волумен 60 (1) 2014 / volume 60 (1)

Transcription

волумен 60 (1) 2014 / volume 60 (1) 2014
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Македонско фармацевтско друштво, ул. Маршал Тито 13б/8, Скопје, Македонија
Macedonian Pharmaceutical Association, Marshal Tito 13b/8, Skopje Macedonia
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Macedonian pharmaceutical bulletin, 60 (1) 3 - 8 (2014)
ISSN 1409 - 8695
UDC: 616.314.17-084
Review paper
Therapeutic effects of local drug delivery systems - PerioChip®
in the treatment of periodontal disease
Liljana Bogdanovska1*, Sahmedin Sali2, Mirjana Popovska2, Ilijana Muratovska2,
Aneta Dimitrovska1, Rumenka Petkovska1
Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, University of “Ss. Cyril and
Methodius”, Mother Theresa 47, Skopje, Republic of Macedonia
2
Department of Oral Pathology and Periodontology, Faculty of Dentistry, University of “Ss. Cyril and Methodius”,
Mother Theresa 47, Skopje, Republic of Macedonia
1
Received: January 2014; Accepted: March 2014
Abstract
The primary goal of periodontal treatment is to stop periodontal disease progression and reduce future risks in disease recurrence. In
order to overcome the limitations of the conventional treatment, controlled drug delivery systems for application in periodontal pockets
have been developed. Their use offers several advantages: the therapeutic agent is targeted directly to the disease site and concentrations
are 10-100 folds higher than the concentrations achieved by systemic administration, with low incidence of side effects.
The PerioChip® is as local controlled-release biodegradable delivery system containing chlorhexidine digluconate. Several multicenter
clinical trials have shown that the application of the PerioChip® in periodontal pockets as adjunct to the conventional periodontal treatment
significantly improved the clinical parameters. In this article, the results from controlled clinical studies aimed to evaluate the clinical and
microbiological efficacy of the PerioChip®, are discussed.
Keywords: periodontal disease, local controlled-release delivery systems, PerioChip®
Introduction
Chronic periodontal disease is an infectious disease
characterized by progressive attachment loss, bone loss,
periodontal pocket formation and/or recession of the gingiva which is a result of the host’s response to a periopathogenic bacterial infection (Kalsi et al., 2011). It is well
known that periodontal disease disturbs the integrity of oral
mucous membranes. In this way periodontal pathogens can
enter the circulation causing serious health problems such
as septicemia, organ abscesses, endocarditis, coronary artery disease, cerebrovascular diseases and stroke. Undoubtly, periodontal disease is a major burden and oral health
* lbogdanovska@ff.ukim.edu.mk
is a WHO priority topic in the 21st century (Petersen et al.,
2005; Petersen, 2005).
The main goal of periodontal therapy is to restore the
homeostatic relationship between the periodontal tissue
and its polymicrobial dental-plaque community (Darveau,
2010). This includes elimination of periodontal pathogens, halting disease progression, obtaining host healing
and gain in clinical attachment level. The oldest and most
widely applied periodontal treatment, scaling and root
planning (SRP), consists of mechanical removal of dental plaque, calculus and stain from the roots and crown
of teeth (Tariq et al., 2012). Clinical investigations have
shown that SRP effectively reduces the microbial levels in
periodontal pockets and improves clinical parameters such
as bleeding on probing; probing depths and clinical attach-
4
Liljana Bogdanovska, Sahmedin Sali, Mirjana Popovska, Ilijana Muratovska, Aneta Dimitrovska, Rumenka Petkovska
ment level (Haffajee & Socransky, 2005). However, this
procedure can be often difficult to perform due to the complex and unfavorable root morphology in deep periodontal
pockets, it can be time consuming and unpleasant for the
patient (Obeid et al., 2004). Additionally, SRP is not able
to eliminate the periodontal pathogens present on oral mucous membranes or in deeper periodontal pockets where
instruments are difficult to reach. In this context, it is evident that local or systemic therapy with an antimicrobial
agent would be a valuable adjunct to mechanical therapy
(Horz & Conrads, 2007; Darveau, 2010). Systemic antimicrobial therapy is especially important in the treatment of
severe forms of periodontitis or in cases where the clinical
outcome is potentially compromised by the patient’s systemic health. Although this is an acceptable route of delivery for the patient, the use of systemic antibiotics is limited by the fact that doses needed to achieve therapeutic concentration of antimicrobials in the periodontal environment
might be associated with undesirable side effects such as
gastrointestinal disturbances, hypersensitivity and bacterial resistance (Horz & Conrads, 2007; Paolantonio et al.,
2008a; Krayer et al., 2010).
Local drug delivery systems for the treatment of
periodontal disease
In order to overcome the disadvantages associated
with the administration of systemic antimicrobial therapy,
systems for controlled release of antibiotics and antiseptics
in periodontal pockets have been intensively studied. The
main goal of these systems is to maintain effective drug
concentrations at the site of action for adequate periods of
time, despite the drug loss from gingival crevicular fluid
clearance. Various local periodontal drug delivery systems
have been designed: fibers, strips, films, gels and microparticles made of both biodegradable and non-biodegradable
polymers (Schwach-Abdellaou et al., 2000). According to
the duration of the drug release, they can be divided into
two categories, “sustained release formulations”, which
deliver the agent for less than 24 hours and “controlled delivery devices”, which release the therapeutic agent over
an extended period of time (Srivastava et. al., 2009). Several antimicrobial agents have been tested for their clinical and microbiological efficacy in periodontal treatment.
Basically, we can distinguish two broad categories of antimicrobial agents: antibiotics such as tetracycline, doxycycline, minocycline and metronidazole and antiseptics such
Fig. 1
as chlorhexidine.
The first local drug delivery system in periodontal treatment was ActiciteTM, a hollow nonabsorbable fiber containing tetracycline. Although quite effective, the
placement of this system in the periodontal pockets required additional skills and a second visit to the dentist in
order to be removed. The disadvantages of the non-resorbable systems led to the development of resorbable systems
for antimicrobial delivery. The first resorbable local delivery system was ATRIDOXTM, gel containing 10 % doxycyline hyclate. Improvements in the formulation of local delivery systems resulted in the manufacture of ARESTINTM,
sustained release product containing minocycline hydrochloride and ElyzolTM, 25% metronidazole gel (Krayer et
al., 2010).
Compared to antibiotics, antiseptic agents have much
broader spectrum of antibacterial activity due to multiple intracellular targets which reduce the possibility of
development of bacterial resistance (Slots, 2002). The
most widely used and studied antiseptic in oral diseases is
chlorhexidine (Fig. 1)
Chlorhexidine shows high affinity towards bacteria
because of the interaction between the positively charged
parts of the molecule and the negatively charged phosphate
groups of lipopolysaccaharides on the bacterial cell walls
(Attin et al., 2006). The main advantages of chlorhexidine
application during SRP or surgical periodontal treatment
include improved wound healing and general plaque control (Imfeld, 2006; Horz &Conrads, 2007). However, despite the advantages, the application may result in staining
of the teeth, taste disturbances and increase of calculus formation (Zanatta et al., 2010).
