groups which data were
considered as ‘unpaired’ for baseline and follow-up period, Chi-square
and/or Fisher’s exact tests were used for nominal data (e.g. patients’ demographics); Mann-Whitney U test was used for ordinal data (e.g. scores
of EID, IDI); Student’s t test or Mann-Whitney U were used for continuous data (e.g.scores of ‘necessity’ and ‘concern’ scales) where appropriate.
Results
During the recruitment process, 59 patients responded to the questionnaire, of which 85% were diagnosed with rheumatoid arthritis, 13.3%
with psoriatic arthritis and 1.7% with polyarthritis. Demographics data
of the patients were summarised in Table I and no statistically significant
difference was found between the study and the control group at baseline
(Chi-square tests or Fisher’s exact test, p>0.05). Subsequent to the follow-up after 6 months, 52 out of 59 (88%) questionnaires were returned.
For the total patient population at the baseline assessment, in regards to the scores for IDI scale (normality test p=0.175) 38% of the patients scored higher than or equal to the mean score (≥36). The mean (SD)
score for the EID scale was 18.0 (3.4) for the total patient population and
31% of the patients scored higher than the mean score (>18). No statistically significant difference was found between the control and the study
37
PHARMACIST
TABLE I
Demographics of patients participating in the study (n=59) at baseline.
Study group
(n=28)
Control group
(n=31)
Total
(n=59)
60.7 ± 11.2
59.5 ± 10.8
60.1 ± 10.9
Gender
Male
Female
7 (25%)
21(75%)
7 (23%)
24 (77%)
14 (24%)
45 (76%)
Carstairs category;
1, 2 and 3 (‘non-deprived’)
4, 5, 6 and 7 (‘deprived’)
11 (39%)
17 (61%)
11 (35%)
20 (65%)
22 (37%)
37 (63%)
Number of other diagnoses*
48 (47%)
54 (53%)
102 (100%)
Cardiovascular
9 (32.1%)
7 (23.3%)
16
Other musculoskeletal conditions
6 (21.4%)
7 (23.3%)
13
Gastrointestinal disorders
6 (21.4%)
5 (16.7%)
11
Peripheral vascular diseases
4 (14.3%)
3 (10.0%)
7
Infectious diseases
3 (10.7%)
3 (10.0%)
6
Skin disorders
2 (7.1%)
2 (6.7%)
4
Endocrine disorders
2 (7.1%)
2 (6.7%)
4
-
3 (10.0%)
3
Renal disorders
2 (7.1%)
3 (10.0%)
5
Respiratory disorders
1 (3.6%)
3 (10.0%)
4
6 (21.4%)
11 (36.7%)
17
Age, Mean ± SD
Haematological disorders
Miscellaneous
* Other diagnoses were indicated in 28 study group’s patients and 30 control group’s
patients.
Comparison of the study and the control group; Chi-square tests or Fisher’s exact test,
p>0.05
group patients (Mann-Whitney U test, U= 394.00, p>0.05) at baseline.
The mean (SD) score for the inhibited desire for knowledge was 7.0 (2.7)
in the total patient population and 55.2% of the patients scored ≤7 in this
study. No statistically significant difference was found between the two
socioeconomic groups (p>0.05) at baseline assessment.
38
According to the results of the BMQ scales at the baseline, half of
the patient population (49% scored ≤19) was convinced that their MTX
medication is ‘necessary’ for maintaining or improving their health now
and in the future, and over a third of the sample (63% scored ≤19) did
not have strong ‘concerns’ about potential side effects and risk of their
MTX treatment. No statistically significant difference was found between
the control and the study group patients in regards to the ‘necessity’
(Student t test, p=0.18; Mann-Whitney U test, U=297.50 p= 0.48) and the
‘concerns’ scale (Mann-Whitney U test, U=311.0; p=0.06; McNemar test,
p>0.05) at the baseline and 6 months follow up assessment respectively.
At the baseline assessment, 86.4% of the total patient population had a
positive value for the necessity and concerns differential.
However, after 6 months, 60% of the total population scored ≤20 on
the ‘necessity’ scale, which indicated that more than half of the patient
population was convinced that their MTX medication was ‘necessary’ for
maintaining or improving their health now and in the future. In the ‘concerns’ scale, 56% of the patient population scored ≤20 which indicated
that nearly over half of the sample did not have strong concerns about
potential side effects and risk of their MTX treatment. In regards to socioeconomic status, no statistically significant difference was found between non-deprived and deprived groups in terms of necessity (p=0.44)
and concern (p=0.25) scale at 6 month follow up.
The general findings did not change noticeably after 6 months. No
statistically significant difference was found between the study and the
control group patients’ responses to the questionnaire at baseline and 6
months after assessment in terms of IDI, EDI and BMQ-necessity and
BMQ-concerns scales (Mann Whitney U test, p> 0.05) (Table II).
According to the patient self-management strategies, 52% of the patients in the study group and 50% of the control group indicated that
they had problems with their medications in the past at the baseline,
compared to 64% and 42% respectively after 6 months assessment.
There was no statistically significant difference between the study and
the control group (Chi-square test, p >0.05).
The patients who had problems with their medications in the past,
also indicated their actions (Table III) and behaviours about seeking ad-
39
PHARMACIST
TABLE II
The comparison of the ‘study’ and the ‘control’ group at the baseline and 6 months after
assessments
Study group; mean score (SD)
Control group; mean score (SD)
Pre
(n=28)
Post
(n=24)
p value*
Pre
(n=30)
Post
(n=28)
p value*
IDI
35.1 (5.3)
36.5 (4.4)
0.085
36.4 (5.9)
36.0 (4.9)
0.642
EID
17.9 (3.5)
17.9 (1.9)
0.817
18.2 (3.4)
17.3 (3.3)
0.216
19.9 (2.9)
20.1 (2.4)
1.000
18.7 (3.6)
19.3 (3.7)
0.250
17.7 (3.8)
19.4 (2.4)
0.030
19.5 (3.7)
20.2 (3.1)
0.435
Satisfaction
15.7 (4.9)
17.8 (4.9)
0.176
19.4 (4.1)
19.4 (5.7)
1.000
Overall
satisfaction
4.3 (1.2)
4.3 (0.9)
0.331
4.2 (1.0)
4.2 (0.9)
0.861
Scales
BMQnecessity
BMQconcerns
*Paired t test; SD: standard deviation; IDI: Intrinsic Desire for Information; EID: Extent of
Information Desired; BMQ: Beliefs about Medicines Questionnaire.
TABLE III
The self-management activities taken by the study and the control group patients in case
of medication problems in the past
Study group
Control group
Baseline
(n=12)
6 months
after
(n=14)
Baseline
(n=14)
6 months
after
(n=11)
Contacted the GPs
3
2
5
4
Contacted
rheumatologist
1
5
1
2
Contacted GP/
rheumatologist
1
3
2
1
Contacted nurse
specialist
-
-
-
1
Stopped medication
2
1
1
-
Changed medication
2
2
1
-
Medication withdrawn
-
-
3
2
Admitted to the hospital
-
1
1
-
No specific action taken
3
-
-
1
*Chi-square test, p=0.48; GP: General Practitioner
TABLE IV
59%
11%
From the GP
surgery
From the
pharmacy
GP: General Practitioner
-
70%
At the GP
surgery
At the pharmacy
67%
At the clinic
Show the card
59%
From the clinic
Advice seeking
Usually
-
4%
15%
4%
15%
15%
Sometimes
Baseline (n=28)
24%
57%
86%
9%
59%
82%
Usually
5%
14%
5%
23%
14%
5%
Sometimes
6 months after (n=24)
Study group
-
61%
74%
6.5%
52%
61%
Usually
-
10%
10%
3%
26%
13%
Sometimes
Baseline (n=30)
19%
78%
63%
19%
56%
59%
Usually
7%
-
11%
7%
15%
11%
Sometimes
6 months after (n=28)
Control group
The self-management activities taken by the study and the control group patients during their monitoring process
40
PHARMACIST
41
vice / showing drug monitoring cards (Table IV) at baseline and follow up
assessment.
Over half of the patient population (56.4%) indicated that they were
‘very satisfied’ with the service that they regularly receive at community
pharmacy, while this rate was 27.3% for those who were ‘fairly satisfied’.
The original 7-item questionnaire (total scores ranged from 6-28) related
to satisfaction on prescription service and provision of information in
the pharmacy was previously piloted30. The mean (SD) value for the patient population was 17.7 (4.85) (normality test, p=0.20) and 51% of the
population scored ≥18. No statistically significant differences were found
between the 'non-deprived' and the 'deprived' patient groups (Student t
test, p=0.71). The control group patients were more satisfied with the service at the pharmacy compared to the study group patients at baseline
(Student t test, p=0.03). Although the patient satisfaction with services at
pharmacy showed a trend to increase after 6 months of the study period,
it did not reach statistical significance (p=0.178).
The data for methotrexate prescriptions were collected by the participating community pharmacists between the period of December 2001
and December 2002 both for the control and the study group patients.
Two hundred and forty seven prescriptions were presented by 37 patients (15 patients from the study group and 22 from the control group)
within 12 months. The data are summarised in Table V and Table VI.
The mean (SD; Median) dose of prescribed methotrexate was 12.4
(3.7; 12.5) mg/week and the mean (SD; Median) dose of prescribed folic
acid was 5.2 (0.9; 5) mg/week for the patient population. The mean (SD;
Median) amount of dispensed tablets of methotrexate per prescription
was 9.8 (8.4; 8) for the 2.5 mg tablet strength and was 7.4 (6.8; 4) for the
10 mg tablet strength. No statistically significant difference was found
between the ‘control’ and the ‘study’ group in terms of the number of prescriptions presented for methotrexate and for other medication during
the data collection period (Chi-square test, p>0.05).
The pharmacists indicated in one hundred and twenty six visits that
the mean (SD; Median) time that they spent with patients on each visit
was 2.6 (2.3; 2) minutes in order to dispense the prescription presented
and/or to discuss any problems that the patient may have at the time of
the visit to the pharmacy.
42
TABLE V
Methotrexate prescription details for the study and the control group patients
Study
group
(n=15)
Control
group
(n=22)
Total
(n=37)
98
149
247
9 (9%)
53 (54%)
36 (37%)
14 (9%)
75 (50%)
60 (40%)
23 (9%)
128 (52%)
96 (39%)
indicated as;
2.5mg/week
7.5mg/week
10mg/week
12.5mg/week
15mg/week
17.5mg/week
20mg/week
22.5mg/week
43
4 (9%)
6 (14%)
15 (35%)
10 (23%)
3 (7%)
3 (7%)
2 (5%)
67
1 (2%)
17 (25%)
8 (12%)
28 (42%)
6 (9%)
5 (8%)
2 (3%)
-
110
1 (0.1%)
21 (19%)
14 (13%)
43 (39%)
16 (15%)
8 (7%)
5 (5%)
2 (2%)
Number of prescriptions with only
2.5mg tablet strength prescribed
38 (86%)
61 (87%)
99 (87%)
-
-
-
Number of prescriptions with
combination of tablet strengths
prescribed
6 (14%)
9 (13%)
15 (13%)
Number of visits to pharmacy
(per person)
98 (5.8)
149 (7.5)
247
Number of prescriptions presented
MTX only
Other medication
MTX and other medication
Dose of MTX in prescriptions
presented
Number of presciptions with only 10
mg tablet strength prescribed
The results indicated that patients from the ‘deprived’ environment
did not present more prescriptions than those from the ‘non-deprived’
environment (observed findings in the ‘deprived’ is 63% (expected 60%;
Chi-square test, p>0.05). However, in regard to the indicated dose of
methotrexate, the proportion of prescriptions presented by patients for
methotrexate at the dose of ≥15 mg/week was 18% in the 'deprived' group
compared to 47.5% in the 'non-deprived' (Chi-square test, p<0.05).
43
PHARMACIST
TABLE VI
MTX prescription details for the patients in the ‘non-deprived’ and the ‘deprived’ group
Non-deprived
(n=15)
Deprived
(n=22)
Total
(n=37)
92
155
247
7 (7.6%)
51 (55.4%)
34 (36.9%)
16 (10.3%)
77 (49.7%)
62 (40.0%)
23 (9%)
128 (52%)
96 (39%)
38
4 (10.5%)
3 (8%)
13 (34%)
9 (24%)
5 (13%)
4 (10.5)
-
72
1 (1%)
17 (24%)
11 (15%)
30 (42%)
7 (10%)
3 (4%)
1 (1%)
2 (3%)
110
1
21
14
43
16
8
5
2
35
65
105
Number of prescriptions with only 10
mg tablet strength prescribed
-
-
-
Number of prescriptions with
combination of tablet strengths
prescribed
6
9
15
Number of prescriptions presented
MTX only
Other medication
MTX and other medication
Dose of MTX in prescriptions presented
indicated as;
2.5mg/week
7.5mg/week
10mg/week
12.5mg/week
15mg/week
17.5mg/week
20mg/week
22.5mg/week
Number of prescriptions with only
2.5mg tablet strength prescribed
Discussion
The study hypothesised that the patients would desire more information about treatment if they would have increased beliefs about the
necessity of their medications and become less concerned about the potential side effects of their therapies, with them being engaged with selfmanagement activities along with the involvement of their pharmacists
in disease management.
The total patient population already indicated a high desire for information about their medicines at the baseline which was not surprising
considering that RA is a chronic condition that the patients have to live
with. Only the ‘study’ group patients maintained desire and attitudes
44
towards willingness to have information, whereas the ‘control’ group patients’ desire slightly decreased after 6 months, which might indicate the
lack of pharmacist’s input into their MTX therapy. Neame and her colleagues have indicated that there is a very high level of need for information about RA and its treatment among patients; however patients’ desire
for involvement in treatment decision making was low compared with the
desire for information; consequently patients want to be informed, but
they do not necessarily want to make decisions about their treatment17.
The original authors of the IDI questionnaire had previously found
that the total EID score was significantly associated with patient’s age
(Pearson r=-0.432), school leaving age (Pearson r=0.115) and occupation
(Spearman rho=-0.15), which indicates that older patients tend to desire
less information than younger patients. Given the fact that the patients
with RA are more likely to be older but still willing to have more information, the crucial point is to maintain their desire for information about
their medicines in the long term. Previous studies have shown that patients from different socio-economic backgrounds may present dissimilar preferences about information or show different attitudes and beliefs towards their medication27. Although the results of the IDI and EID
scales from the pre- and post- assessments showed varying patterns, the
slightly observed trend may indicate that the patients from the deprived
areas are more likely to demand information regarding their treatment
and may need more attention from the health care professionals. However, the interpretation of these findings is limited by the small sample
size. This is worthy of more investigation in a larger study.
The original authors of the BMQ indicated that the patients with
stronger beliefs in the necessity were significantly more adherent, whereas the patients with stronger concerns were significantly less adherent
(31). Considering the necessity scale, 51% of the patients in this study
seem to be more likely to adhere to the treatment, however 37% of the
patients are less likely to adhere to their MTX therapy considering the
concerns, where pharmacist should be more vigilant to identify those
patient groups. The study group patients expressed increased concerns
about the MTX potential side effects and risk of the treatment after the
6 months period. In other words, involvement of the pharmacists in the
monitoring processes might have caused patients to become more concerned about their treatment which presented an existing or even an
PHARMACIST
45
increased awareness about their therapy. Only 14% of the total patient
population have a negative value (less than ‘0’) for the ‘necessity and concerns differential’, at baseline and 6 months after, which indicated that
the majority of the patients believe that the potential benefits of methotrexate therapy outweigh the risk of the treatment, indicating a direction
of change associated with an increased likeliness to adhere to the therapy.