PerioChip® is a controlled-release drug delivery system that delivers chlorhexidine in the periodontal pockets. It is the only locally applied antiseptic approved by
the FDA as adjunct to SRP procedures for the reduction of
probing pocket depth or as a part of a routine periodontal
maintenance treatment (Ryan, 2005). PerioChip® was developed by Dexcel Pharma (Jerusalem, Israel). It contains
2.5 mg chlorhexidine digluconate in a biodegradable matrix of gelatin and glutaraldehide which releases 40 % of
chlorhexidine within the first 24 hours and the rest over
the one week treatment period. While the chip is degraded, chlorhexidine is released gradually for approximately
7-10 days with concentrations above 125 µg mL-1 in gingival crevicular fluid (GCF). When in situ, there is an intial burst in concentration of 2000 µg mL-1 chlorhexidine
in GCF. After 2-4 days the concentration of drug reaches
Chemical structure of chlorhexidine digluconate.
Maced. pharm. bull., 60 (1) 3 - 8 (2014)
Therapeutic effects of local drug delivery systems - PerioChip® in the treatment of periodontal disease
1300 µg mL-1 and then decreases but remains above 125 µg
mL-1 in the first 7 days (Soskolne et al., 1998). Another in
vivo study on 12 patients reported chlorhexidine concentrations of 800-1000 µg mL-1 in GCF in the first 48 hours
after chip placement. This first burst was followed by lower concentrations of 100-500 µg mL-1 in the next 6 days.
Concentrations above the minimal inhibitory concentration
(MIC) were observed for at least 7 days (Schwach-Abdellaou et al., 2000).
Clinical studies performed to assess the effectiveness
of PerioChip® in periodontitis compared two treatment modalities: SRP as monotherapy and SRP+ PerioChip®. They
were designed so that the target sites in experimental group
of patients received PerioChip® whereas the same sites in
the control group SRP only. Patients included in the studies were considered eligible if they had periodontal pockets ≥ 5 mm in depth that bled on probing. The clinical parameters used to evaluate the treatment were the following:
plaque index, bleeding on probing, probing depth and clinical attachment level.
Soskolne et al., evaluated the safety and efficacy of
the PerioChip® in a multicenter study of 118 patients with
moderate forms of periodontitis. After 3 and 6 months, the
selected pockets in the experimental quadrants showed significant reduction in periodontal pocket depth compared to
the pockets treated with SRP only (p<0.0001). Clinical attachment level showed similar, but less marketed improvement over the study period, although the gain was greater
compared to the pocket that received SRP only. One year
later, greater improvements were found in clinical attachment level values in the group with pockets that received
SRP as the only treatment. The probing depth distribution
per patient was similar in both treatment groups. There was
a significant reduction in the number of sites with visible
bleeding on probing, but they did not reach significant difference (Soskolne et al., 1997).
Jeffcoat at al., performed two double-blind, randomized, placebo controlled multi-center clinical trials on
447 patients. Significant reduction of probing depth at 9
months was observed in the PerioChip® group + SRP compared both with control treatment group, SRP alone and
placebo chip + SRP (p < 0.001). A significant improvement
in clinical attachment level was observed at 9 months in the
PerioChip® + SRP treatment group compared to the other
treatment groups (p<0.05). These results in those sites that
received SRP + PerioChip® were in accordance with the results reported by Soskolne et al. There was a statistically significant reduction in bleeding on probing in the control group compared to the test group at 9 months (p<0.05)
(Jeffcoat et al., 1998).
In a study conducted by Heasman et al., the efficacy
of PerioChip® in maintenance patients was evaluated. 26
patients (non-smokers) were enrolled in the study. Important clinical parameters were recorded at baseline and after 1, 3 and 6 months. The study suggests that the application of the PerioChip® is beneficial for patients on mainМакед. фарм. билт., 60 (1) 3 - 8 (2014)
5
tainence therapy although the benefit is not apparent until
six months after placement (Heasman et al., 2001).
In the study by Grisi et al., it was observed that the
gingival recession obtained by SRP + PerioChip® treatment
was greater than the one obtained by SRP alone. That may
be related to greater reduction in gingival margin inflammation, as well as to the mechanical trauma caused by additional applications of the chip after 3 and 6 months. Both
treatment groups presented marketed reduction in the percentage of sites with visible bleeding on probing throughout the study. The pocket depth reduction in the group
treated with SRP + PerioChip® was even greater than the
one observed in the studies performed by Soskolne et al.,
and Jeffcoat et al. The study also reported that there were
no statistically significant differences between the SRP and
SRP + PerioChip® groups for clinical parameters after a
9-month period. This might be due to the lack of the statistical power of the study as a result of the insufficient number of subjects enrolled which might lead to statistical errors. The overall conclusions of this study indicate that application of the PerioChip® as adjunct to SRP may be beneficial in improving clinical periodontal parameters and
may help reduce the risk of disease recurrence during routine supportive periodontal treatment (Grisi et al., 2002).
Salvi et al., reported an immediate effect of PerioChip®
on the total bacterial count, proposing a greater potential
for reduction of bacterial colonization soon after the application of this type of local delivery system. However, Salvi
reported that the dimensions of the PerioChip® may not allow efficacy in the deepest pockets; this may explain why,
in the presence of thorough SRP, the deeper pockets did
not show better results than all of the experimental sites together (Salvi et al., 2002).
The efficacy of PerioChip® on the clinical parameters and on GCF matrix metalloproteinase (MMP)-8 levels in chronic periodontitis patients was evaluated in the
study conducted by Azmak et al. 20 patients with chronic periodontitis were screened for 6 months. In each patient, PerioChip® was inserted into a randomly selected site
following scaling and root planing (SRP + PerioChip®),
while the other selected site received only SRP. Probing
depth, clinical attachment level, plaque index, and papilla bleeding index were recorded at baseline and at 1, 3,
and 6 months. GCF MMP-8 levels were analyzed at baseline; 2 and 10 days; and at 1, 3, and 6 months. At baseline, there were no statistically significant differences in
the mean probing depth, clinical attachment level, papilla bleeding index and plaque index scores between SRP +
PerioChip® and SRP alone groups. At 1, 3, and 6 months,
all clinical parameters in each group significantly decreased (P <0.0167) when compared to baseline. The reduction of probing depth and improvement in attachment
level were higher in the SRP + PerioChip® group compared
to SRP alone at 3 and 6 months. However, the differences between the 2 groups were not statistically significant.
Papilla bleeding and plaque index scores were not signifi-
6
cantly different between SRP+ PerioChip® and SRP alone
groups at any visit. GCF MMP-8 levels were similar in
both groups at baseline. Intragroup analysis showed significant decreases in the GCF MMP-8 level for the SRP+
PerioChip® group between baseline and 1, 3, and 6 months
(p < 0.01). Intergroup analysis demonstrated significantly
lower mean levels of GCF MMP-8 at 1 month in the SRP
+ PerioChip® group compared to the SRP alone group (p <
0.05). These data suggest that PerioChip® application following SRP is beneficial in improving periodontal parameters and reducing GCF MMP- 8 levels for 6 months’ duration. The use of a chairside MMP-8 dipstick periodontitis test might be a useful adjunctive diagnostic tool when
monitoring the course of PerioChip® treatment (Azmak et
al., 2002).
Mizrak et al., conducted a randomized, single blind
study in order to determine the effect of a PerioChip® on
crevicular prostaglandin E2 (PGE2) levels and on the clinical and microbiological parameters of periodontitis when
used as adjunctive therapy to SRP in patients with chronic periodontitis. Clinical indices, microbiological samples
and GCF samples were evaluated at baseline and after 1,
3, and 6 months. Microbiological samples were evaluated
under a light microscope. Significant improvements could
be found for all clinical variables in both groups over the
study period. The mean changes in probing depth obtained
by SRP plus PerioChip® were greater than those obtained
by the SRP alone group at 3 and 6 months. In the test group,
there was also significant gain in clinical attachment level
at 6 months. When data were combined from all groups,
significant reductions in GCF PGE2 levels and number of
microorganisms were noted at all time points. However,
in the test group, reduction was greater at 6 months for
crevicular PGE2 level and at 3 and 6 months for proportions of spirochetes. Based on the findings of this study,
the PerioChip® reduced GCF PGE2 levels and had positive
effects on clinical parameters and subgingival flora when
used as adjunctive therapy to SRP in patients with chronic
periodontitis (Mizrak et al., 2006).
The objective of the study conducted by Rodrigues et
al., was to evaluate the effectiveness of a PerioChip® clinically, in sites still showing signs of disease during periodontal maintenance therapy. Forty-two maintenance nonsmoking patients (previously treated with SRP), presenting at least one probing depth of 5-8 mm, and bleeding on
probing at single-rooted teeth were assigned randomly to
two groups: treated with a PerioChip® (CHIP group) and
treated with SRP (SRP group). Patients were assessed for
plaque index, gingival index, bleeding on probing, probing depth, clinical attachment level and gingival recession
at baseline, 6 weeks, and 3 and 6 months.Both treatments
resulted in improvements in all parameters evaluated. After 6 months, a reduction in pocket depth of 2.64 +/- 0.02
mm and 2.12 +/- 0.02 mm was observed for CHIP and SRP
groups, respectively (p > 0.05). The observed gain in CAL
was 2.19 +/- 0.87 mm and 2.07 +/- 1.53 mm for CHIP and
SRP groups, respectively (p >0.05). In deep pockets, depth
reduction was 3.60 +/- 0.70 mm for CHIP group and 2.83
+/- 0.62 mm for SRP group (p = 0.01). Both treatments
were equally effective in periodontal health reestablishment in inflamed single-root sites of maintenance patients.
However, for deep pockets, the PerioChip® was more effective than SRP in reducing probing depth (Rodrigues et
al., 2007).
A randomized, single-blind, controlled, split mouth
study conducted by Paolantonio et al., investigated the
effects of PerioChip® on the clinical parametars and the
levels of alkaline phosphatase in GCF. The results from
the controlled study confirmed significant reduction of
pocket depth with administration of the PerioChip®; this
treatment yielded significantly lower pocket depth values
at the 6-month evaluation in comparison with SRP alone.
Similar trends were observed for clinical attachment level.
These results suggest that the PerioChip® can be used to
improve clinical results obtained by SRP (Paolantonio et
al., 2008a).
In order to provide with further data on clinical and
microbiological effects of PerioChip® when used as an adjunct to SRP, a multicenter study, by Paolantonio and coworkers, was carried out at four Italian universities. The
main clinical effects of periodontal treatment depend mainly on a reduction in the subgingival bacterial mass. In this
regard, an effective subgingival chemical device is expected to affect the amount and/or the composition of the
subgingival microbiota. In the present study, the SRP +
PerioChip® treatment resulted in total bacterial count that
was significantly lower than that of SRP alone at 15 days
and after 1 month of therapy. In conclusion, the adjunctive
use of PerioChip® with SRP resulted in a clinically meaningful improvement in pocket depth reduction and clinical attachment gain compared to SRP alone. These results
were concomitant with the significantly greater effect that
the SRP + PerioChip®, treatment exerted on the subgingival microbiota compared to SRP alone. These clinical and
microbiological effects were obtained by a single episode
of chip placement inside the periodontal pocket (Paolantonio et al., 2008b).
In a study performed by Machtei et al., the safety and
efficacy of frequent application of the PerioChip® and Flubiprofen Chip® (FBP) in chronic periodontitis patients was
examined. The frequent application of PerioChip® and FBP
Chips resulted in a pocket depth reduction, gain in clinical
attachment level and decrease in bleeding on probing. The
PerioChip® gave greater response compared to FBP chip.
The principal findings of this study would tend to suggest
that if both chips are used consecutively or simultaneously,
it might result in even greater improvement in clinical parameters compared to when each of them is used individually (Machtei et al., 2011).
The efficacy of the PerioChip® for a period of three
months has been analyzed by Puri and coworkers. The
three month interval was chosen because of the effects of
locally delivered chlorhexidine which has been shown effective for eleven weeks and 3 months corresponds to a
Maced. pharm. bull., 60 (1) 3 - 8 (2014)
Therapeutic effects of local drug delivery systems - PerioChip® in the treatment of periodontal disease
typical recall interval for patients after periodontal treatment. In this study, the higher improvement in the clinical parameters was found to be attributed to PerioChip®,
which is known to inhibit microbial proteases from periodontal pathogens, which play a key role in the destruction of periodontal tissues during disease progression. Although, the use of systems for local delivery does not replace the need for SRP, the use of the PerioChip® along
with SRP represents a simple and non-invasive technique
adjunctive to classical treatment. In addition it reduces and
delays the recurrence of periodontal pathogens in periodontal pockets (Puri et al., 2013).
Jagadish et al., evaluated the efficacy of two chlorhexidine containing systems for local delivery, namely EC40
CHX varnish and PerioChip®, over SRP for a period of
three months. The study indicated that the application of
the PerioChip® as adjunct to SRP produced clinically significant reduction of pocket depth, bleeding on probing and
gains in clinical attachment level compared to baseline although the results weren’t statistically significant. However, the small sample size and a single application/insertion
of the PerioChip® could be considered as limitations of this
study (Jagadish et al., 2013).
Overall, PerioChip® application has a well documented safety profile and does not cause any visible staining of
the teeth. The most frequently observed adverse effects in
clinical trials were mild to moderate toothache which resolved spontaneously in most cases and required no further
medical treatment (Ryan, 2005).
Summary
The adjunctive use of controlled-release drug delivery
systems represents a valuable treatment modality which
improves the effects of the conventional periodontal
treatment.
The results from numerous studies have shown that
the application of the PerioChip® results in markedly
suppressed subgingival bacterial flora, with effects evident
up to 11 weeks after administration. The additional benefits
are even more evident when this local drug delivery
system is replaced every 3 months, during the course of
maintenance phase therapy. Therefore, the use of the
PerioChip® as adjunct to SRP represents a simple and noninvasive treatment modality to enhance periodontal health.