It was recently shown that the concerns about MTX treatment seemed to
play minor role whereas the necessity is closely associated and compliance with MTX therapy was unrelated with functional capacity, however
compliance behaviour changes with the disease duration and use of folic acid supplementation32. Furthermore, patients who have greater concerns about arthritis medication seem to experience more side effects,
especially when starting a new drug or tend to discontinue the current
treatment33,34. Therefore being alert for concerned patients especially at
earlier stage of treatment process would enable health care professionals
to help patients in order to lessen their concerns and increase vigilance
about drug treatment.
The patients become more involved with MTX monitoring process
which was indicated by an increased proportion of showing of their drug
monitoring card in the clinic, general practitioner (GP) surgery and at
the pharmacy and seeking advice when they have problems with their
medication. The patients perceive the hospital rheumatology clinic as
a first call for information or help request regarding their diseases or
treatments. This attitude is mainly because of the implementation of an
established telephone helpline with a specialist nurse in order to help
the patients with arthritis. The nurse specialist is delegated to help and
solve any problems that the patients may have experienced during their
treatments. Thus, the conventional system places a centralised workload, perhaps disproportionately, on one particular health care profession which may have the effect of restricting the initiative of patients to
seek any help from other health care providers. The greater recognition
of the community pharmacist’s role, by the patients as well as the health
care providers, within the system of monitoring of the patients would create an opportunity for responsibilities to be shared by the patients and
the care providers.
The study also has number of limitations. At first, small sample size
might have affected the ability of the study to demonstrate significant
46
differences in outcome measures because sample size calculation was
based on the previous results of the questionnaire scales. Secondly, a
certain amount of time should be allowed for the patient population to
demonstrate detectable changes that might occur in the outcome measures. This study was initially designed to last for 12 months, whereas
the intervention period had to be limited to 6 months. Therefore, the
intended time period might have not been sufficient to recognise the
changes in the patients’ beliefs, attitudes and opinion about the new care
model proposed from the health care providers.
Conclusion
The patients are willing to receive information about their treatment,
although they hold concerns over the side effects of the MTX therapy.
Given the fact that, most of the patients with chronic diseases become
an expert in their condition after living with it for a long period of time,
the majority of them encompass potential side effects in long and/or
short term therapy. The education of patients and maintenance of their
knowledge about treatments are crucial for chronic disease management.
Therefore, it is important to verify and assess the patients’ desire for information about their therapies and their beliefs about their medications
which would provide an explicit framework for the self-management strategies. The study serves as a descriptive analysis of patients’ behaviour
and attitudes during chronic disease management, which impact on patient empowerment, the patient’s awareness and self- management role.
Conflict of interest: None.
Summary
Objectives: To evaluate patient’s attitudes, beliefs and desire for information on methotrexate treatment in arthritis and to assess the influence of involvement of a pharmacist on these aspects. Patient’s selfmanagement activities within community settings and any influence of
socio-economic environment were also investigated.
PHARMACIST
47
Methods: The study was undertaken at the hospitals and community
settings in Glasgow, Scotland where patients were categorised as ‘affluent (non-deprived)’ and ‘deprived’ areas according to the postal codes of
their residence. After randomisation for the study and the control group,
the patients were given the questionnaire to fill in and a monitoring card
to bring in to the pharmacy at the time of recruitment and 6 months
thereafter. ‘Study’ group patients were received a pharmaceutical care
plan which was issued by rheumatologist and pharmacist at the clinic
and a copy of care plan was given to the patient him/herself, to their community pharmacists and to the GPs. Therefore, pharmaceutical care plan
was considered a helpful tool for a community pharmacist in order to
monitor those patients more closely and involve in disease management.
‘Control’ group patients were received their care in a routine way.
Results: Fifty nine patients consented and were randomised into the
study and the control group. Fifty two patients who had complete data
were further analyzed. At the baseline 49% of the patient population
was convinced that methotrexate medication was ‘necessary’ for maintaining or improving their health, and 63% of the sample did not have
strong ‘concerns’ about potential side effects and risk of treatment; after
6 months these rates were %60 and %56, respectively. No statistically
significant difference was found between the study and the control group
in terms of beliefs on ‘drug necessity’ and ‘concerns about drug side effects’ (p> 0.05). The hypothesised effect of social deprivation has not been
shown in this study.
Conclusion: Patients’ desire for information should be balanced according to patients’ beliefs on ‘drug necessity’ and ‘concerns about drug
side effects’. The recognition of patient’s willingness to take self-care
activities would create opportunity for pharmacists in monitoring to be
shared by patients and care providers.
Keywords: Beliefs about medicines questionnaire (BMQ), Intrinsic
Desire for Information (IDI), methotrexate, rheumatoid arthritis, pharmacist, primary care
48
Özet
Artrit Tedavisinde Hastaların Oral Metotreksat Tedavisi
Hakkında Bilgi İstekleri, İnançları ve Yaklaşımları: Eczacının
Katılımı ve Sosyal Deprivasyonun Etkisi
Amaç: Bu çalışma; artrit tanısı konmuş, oral metotreksat tedavisi alan hastaların, ilaç tedavileri hakkındaki bilgi isteklerini, inanç
ve yaklaşımlarını değerlendirmek ve bu parametreler üzerine eczacı
katılımının etkisini incelemek üzere yapılmıştır. Hastaların, toplum
içerisinde kendi-kendine bakım amacıyla yaptıkları aktiviteler ve sosyal
deprivasyonun etkisi de ayrıca irdelenmiştir.
Metod: Çalışma, İskoçya-Glasgow’daki iki hastane ve seçilen serbest eczanelerde yapılmıştır. Hastalar yaşadıkları bölgenin posta koduna göre ‘varlıklı (refah içinde)’ ve ‘yoksul (mahrum)’ gruplar olarak
sınıflandırılmıştır. ‘Çalışma’ ve ‘kontrol’ grubuna randomizasyon
sonrasında, hastalara doldurmaları için bir anket verilmiş ve hastalardan 6 ay sürecek olan çalışma süresince ilk vizitte ve 6 ay sonraki vizitte,
izlem kartlarını yanlarında taşıyarak eczacılarına göstermeleri istenmiştir.
‘Çalışma’ grubundaki hastalar için, hastanede romatoloji uzmanı ve
eczacı tarafından hazırlanan farmasötik bakım planı oluşturularak, birer
kopyasının da hastanın kendisine, serbest eczacısına ve aile hekimine
verilmesi sağlanmıştır. Böylelikle hastanın serbest eczacı tarafından
yakından izlemine yardımcı olunmuştur. ‘Kontrol’ grubundaki hastalar
ise tedavilerini rutin şekilde almaya devam etmiştir.
Bulgular: Elli dokuz hasta onam formunu imzalayarak çalışmaya
katılmış ve ‘çalışma grubu’ ve ‘kontrol grubu’ olarak randomize
edilmişlerdir. Verilerinin eksiksiz olduğu belirlenen 52 hasta üzerinde analiz yapılmıştır. Çalışma başında hastaların %49’u sağlık
durumlarını iyileştirmek ve iyi hali sürdürmek için metotreksat tedavisinin ‘gerekliliği’ne inanmış; %63’ünün ise tedavi riski ve olası yan etkiler konusunda yoğun ‘endişeler’inin olmadığı belirtilmiştir; bu oranlar
6 ay sonrasında sırasıyla %60 ve %56 olarak belirlenmiştir. Çalışma’ ve
‘kontrol’ grubu arasında ‘ilacın gerekliliği’ ve ‘ilaç yan etkileri hakkındaki
endişeler’ bakımından istatistiksel açıdan önemli bir fark bulunmamıştır
(p>0.05). Öne sürülmüş bir tez olan sosyal deprivasyonun etkisi ise bu
çalışmada ne yazık ki gösterilememiştir.
PHARMACIST
49
Sonuç: Hastaların tedavileri hakkında bilgi istekleri hastanın ‘ilacın
gerekliliği’ ve ‘ilaç yan etkileri hakkındaki endişeleri’ konusundaki
inançlarına göre dengelenmelidir. Hastanın kendi-kendine bakım aktivitelerini yapma konusundaki istekliliğinin farkına varılması, eczacının
hasta ve diğer sağlık personeli ile ortak bir izlem/bakım hizmeti sunması
açısından fırsatlar yaratacaktır.
Anahtar kelime: metotreksat, artrit, serbest eczacı
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28.Duggan C, Bates I, Sturman S, Andersson E, Astrom K, Carlsson J. Validation of a
‘desire for information’ scale, International Journal of Pharmacy Practice,10,31-7,2002.
PHARMACIST
51
29. Horne R, Weinman J, Hankins M. The Beliefs about Medicines Questionnaire: the development and evaluation of a new method for assessing the cognitive representation
of medication, Psychology and Health,14,1-24, 1999.
30.Whithead P, Atkin P, Krass I, Benrimoj SI. Patient drug information and consumer
choice of pharmacy, International Journal of Pharmacy Practice,7,71-9,1999.
31.Horne R. Representations of medication and treatment:advances in theory and measurement. In: Weinman J, Petrie KJ, editors. Perceptions of health and illness. p. 15588.
32. de Thurah A, Nørgaard M, Harder I, Stengaard-Pedersen K. Compliance with methotrexate treatment in patients with rheumatoid arthritis: infuence of patients’ beliefs
about the medicine. A prospective cohort study, Rheumatol Int, 30,1441-8, 2010.
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Research,62(6),791-9, 2010.
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Adherence of Drug Advertisements to the
International Marketing Codes
Received:06.11.2012
Revised:
07.12.2012
Accepted:21.12.2012
Bilge Sözen Şahne*0, Selen Yeğenoğlu*, M. Mithat Üner**,
Albert I. Wertheimer***
Introduction
Misleading information about drugs may lead to inappropriate drug
use which can damage patients’ health and cause an increase in medical
costs1-3. On the other hand, some of the promotional activities implemented by pharmaceutical companies provide useful information about
drugs.
In this context, the World Health Organization (WHO) has defined
the promotional activities as follows: “Promotion refers to all informational
and persuasive activities by manufacturers and distributors, the effect of
which is to induce the prescription, supply, purchase and/or use of medicinal drugs” 4.
Hacettepe University, Faculty of Pharmacy, Department of Pharmacy Management,
06100, Sihhiye - Ankara TURKEY
**
Gazi University, Faculty of Economics and Administrative Sciences, Department of
Business Administration, 06900, Beşevler - Ankara TURKEY
***
Temple University School of Pharmacy, Department of Pharmacy Practice, PA 19140
Philadelphia / USA
o
Corresponding author: e-mail: [email protected] Tel: +90 (312) 305 42 26
*
54
“Personal selling, Advertising, Public Relations and Sales Promotion”
are the four major promotion activities5. Among them advertising is one
of the most used promotional methods for drugs. A drug advertisement
often placed in objectively and verifiable medical journals is the easiest
available drug promotion method to healthcare professionals6,7. Besides
drug advertisements are one of the most detailed information sources for
updating physicians’ drug knowledge8. Moreover it is indicated that the
main reason for placing drug advertisements in medical journals is to
finance them9.
All types of information activities; drug recommendation to patients,
some stages of selling and supplying efforts can be included in pharmaceutical advertising10. Announcing a new product, creating a product or
rising brand awareness, providing information about the brand, its benefits and superior features and increasing sales are the main aims of drug
advertisements11. In many countries, drug advertising is allowed only to
the healthcare providers (i.e. physician, pharmacist, nurse, dentist, etc)
who are the decision makers on the pharmaceutical product as a prescription writer12. In most of the countries physicians are the only qualified professionals who have the authority to prescribe. However, drug
advertisements can affect physicians’ prescribing habits and diagnosis13,
14
. Thus advertisements aim to give information to the physicians about
evidence-based practices and to follow these practices15.
In Turkey, pharmacists also have an important role on the healthcare system. Community pharmacies are the first port for the people to
counsel on various health issues. Ailments in minor diseases are one of
the most frequently counseled issues16.
In a Finland based study Närhi found that physicians and pharmacists are the most common information sources for medicine users17.
Therefore, drug advertisements are significant both for the physicians
and for the pharmacists.
Considering all these, it is important to be attentive while preparing and publishing drug advertisements. Some international organizations’ codes such as WHO’s “Criteria for Medicinal Drug Promotion”4 and
International Federation of Pharmaceutical Manufacturer’s Association
(IFPMA)’s “IFPMA Code of Pharmaceutical Marketing Practices”18 have
some criteria for drug advertisements.
ADHERENCE OF DRUG ADVERTISEMENTS TO THE INTERNATIONAL MARKETING CODES
55
WHO’s “Criteria for Medicinal Drug Promotion” which was accepted
in 1988 is one of the first documents related to drug advertisements.
These criteria are the basis of later regulations. On the other hand IFPMA
updated the “IFPMA Code of Pharmaceutical Marketing Practices” which
regulates drug promotion on an international scale in 2006 4, 7.
The purpose of this study is to assess the characteristics and adherence of drug advertisements published in medical journals taking into
consideration WHO’s and IFPMA’s criteria. Also we aimed to see whether
any differences exist among the analyzed publications in terms of drug
advertisements.
Materials and Methods
The drug advertisements published in “Journal of American Medical Association (JAMA)”, “JAMA Türkiye” and “Eczacı (Pharmacist)” were
evaluated in this study. The rationale for choosing these journals is that
all of the reviewed journals are related to healthcare and they are both
for physicians and pharmacists. “JAMA” is a scientific general medicine
journal which reaches to a large number of healthcare professionals in
the world19. At the beginning of the study, to compare with “JAMA”, a SCI
(Science Citation Index) journal in Turkey was tried to be selected. But
none of these SCI general medicine journals contained drug advertisements. As a result, “JAMA Türkiye” is selected to make a comparison
with “JAMA”. “JAMA Türkiye” contains translated articles published in
JAMA. But the drug advertisements printed in “JAMA Türkiye” is totally
different from the original “JAMA”. The journal “Eczacı” which is a professional journal reaches to a large number of pharmacists in Turkey too.
Data collection
All drug advertisements published in 2006 and 2007 issues of the
journals “JAMA”, “JAMA Türkiye” and “Eczacı” were retrospectively examined. As the publication of “JAMA Türkiye” ended in the beginning
of 2008 like “JAMA Français”20, 21 the selected journals examination period was limited to only two years (2006-2007). All these journals were
reached by the first author of this manuscript visiting the Libraries of
Hacettepe, Ankara and Gazi University Faculties of Medicine and the
56
Library of Hacettepe University Faculty of Pharmacy. And then each issue of these journals was reviewed one by one. The missing issues of
“JAMA Türkiye” and “Eczacı” were obtained through contact with the
publishers by e-mail. Some journal issues had their own advertisement
indices, missing indices were created by the first author. Photocopies of
these drug advertisements were taken in the light of these indices. Journal collection process started in October 2008. Completing the missing
issues with drug advertisements’ photocopies was finished in April 2009.
The number of allocated pages for all advertisements, the number of
all advertisements and both the number of allocated pages for drug advertisements and the number of drug advertisements were determined to
understand the distribution of advertisements in journals. Then, the ATC
(Anatomical Therapeutical Chemical Classification) system was used to
classify the drugs in advertisements into therapeutic categories by taking
into account Vademecum22 .
WHO’s “Criteria for Medicinal Drug Promotion” and IFPMA’s “Code of
Pharmaceutical Marketing Practices” were transformed to checklists and
these were used to evaluate the drug advertisements’ adherence to the
international codes. WHO’s codes are more comprehensive but IFPMA’s
codes include two more criteria other than the WHO’s, that is why we
combined the two international codes.
Statistical analysis
Data analysis was performed by using SPSS version 15.0 (SPSS, Inc.,
Chicago IL). When chi-square tests were done, the codes of the two organizations (WHO and IFPMA) were combined. Tables were established by
considering these two codes.
Results
The distribution of the advertisements:
The number of published issues for “JAMA”, “JAMA Türkiye” and
“Eczacı” are as follows respectively 94; 23; 24 during 2006 and 2007.
Table I shows the number of total pages in each Journal, number of all
Journal
name
Total
ECZACI
2006
2007
Total
11245
5606
5639
2006
2007
1209
583
626
Total
2006
2007
948
828
Publishing year
1776
Number of
Total Pages
N1
JAMA
JAMA TÜRKİYE
TABLE I
1956
1033
923
207
117
90
615
329
286
Number of
Advertisements
N2
1374
768
606
184
105
79
104
59
45
70.24
74.35
65.66
88.88
89.74
87.77
16.91
17.93
15.73
Number of Drug
Advertisements
N3
(N3/N2)
Quantitative characteristics of journal advertisements by years
2094
1172
922
182
109
73
98
56
42
8.62
20.91
6.35
15.05
8.69
11.66
5.52
5.91
5.07
Allocated Total
Pages for Drug
Advertisements
N4 (N4/N1)
57
58
the advertisements, number of drug advertisements and allocated total
pages for drug advertisements.
The percentage of the drug advertisements printed in “Eczacı”, “JAMA”
and “JAMA Türkiye” are respectively %16.91, %70.24 and %88.88 among
all advertisements.
According to the results of the chi-square tests for the distribution
of drug advertisements in “Eczacı” and “JAMA Türkiye”, the two journals
are statistically different in terms of the number of drug advertisements
and the allocated number of pages for drug advertisements. (The number
of drug advertisements - Likelihood Ratio=0, p < 0.05; the allocated page
number for drug advertisements – Likelihood Ratio=0, p < 0.05).
The results of chi-square tests for the distribution of drug advertisements in “JAMA” and “JAMA Türkiye” shows that these two journals are
statistically different in terms of the number of drug advertisements and
the allocated number of pages for drug advertisements (The number of
drug advertisements - Likelihood Ratio=0, p < 0.05; the allocated page
number for drug advertisements – Likelihood Ratio=0.002 p < 0.05).
Drug advertisements’ categories:
In the Figure 1, the percentage of drug advertisements category in
the reviewed journals are shown. 25% of drug advertisements in “Eczacı
Journal” belonged to the “Genito urinary system and sex hormones”,
23.91% of drug advertisements in “JAMA Türkiye” belonged to the “Alimentary tract and metabolism” and 30.35% of drug advertisements in
“JAMA” belonged to “Nervous system”.
The adherence of drug advertisements to the international codes:
Five of the published advertisements in “Eczacı” and only one of the
published advertisement “JAMA Türkiye” met all criteria (Table II). These
two journals are not statistically different from each other in terms of the
adherence to the criteria (Fisher’s Exact Test=0.411, p > 0.05). None of
the drug advertisements in “JAMA” met all the criteria.
Only “The date of production of the advertisement” and “The approved
therapeutic uses” criteria are statistically different between “JAMA Türkiye” and “Eczacı” (The date of production of the advertisement – Likelihood Ratio=0.032, p < 0.05; the date of production of the advertisement
The Histogram of the ATC classified drug advertisements in journals
FIGURE 1
59
TABLE II
28
42
38
14
14
16
Providing the address of the
manufacturer
Providing references
The date of production of the
advertisement indication
The method of use indication
JAMA TR: JAMA Türkiye
A: Available
NA: Not Available
4
30
Major interactions indication
Providing the name of the
pharmaceutical company
4
26
28
0
12
7
38
35
Contra-indications indication
4
0
1
23
2
0
Precautions indication
42
38
Dosage form indication
41
Indication of approved therapeutic
uses
Side-effects indication
40
19
Indication of the active ingredients
Indication of the other ingredients
42
16
14
16
22
23
18
21
22
20
22
22
9
23
23
7
9
7
1
0
5
2
1
3
1
1
14
0
0
NA
A
A
NA
JAMA TR
ECZACI
Indication of the name of the
product
Required Characteristics in
Drug Advertisements
6,066
4,603
7,990
-
-
0,366
-
-
-
-
-
0,227
-
-
X2
0,014
0,032
0,05
0,648
-
0,545
0,474
0,648
0,691
0,354
1,00
0,634
0,536
-
p
24
31
34
35
53
39
41
40
40
44
44
0
51
54
A
30
23
20
19
1
15
13
14
14
10
10
54
3
0
NA
JAMA
16
14
16
22
23
18
21
22
10
22
22
9
23
23
A
7
9
7
1
0
5
2
1
13
1
1
14
0
0
NA
JAMA TR
4,169
0,080
0,313
-
-
0,190
-
-
6,470
-
-
-
-
-
X2
Comparison between JAMA Türkiye – Eczacı and JAMA – JAMA Türkiye in terms of adherence to the codes
0,041
0,777
0,576
0,04
1,00
0,663
0,207
0,031
0,111
0,158
0,158
0
0,550
-
p
60
61
– Likelihood Ratio=0.014, p < 0.05). All advertisements have “The name of
the product” and “The name of the pharmaceutical company”. Therefore,
chi-square tests were not applied for these criteria (Table II).
There are statistically significant differences between “JAMA” and
“JAMA Türkiye” in terms of the “Name of other ingredients”, “Contra-indications”, “Address of manufacturer” and “Approved therapeutic uses”.
(Name of other ingredients – Fisher’s Exact Test = 0, p < 0.05; Contraindications – Fisher’s Exact Test = 0.031, p < 0.05; the address of manufacturer - Fisher’s Exact Test=0.04, p < 0.05; Approved therapeutic uses
– Likelihood Ratio=0.041, p < 0.05). All advertisements have the “Name
of the product”. Therefore, chi-square tests were not applied for these
criteria (Table II).
Discussion
Nowadays it is known that the increased use of medication continues . This increase was detected in North America, which is the largest
pharmaceutical market in the world at the same time24. Therefore it is not
surprising to find out that “JAMA” has the highest drug advertisement
ratio among other analyzed journals. Also Turkey is one of the largest
pharmaceutical markets in Europe 25. According to the Turkish Pharmacists’s Association in 2007 (TEB), 114.583 physicians are available in
Turkey26. This high number of decision-makers on drugs makes Turkey
an attractive market for pharmaceutical companies. This situation can
be explained by the high drug advertisement ratio in medical journals
printed in Turkey. But on the other hand the lowest drug advertisement
ratio belongs to “Eczacı” when it is compared with “JAMA” and “JAMA
Türkiye”.
23
This can be explained by Turkish legislation. According to this legislation, pharmacists are not allowed to prescribe. So pharmaceutical companies may choose to advertise in the journals like “JAMA” and “JAMA
Türkiye” as their target groups are directly the physicians.
The most advertised and used drug category in Turkey is “Antiinfectives for systemic use” 27. That indicates the connection between drug
advertisements and drug use. In our study we also found that this drug
62
class is also have a high number of advertisements in local medical journals (“Eczacı” and “JAMA Türkiye”). It is interesting that the “Genito urinary system and sex hormones” are the most advertised drug categories
in “Eczacı” although this category is not in the best-seller group. The
reason for this can be the patient choice to take these drugs only in consultation with a pharmacist.
As a communication and information channel, medical journals and
drug advertisements in them have a significant role. Drug advertisements
in medical journals are one of the important components of drug marketing. Their target groups are journal readers, in other words healthcare
professionals28.
Lua et al. found in their study that some questions from patients to
the physicians are about drug use and effect29. However this information
is missing on some advertisements in these three journals.
Drug advertisements may have bias, and there are doubts about
the accuracy of the information provided in drug advertisements30, 31. To
eliminate this suspicion, scientific references are given in drug advertisements. Providing references in drug advertisements is a marketing
strategy32. WHO and IFPMA criteria have also emphasized the need for
references in drug advertisements. There are many published studies
about reference citing in advertisements11, 15, 33-35. According to a study
by Cooper and Schringer, 126 of 438 advertisements in 10 American
Journals have no references33. In a study conducted in 2010, of the 614
advertisements 52 had no references 34. In our study, of 62.9% of advertisements in “JAMA”, 69.9% in “JAMA Türkiye” and 33.3% in “Eczacı”
have references.
It is stated that the adherence of drug advertisements to the WHO
criteria in less developed countries is low36. This situation is quite contrary to these findings. In this study, it was hypothesized that the advertisements especially in “JAMA” would adhere to the international bodies’
criteria more than the advertisements in other journals. However, the results show that the advertisements in local journals in Turkey adhere to
all the criteria more than “JAMA”. Only 5 of the advertisements published
in “Eczacı” and 1 in “JAMA Türkiye” met all the criteria of WHO and IFPMA. The lack of fulfilling all the criteria in advertisements published in
“JAMA” is remarkable. It is thought that the strictness of the regulation
on drug advertisements in Turkey can be effective for this result.
63
Unethical drug promotion, especially in less developed countries, increases the rate of irrational drug use37. The excessive number of drug
advertisements in journals and their impartiality are a subject of discussion38. Moreover journal editors should abide by criteria and these criteria should be up to date39.
Drug advertisements are important sources for reliable drug information to healthcare professionals and they are expected to provide
impartial, scientific and complete information for prescribers and other
decision-makers. In this context it is significant to adhere to the international marketing codes of the well known organizations like WHO and
IFPMA. It is required to be neutral in publishing drug advertisements
for the protection of public health. The healthcare professionals should
also pay attention while evaluating advertisements in the journals as
sometimes adherence to the international codes can be ignored. This is
important both when they provide any information about drugs to their
patients and give an unbiased feedback to the editors of the journals.
Summary
Drug advertisements provide information about drugs. The aim of
this study is to explore the adherence of drug advertisements in medical journals to the international marketing codes. Drug advertisements
printed in “Journal of American Medical Association (JAMA)”, “JAMA
Türkiye” and “Eczacı (Pharmacist) Journal” were analyzed. Adherence of
the analyzed advertisements to the international drug promotion codes
was the main outcome measure. The lowest compliance ratio of drug advertisements belongs to the “JAMA” Journal. The rate of cited references
in drug advertisements is very low and the majority of these advertisements do not adhere to the international codes. In order to avoid this
situation, adherence of drug advertisements to the international codes is
extremely significant.
Keywords: Advertisement, Medical journal, Codes, Adherence
64
Özet
İlaç Reklamlarının Uluslararası Pazarlama Kurallarına
Uygunluğu
İlaç reklamları, ilaçlar hakkında bilgi sağlamaktadır. Bu çalışmanın
amacı, medikal dergilerdeki ilaç reklamlarının uluslararası reklam kurallarına uygunluğunun ortaya konmasıdır. “Amerikan Tıp Birliği Dergisi
(Journal of American Medical Association - JAMA)”, “JAMA Türkiye” ve
“Eczacı” dergilerindeki ilaç reklamları incelenmiştir. İlaç reklamları için
hazırlanan uluslararası kurallar belirlenmiş ve reklamların bu kurallara
uygunluğu incelenmiştir. En düşük uygunluk oranının “JAMA” dergisine ait olduğu belirlenmiştir. İlaç reklamlarında referanslara yer verilme
oranının çok düşük olduğu ve bu reklamların çoğunun uluslararası kurallara uymadığı tespit edilmiştir. Bu durumun önüne geçilmesi için, ilaç
reklamlarının uluslararası kurallara uyması son derece önemlidir.
Anahtar Kelimeler: Reklam, Medikal dergi, Kurallar, Uygunluk
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Hacettepe Üniversitesi Eczacılık Fakültesi Dergisi
Cilt 32 / Sayı 1 / Ocak 2012 / ss. 67-90
Kanser Tedavisinde Lenfatik
Hedeflendirme
Received:18.07.2011
Revised:
31.10.2011
Accepted:04.11.2011
Özge Çevik1, Uğur Aydın1, R. Neslihan Gürsoy1
Giriş
Kanser günümüzün en önemli sağlık sorunlarından biridir. Sık görülmesi ve ölümcül olması nedeniyle de bir halk sağlığı sorunudur. Dünya Sağlık Örgütü’nün 2008 Kanser Raporuna göre dünyada 2008 yılında
12,8 milyon yeni kanser vakası görülmüştür1. Kanserin tahmini küresel
etkisi Şekil 1’de gösterilmiştir. Kansere yakalanan kişi sayısının yıllar
içinde gösterdiği artış nedeni ile 2025 yılında kanser vakalarının sayısının dünya çapında 25 milyonu aşacağı öngörülmektedir. Bu nedenle kanser günümüzde en çok çalışılan konular arasında yer almakta ve
kanser üzerine yapılan çalışmalar gün geçtikçe artmakta, daha etkin ve
daha az yan etkili tedavi arayışı sürmektedir.
Türkiye’de ise 2004 – 2006 yılları kanser istatistiklerine bakıldığında
erkeklerde deri hariç tüm kanser türleri için kaba hız yüz binde 222.6,
YSH (Yüz binde, dünya standart nüfusu) 223.2’dir2. En sık görülen kanserler akciğer, prostat, mesane, kolorektal ve mide kanserleri olarak
sıralanmaktadır. En sık görülen beş kanserin sıralaması üç yılda da
(2004, 2005, 2006) değişmemiştir.
Kadınlarda, 2004 – 2006 yıllarında tüm kanserler için kaba hız yüz
binde 164.4, YSH 150.3; deri hariç tüm kanserlerde ise kaba hız yüz
1
o
Hacettepe Üniversitesi Eczacılık Fakültesi, Farmasötik Teknoloji A.D.
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HACETTEPE ÜNİVERSİTESİ ECZACILIK FAKÜLTESİ DERGİSİ
Şekil 1
Kanserin Tahmini Küresel Etkisi
(Yıllık Yeni Vaka Sayısı)
binde 145.4, YSH 133.3’tür2. İlk beş sırada meme, kolorektal, tiroid, uterus korpusu ve akciğer kanserleri yer almaktadır. 2004, 2005 ve 2006
yıllarında en sık görülen ilk üç kanser meme, kolorektal ve tiroid kanserleri olarak sıralanmaktadır2. Akciğer kanseri, 2006 yılında en sık görülen
ilk beş kanser içinde yer almazken, 2005 yılında dördüncü, 2004 yılında
ise beşinci sıraya yerleşmiştir2.