Furthermore, the platform of the PerioChip® provides a
technology of delivering other chemotherapeutic agents in
the periodontal pockets.
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Резиме
Терапевтски ефекти од примената на системи со
контролирано ослободување за локална апликација PerioChip® во третманот на пародонталната болест
Лилјана Богдановска1, Сахмедин Сали2, Мирјана Поповска2, Илијана Муратовска2,
Анета Димитровска1, Руменка Петковска1
Институт за применета хемија и фармацевтски анализи, Фармацевтски факултет,
Универзитет „Св. Кирил и Методиј“, Мајка Тереза 47, Скопје, Република Македонија
2
Клиника за болести на уста и пародонт, Стоматолошки факултет, Универзитет „Св. Кирил и Методиј“,
Скопје, Република Македонија
1
Клучни зборови: пародонтопатија, формулации со контролирано ослободување за локална апликација, PerioChip®
Основната цел на третманот на пародонталната болест е да се запре прогресијата на болеста, како и да се
спречи нејзина повторна појава. Со цел да се надминат ограничувањата на конвенционалниот третман, развиени се
формулации со контролирано ослободување наменети за локална апликација во пародонталните џебови. Примената
на овие формулации нуди низа предности: терапевтскиот агенс се ослободува во пародонталниот џеб со што се
постигнуваат концентрации 10-100 пати повисоки од оние кои би се постигнале по системска администрација на
истиот и истовремено значително е намалена појавата на несакани ефекти.
PerioChip® претставува формулација со контролирано ослободување која содржи хлорхексидин диглуконат. Голем
број на мултицентрирани клинички студии покажуваат дека апликацијата на PerioChip® заедно со конвенционалниот
третман резултира со значително подобрување на клиничката слика. Во овој труд ќе биде направен преглед на
резултатите од контролирани клинички студии што ги проценуваат клиничките и микробиолошките ефекти од
примената на PerioChip®.
Maced. pharm. bull., 60 (1) 3 - 8 (2014)
ISSN 1409 - 8695
UDC: 615.322.099
Review paper
Toxicоlogical evaluation of the plant products using Brine
Shrimp (Artemia salina L.) model
Меntor R. Hamidi, Blagica Jovanova, Таtјаnа Kadifkova Panovska*
Faculty of Pharmacy, Institute of Applied Biochemistry, Department of Toxicology, Ss. Cyril and Methodius University,
Majka Tereza 47, Skopje, Republic of Macedonia
Received: February 2014; Accepted: April 2014
Abstract
Many natural products could serve as the starting point in the development of modern medicines because of their numerous biological
and pharmacological activities. However, some of them are known to carry toxicological properties as well. In order to achieve a safe treatment with plant products, numerous research studies have recently been focused on both pharmacology and toxicity of medicinal plants.
Moreover, these studies employed efforts for alternative biological assays. Brine Shrimp Lethality Assay is the most convenient system for
monitoring biological activities of various plant species. This method is very useful for preliminary assessment of toxicity of the plant extracts. Rapidness, simplicity and low requirements are several advantages of this assay. However, several conditions need to be completed,
especially in the means of standardized experimental conditions (temperature, pH of the medium, salinity, aeration and light). The toxicity of herbal extracts using this assay has been determined in a concentration range of 10, 100 and 1000 µg/ml of the examined herbal extract. Most toxicity studies which use the Brine Shrimp Lethality Assay determine the toxicity after 24 hours of exposure to the tested sample. The median lethal concentration (LC50) of the test samples is obtained by a plot of percentage of the dead shrimps against the logarithm
of the sample concentration. LC50 values are estimated using a probit regression analysis and compared with either Meyer’s or Clarkson’s
toxicity criteria. Furthermore, the positive correlation between Meyer’s toxicity scale for Artemia salina and Gosselin, Smith and Hodge’s
toxicity scale for higher animal models confirmed that the Brine Shrimp Lethality Assay is an excellent predictive tool for the toxic potential of plant extracts in humans.
Keywords: brine shrimp lethality assay; toxicity testing; plant extracts; probit analysis; LC50
Introduction
The search for new drugs which are plant-derived has
been receiving renewed interest among researchers throughout the world in view of discovering new drugs that possess
potency to combat the menace of drug resistant pathogenic
microorganisms, antitumor and anticancer agents (Mirzaei
and Mirzaei, 2013; Santos Pimenta et al., 2003).
Plants can be useful either in their crude or advanced
forms, offering a source of drugs in their pure state (Farnsworth and Soejarto, 2009). According to the World Health
* Tel.: +389 2 3126024 ext. 201;
Fax: +389 2 3123054;
e-mail: taka@ff.ukim.edu.mk
Organization’s questionnaire, it is announced that 70-80%
of the population in the world are relying on unconventional medicine, mainly in plant sources, in the primary health
protection (WHO, 2007). Recognized for their ability to
produce a wealth of secondary metabolites, many of these
natural products have been shown to present interesting biological and pharmacological activities, which could serve
as the starting point in the development of modern medicines (Abubakar et al., 2010).
Well-known drugs which were developed from plant
species are Vinblastine and Vincristine (first cures in human
cancer) from Catharanthus roseus, Quinine (anti-malarial agent) from Cinchona species, Scopolamine (sedative)
from Datura metel L., and many others which remained in
use until present day (Farnsworth and Soejarto, 2009).
10
Меntor R. Hamidi, Blagica Jovanova, Таtјаnа Kadifkova Panovska
Although many plants have valuable properties, some
of them are known to carry toxicological properties as well.
Recent studies indicate that although numerous plants are
used as food sources, some of them may have mutagenic or
genotoxic potential (Tülay and Özlem, 2007). Numerous
research studies have recently focused on both pharmacology and toxicity of medicinal plants used by humans. This
is of high importance in order to achieve a safe treatment
with plant products (Parra et al., 2001).
The toxicity of the plants may originate from different
contaminants or from plant chemical compounds that are
part of the plant. Various assays are used for the research
of potential toxicity of herbal extracts based on different
biological models, such as in vivo assays on laboratory animals. However, recent studies employed efforts for alternative biological assays that include species of Artemia salina, Artemia franciscana, Artemia urmiana and Thamnocephalus platyurus. These toxicity tests are considered a
useful tool for preliminary assessment of toxicity (Carballo et al., 2002; Veni and Pushpanathan, 2014; Mayorga et
al., 2010).
During the past 30 years, the Brine Shrimp Assay has
been widely used to test the toxicity of a great variety of
plant products. Brine shrimp (A. salina) is most extensively studied of the Artemia species, estimated to represent
over 90% of the studies in which Artemia is used as an
experimental test organism (Campbell et al., 1994). The
Brine Shrimp Toxicity Assay was proposed and developed
by Michael et al. (1956) and later adapted by Vanhaecke
et al. (1981), Meyer et al., (1982), and Sleet and Brendel
(1983).