Kanser; bir hücre hastalığıdır. İnsan vücudu; milyonlarca hücreden
oluşur3. Büyüyebilmek, ölü hücreleri yenilemek ya da yaralanma sonucu zedelenmiş hücreleri onarmak amacıyla vücudumuz sürekli yeni
hücreler üretmektedir3. Kanser en kısa tanımıyla, hücrelerin kontrolsüz
şekilde çoğalmaları demektir3. Anormal şekilde çoğalmaya başlayan bu
hücreler bulundukları yerdeki ve hatta uzaklarında bulunan doku ve
organları işgal eder ve bu bölgelerde işlevsel bozukluklara yol açarlar.
Kanserde ölüm oranlarının yüksek olması konunun önemini daha da
artırmaktadır3.
Genler, hücrelerin işlevlerini yönlendiren parçalardır4. Hücreler, bazı
genlerden aldığı bilgi ve komutlara uygun olarak çoğalırlar. Kansere bo-
KANSER TEDAVİSİNDE LENFATİK HEDEFLENDİRME
69
zuk genler yol açar4. Genlerin yapıları aşırı güneş ışınları, sigara içme ya
da yediğimiz yiyecekler gibi faktörler sebebiyle değişir. Hücrelerin çoğalacağı ve öleceği zamanı normal genler belirler. Bu, vücudumuzda sürekli
olan bir olaydır. Ancak bozuk genler bazen hücrelerin aşırı çoğalmasına neden olur ya da ölmesi gereken hücrelerin ölümünü engeller. Bu
fazla olan hücreler, çoğalmaya devam ederek tümör olarak adlandırılan
kitleleri oluştururlar4.
Normal hücre döngüsü, hücrenin birebir kopyasının oluştuğu kritik bir işlemdir5. Birçok kanser türünde hücrenin büyüme ve bölünme
sürecinde ortaya çıkan mutasyonlar etkin rol oynar. Yaşlanan hücrelerin ölümü üzerine hücre bölünmesi zorunlu hale gelir5. Apoptoz (programlanmış hücre ölümü) ve hücre çoğalması arasındaki denge kansere yol açan genler (onkogen) tarafından bozulabilir6. Onkogenler; hücre
çoğalması, hücre bölünmesi ve hücre ölümü gibi olayları kontrol eden
ve protoonkogen olarak bilinen normal hücresel genlerin mutasyona
uğramış halleridir6. Tümör baskılayıcı genler, normal bir hücrenin tümör
hücresine dönüşme ihtimalini azaltır. Birçok tümör baskılayıcı gende etkinin görülebilmesi için her iki allelde de mutasyon görülmelidir6. Kanserlerin büyük bir kısmında bir tümör baskılayıcı gen olan p53 geninin
mutasyonu mevcuttur. p53 ya da diğer adıyla tümör protein 53 (TP53),
hücre döngüsünü düzenleyen bir transkripsiyon faktörüdür7. Birçok organizmada kanseri baskılamak için çok önemli bir proteindir. p53, genomda mutasyon olmasını önleyerek genom stabilitesini korur. p53, hücre içerisinde dörtlü (tetramer) bağ yapmış halde işlevseldir. p53’ün kanseri önlemedeki en önemli görevleri; DNA zarar gördüğünde DNA tamir
proteinlerini harekete geçirmek ve DNA tamir edilemeyecek kadar zarar
gördüğünde ise apoptozu başlatmaktır7.
Kanser, herhangi bir dokuda başlayabilir ve hangi dokuda başlamışsa o isimle anılır8. Metastaz, kanserin bir organdan, doğrudan bağlantısı olmayan diğer bir organa taşınması olarak tanımlanır ve kötü huylu
kanserin en belirgin klinik özelliklerindendir8. Kanser hücreleri birincil
bölgeden kan yoluyla ya da lenf sistemi aracılığıyla çevre dokulara yayılım yapar. Yayılan tümörler artık metastatik tümördür. Ama kökeni
birincil dokudaki hücrelerdir. Bu tümörlerin yayılışı sıklıkla birincil bölgedeki lenf düğümleri aracılığıyla olur8. Yeni damar oluşumunu takiben
birincil tümör bölgesindeki hücre tutunması azalır ve hücrelerden bazıları serbest kalır. Serbest kalan bu hücreler, tümörü çevreleyen stroma
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içine invaze olarak dolaşıma veya lenf sistemine katılırlar. Sisteme giriş
sonrası, dolaşımda hayatta kalabilen hücreler veya küçük embolilerin
uzak bölgelerdeki organların kapiller yatağında tutulması gerçekleşir9.
Bu aşamadan sonra, serbest hücre penetre olur ve bulunduğu kan veya
lenf damarından dışarıya doğru akışı gerçekleşir. Son olarak, yeni oluşan
tümör, gelişmek için yeni kan damarları oluşumunu tetikler ve ikincil
kanseri oluşturur9.
Kanser tedavisinde kullanılan başlıca mevcut yöntemler; cerrahi işlemler, radyoterapi, kemoterapi, immünoterapi, hormon tedavisi ve lazer
tedavisi olarak sıralanabilir3. Kanser tedavisinde en sık başvurulan tedavi şekli; tümörün uzaklaştırılmasını sağlayan cerrahi yöntemleri kapsar.
Bu, yayılım yapmamış birçok kanser için iyi bir tedavi seçeneğidir. Eğer
kanser ilerlemişse ya da çevre dokulara yayılmışsa; cerrahi müdahale,
radyoterapi ve kemoterapi birlikte uygulanabilir10. Radyasyon tedavisi;
kanser tedavisinde X ışınları, gama ışınları ve elektronlar gibi iyonize
ışınların kullanılmasını içerir. Bu ışınlar; kanserli hücreyi tahrip ederek
etki etmektedirler. İmmünoterapi, BCG aşısı, interlökin ve interferonlar
gibi biyolojik moleküller kullanılarak bağışıklık sisteminin uyarılmasıdır3.
Hormon tedavisi ise; hormon sekresyonuna bağlı gelişen meme ve prostat
kanseri gibi kanserlerde özel bazı hormonların kullanımıyla uygulanan
tedavidir3. Lazer tedavisi ise, cerrahi müdahalelerde yararlı olabilmektedir. Bu nedenle, beyin tümörleri ve gırtlak kanserinde kullanılmaktadır3.
Kemoterapi ise, kanserin ilaçla tedavisi demektir. Kanser kemoterapisi,
yerleşmiş bir tedavi yöntemidir. Kemoterapide kullanılan ilaçlar, kanserli hücrelerin çoğalmalarını durdurmakta ve yok etmektedir. Ancak; bu
ilaçlar vücuttaki normal hücrelere de etki edebilir ve ciddi yan etkilere
yol açabilirler3.
Kanser Tedavisinde Hedeflendirme
Son 20 yılda kanser dünya çapında birçok ülkede ölüm nedenleri arasında ilk sıralara yükselmiştir11. Bu sebeple, kanser tedavisi için
büyük çaba harcanmaktadır. Ancak, kanser tedavisinde başarıyı kısıtlayan en önemli faktör, geleneksel tedavide kullanılan antikanser ajanların tümörlü hücre ve dokular için seçici olmamasıdır11. Neredeyse, tüm
kemoterapötik ajanların normal doku ve organlara yan etki gösterdiği
bilinmektedir12.
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Normal kılcal damarlar; arteriol, kapiler ve venüllerden oluşan düzenli ve fonksiyonel bir yapıya sahiptir13. Tümör damarları ise; genel olarak, sıralı olmayan, hasar görmüş ve geniş açıklıklara sahip endotelyal
hücrelerden oluşur. Oldukça geniş lümene ve özellikle vazoaktif
mediatörlerden AT II (Anjiyotensin II)’ye karşı bozulmuş reseptör fonksiyonuna sahiptirler14,15. Buna ek olarak; bradikinin, nitrik oksit, vasküler
büyüme faktörleri ve prostaglandinler gibi vasküler permeabilite artırıcı
faktörlerin üretiminin artması nedeniyle makromoleküllü ilaçların normal dokuya göre tümörlü dokuya geçişleri kolaylaşmaktadır16. Tüm bu
faktörler tümör kapilerlerinden geçişi artırmanın yanısıra, özellikle nanoboyuttaki makromoleküler ilaçların tümörlü dokulara selektif olarak
gönderilmesini mümkün kılar17,18. Ayrıca, tümörlü dokuda lenfatik sıvı
akışının az olması, ilacın tümörlü dokuda kalış etkisini artırarak uzatılmış etki sağlar. Bu etkiye; artırılmış geçiş ve alıkonma etkisi (EPR, enhanced permeability and retention effect) denir19.
Tümör çevresinde kan ve lenf damarlarının anormal yapı ve işlev
göstermesi nedeniyle dokulararası sıvı basıncında artış görülür20. Tümör
damarlarında seçicilik azaldığı için permeabilite artar20. Anormal metabolik çevreleri nedeniyle tümörlerde hipoksi ve asidoz oluşur21,22. Ayrıca;
damar ağlarının dengesiz gelişimi ve tümör hücresinin aşırı çoğalması
nedeniyle doku içinde hipovasküler bölgeler oluşur. Kan damarlarının
uzağında kalan bölgelerde de kronik hipoksi görülür. Anaerobik glikoliz sonucu oluşan laktik ve karbonik asitler nedeniyle tümör çevresinin
pH’sı oldukça düşük değerlerdedir20. Oksijen basıncı ve pH değerleri tümör büyümesi, metabolizması ve çeşitli tedavilere verilen yanıtları etkilemektedir. Bu iki özellik, anjiojenik faktörlerin etkisini artırarak tümör
büyümesi ve metastazına yol açar20.
Kanser tedavisinin asıl amacı; normal dokuları etkilemeden kanser
hücresini yok etmektir23. Bu durum ise, kanser hücresinin selektif olarak
hedeflendirilmesi ile mümkündür. Tümöre hedeflendirmedeki ideal özellikler; 1) ilacın tümördeki yerleşiminin aktif veya pasif hedeflendirilme ile
artırılması, 2) hedeflendirilmemiş hücrelerdeki yerleşiminin azaltılması,
3) geçiş bölgelerinden olabilecek ilaç sızıntısının en aza indirilmesi, 4)
ilacın parçalanmadan korunması, 5) ilacın hedeflenen bölgede istenilen
süre boyunca kalabilmesi, 6) hücre içine alımın kolaylaştırılması, ve 7)
taşıyıcı sistem bileşenlerinin biyouyumlu ve biyoparçalanabilir olması
şeklinde sıralanabilir23.
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Değişik formlardaki farmasötik taşıyıcı sistemlerin (partiküler
sistemler, lipozomlar, emülsiyon sistemleri gibi) doğrudan etkin maddeye bağlanması veya etkin maddeyi hapsetmesi ve bu şekilde ilaçların
hedeflendirilmesine olan ilgi gün geçtikçe artmaktadır24. Hedeflendirme
ile konvansiyonel, biyoteknolojik ve gen kökenli ilaçlar, vücudun organ,
doku ve hücre gibi spesifik bölgelerine seçici olarak taşınabilmektedir.
Bu seçici hedeflendirme ile, istenmeyen yan etkiler azalmakta, en uygun
terapötik yanıt elde edilmekte ve yüksek dozlarda toksik etkileri gözlenen
maddeler güvenli olarak kullanılabilmektedirler24.
İlaçların hedeflendirilmesi iki şekilde sağlanır; bunlardan biri pasif
hedeflendirme, diğeri de aktif hedeflendirmedir24. Pasif hedeflendirme,
intravenöz enjeksiyondan sonra taşıyıcının doğal olarak hedeflendirilmesi; organ, doku ve hücreye yerleşmesidir24. Aktif hedeflendirme; manyetik
hedeflendirme, ultrasonik hedeflendirme ve ligant – reseptör aracılı hedeflendirme olmak üzere 3 ana başlık altında incelenebilir24.
Manyetik hedeflendirme, dışarıdan manyetik alan uygulanmasıyla
istenilen bölgeye etkin maddenin taşınmasıdır. Ultrasonik hedeflendirmede, ultrasonik dalga kontrollü sistemlerde biyolojik olarak aşınan veya
aşınmayan polimerler ilaç taşıyıcısı olarak kullanılır ve hedeflendirilirler.
Ligant – reseptör aracılı hedeflendirmede, farklı yapıda ligantlar kullanılmaktadır. Burada, taşıyıcı sistemler hücre yüzeyindeki spesifik reseptörlere yönlendirilerek hücre içine alınma (internalizasyon) ligandın ve
reseptörün yapısına bağlı olarak reseptör aracılı endositoz veya transitoz
ile gerçekleşmektedir. Ligant – reseptör aracılı hedeflendirmede kullanılan ligantlar arasında monoklonal antikorlar, vitaminler, peptid yapıdaki
büyüme faktörleri, sitokinler, glikoproteinler sayılabilir24. Etkin madde,
hedef hücrenin yüzeyindeki reseptöre bağlanabilen liganda (hormon,
peptid gibi) konjuge edilir. Böylece; etkin madde spesifik olarak hedef
hücreye yönlendirilmiş olur25.
Hedeflendirme amacı ile kullanılan yeni ilaç taşıyıcı sistemlerden
bazıları; lipozomlar, miseller, nanopartiküller, dendrimerler ve nanokapsüllerdir26. Spesifik olmayan taşıyıcılardan dekstran, albumin, DNA, poliaminoasitler ve diğer polimerler gibi makromoleküller kullanılarak hazırlanan nanopartikül, mikrokapsül, mikroküre ve lipozom gibi sistemler,
tümör çevresinde lenfatik drenajın az olmasından kaynaklanan artırılmış
tutulma etkisi ve tümör boyutu gibi çevresel özelliklerden dolayı tümörde
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Şekil 2
Tümöre aktif ve pasif hedeflendirme27
toplanırlar. Ayrıca kullanılan makromolekülün molekül ağırlığı ve yükü
gibi fizikokimyasal özellikleri de tümörde toplanmada etkilidir26. Bu sistemler pasif hedeflendirme ile tümörlere yönlendirilebileceği gibi, antikor, ligant, peptit gibi moleküllerle kombine edilerek aktif hedeflendirme
amacı ile de kullanılabilirler (Şekil 2).
Günümüzde, özgün monoklonal antikorlar ile tümöre hedeflendirme
çalışmaları yapılmakta olup28, anti-VEGF (vasküler endotelyal büyüme
faktörü) ve anti-EGFR (epidermal büyüme faktörü reseptörü) monoklonal antikorlarının ilerlemiş kolorektal kanserli hastalarda kemoterapinin etkinliğini artırdığı gösterilmiştir29. Bu antikorlar; Amerikan Gıda ve
İlaç Dairesi’nin (FDA) onayını almış, Avastin® (Bevacizumab) ve Erbitux®
(Cetuximab) ticari isimleri ile kolorektal kanser tedavisinde kullanılmaktadırlar30. Ayrıca, antianjiojenik tedavide kullanılan Panitumumab (Vectibix®) ve Trastuzumab (Herceptin®) gibi onaylı monoklonal antikorlar,
Erlotinib (Tarveca®), Sorafenib (Nexavar®), Sunitinib (Sutent®) gibi küçük
molekül yapıda tirozin kinaz inhibitörleri, Temsirolimus (Torisel®) gibi
mTOR inhibitörleri ve Bortezomib (Velcade®) gibi diğer hedeflendirilmiş
antianjiojenik ajanlar da FDA onayı almış olup klinikte kullanılmaktadırlar30. Öte yandan, hedeflendirilmiş antikor tedavisini kısıtlayan birta-
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kım faktörler bulunmaktadır32. Antikorların büyük boyutlu olmaları ve
bu moleküllerin hedef bölgede düşük penetrasyon ve hücre içine alımı ile
karaciğer ve RES (Retiküloendotelyal Sistem) tarafından özgün olmayan
alımları, bu tip moleküllere ilişkin en önemli sakıncadır32. Bunun sonucunda, tümöre zayıf penetrasyon ile karaciğer ve kemik iliğinde dozun sınırlanmasına yol açan toksisite görülebilir. Antikorların; radyonüklit, sitotoksik ilaç ve toksinlerin tümör bölgesine taşınması için kullanımında
bu kısıtlayıcı faktörler etkili olabilir30. Antikor hedeflendirilmesinde çıkan
sorunları azaltmak açısından, peptitler tümöre hedeflendirmede mükemmel alternatifler olarak düşünülmektedir30. Peptitlerin antikorlara göre
küçük boyutlu olmaları, kimyasal olarak dayanıklı ve türevlendirmeye
uygun olmaları ile genel olarak RES’e yakalanmamaları gibi üstünlükleri
bulunmaktadır30. Ayrıca; kanser hücrelerine özgü aminoasit dizilimine
sahip peptitler ile kanserde hedeflendirme mümkün olabilmektedir31–34.