Brine Shrimp Lethality Assay (BSLA) has been applied as an alternative bioassay technique to screen the
toxicity of plant extracts (Meyer et al., 1982; McLaughlin et al., 1998a; Moshi et al., 2010; Ogugu et al., 2012;
Gadir, 2012; Solanki and Selvanayagam, 2013; Sharma
et al., 2013), toxicity of heavy metals (Sleet and Brendel,
1985; Martínez et al., 1999) and metal ions (Kokkali et al.,
2011), toxicity of cyanobacteria (Jaki et al., 1999) and algae (Mayorga et al., 2010), cytotoxicity of dental materials (Pelka et al., 2000), toxicity of nanoparticles (MaurerJones et al., 2013), as well as screening of marine natural
products (Carballo et al., 2002).
The technique is economic and utilizes small amount
of test material (Pisutthanan et al., 2004). Since its introduction, this in vivo test has been successively employed
for bioassay-guide fractionation of active cytotoxic and
antitumor agents (Ahmed et al., 2010; Ramachandran et
al., 2011). Additionally, several studies demonstrated that
there is a good correlation between the results for the lethal concentration that kills 50% of the exposed population (LC50) obtained with the Brine Shrimp Lethality Assay using A. salina and the results of the Acute Oral Toxicity Assay in Mice (Parra et al., 2001; Arlsanyolu and Erdemgil, 2006).
Plant collection, storage and preparation of
herbal extracts
Collected plant materials (root, bark, branches, stipules, leaves, flowers, fruits or whole plant) were authenticated by comparing with herbarium specimens. After identification, they were washed and dried before examination (Ogugu et al., 2012; Lalisan et al., 2014), either airdried or dried in an air stove to higher temperature (~40
°C) (Ahmed et al., 2010; Parra et al., 2001; Olowa and Nuñeza, 2013). All were chopped in a grinder mill and stored
in desiccators at room temperature (Parra et al., 2001; Veni
and Pushpanathan, 2014). For antimicrobial tests, the material was pulverized in sterile electric blender (Lalisan et
al., 2014).
The powdered plant sample was soaked in one solvent
or mixture of solvents for a longer period of time, either
by using laboratory extraction flasks or Soxhlet extractor
(Veni and Pushpanathan, 2014), followed by filtration and
evaporation, concentrated to dryness under reduced pressure using rotary evaporator (Mayorga et al., 2010). Several other extraction techniques are repercolation with multiple extractions, maceration of the plant material or decoction of a fresh plant material over a period of time (Parra
et al., 2001). Depending on the nature of the plant material, some steps of the extraction process were optional (for
example, the Soxhlet extraction of plant samples has been
employed in order to extract substances of low and medium volatility as well as thermally stable constituents based
on polarity gradient of the solvent) (Veni and Pushpanathan, 2014).
Choosing the type of solvent for the extraction process is vital for the toxicity testing, because different solvents show different extracting potential. If possible, extraction should be carried out under mild conditions utilizing solvents of low reactivity (Ghisalberti, 1993). Methanol, 96% ethanol, n-hexane, ethyl acetate, petroleum ether,
carbon tetrachloride, dichloromethane and acetone are the
most commonly used extracting solvents for this purpose
(Ahmed et al., 2010; Ogugu et al., 2012).
In case of air or freeze-dried samples, a polar solvent
such as ethyl acetate or methanol is preferable. Alcoholic
solvents rupture cell membranes and extract greater amount
of endocellular materials, and in this case the separation of
pure compounds is left for a later chromatographic separation (Arlsanyolu and Erdemgil, 2006; Ghisalberti, 1993).
If a mixture of solvents is a more suitable option for
extraction of some compounds, then a good system with
high extraction potential is a 1:1 mixture of two solvents,
but the ratio highly depends on the preferred polarity of the
mixture (Pimentel Montanher et al., 2002). In case of fresh
plant materials, dichloromethane-methanol solvent mixture is ideal for extracting purposes. This is usually followed by re-extraction using less polar solvents in order to
separate the lipophilic fraction from the water-soluble fraction (Ghisalberti, 1993).
Maced. pharm. bull., 60 (1) 9 - 18 (2014)
Toxicоlogical evaluation of the plant products using Brine Shrimp (Artemia salina L.) model
11
Studies have shown that there is a significant difference in the obtained LC50 results for different solvents’ extracts, mainly because some solvents are a poor medium
for obtaining specific bioactive components (responsible
for the toxicity) from the plant sample, than others (Lalisan et al., 2014).
Dimethylsulfoxide is widely used solvent for the reconstitution of evaporated plant extracts, because brine
shrimp nauplii show no significant sensitivity to this solvent up to 11% concentration (Ahmed et al., 2013; Musa,
2012; Kamba and Hassan, 2010). Preparation of stock solution and several dilutions (suggested by McLaughlin,
1991) provide different dose levels, which gives opportunity to determine the linear increase of the toxicity by increasing the concentration of the toxic compound(s) present in the plant extract.
Apart from the great number of advantages this method has, several criteria need to be completed in order to
achieve a broad applicability of the Brine Shrimp Assay.
This includes the following:
- Standardized experimental conditions in the
means of temperature, salinity, aeration, light and
pH,
- Same geographical region of the cysts,
- Same age of Artemia nauplii at the start of every test,
- Positive and negative controls are essential part
of the assay, in order to check the sensitivity of
the larvae and the conformity with the standard
procedure (Vanhaecke et al., 1981).
Advantages vs disadvantages of the Brine Shrimp
Assay
The container used for brine shrimp hatching, consists
of two unequal chambers with several holes on the divider in between (Fig.1). This will enable the hatched Artemia
nauplii (Fig.2) to migrate from the hatching compartment
into the illuminated compartment (Sharma et al., 2013;
Pisutthanan et al., 2004). Both chambers of the container are filled with artificial sea water (3.8%) (Gadir, 2012;
Ogugu et al., 2012). In some Brine Shrimp Assays, the
seawater might contain other salts such as MgCl2· 6H2O,
Na2SO4, CaCl2 · 2H2O or CaCl2 · 6H2O (Parra et al., 2001).
After filtration through a 1 µm filter under vacuum, the artificial sea water is ready for hatching of the Artemia cysts
(Vanhaecke et al., 1981).
The container needs to be spacious enough to contain an air pump. Supplying a regular air flow with average pressure and proper light is essential for the hatching
process (Veni and Pushpanathan, 2014; Mirzaei and Mirzaei, 2013). This is important because the artificial seawater
should be at least 90% saturated with oxygen for a successful hatching (Vanhaecke et al., 1981). In addition, equal
distribution of the oxygen in the seawater is achieved by a
constant aeration and pressure in the solution for 48 hours
in order to reach Artemia nauplii mature state.
Small quantities of dry cysts of Artemia salina were
sprinkled into the dark, larger chamber. Yeast solution
0.06% was added to the hatching chamber for every liter of
salt water to feed the larvae after 24 hours, otherwise the
Artemia larvae would die during the III or IV instar stage
(Parra et al., 2001; Sorgeloos et al., 1978). The molting
into the further larval stages is dependent on temperature
(37 °C) as well as quality and quantity of food (Lalisan et
al., 2014). After 36 hours of incubation at room temperature (22-29 °C), the active nauplii free from egg shells
were attracted by the light source into the smaller chamber (Sharma et al., 2013; Pisutthanan et al., 2004). It is important that nauplii from the same generation are applied
for each concentration of the tested compound (Ogugu et
al., 2012).