Faj sunumu tekniği kullanılarak elde edilen aminoasit dizilimleri ile belirlenen peptitlerin, çeşitli ilaç veya ilaç taşıyıcı sistemler ile kombine edilerek kanser hücrelerine seçici olarak hedeflendirilebildiği çeşitli çalışmalarla gösterilmiştir31–34.
Tümöre hedeflendirme çalışmalarında kullanılan ilaç taşıyıcı sistemlerden olan lipozomlar; sulu bir kısım ve bunu çevreleyen fosfolipit
tabakadan oluşmuş membran yapısını andıran kesecikler olarak tanımlanırlar35. Lipozomlarda hedeflemeyi sağlamak amacıyla da immunoglobulinler kullanılır. Lipozomlar; özgün bir antikorla konjuge edilerek (immünolipozom) verildiklerinde hedeflenmeyi sağlarlar36–38. Diğer bir sistem
olan miseller, partikül büyüklüğü 5 – 100 nm arasında olan, hidrofobik
ve hidrofilik kısımlardan oluşan moleküllerin oluşturduğu kolloidal dağılımlardır24. Miseller kullanılarak aktif hedeflendirme çalışmaları yürütülmüştür39. Aktif hedeflendirmede antikorlar, küçük organik moleküller
(vitaminler), aptamerler ve karbonhidratlar görev alabilir. Örnek olarak;
folik asit reseptörü ovaryum ve akciğer gibi birçok kanser türünde aşırı
çoğalmaya neden olan bir proteindir40–42. Normal bir dokuya oranla tümörlü dokuda yaklaşık 100 – 300 kat daha fazla çoğalabildiği gözlenmiştir42.
Folik asit reseptörünü hedef alan doksorubisin yüklü miseller kullanılarak yapılan in vivo çalışmalarda hedeflenmiş misellerin hedeflenmemiş
olan misellere göre tümör büyüme hızını iki kat azalttığı görülmüştür43.
Aktif hedeflendirmede kullanılan ligantlardan biri de antikorlardır. Yapılan bir araştırmada, GRGDS-modifiye misellerin (GRGDS; çeşitli metas-
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tatik kanser hücrelerinde aşırı çoğalmaya uğrayan αvβ3-integrine özgü
bir peptit) hedeflenmemiş yapılara oranla metastatik melanoma B16F10
hücrelerine karşı daha sitotoksik olduğu kanıtlanmıştır39.
FDA onayı almış formülasyonlardan nanopartiküler sistemler halihazırda piyasada bulunmaktadır. İlk olarak da lipozomal formülasyonlar (Doxil®, Daunoxome®) onay almıştır. Doxil®; partikül boyutu 100
nm'den küçük lipozomal keseciklerden oluşur. İlacın opsonizasyonunu
engellemek için pegilasyon yapılmıştır44. Bu işlem; ayrıca; sterik stabilizasyon sağlayarak plazma yarı ömrünü uzatır. Lipozomal formülasyon
içerisinde verilen ilaç tümörlü bölgede normal verilişe göre 10 kat daha
fazla ölçülmüştür. Normal verilen doksorubisinle doxil® karşılaştırıldığında Doxil®’in kardiyotoksisite, kemik iliği baskılanması, saç dökülmesi,
bulantı ve kusma gibi yan etkilere daha az neden olduğu görülmüştür44.
Kansere hedeflendirme alanında diğer bir yeni ilaç taşıyıcı sistem ise 130
nm boyutunda paklitaksel bağlı albümin nanopartikül formülasyonu
olan Abraxane®’dır27. Abraxane® insanda kullanım için FDA onayı alan
(2005) ilk nanopartikül formülasyonu ilaçtır ve metastatik meme kanseri
tedavisinde kullanılmaktadır27. Abraxane® ile paklitaksel etkin maddesini çözünürleştirmede kullanılan Cremophor® sürfaktanının kullanımı
ortadan kaldırılmakta ve böylece uzun süreli tedavide kombine steroid ve
antihistaminik tedavisine gerek kalmamaktadır. Ayrıca; bu şekilde hastaya daha yüksek dozda ilaç uygulanabilmektedir45.
İlaçların hedeflendirilmesindeki başarı; taşıyıcının hedef dokuya
olan ilgisi ile değerlendirilebilir; bu bağlamda lenfatik sisteme hedeflendirme; öncelikli olarak kanser tedavisi sağladığı, makromolekül yapıdaki
ilaçların oral emilimini iyileştirdiği ve ayrıca mukozal immünitede başarı
sağladığı için üzerinde çokça çalışılan bir konu olmaktadır46.
Kanser Tedavisinde Lenfatik Hedeflendirmeden Nasıl Yararlanılır?
Lenfatik sistem; lenf damarları, lenf kanalları, dalak, timus bezi ve
lenf nodları gibi lenfatik organlardan oluşur47. Lenf ve kan damarlarının
her ikisi de endotel hücrelerle kaplı olsa da temelde bu iki sistem belirgin
farklılıklar gösterir. Kan damarı sistemi, kalbin pompaladığı kanın kapalı
bir sistem içinde taşınmasını sağlar. Lenf sistemi ise, dokulararası sıvının dokudan kana tek yönlü taşındığı açık uçlu yapılardan oluşur47. Lenf
sisteminin birinci görevi; periferal dokulardan dokulararası sıvıyı top-
76
layıp tekrar kan dolaşımına kazandırmaktır. Bir diğer görevi de; diyetle
alınan yağların, yağda çözünen vitaminlerin ve bağışıklık sisteminde görevli bazı moleküllerin (A, D, E, K vitaminleri, trigliseritler, lipoproteinler)
taşınmasını sağlamaktır48. Bağırsakta lenf damarları ve kan damarları
yan yana bulunur49. Portal kan akışının lenf sistemine göre yaklaşık 500
kat fazla olması sebebiyle emilimi gerçekleşen maddelerin çoğu portal
kandan sistemik dolaşıma katılır49. Ancak, yüksek molekül ağırlıklı maddeler ve kolloidal yapılar lenfatik kapiller yapının daha geçirgen olması
sebebiyle lenf dolaşımına katılırlar49. Ayrıca, lipofilisitesi fazla olan (log
P>5, lipit çözünürlük> 50 mg/g) ilaçlar da intestinal lenf aracılığıyla dolaşıma katılır (Şekil 3)50.
Lenf sisteminin anatomik ve fizyolojik yapısı gereği ilaçların lenf
yoluyla taşınmasının bazı üstünlükleri vardır50. Şöyle ki; lenf sistemiyle
sistemik dolaşıma katılan ilaçlar karaciğere uğramazlar. Dolayısıyla
da ilk geçiş metabolizmasından korunmuş olurlar. Bu da, karaciğerde
önemli ölçüde yıkılan ilaçların oral biyoyararlanımının artmasını sağlar50.
Lenfatik sistem B ve T lenfositlerin sistemik dolaşıma katılmasında da
etkin rol oynar. Lenfatik dokuların HIV (Human Immunodeficiency Virus)
virüsünün gelişiminde de önemli bir rolü olduğu öne sürülmektedir 51,52.
HIV virüsü; hastalığın erken safhalarında lenfoid organları (dalak, lenf
nodları ve tonsiller) depo olarak kullanmaktadır. En etkili antiviral te-
Şekil 3
Gastrointestinal kanaldan kan ve lenf dolaşımına geçiş
(TG: Trigliserit, LP: Lipoprotein, D: İlaç molekülü)
77
davi uygulamasında bile periferal kan virüsten temizlenirken, virüs lenf
organlarında belirti göstermeden çoğalmaya devam etmektedir53. Bu
nedenle; antiviral ajanların lenfatik sisteme hedeflenmesi ile virüsün yok
edilmesi daha etkin bir şekilde sağlanabilecektir54,55. Lenfatik sistemin
tümörlerin metastazında da etkili olduğu bilinmektedir49,50. Dolayısıyla;
lenfe hedeflenen antitümör ve antiviral ilaçlar kanser ve AIDS tedavisinde
etkili olabilecektir52.
Lenf damarları; insanlarda kanserin yayılım yapmasına olanak sağlayan önemli bir yolaktır. Birçok metastatik kanser türü lenf yollarından
geçerek çevre dokulara yayılırlar56. Son yıllarda yapılan araştırmalar, kanserde lenfanjiyojenezin rolünün ve lenf damarlarının gelişiminin altında yatan moleküler mekanizmaların anlaşılmasını sağlamıştır. Bu çalışmalarda
VEGF-C, VEGF-D (damar endotel büyüme faktörü) ve bunların reseptörü
olan VEGFR-3’ün keşfedilmesi temel basamak olmuştur57. Hayvanlar üzerinde yapılan deneysel çalışmalar sonucunda VEGF-C/VEGF-D/VEGFR-3
arasındaki sinyal iletimine bağlı olarak tümörlü bölgede yeni damarların
oluştuğu (lenfanjiyojenez) belirlenmiştir57. Kültür ortamındaki endotelyal
hücrelerde VEGFR-3 sinyal mekanizması incelendiğinde, VEGFR-3’ün tek
başına uyarılmasının hücrelerin apoptozdan kaçmasına ve devamında
da kontrolsüz çoğalmasının engellenmesine yeterli olduğu görülmüştür58.
Günümüze kadar yapılan patolojik çalışmalar, kanser hücrelerinin lenfde
yayılmasının birçok kanser türünde erken evrede gerçekleştiğini ortaya
koymaktadır59. Kısaca, gelecekte muhtemel yeni hedefler ortaya çıkacak
olsa da, şu an itibariyle VEGF-C/VEGF-D/VEGFR-3 sinyal mekanizması
kanser yayılımını sınırlandırmak için yeni terapötik sistemlerin geliştirilmesinde en cazip hedef olarak görülmektedir60.
VEGF-C, tümör hücrelerinin yakınında yeralan fibroblastlar, makrofajlar, keratinositler ve plateletler tarafından salgılanır61. VEGF-C sadece tümör oluşumunu tetiklemekle kalmayıp, lenf damarlarına sıvı dönüşünü de artırarak tümör hücrelerinin lenf yoluyla taşınmasını artırır.
VEGF-C/VEGF-D/VEGFR-3 üçlü sisteminde VEGF-D ve VEGF-C’ye yönelik antikorlar kullanılır. VEGF-C gibi tek bir ligandı hedeflemek birkaç
reseptör (VEGFR-3, VEGFR-2 ve NPR-2) üzerinde meydana gelen etkileri
nötralize eder. Ancak; bu reseptörleri etkileyen VEGF-D gibi diğer ligantları etkilemez. Tek bir reseptör hedeflendiğinde ise; onun ligantlarının
oluşturduğu etki engellenir. Ancak; bu durumda da diğer reseptörlere
bağlı etkide bir değişme olmaz. sVEGFR-3 (çözünür VEGFR-3) prote-
78
inleri ya da adenoviral yapılar tümör lenfanjiyojenezini hedeflemede en
yaygın kullanılan yöntemlerdir62. sVEGFR-3 globulin proteininin insan
meme kanserinde lenfanjiyojenezi ve lenf nodlarına metastazı önlediği
gösterilmişitir63. sVEGFR-3 tedavisinde zamanlama da oldukça önemlidir. SVEGFR-3 erken dönemde verildiğinde tümör oluşumunu engeller.
Geç kalınırsa, tümör hücreleri lenf nodlarına ulaşmış olacağı için tedavi
yetersiz kalır64. Bir diğer ligant olan semoforin 3F (SEMA3F); VEGF-A
ve VEGF-C ile nörofilin-2 (NRP-2) reseptörü için yarışmaya girerek inhibisyon sağlar. NRP-2, VEGFR-2 ile birlikte VEGF-A için; VEGFR-3 ile
birlikte de VEGF-C için yardımcı reseptördür59. Ayrıca; SEMA3F’nin
tümör baskılayıcı olarak da kanseri önlediği öne sürülmektedir. Bu protein anjiyojenezi, lenfanjiyojenezi ve tümör yayılımını engellemede yeni
bir tedavi yaklaşımı olarak görülmektedir65.
Günümüze kadar lenf endotelinde yapılan reseptör hedefleme çalışmaları genellikle VEGFR-3 üzerinedir66–68. Antikor yardımıyla VEGFR3’ün hedeflenmesi lenfanjiyojenezi engeller66–68. VEGFR-3 reseptörü lenf
endotelinde olduğu gibi kan damarlarında da aşırı çoğalma gösterebilir. Dolayısıyla; VEGFR-3’ü hedef alan antikorlar normal anjiyojenezi ve
birincil tümör oluşumunu da engellerler68,69. VEGFC; VEGFR-3’ün yanı
sıra VEGFR-2’nin de uyarılması sonucu anjiyojeneze sebep olur. DC101
antikoru yardımıyla VEGFR-2’nin hedeflenmesi VEGFC’e bağlı anjiyojenezi engeller59,70. Bu iki reseptörü hedef alan yöntemler karşılaştırıldığında; VEGFR-3’ü hedef alındığı durumda tümör hücresinin bölgesel
ve uzak bölgelere yayılımının, VEGFR-2’nin hedef alındığı durumda ise,
tümör gelişiminin ve anjiyojenezin daha etkin bir şekilde engellenebildiği
görülmüştür68.
Kanser tedavisinde kullanılmak üzere büyüme faktör reseptörlerini
hedeflemek ve kinaz etkisini engellemek için küçük bileşikler kullanılır71.
Bu kinaz inhibitörleri, tümör büyümesinde rol alan büyüme faktörlerine
ait sinyalleri kontrol ettikleri için kanser terapisinde önemli yere sahiptirler. Birçok tirozin kinaz inhibitörü, onkojenik tirozin kinazların katalitik bölgelerinde ATP bağlanma bölgelerini yarışmalı olarak inhibe ederler.