Brine Shrimp Lethality Assay is a convenient system
for monitoring biological activities of various plant species.
Although this method does not provide any adequate information regarding the mechanism of toxic action, it is a
very useful method for the assessment of the toxic potential
of various plant extracts (Gadir, 2012; Naidu et al., 2014).
This method provides preliminary screening data that can
be backed up by more specific bioassays once the active
compounds have been isolated (Pisutthanan et al., 2004).
This test has several advantages which can be summarized as follows:
- Rapidness (the monitoring could be extended to
maximum 60 hours, but in most cases, relevant
data for the LC50 calculations are obtain after 24
hours of exposure),
- Simplicity (no special training needed for the
equipment and the assay),
- Low requirements (no aseptic techniques, no special equipment, relatively small amount of test
sample is needed and a large number of test organisms of exactly the same age and physiological condition can be easily obtained to start the
test),
- Robustness (the cysts are commercially and readily available so that the tests can be carried out
worldwide with the same original material and
without any problem of provisioning),
- Inexpensiveness (the quantity of cysts required
per test is very small so that the price of the biological material is negligible),
- High degrees of repeatability – according to ARC
– test (Artemia Reference Centre) (Vanhaecke et
al., 1981; Meyer et al., 1982; McLaughlin and
Rogers, 1998b; Vanhaecke and Persoone, 1984).
Макед. фарм. билт., 60 (1) 9 - 18 (2014)
Hatching of Artemia cysts
12
Fig. 1 Container used for hatching Artemia nauplii (Pellosi et al., 2013).
Fig. 2 Artemia salina nauplius (by Juan Camilo Jaramillo).
Influence of the experimental conditions over the
sensitivity of Artemia nauplii
The pH value of the seawater is a very important factor for the hatching of the Artemia eggs. Optimal pH range
is 8.0 ± 0.5. If necessary, the pH should be adjusted using
NaOH or Na2CO3, to avoid lethality of the Artemia larvae
caused by decrease of pH during incubation (Vanhaecke et
al., 1981; Parra et al., 2001).
Temperature variations affect the hatching process of
Artemia cysts. Warmer medium temperature enables faster
hatching of the cysts (Table 1).
It has been shown that 50% of the cysts hatch within
30 hours at 20 °C, while at 24 °C this percentage is already
obtained after 21 hours. With regard to the molting rate,
it appears that at 24 °C more than 60% of the larvae molt
into the II instar stage 16 hours after hatching, whereas at
20 °C it takes 35 hours to arrive at the same stage (Sorgeloos et al., 1978).
The light stimulus influences the hatching of Artemia
cysts significantly. Sorgeloos explained how the embryological development of hydrated embryos that are not stimulated by light, can be delayed until the light trigger is applied. Apparently, the cysts started hatching after being exposed to light, which gave statistically significant differences between the dark and the light series (Sorgeloos,
1973).
Additionally, Vanhaecke et al. (1981) indicated that
the most sensitive age for the majority of the tested compounds were the 48 hour-old nauplii at the stage of instar
II-III.
Maced. pharm. bull., 60 (1) 9 - 18 (2014)
13
Table 1. Percentage of hatched and molted cysts depending on the temperature of the medium*
Temperature of the
medium
20 °C
24 °C
Percentage of
hatched cysts
50%
50%
Temperature of the
medium
Percentage of molted
larvae
20 °C
24 °C
~60%
>60%
Hatching time
Developmental stage reached
30 hours
21 hours
Hatching time
+
molting time
30 hours+35 hours
21 hours+16 hours
I (first) stage
I (first) stage
Developmental stage reached
II (second) stage
II (second) stage
*Based on the results in the research paper of Sorgeloos et al. (1978)
Furthermore, the larvae of I instar stage only consume
their yolk as a food source and they are more resistant to
positive control (chromic acid) due to their poorly developed epithelium of the digestive tract that disables the normal absorption of nutrients and toxic compounds from the
external medium (Sorgeloos et al., 1978; Vanhacke and
Persoone, 1984; Sleet and Brendel, 1985; Kokkali et al.,
2011).
Therefore, even under given constant experimental
temperature, salinity, light, pressure and pH conditions,
the sensitivity of Artemia nauplii in different instar developmental stages differs markedly among different strains
from different geographical regions.
Preparation of the Brine Shrimp Assay
The Brine Shrimp Lethality Assay has been developed
for toxicity testing of various concentrations of pure compounds and crude plant extracts. Firstly, pure compounds
and crude plant extracts were dissolved in a solvent suitable for the tested herbal sample. However, a mixture of
several solvents may be a more suitable solvent system for
some plant samples.
By dissolving the plant extract in a convenient solvent,
a stock solution is made, which can be used for serial dilutions to prepare different concentrations. This is useful in
the means of toxicity testing, because it is of high importance to determine the concentration range in which there
is a linear correlation between the concentration and the lethality of the brine shrimps. Most experiments that involve
the Brine Shrimp Lethality Assay for toxicity assessment
of herbal extracts include a concentration range of 10, 100
and 1000 µg/ml (Parra et al., 2001).
The results for the toxicity of tested herbal preparations gained by using crude plant extracts were more accurate than by testing pure compounds isolated from the
same plant. This difference in the toxicity results is probably due to the chemical complexity of the crude or partially purified extract, which seemed to be essential for the
Макед. фарм. билт., 60 (1) 9 - 18 (2014)
bioavailability of the active constituents of the examined
plant. On the other hand, isolated pure compounds seemed
to lose this specific bioactivity (Mayorga et al., 2010).
Prepared crude plant extracts in various concentrations
can be tested by applying certain volume of the extract in
vials containing brine solution or by applying the tested
volume of extract on filter paper discs which are placed
in vials afterwards (Meyer et al., 1982). Each vial contains the tested crude plant extract, artificial sea water and
10-15 brine shrimp nauplii. Ten nauplii were selected and
transferred into each sample vial, and the final volume in
each vial is adjusted with artificial seawater (Gadir, 2012).
If there is no possibility to apply exact number of brine
shrimps, all remaining alive nauplii are immobilized and
the calculations could be made by counting the total number of dead shrimps in each vial (Pisutthanan et al., 2004;
Ghisalberti, 1993).
Adding dry yeast suspension as food for the brine
shrimps is optional (Gadir, 2012), because feeding the
brine shrimps during the assay is insignificant when it
comes to determining toxicity.
The negative control enables elimination of other factors that contribute to the total number of dead nauplii. The
solvent used to dissolve the crude plant extracts is a relevant negative control for this purpose.
The toxicity of tested plant samples was determined
by comparing their LC50 values with highly toxic substances suitable to be used as positive controls for this test, such
as: vincristine sulphate (Ahmed et al., 2010), potassium dichromate (Naidu et al., 2014), thymol (Sharma et al., 2013;
Mirzaei and Mirzaei, 2013), cyclophosphamide (Moshi et
al., 2010), pure DMSO (Arlsanyolu and Erdemgil, 2006),
caffeine (Gadir, 2012) etc.
Data analysis
Depending on the conditions of the experiment, survivors have been counted by magnifying glass or a microscope after certain period of time (6, 12, 24, 36, 48, 54 and/
14
or 60 hours), although most toxicity studies which use the
Brine Shrimp Assay determined the toxicity by counting
the survived nauplii after 24 hours of exposure to the tested sample. Usually, no deaths were observed to occur in
the negative control after 24 hours.
After 24 hours of exposure, the median lethal concentration (LC50) of the test samples has been obtained by a
plot of percentage of the shrimps killed against the logarithm of the sample concentration (Meyer et al., 1982;
Ahmed et al., 2010; Moshi et al., 2010). LC50 values were
estimated using a probit regression analysis (Finney,
1971). Finney’s statistical method (Table 2) was incorporated in several software packages such as Stata, MatLab,
R and IBM SPSS, which enable computerized calculations
of LC50 with confidence intervals (Fig.3). These calculations may not give the exact lethal concentration of the examined compound or extract that kills 50% of the population, but without doubt it represents a significant prelimi-
nary data for further toxicity testing assays. In most studies, the experiment has been conducted in triplicate, so that
gained results were statistically reproducible (Sharma et
al., 2013; Pisutthanan et al., 2004; Parra et al., 2001).
Toxicity testing criteria
The toxicity of herbal extracts expressed as LC50 values is commonly valorized either by comparison to Meyer’s or to Clarkson’s toxicity index.
According to Meyer’s toxicity index, extracts with
LC50 < 1000 µg/ml are considered as toxic, while extracts
with LC50 > 1000 µg/ml are considered as non-toxic (Meyer et al., 1982).
Clarkson’s toxicity criterion for the toxicity assessment
of plant extracts classifies extracts in the following order:
extracts with LC50 above 1000 μg/ml are non-toxic, LC50 of
Table 2. Finney’s table for transformation of percentage of mortality to probit values (Finney, 1952).
%
0
10
20
30
40
50
60
70
80
90
__
99
0
__
3.72
4.16
4.48
4.75
5.00
5.25
5.52
5.84
6.28
0.0
7.33
1
2.67
3.77
4.19
4.50
4.77
5.03
5.28
5.55
5.88
6.34
0.1
7.37
2
2.95
3.82
4.23
4.53
4.80
5.05
5.31
5.58
5.92
6.41
0.2
7.41
3
3.12
3.87
4.26
4.56
4.82
5.08
5.33
5.61
5.95
6.48
0.3
7.46
4
3.25
3.92
4.25
4.59
4.85
5.10
5.36
5.64
5.99
6.55
0.4
7.51
5
3.36
3.96
4.33
4.61
4.87
5.13
5.39
5.67
6.04
6.64
0.5
7.58
6
3.45
4.01
4.36
4.64
4.90
5.15
5.41
5.71
6.08
6.75
0.6
7.65
7
3.52
4.05
4.39
4.67
4.92
5.18
5.44
5.74
6.13
6.88
0.7
7.75
8
3.59
4.08
4.42
4.69
4.95
5.20
5.47
5.77
6.18
7.05
0.8
7.88
9
3.66
4.12
4.45
4.72
4.97
5.23
5.50
5.81
6.23
7.33
0.9
8.09
Fig. 3 Obtaining the logarithmic value of the concentration by interpolation from the linear correlation between probits
and log(c) (Saha et al., 2014).
Maced. pharm. bull., 60 (1) 9 - 18 (2014)
500 - 1000 µg/ml are low toxic, extracts with LC50 of 100 500 μg/ml are medium toxic, while extracts with LC50 of 0 100 µg/ml are highly toxic (Clarkson et al., 2004).
Correlation between Brine Shrimp Assay and
other animal models
Due to the ethical issues in toxicological tests, substituting animals with alternative models is very important.
The effectiveness of the Artemia salina bioassay for predicting the toxicity of plant extracts was evaluated by comparing the LC50 results for the brine shrimps with the LD50
results for acute toxicity in rats and mice (Sharma et al.,
2013; Naidu et al., 2014; Parra et al., 2001).
Syahmi et al. have shown that LC50 values obtained
by Brine Shrimp Lethality Assay are in good correlation
15
with LD50 values obtained by examination of the acute toxicity of E. guineensis methanolic extracts using laboratory Swiss albino mice (Syahmi et al, 2010). Furthermore,
using Sprague Dawley rats showed that LD50 values for
the acute toxicity examined by oral route of Mentha spicata methanolic extract are significantly correlated with the
LC50 results obtained by the Brine Shrimp Lethality Assay
(Naidu et al., 2014). The LD50 values for the mice and rats
models were above 5000 mg/kg, and the LC50 values for
the brine shrimps were above 1000 µg/ml, indicating that
these extracts are non-toxic.
It has been shown that a good correlation exists between
the LC50 results obtained by force feeding female Sprague
Dawley rats with a feeding needle of aqueous extracts of
various plant species and the LC50 results obtained by 24
hour exposure of brine shrimps to those aqueous extracts.
The LC50 results for the brine shrimps were above 5 mg/ml,
Table 3. LC50 versus LD50 values in Brine Shrimp Lethality Assay and in vivo study - Swiss albino mice; toxicity classification according to Meyer’s and Gosselin, Smith and Hodge’s criteria
Plant extract
Aloe vera (L.) Burm.
Artemisia absinthium L.
Citrus aurantium L.
Cymbopogon citratus (DC. Ex Nees)
Stapf
Datura stramonium L.
Justicia pectoralis Jacq.
Musa x paradisiaca L.
Ocimum basilicum L.
Ocimum gratissimum L.
Ocimum tenuiflorum L.
Orthosiphon aristatus (Blume) Miq.
Pimenta dioica (L.) Merr.
Piper auritum Kunth
Plantago major L.
Plectranthus amboinicus (Lour.)
Spreng.
Plectranthus amboinicus (Lour.)
Spreng. Aqueous extract
Ruta graveolens L.
Senna alata (L.) Roxb.
Stachytarpheta jamaicensis (L.) Valh
Thuja occidentalis L.
LC50 (Mg/ml) obtained
by Brine Shrimp Lethality Assay
3.59
15.74
3.99
LD50 (mg/kg) obtained by
using Swiss albino mice
as experimental models
120.65
2499.10
476.94
Toxic/Very toxic
Toxic/Moderately toxic
Toxic/Very toxic
9.83
460.00
12.86
60.14
15.10
9.92
18.76
18.75
16.72
32.78
26.67
4.74
821.93
3531.11
383.97
956.50
2081.00
1540.00
5026.31
2560.00
1802.00
182.54
Toxic/Very toxic
Toxic/Slightly toxic
Toxic/Very toxic
52.29
4902.92
82.27
8193.00
Toxic/Slightly toxic
5.39
7.74
14.51
11.94
219.45
1459.32
2035.12
440.00
Toxic/Very toxic
Toxic/Very toxic
*according to Meyer’s toxicity index for BSLA (Meyer et al., 1982)
**according to Gosselin, Smith and Hodge scale (Gosselin et al., 1984)
Макед. фарм. билт., 60 (1) 9 - 18 (2014)
Toxicity class
Toxic/Very toxic
16
and the results obtained for the acute oral toxicity in laboratory rats also indicated no evidence of toxicity. The results have shown that in both the brine shrimp and the laboratory animals there were no signs of acute toxicity (Shafii
et al., 2011).