Ayrıca; küçük boyutlu olmaları, oral verilişe uygun olmaları ve radyasyon
terapisi ve kemoterapi ile kombine tedavide kullanıma uygun olmaları
önemli üstünlüklerindendir. Bu nedenle; FDA tarafından onaylanmış
ve piyasada bulunan tirozin kinaz inhibitörleri günümüzde kanser tedavisinde kullanılmaktadırlar71. Bunlardan ilki 2001 yılında FDA onayı
79
alan İmatinib Mezilat tabletleri, piyasa adı ile Gleevec®’tir. Kronik Myeloid
Lösemi tedavisinde kullanılan bu ilaç, bu hastaların çoğunda bulunan
BCR-ABL onkogenine ait protein kinaz inhibitörlerindendir. EGFR’yi hedefleyen diğer preperatlar Iressa® (Gefitinib) ve Tarceva® (Erlotinib)’dır. Bu
ilaçlar EGFR ekspresyonunun sıklıkla görüldüğü küçük hücreli olmayan
akciğer kanseri tedavisinde kullanılmaktadırlar. Vasküler büyüme faktörü olan ve tümör anjiyojenezinde önemli rolü olan VEGF büyüme faktörü reseptör tirozin kinazlarına örnek olarak ise; Sorafenib (Nexavar®)
ve Sutent (Sunitinib®) verilebilir. Bu inhibitörler ise renal kanserlerde ve
gastrointestinal tümörlerin tedavisinde kullanılmaktadırlar.
Kullanılan monoklonal antikorların reseptöre seçiciliği yüksek
olmasına rağmen molekül ağırlıklarının yüksek olması bir sakıncadır.
Dolayısıyla; bu moleküllerin in vivo kullanımında ilacın dokuya geçişi ve
hücreler tarafından alımı düşük düzeydedir30. Tümör hücresini tanıyan
küçük peptit molekülleri bu sıkıntının üstesinden gelebilir. Ancak, tedavide peptitlerin kullanımı kısa yarı ömürleri nedeniyle kısıtlanmıştır72.
Ayrıca, bu peptitlerin doku ve organlardaki biyoyararlanımı da düşük
düzeydedir. Dolayısıyla, peptidin hedef bölgeye ulaşmadan atılma oranını
en aza indirmek gerekir. Bu yapıların ilaç olarak kullanılabilmeleri için
kimyasal modifikasyonlar gerekebilir. Bu bağlamda; D-aminoasitler,
psödo-aminoasitler ve peptid siklizasyonu peptidlerin stabilizasyonlarını
artırmada en yaygın kullanılanlardır73. Diğer yandan, peptitlerin uzun
ve yeterli bir etki oluşturabilmesi için bazı doku ve organlarda bulunan endopeptidaz ve aminopeptidaz gibi proteolitik enzimlere karşı da
koruyucu önlemler almak gerekir. Nanotaşıyıcı sistemler genellikle bunun üstesinden gelebilir; peptitlerin lenf dolaşımında daha uzun süre
kalmasını ve dolayısıyla biyoyararlanımının artmasını sağlayabilirler.
Dekstran, dekstran sülfat ve siklodekstrinler gibi çeşitli makromoleküller, jelatin mikroküreleri, lipozomlar, emülsiyonlar ve miseller gibi ilaç
taşıyıcı sistemler peptit yapılı ilaçların lenfatik sisteme taşınması için
çalışılan bazı sistemlerdir74.
Peptitlerle kanserin hedeflendirilmesi ile ilgili literatürde
çalışmalar bulunmaktadır75–77. Faj sunum tekniği ile elde edilen peptit
kütüphanelerinde bulunan tümör kan veya lenf damarlarına özgü peptitlerle (tumor homing peptides) kanserde hedeflendirme çalışmaları
yapılmaktadır34. Bu peptitlerden özellikle tümör lenf damarlarına özgünlük gösteren Lyp-1 peptidi ile kuantum noktaları ve nanopartiküller gibi
80
sistemler çalışılmıştır33,78. Diğer bir çalışmada antikanser etkili ve meme
kanserine ait lenf damarlarına spesifik Lyp-1 peptidinin yağ bazlı ilaç
taşıyıcı sistemlerin içine hapsedilmesi amaçlanmış ve bu amaçla SMEDDS
(Kendiliğinden mikroemülsifiye olabilen sistemler; Self-microemulsifying
drug delivery systems) tasarlanmıştır79. Bu peptit kullanılarak yapılan
hücre kültürü çalışmalarında; Lyp-1 peptidinin, sıklıkla metastaz görülen
meme kanser hücrelerine olan özgünlüğü gösterilmiştir80,81. Tümör lenf
damarlarına özgün olan ve hücreye bağlandığında aynı zamanda apoptozu da tetikleyen bu peptidin meme kanserinde lenfatik sistem yoluyla
olan metastazın önlenmesi ve tedavisinde oldukça ümit verici olduğu
söylenebilir.
Manyetik karbon nanotüpler (MNT) lenfatik bölgeye etkili bir şekilde ilaç taşınmasında kullanılan sistemlerdendir82. İlaçların manyetik
olarak hedeflendirildiği sistemler bir manyetik alan yardımıyla ilaçların
etki bölgesine taşındığı umut verici sistemlerdir. Bu manyetik alan ilacı
RES’den uzak bir bölgeye sabitler. Hedeflendirmenin derecesi, kanın
akış hızı ve manyetik alanın şiddetine bağlıdır83. Dolayısıyla, kan akış
hızının daha az olduğu dokularda ilaç birikimi daha fazladır. Kitosan,
poliglikolitler, polialkilakrilosiyanatlar gibi biyolojik olarak parçalanabilen polimerlerle ilaç ve manyetik partiküller çeşitli teknikler kullanılarak birleştirilerek hedeflendirme çalışmaları yapılmıştır84. Bu polimerler
sayesinde, ilaçlar RES’e yakalanmadan uzun süre kanda kalabilirler84.
Lenfatik hedeflendirmede ise, 5-flurourasil ve sisplatin gibi maddeler iç
yüzeyinde Fe3O4 gibi manyetik partiküller bulunan nanotüplerin gözeneklerine dahil edilerek nanoteknolojik yöntemlerle üretim yapılmıştır82.
Bu sistemden yararlanmak için fosfolipitler, polietilenglikol (PEG) ve folik asit kovalan olmayan bir yolla MNT bağlanır. MNT’lerin lenf hücreleri
tarafından kolayca alındığı ve seçici olarak kanser hücrelerini tahrip
ettikleri gösterilmiştir82. Yüzey modifikasyonu ile MNT’lerin, kansere
lenfatik hedeflendirme de kullanılabilecek uygun taşıyıcılar olduğu
söylenebilir.
Teşhis ve tedavi amacıyla ilaçları lenfatik sisteme hedeflendirmede
kullanılan taşıyıcılardan bir diğeri lipozomlardır ve bu amaçla subkütan (s.c.) uygulamaları üzerinde çalışılmaktadır85. Lipozomun subkütan
uygulamayı takiben lenfatik emilimini ve lenf nodları tarafından alımını
etkileyen belirleyici faktörlerin lipozom boyutu ve uygulama bölgesi olduğu belirlenmiştir. Lipit bileşimi ve PEG kaplamanın s.c. uygulamayı taki-
81
ben emilimi etkilemediği gösterilmiştir. Öte yandan, lipozom boyutu 0,1
µm’den küçük olduğunda, emilim miktarının uygulanan dozun %70' ine
kadar çıkabildiği tespit edilmiştir (Şekil 4). Lipozomlar yağ bazlı sistemler olmaları sebebi ile lenfatik emilimi artırmaktadır. Boyutun küçülmesi
ile de lenfatik alımın artması sağlanmış, böylece nano boyutlu yağ bazlı
sistemlerin özellikle suda çözünürlüğü az olan ilaçların lenfatik hedeflendirmesi için kullanılabileceği görülmüştür.
Diğer bir yaklaşım ise, poli-laktit-ko-glikolit–paklitaksel (PLGA–
PTX)’le jelatin sünger matrisin birleştirilmesi ile oluşmuş sistemlerle
ilgilidir86. Bu kombine sistem, jelatin matrisin yapısal üstünlüğü sayesinde sürekli salım sağlar. Tedavi edici ve koruyucu olarak lenfatik bölgeye
doğrudan yerleştirilebilir (implantasyon). PLGA mikroküreleri; püskürterek kurutma yöntemi kullanılarak hazırlanmıştır. Bu çalışmada; partikül
boyutları intraperitoneal ve intraplevral olarak lenfatik hedeflendirilmeye
uygun olacak şekilde 1 – 8 µm aralığında olacak şekilde hazırlanmıştır.
PTX salımı; PLGA parçalanmasına bağlı olarak kontrol edilmiştir. Subkütan uygulamadan sonra ilacın lenf nodlarına ulaşabilmesi için 0,1
µm’den küçük partiküllere ihtiyaç varken, intraplevral ve intraperitoneal uygulamadan sonra ilacın lenf nodlarına ulaşabilmesi için partikül
boyutunun 1 – 5 µm arasında olmasının yeterli olduğu belirtilmiştir86.
Sağlıklı ve akciğer kanseri geliştirilen farelerde yapılan in vivo çalışmada,
Şekil 4
Lipozom boyutuna bağlı olarak lenf nodlarında % emilen miktar85
82
intraplevral ve intraperitoneal olarak implante edilen sistemlerden PLGA
mikropartiküllerin lenfatik sisteme absorpsiyonunun sağlandığı görülmüştür. Bu sistemin özellikle kanserde lenfatik metastazın önlenmesinde faydalı olabileceği öngörülmektedir.
Mitomisin C’nin intramuskuler ve intraperitoneal yolla uygulanan
yağ içinde su (s/y) ve su içinde yağ (y/s) tipi emülsiyonların vasıtasıyla
lenfatik taşınması kanıtlanmıştır. Bölgesel enjeksiyonu takiben y/s tipi
emülsiyonların lenflere en çabuk, s/y tipi emülsiyonlar lenflere daha geç
ulaştığı görülmüştür87. Buna bağlı olarak mide kanserinde kullanılmak
üzere bir antikanser ilacı, pirarubisin (Lipiodol), içeren emülsiyon formülasyonu geliştirilmiştir. Bu formülasyon içinde ilacın lenf nodunda ve
enjeksiyon bölgesinde yaklaşık 7 gün kalabildiği gösterilmiştir87.
Bir başka çalışmada ise farklı PEG (200, 500, 2000 Da) türevleri ve 4benzen sülfonat ile modifiye edilen polilizin dendrimerlerin lenfatik emilimi ve taşınımı karşılaştırılmıştır88. PEG 200’le hazırlanan dendrimerin
s.c. uygulanmasını takiben hızlı bir şekilde ve tamamına yakını kan dolaşımına geçmiştir. Yalnız % 3 kadarı 30 saat içinde torasik lenfe geçmiştir.
PEG zinciri artırılarak kana geçiş azaltılmış lenfe geçiş ise 30 saat içinde
% 29’a çıkartılmıştır. 4-benzen sülfonatlı dendrimerin ise kandan ve lenften iyi emilmediği görülmüştür. Ayrıca; pegile edilmiş polilizin dendrimerlerin s.c. enjeksiyon bölgesinden iyi emilim gösterdiği görülmüştür.
Bu çalışmanın sonucunda; PEG boyutunun artmasıyla lenfatik emilimin
arttığı ortaya konulmuştur.
Sonuç ve Tartışma
Vücutta etkin maddenin, istenen hedef bölgeye gönderilmesinde, taşıyıcı sistemlerin kullanımı yeni bir yaklaşım değildir. Geçen yüzyılın sonlarına doğru, Paul Ehrlich, sihirli mermi “Magic bullet” diye bir kavramı ilk
kez ortaya atan kişi olmuştur89. Ehrlich ilk çalışmasında, frengi hastalığının tedavisinde kullanılan “Salvarsan” (Asfenamin) isimli ilacın bu hastalık etkenine olan seçici etkinliğinden esinlenmiştir ve “sihirli mermisini”
geliştirmeyi başarmıştır. Bundan sonra da bu konuda önemli gelişmeler
yaşanmıştır. Nanoteknolojideki hızlı gelişmeler sayesinde önümüzdeki yıllarda kanser tedavisi daha akılcı ve daha etkili bir şekilde yapılabilecektir.
Hedeflendirmede yeni hedeflerin, yeni ligantların bulunması, nanotaşıyı-
83
cıların özelliklerinin iyileştirilmesi sayesinde, ilacın tümörlü bölgeye daha
etkili bir şekilde taşınması ve daha az yan etkiye neden olması amaçlanmaktadır. Lenfatik hedeflendirme, kanserin yayılımını önlemek için
son derece önemlidir. Lipitlerin emilim mekanizmalarının ve intestinal
bölgenin fizikokimyasal özelliklerinin daha iyi anlaşılması sayesinde lenfatik sisteme hedeflendirmede daha etkili formülasyonların geliştirilmesi
mümkün olmuştur. Böylece, kanser tedavisinin daha etkin bir biçimde
yürütülmesi için önemli bir adım atılmıştır. Gelecekte de, bu çalışmaların
kanser tedavisinde başarıyı daha da artırması beklenmektedir.
Özet
Kanser; kontrolsüz bölünen ve diğer dokulara yayılabilme özelliği
olan anormal hücrelerin oluşturduğu hastalıklar için kullanılan bir terimdir. Kanser tedavisinde radyoterapi, ameliyat ve kemoterapi gibi yöntemler kullanılır. Kemoterapi; kanser hastalarında önemli yan etkilere
neden olur. Bu nedenle; tümörlü dokuya özgü ilaç taşıyıcı sistemlere
ihtiyaç duyulur. İlacın sağlıklı dokularda daha az birikmesinin ve tümörlü
bölgede tercihen yoğunlaşmasının bir sonucu olarak ilaç toksisitesi
azalır. Bu sistemlere örnek olarak; lipozomlar, miseller, mikro-nanopartiküler sistemler verilebilir. Kanser hücreleri vücudun diğer organlarına
kan ve lenf yoluyla yayılırlar. İlacın lenfatik sisteme hedeflendirilmesinin
bir sonucu olarak kanser hücrelerinin çevre dokulara yayılması önlenebilir. Bu derlemede öncelikle; kanser hakkında temel bilgiler verilmekte;
daha sonra ise; bu konuda yapılmış güncel çalışmalar ve araştırmalar
ışığında kanser tedavisinde ilacın hedeflendirilmesinin önemi ve lenfatik
hedeflendirmeden nasıl yararlanılabileceği anlatılmaktadır.
Anahtar Kelimeler: Kanser, Kanserde Hedeflendirme, Yeni İlaç Taşıyıcı
Sistemler, Lenfatik Hedeflendirme
84
Abstract
Lymphatic targeting in Cancer Therapy
Cancer is a term used for diseases of uncontrollable self-division of
abnormal cells, which are able to invade other tissues. Radiotherapy,
surgery and chemotherapy applications are in cancer treatment. Chemotherapy can cause many side effects in cancer patients. Therefore,
tumor tissue-specific drug carrier systems are needed. Drug toxicity is
reduced as a consequence of preferential accummulation at target sites
and lower concentration in healthy tissues. Examples of these systems
are liposomes, micelles, micro-nanoparticulate systems. Cancer cells
can spread to other parts of the body through the blood and lymphatic
systems. Spread of cancer cells into surrounding tissues can be prevented by drug targeting to the lymphatic system. In this context, basic
information about cancer is given then, in the view of current studies,
importance of targeting and advantages of lymphatic targeting in cancer
therapy are explained.
Keywords: Cancer, Targeting in Cancer Therapy, New Drug Delivery
Systems, Lymphatic Targeting.
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Hacettepe Üniversitesi Eczacılık Fakültesi Dergisi
Cilt 32 / Sayı 1 / Ocak 2012 / ss. 91-106
Arnebia Forssk. Türlerinin Kullanışları ve
Biyolojik Aktiviteleri
Received:22.09.2011
Revised:
03.10.2011
Accepted:16.11.2011
Merve Yüzbaşıoğlu*, Ayşe Kuruüzüm-Uz*0
Giriş
Boraginaceae familyasında yer alan Arnebia Forssk. cinsi ülkemizde 5 tür ile temsil edilmektedir1,2. Türkiye’de halk arasında eyilik otu,
kırmızı kök, eğnik, enlik, havaciva olarak da bilinen A. densiflora’ nın
kök kabukları haricen yara iyileştirmede ve yanık tedavisinde kullanılmaktadır3,4. Hem halk arasında kullanılışı hem de özellikle köklerinde
bulunan naftokinonlar5, benzokinonlar6, diterpenler7, steroitler8, triterpenler9 ve flavonoitler10 gibi aktif bileşenler taşımaları nedeniyle son
zamanlarda birçok araştırmanın konusu olmuşlardır.
Halk Arasında Kullanılışları
Kars, Iğdır yöresinde yapılan bir etnobotanik çalışmada Arnebia densiflora’ nın kök kabuklarının ve çam türlerinin odunlarının parçalanıp
karıştırılarak tereyağ içerisinde pişirilmesi ile hazırlanan merhemin yara
iyileştirme ve yanık tedavisinde kullanıldığı kayıtlanmıştır11,12.
Boraginaceae familyasına ait Alkanna, Arnebia, Maharanga ve Onosma cinslerinde bulunan toplam 8 türün kök ve rizomlarından oluşan
Ratanjot’un ekzama, yara ve isilikte etkili olduğu, Hindistan’da göz has* Hacettepe Üniversitesi, Eczacılık Fakültesi, Farmakognozi Anabilim Dalı, 06100
Sıhhiye, Ankara, TÜRKİYE
o
Corresponding author: e-mail: [email protected]
92
talıkları, bronşit, karın ağrıları ve kaşıntı tedavisinde kullanıldığı bilinmektedir13. Hindistan’da yapılan etnobotanik bir çalışmada Arnebia benthamii’ nin hardal yağında bekletilmiş köklerinin saç çıkartıcı amaçla halk
arasında kullanıldığı bildirilmiştir14.
Nepal’in batısında Arnebia benthamii’ nin kurutulmuş ve toz edilmiş
köklerinin kan temizleyici, soğuk algınlığı ve vücut ağrılarını iyileştirici;
yağda bekletilmiş köklerinin ise saç çıkartıcı amaçla ve saç için kırmızı
boya olarak kullanıldığı kayıtlanmıştır15.
Biyolojik Aktiviteleri
Arnebia türleri üzerinde günümüze kadar yapılan araştırmaların
derlenmesi sonucunda bulunan, Arnebia türlerinin ve bu türlerden
elde edilen bazı bileşiklerin gösterdiği biyolojik aktiviteler şu şekilde
sınıflandırılabilir:
Sitotoksik ve Antitümöral etki
A. euchroma’ nın köklerinden elde edilen rabdosiin ve izomeri, memeli DNA topoizomerazlarının in vitro non-selektif inhibitörleri olarak etki
göstermişlerdir16.
A. euchroma köklerinden elde edilen 3 naftokinon türevinin (asetilalkannin, izobutilalkannin, 2',3'-epoksialkannin) insan kolorektal kanser
hücrelerinde (HCT116) ve insan hepatoma (Hep G2) hücrelerinde yapılan
MTT deneyi sonucunda IC50 değerleri, HCT hücreleri üzerinde sırasıyla
(0,34 ± 0,09 μM, 0,30 ± 0,09 μM, 0,39 ± 0,08 μM) Hep-G2 hücreleri üzerinde sırasıyla (0,39 ± 0,07 μM, 0,42 ± 0,03 μM, 0,34 ± 0,04 μM) olarak
bulunmuştur (Şekil 1). Bu sonuçlar hidroksinaftokinon türevlerinin her
iki hücre üzerinde de güçlü hücre büyümesini engelleyici etkileri olduğunu göstermiştir17.
A. euchroma’ nın köklerinin antitümör etkisi MTT (3-(4,5-dimetiltiazol2-il)-2,5-difeniltetrazolyum bromür) deneyi ile in vitro olarak insan akciğer adenokarsinoma hücreleri (A549), insan karaciğer karsinoma hücreleri (Bel-7402), insan meme adenokarsinoma hücreleri (MCF-7), fare Lewis akciğer karsinoma (LLC) hücreleri üzerinde; in vivo ise LLC modeli ile
karsinoma oluşturulmuş C57BL/6 farelerinin kullanıldığı çalışma ile de
araştırılmıştır. LLC dokusunda bax, bcl-2 ve kaspaz-3 proteinlerinin ekspresyonu immunohistokimyasal boyama ile teşhis edilmiştir. A549, Bel7402, MCF-7 ve LLC hücre hatlarında asetilşikonin hücre çoğalmasını
ARNEBİA FORSSK. TÜRLERİNİN KULLANIŞLARI VE BİYOLOJİK AKTİVİTELERİ
93
doza bağımlı olarak inhibe etmiştir (Şekil 1). Bu hücrelerdeki IC50 değerleri sırasıyla 5,6 ± 0,86 μg/ml, 6,82 ± 1,5 μg/ml, 3,04 ± 0,44 μg/ml ve
2,72 ± 0,38 μg/ml olarak bulunmuştur. Ayrıca asetilşikonin, LLC ile tümör oluşturulmuş C57BL/6 farelerindeki tümör büyümesini baskılamıştır. Asetilşikoninin 2 mg/kg dozdaki inhibisyon oranının % 42,85 olduğu
bulunmuştur. İmmunohistokimyasal boyama sonuçları asetilşikoninin
bax ve kaspaz-3 ekspresyonu yaptığını; bcl-2 expresyonunu azalttığını
göstermiştir ki bu durum asetilşikoninin pro-apoptotik bcl-2 ve kaspaz-3
ü aktive ederek tümör hücre apoptozisini indüklediğini göstermiştir18.
Doymamış pirolizidin alkaloitleri içeren A. hispidissima topraküstü
ekstresi Ehrlich asit karsinoma hücrelerinde in vitro olarak sitotoksik
aktivite göstermiştir19.
Yapılan çalışmalarda Arnebia nobilis’in % 50’lik alkoldeki kök ekstresi ve bu ekstreden elde edilen arnebin 1 ve arnebin 3 maddeleri, Walker
karsinosarkoma 256 farelerinde in vivo olarak etkili bulunmuştur ve naftokinonlar tümör hücrelerini in vitro olarak inhibe etmiştir20.
A. euchroma’dan elde edilen polisakkaritlerin farelerde oluşturulmuş
uterus servikal karsinoma tümör modelindeki (U14) antitümoral etkinliği
araştırılmıştır. Sonuçlar, A. euchroma’dan elde edilen polisakkaritlerin
U-14 fare tümör modelinde yüksek antitümöral etkinlik gösterdiğini
(p<0.05), U-14 fare tümör modellerinin ömürlerini uzattığını, ortalama
dozlarda dalak indeksini artırdığını, orta ve yüksek dozlarda ise timüs
indeksini azalttığını göstermiştir21.
A. purpurea’ dan elde edilen ve yapısı saptanan maddelerin sitotoksik
aktivitelerinin MTT yöntemi ile fare kaynaklı kanser hücre kültüründe
(L929) araştırılması sonucunda sitotoksik aktivite, köklerden elde edilen naftokinon türevi bileşiklerde (izovalerilalkannin, α-metil-n-butil
alkannin ve izobutilalkanninden oluşan karışım, asetilalkannin, alkannin) en yüksek (sırasıyla 50 μg/ml’deki % inhibisyon=% 98,57; %
97,41; % 97,67), β-sitosterolde naftokinonlarla kıyaslanabilir derecede
yüksek (50 μg/ml’deki % inhibisyon=% 65,93) ve triterpen türevi olan
3-O-asetiloleanolik asit’de de anlamlı derecede (50 μg/ml’deki % inhibisyon= % 43,70) bulunmuştur22.
Yaşlanmayı engelleyici etki
A. euchroma’ nın yağda çözünen ekstresinin (AE-I) yaşlanmaya karşı etkisi farelerdeki D-galaktoz ile oluşturulmuş subakut yaşlanma
94
modeli üzerinde denenmiştir. D-galaktoz ile yaşlanan fare modeli % 1-2
D-galaktozun (10 mL/kg) enjeksiyonu ile oluşturulmuştur. Timus ve dalağın aktivite katsayıları, kan üre azotu (BUN) içeriği ve serumdaki total
antioksidan kapasite (T-AOC); karaciğer dokusundaki süperoksit dismutaz (T-SOD), glutatyon peroksidaz (GSH-Px) aktiviteleri, malonildialdehit
miktarı ve beyin homojenatındaki monoamin peroksidaz (MAO), nitrik
oksit sentaz aktiviteleri ölçülmüştür. Tüm dozlarda AE-I’in farelerdeki serum BUN düzeyini azalttığı (p<0,01); karaciğer homojenatındaki T-SOD
aktivitesini anlamlı ölçüde artırdığı; GSH-Px aktivitesini güçlendirdiği
(p<0.05); fakat timus karaciğer ve beyin homojenatları üzerinde belirgin
etkisinin bulunmadığı gözlenmiştir (p>0.05). Elde edilen biyokimyasal sonuçlara göre AE-I in farelerde D-galaktoz tarafından oluşturulan subakut
yaşlanma üzerinde yaşlanmayı engelleyici etkisinin olduğu görülmüştür23.
Şekil 1
Asetilalkannin ve Asetilşikonine ait formüller
Antienflamatuvar etki
A. euchroma’nın köklerinden elde edilen asetilşikonin histaminle indüklenen kapiller permeabilite, fare pençe ödemi ve koton pellet ile oluşturulan granulomu inhibe etmiştir. Ayrıca asetilşikonin, böbreküstü bezi
çıkartılmış farelerde de antienflamatuvar etki göstermiştir. LD50 değeri
22,75 mg/kg olarak bulunmuştur24.
A. euchroma’ nın köklerinin petrol eteri, kloroform, alkol ve sulu
ekstrelerinin antienflamatuvar etkinliği karragen indükleyici fare pençe ödemi metodu ile araştırılmıştır. 100-150 g ağırlığında albino porton
95
farelerinin kullanıldığı deneyde kontrol grubuna % 1'lik tuz içeren taşıyıcı çözelti, standart olarak kullanılacak gruba ise ibuprofen 50 mg/kg
oral dozda verilmiştir. Her bir ekstrenin farelere 500 mg/kg oral dozda
verildiği deneyin sonucunda maksimum ödem inhibisyonları % 61,2, %
45, % 27,5 ve % 60 olarak bulunmuştur. Bulunan bu sonuçlar karragen
ile indüklenmiş fare pençe ödemine 300 dakikalık zaman aralığında ekstrelerin aktivitelerinin referans ilaç olan ibuprufenle kıyaslanabilir oranda olduğu gösterilmiştir (ibuprofen 50 mg/kg, p.o., % 61,6 inhibisyon)25.
A. hispidissima kök etanolik ekstresi ve bu ekstreden kromatografik
metotlar sonucu elde edilen arnebin-5, arnebin-6, terakrilşikonin, arnebinon ve asetilşikoninin antienflamatuvar aktiviteleri farelerde oluşturulan karragen ile indüklenmiş pençe ödemi ve komplet Freund adjuvanı
(CFA) ile indüklenmiş kronik artrit ile araştırılmıştır. Karragen ile indüklenen fare pençe ödemi deneyinde 150–200 g ağırlığında erkek Wistar
albino fareleri, CFA ile indüklenmiş kronik artrit deneyinde 25-30 g ağırlığında Swiss albino fareler kullanılmıştır. Kontrol grubuna sadece % 2
lik tuz çözeltisi verilmiş; standart olarak ise asetilsalisilik asitin etanoldeki % 1'lik çözeltisi kullanılmıştır. Sonuçlar A. hispidissima’nın etanol
ekstresinin ve tüm naftokinon türevlerinin istatistiksel olarak anlamlı
bir şekilde antienflamatuvar etkinliğinin olduğunu göstermiştir (p<0,01).
Karragen ile indüklenmiş akut artritin inhibisyonunda 8 saat sonunda
5 mg/kg dozda arnebinon % 27,17 inhibisyonla en etkili, CFA ile indüklenmiş kronik artritin ilerlemesinin baskılanmasında ise yine arnebinon
1. günün sonunda 5 mg/kg dozda % 62,45 inhibisyon ile en yüksek etkili
bulunmuştur26.
A. hispidissima’ nın kök n-hekzan ekstresinin ve bu ekstreden elde
edilen naftokinonların (arnebin-1, arnebin-7, tiglik asit, alkannin, arnebinol ve sikloarnebin-7) antienflamatuvar etkisi, farelerde oluşturulan
karragen ile indüklenmiş akut artrit ve komplet Freund adjuvanı (CFA)
ile indüklenmiş kronik artrit ile araştırılmıştır. Karragen ile indüklenmiş
akut artrit enflamasyon deneyinde 4–6 haftalık, 150–200 g, erkek Wistar
albino fareler; CFA ile yapılan deneyde ise 4-6 haftalık, 25-30 g, erkek
Swiss albino fareler kullanılmıştır. n-hekzan ekstresi 100 ve 500 mg/
kg, p.o; naftokinonlar ise 50-100 mg/kg, p.o dozlarda farelere verilmiştir.
Her iki deneyde de kontrol grubuna sadece tuz verilmiş, standart olarak
da %1 karboksimetilselüloz içinde 50 ve 100 mg/kg, p.o. dozlarda asetilsalisilik asit verilmiştir. Sonuçlara göre her 2 metotta da n-hekzan ekstresi ve naftokinonların anlamlı ölçüde antienflamatuvar etki gösterdiği
96
bulunmuştur (p<0.01). Karragen ile indüklenmiş akut artritin inhibisyon
deneyinde 4. saatin sonunda 50 mg/kg dozda sikloarnebin-7’nin % 56,3
inhibisyonla standarttan sonra en etkili, CFA ile indüklenmiş kronik artritin ilerlemesinin baskılanmasında ise 1. günün sonunda 50 mg/kg dozda arnebin-1’in % 60,4 inhibisyonla standarttan sonra en etkili olduğu
görülmüştür27.
Prostaglandin biyosentez inhibitör etki
Yapılan deneyde A. euchroma’ nın köklerinden elde edilen major naftokinonların prostaglandin biyosentez inhibitörü olmadığı gösterilmiştir.
Yine bu bitkiden elde edilen şikonafuran B, C ve de-O-metilasidiploidinde
en fazla, arnebinol ve arnebinonda ise daha az olmak üzere prostaglandin biyosentez inhibitörü etki gözlenmiştir28.
Yapılan bir diğer çalışmada, A. euchroma’ nın kök metanol ekstresinden elde edilen arnebinol ve de-O-metilasiodiplodinin prostaglandin
biyosentez inhibitörü olduğu bulunmuştur. Arnebinolün IC50 29,5 μM,
ve de-O-metilasiodiplodinin 20 g/ml de % 62,7 inhibitör etkisi olduğu
görülmüştür29.
Platelet agregasyonu inhibe edici etki
A. euchroma’ nın kök kloroform ekstresinden elde edilen şikonin ve
türevlerinin (β-β dimetilakrilşikonin, asetilşikonin, terakrilşikonin) antiplatelet etkinlikleri, yıkanmış tavşan plateletlerinde ayrı ayrı kollajen,
araşidonik asit, platelet aktive edici faktör (PAF) veya trombin nedeni ile
oluşan % agregasyonun ölçülmesiyle araştırılmıştır. 2 μg/ml dozda trombin ile indüklenmiş platelet agregasyonunun terakrilşikonin ve asetilşikonin tarafından kısmen inhibe edildiği, araşidonik asit ile indüklenmiş
platelet agregasyonunun asetilşikonin tarafından önemli oranda inhibe
edildiği, kollajen ile indüklenmiş platelet agregasyonunda terakrilşikonin
ve asetilşikoninin tamamıyla inhibisyon gösterdiği, PAF ile indüklenmiş
platelet agregasyonunda ise terakrilşikonin, asetilşikonin ve şikoninin
çok az oranda inhibisyon gösterdiği bulunmuştur30.
Yara iyi edici etki
A. densiflora kök ekstresinin fare damak mukozasındaki yara iyi edici etkisini gözlemek amacıyla yapılan çalışmada, farelerin damaklarının
97
orta hattında skalpek kullanılarak 10 mm kalınlığında mukozal yaralar
oluşturulmuştur. Deney grubunda oluşturulan yaralara günde bir defa
topikal olarak % 10 (h/h) 5 gram A. densiflora kök ekstresi ile hazırlanmış 45 gram merhem tatbik edilmiştir. 4., 7., 14. ve 21. günlerde yaraların üzerinden doku örnekleri alınarak histolojik incelemeler yapılmıştır.
Deney grubunda % 10 A. densiflora kök ekstresinin yara iyileşmesinde
giderek artan etkisi olduğu gösterilmiştir31.
A. densiflora’ nın kök n-hekzan, kloroform, etilasetat ve metanol ile
ekstrelerinin erkek Sprague–Dawley fareleri (160–180 g) ve Swiss albino
fareleri (20–25 g) üzerinde linear insizyon ve eksizyon yara modelleri oluşturularak yara iyi edici aktiviteleri araştırılmıştır. % 1 n-hekzan ekstresi
içeren ve glikol stearat: propilen glikol: likid parafin (3:6:1) ile hazırlanan
merhem her iki modelde de referans merhem olan 0,5 g Madecassol® ile
karşılaştırılabilir düzeyde yüksek etkinlik göstermiştir32.
A. nobilis’ köklerinden elde edilen arnebin-1 in kütanöz punch yara
modeli üzerinde yara iyi edici etkisi araştırılmıştır. %0,1 fosfat tampon
tuzunda hazırlanmış Arnebin-1 süspansiyonu, hidrokortizon ile muamele edilmiş veya edilmemiş olan yaralar üzerine günlük topikal olarak
uygulanmıştır. Arnebin-1 hidrokortizonlu veya hidrokortizonsuz yaralar
üzerinde yaranın genişliği ve boşluk uzunluğunu açıkça azaltarak kontrollerle karşılaştırılabilir düzeyde önemli yara iyi edici etki göstermiştir.
Arnebin-1 tedavisi hücre proliferasyonu, hücre göçü ve kalın granüle dokudan damar oluşumunu ve yaralarda yeniden epitel doku oluşumunu sağlamıştır. Hidrokortizonsuz kontrol grupları ile karşılaştırıldığında
arnebin-1 ile muamele edilmiş yaralarda kollajen, fibronektin ve büyüme faktörü β-1 (TGF-β1) in sentezinde artış görülmüştür. TGF-β1in yara
iyileşmesinin hızını artırması ve hidrokortizon ile tedavi edilmiş yaralardaki bozuklukların tedavisinde de etkili olması; arnebin-1 in de steroit
ile bozulmuş yaralardaki iyileşmeyi sağlamak için iyi bir terapötik ajan
olabileceğini düşündürmüştür33.
Antimikrobiyal etki
Metisiline dirençli Staphylococcus aureus (MRSA) ve Vankomisine
dirençli enterekoklara (VRE) karşı etkinliği araştırılan A. euchroma nın
kök kabukları kullanılarak yapılan biyolojik aktivite rehberli çalışmasında elde edilen altı naftokinon türevinin (minimum inhibitör konsantrasyonları (MICs) 1.56 ve 3.13 µg/mL arasında), alkannin ve şikonine
98
(MIC = 6.25 μg/mL) göre daha yüksek anti-MRSA aktivite gösterdiği bulunmuştur. Bu türevler aynı zamanda anti-MRSA ya benzer minimum
inhibitör konsantrasyonlarda anti-VRE etkinlik göstermişlerdir5.
A. hispidissima’nın topraküstü petrol eteri ekstresinin ve bu bitkiden
elde edilen triterpenler olan β-amirin, β-amirin asetat, lupeol ve betulinin
(Şekil 2) Escherichia coli, Staphylococcus aureus, Klebsiella pneumoniae,
Bacillus thuringiensis’e karşı antibakteriyel; Aspergillus niger, A. flavus,
Rhizoctonia phaseoli ve Penicillium chrysogenum’e karşı antifungal etkileri disk difüzyon metodu kullanılarak karşılaştırılmıştır. Bakteriler için
referans olarak gentamisin; mantarlar için ise referans olarak mikostatin kullanılmıştır. Petrol eteri fraksiyonu K. pneumoniae (İnhibisyon zon
çapı;=IZ=14 mm) ve P. chrysogenum (IZ=7 mm) e karşı maksimum aktivite göstermiştir. Elde edilen triterpenlerden β –amirin’in E. coli’ ye karşı;
(IZ=16 mm) β -amirin asetatın ise R. phaseoli’ ye (IZ =10 mm) karşı maksimum aktivite gösterdiği bulunmuştur34.
A. decumbens’ ten elde edilen şikoninin, Pseudomonas aeruginosa,
Escherichia coli, Staphylococcus aureus, ve Klebsiella pneumonia’ya karşı
yüksek antibakteriyel etkinlik gösterdiği bulunmuştur35.
Antiviral etki
AIDS tedavisinde yeni, tolerans gelişmemiş ve terapötik indeksi geniş
olan ilaç olabilecek maddelerin araştırıldığı bir çalışmada; Çin tıbbında
AIDS’e karşı kullanılan ve daha önce yapılan çalışmalarda aseton ektresinin infekte edilmiş H9 hücrelerinde HIV replikasyonunu engelleyici etkinliği bulunan Arnebia euchroma’nın kök n-hekzan ekstresinden hareketle aseton ekstresi hazırlanmış ve biyolojik aktivite rehberli çalışılması
sonucu en aktif fraksiyonundan kafeik asit tetramer tuzları ve kafeik asit
tetramer glukozitlerinin dipotasyum ve potasyum tuzları elde edilmiştir.
Bu bileşiklerin anti-HIV etkinlikleri ELISA testi ile araştırılmış ve rabdosiinin monosodyumlu, monopotasyumlu ve rabdosiinin optik izomerinin
monosodyumlu türevlerinin sırasıyla EC50 değerleri 2,8, 4 ve 1,5 μg/ml
bulunmuştur ve bu durum kafeik asit tetramerlerinin monosodyum ve
monopotasyum tuzlarının; glukozlu veya disodyum/dipotasyumlu türevleri ile karşılaştırıldığında en güçlü anti-HIV aktivite gösterdiğini kanıtlamıştır36.
A. euchroma’nın köklerinden elde edilen glikozaminoglukanların
(AEG) papillomavirüse karşı etkinliği RT-PCR metodu ile araştırılmıştır.
99
Şekil 2
β-amirin, Lupeol ve Betuline ait formüller
Verruka dokularının kullanıldığı bu PCR tekniğinde kontrol grubunda
serum fizyolojik çözeltisi, deney grubunda ise AEGnin 8 farklı dozu kullanılmıştır. Deney sonuçları AEG'nin anti-HPV etkisinin olduğunu ve en
düşük etkili konsantrasyonun 0,781 mg/ml olduğunu göstermiştir37.
Kontraseptif etki
A. euchroma’ nın köklerinden izole edilen dipotasyum rabdosiin, potasyum rozmarinat, sodyum rozmarinat ve sodyum ferulatın kontraseptif
etkileri incelenmiş ve dipotasyum rabdosiinin yüksek oranda kontraseptif etki gösterdiği bulunmuştur (Şekil 3)38.
A. euchroma’ nın glikozaminoglukanlarının (AEG) fare ovaryumundan elde edilen granulosa hücreleri üzerindeki steroidojenez etkisi
incelenmiştir. Sonuçlar, ortalama dozlarda AEG’nin insan koriyonik
gonadotropinleri muamele edilmiş granulosa hücrelerinde doz bağımlı
olarak progesteron ve östradiol üretimini inhibe ettiğini; yüksek dozlarda
ise (>3.00 g/L-1) granulosa hücrelerindeki temel progesteron ve estradiol
üretimini inhibe ettiğini göstermiştir39.
100
Şekil 3
Rabdosiine ait formül
Antioksidan ve Antikolinesteraz İnhibe Edici Aktivite
Arnebia densiflora’nın kök, gövde ve çiçeklerinin kloroform, etil asetat,
metanol ve sulu ekstresinin asetilkolinesteraz inhibisyon değeri, ELISA
mikroplak okuyucu kullanılarak; antioksidan aktivitesi ise DPPH radikal süpürme testi ve Fe+2-ferrozin test sistemi kullanılarak belirlenmiştir.
Sonuçlar, kök metanol ve sulu ekstrelerinin 200 µg/ml de orta derecede
DPPH radikal süpürücü etki gösterirken, tüm ekstrelerin standart olarak
kullanılan bütilhidroksianisole oranla daha iyi demir iyonu bağlayıcı etkiye sahip olduğunu göstermiştir. Sadece kök kloroform ekstresi 62,5 µg/
ml de asetilkolinesteraza karşı orta derecede inhibisyon göstermiştir40.
İmmün Sistem Fonksiyonları Üzerinde Etki
Siklofosfamid ile immün supresyonu indüklenmiş farelerde A.
euchroma’dan elde edilen polisakkaritlerin koruyucu etkisi araştırılmıştır.
Sonuçlar bu polisakkaritlerin timüs ve dalak indekslerini, klerens indeksini ve dalak lenfositlerinin proliferasyonunu artırdığını göstermiştir.
A. euchroma’dan elde edilen polisakkaritler immün supresyonu
gerçekleştirilmiş farelerde önemli ölçüde spesifik ve non- spesifik immünolojik fonksiyonları iyileştirmiştir41.
Toksisite
A. euchroma’nın akut toksisitesi, bu bitkinin köklerinden hazırlanan
petrol eteri, etilasetat, etanol ve su ekstrelerinin fareler üzerinde yapılan
101
toksisite deneyleriyle araştırılmıştır. Deney sonuçlarına göre petrol eteri,
etilasetat, etanol ve su ekstrelerinde farelerdeki maksimum tolere edilen
doz sırası ile 2,0 g/kg, 12,5 g/kg, 19,5 g/kg, ve 23,4 g/kg bulunmuştur.
Bu durum UV spektrofometresi ile yapılan şikonin miktar tayini ile paralellik göstermiştir42.
A. hispidissima’nın taşıdığı monokrotalin ve ekhimidin adındaki pirolizidin tip alkaloitlerinden dolayı memelilere hepatotoksik etkili olabileceği öngörülmüştür43.
Sonuç
Dünya üzerinde yaklaşık 2000 türü bulunan Boraginaceae
familyasına ait Arnebia Forssk. cinsinin biri endemik olmak üzere 5 türü
ülkemizde yetişmektedir. Özellikle Uzakdoğu tıbbında sıkça kullanılan,
Çin Farmakopesinde kayıtlı türleri bulunan, patentli birçok preparatın
terkibine giren bu cins; yıllardır Anadolu’da ‘Eyilik’ adı ile bilinmekte
ve zeytinyağında bekletilmiş kök ve kök kabukları açık yaraların ve
kesiklerin tedavisinde kullanılmaktadır. Arnebia türlerinin etnofarmakolojideki kullanılışının yanı sıra önemli sitotoksik etkilere de sahip olduğunun bulunması ile son yıllarda üzerinde yapılan aktivite
çalışmaları artış göstermiştir. Yapılan in vitro ve in vivo çalışmalar bu
türlerin ve bu türlerden elde edilen bileşiklerin önemli antitümoral, antienflamatuvar, prostaglandin sentez inhibitörü, antimikrobiyal, antiviral,
platelet agregasyonunu inhibe edici, yara iyi edici ve kontraseptif etkileri
olduğunu kanıtlamıştır. Bu çalışmada Arnebia türleri üzerinde yapılmış
olan araştırmaların derlenmesi sonucunda, ülkemizde yetişen türler
üzerinde yeterince çalışma bulunmaması nedeniyle, kanser tedavisinde
öncü, kanserdeki mekanizması ve etkinliği belirlenmiş bileşikler taşıyan
Arnebia türlerinin henüz üzerinde çalışma yapılmamış kanser hücrelerinde denenmesinin gerekliliği düşünülmüştür. Ayrıca yara iyi edici aktivite yanında antienflamatuvar ve antimikrobiyal etkinliği yüksek olan
Arnebia türlerinin ve taşıdıkları etkili madde gruplarının yara iyi edici
ilaçların bileşiminde yer alabileceği öngörülmüştür.
102
Özet
Boraginaceae familyasında yer alan Arnebia Forssk. cinsi ülkemizde
biri endemik 5 tür ile temsil edilmektedir. Halk arasında eyilik otu, kırmızı
kök, eğnik, enlik, havaciva olarak da bilinmektedirler. Çalışmamızda, Arnebia türlerinin ve bu türlerden elde edilen bileşiklerin daha önce yapılan
çalışmalarda belirtilen antitümoral, antienflamatuvar, prostaglandin
sentez inhibitörü, antimikrobiyal, antiviral, platelet agregasyonunu inhibe edici, yara iyi edici ve kontraseptif etkileri derlenmiştir.
Anahtar Kelimeler: Arnebia Forssk., Boraginaceae, Biyolojik Aktivite,
Naftokinonlar.
Summary
Usage and Biological Activities of Arnebia Forssk. species
The genus Arnebia, which belongs to Boraginaceae is represented
by 5 species in Turkey as well as one of them is an endemic plant. The
roots of this plant were known as “eyilik otu, kırmızı kök, eğnik, enlik,
havaciva” in Turkey. In our study; antitumoral, antiinflammatory, prostaglandin synthesis inhibitory, antimicrobial, antiviral, platelet agregation inhibitory, wound healing and contraceptive effects indicated in the
previous studies of Arnebia species and compounds isolated from this
species have been compiled.
Key Words: Arnebia Forssk., Boraginaceae, Biological Activity, Naphthoquinones.
KAYNAKLAR
1. Edmondson, J.R., “Arnebia Forssk.” P. H. Davis (Ed.). Flora of Turkey and the Aegean
Islands, Edinburgh, Edinburgh University Press, (1978), Cilt VI.
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107
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