The cytotoxic effect of the bioactive compound Quassin present in different fractions of Quassia amara has been
studied using Brine Shrimp Lethality Assay and experimental rats. From the examined fractions of this plant, the
methanol fraction has shown the least toxic activity in brine
shrimps, as well as in experimental rats which all survived
after dosage with single limit test dose of 2000 mg/kg and
5000 mg/kg (Obembe et al., 2014).
A cytotoxicity study using Brine Shrimp Lethality Assay and Acute Oral Toxicity in Mice for the toxicity testing
of Swietenia mahagoni (Linn.) seed methanol extract has
shown that the plant is nontoxic. The LC50 results obtained
for this plant extract using the Brine Shrimp Lethality Assay
correspond to LD50 values between 2500 and 8000 mg/kg
indicating that according to the Brine shrimp Lethality Assay it is nontoxic. The correlation between the LC50 results
obtained by the BSLA and the LD50 results obtained in mice
has been confirmed with the obtained LD50 values for mice
above 5000 mg/kg, meaning the plant is nontoxic for mice
as well (Sahgal et al., 2010).
Parra et al. 2001 examined the toxicity effects of 20
plant extracts from different species using the Brine Shrimp
Lethality Assay (BSLA) and in vivo studies with rats (Table 3).
The positive correlation between LC50 results obtained
by the BSLA and LD50 results for in vivo animal models
is confirmed by comparing the toxicity class of each plant
extract according to Meyer’s versus Gosselin, Smith and
Hodge’s toxicity scales (Table 3). The predicted oral LD50
dose for humans in Gosselin, Smith and Hodge’s toxicity
scale is based on the oral LD50 doses for rats as follows: less
than 5 mg/kg is considered super toxic, 5-50 mg/kg is extremely toxic, 50-500 mg/kg is very toxic, 500-5000 mg/
kg is moderately toxic, 5000-15000 mg/kg is slightly toxic
and above 15000 mg/kg is considered practically non-toxic
(Gosselin et al., 1984).
This comparison confirmed that the Brine Shrimp Lethality Assay is an adequate method for preliminary toxicity
testing in humans by using the brine shrimps as alternative
model for the mice and rats in vivo models.
Conclusion
Many plant species possess pharmacologically active
constituents which contribute to their wide use in folk medicine and in the design of drugs. On the other hand, many
plants which have been described as being curative can also
be associated with harmful effects. The concentration of a
substance is the most important determinant of the outcome:
if it reaches a sufficiently high concentration in the susceptible biological system, it could lead to toxic effects.
In order to gain relevant data which can be extrapolated to human population, it was of high need that in vivo testing using animal models was involved. For years, researchers have been using rats and other animal models that show
a high correlation with the human population. Recently, this
type of testing has been limited because of ethical and economic aspects. Therefore, alternative toxicity assays are
widely used for the testing of the toxicity potential of plant
products. Brine Shrimp Lethality Assay seemed like an appropriate solution, especially because it could still classify
as in vivo testing. Artemia salina nauplii is one of the alternatives for the biological toxicity assays of herbal extracts,
and this test turned out to be significantly correlated with
several other animal models. The preliminary toxicity data
obtained by conducting the Brine Shrimp Lethality Assay
gives LC50 values which are a convenient platform for further toxicity studies.
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Резиме
Токсиколошка евалуација на растителни производи со
примена на модел со солени ракчиња (Artemia salina L.)
Ментор Р. Хамиди, Благица Јованова, Татјана Кадифкова Пановска*
Фармацевтски факултет, Институт за применета биохемија, Катедра за токсикологија,
Универзитет „Св. Кирил и Методиј“, Мајка Тереза 47, 1000 Скопје, Република Македонија
Клучни зборови: тест за леталност на солени ракчиња, тестирање на токсичност, растителни екстракти, пробит анализа, LC50
Природните производи наоѓаат голема примена во развојот на нови лекови, поради бројните биолошки и
фармаколошки својства кои ги поседуваат. Од друга страна, тие може да поседуваат и токсиколошки својства. Затоа,
во терапијата со овие производи, податоци за безбедност се обезбедуваат преку бројни студии чија што главна цел
на истражување се однесува на испитување на фармаколошките и на токсиколошките карактеристики. Овие студии
истовремено се насочени и кон пронаоѓање на алтернативни биолошки тестови, од кои Тестот за леталност со солени
ракчиња (Brine Shrimp Lethality Assay) се покажал како најсоодветен за оваа цел.
Овој метод дава можност за прелиминарна проценка на токсичноста на растителните екстракти, така што има
многубројни предности, од кои што брзината, едноставноста и минималните услови за изведување на тестот се само
дел од нив. Сепак, за да биде соодветна неговата примена во токсиколошки цели, потребна е стандардизација на
експерименталните услови (температура, pH и концентрација на сол на медиумот, аерација и светлина).
Одредувањето на токсичноста на хербални екстракти со примена на овој метод е во концентрациски опсег од 10,
100 и 1000 µg/ml. Смртноста на ракчињата се определува најчесто после 24 часа експозиција, преку пресметување
на средната летална концентрација (LC50). Средната летална концентрација (LC50) на испитуваните примероци
се пресметува преку графички приказ на зависноста меѓу процентот на мртви ракчиња наспроти логаритамските
вредности од соодветните концентрации на испитуваниот примерок и примена на пробит регресиона анализа.
Потоа овие вредности се споредуваат со Мејер-овиот или Кларксон-овиот критериум за токсичност. Позитивната
корелација помеѓу Мејеровата скала за токсичност на A. salina и Госелин, Смит, Хоџовата скала за токсичност за
виши животински модели потврдува дека Тестот за леталност со солени ракчиња претставува одлична алатка за
предвидување на токсичниот потенцијал на растителните екстракти кај луѓето.
Maced. pharm. bull., 60 (1) 9 - 18 (2014)
ISSN 1409 - 8695
UDC: 615.015
Original scientific paper
Statistical approach for selection of regression model during
validation of bioanalytical method
Natalija Nakov*, Jasmina Tonic-Ribarska, Aneta Dimitrovska, Rumenka Petkovska
Institute of Applied Chemistry and Pharmaceutical Analysis, Faculty of Pharmacy, University of “Ss Cyril and Methodius”,
Majka Tereza 47, 1000 Skopje, Republic of Macedonia
Received: May 2014; Accepted: June 2014
Abstract
The selection of an adequate regression model is the basis for obtaining accurate and reproducible results during the bionalytical
method validation. Given the wide concentration range, frequently present in bioanalytical assays, heteroscedasticity of the data may be
expected. Several weighted linear and quadratic regression models were evaluated during the selection of the adequate curve fit using nonparametric statistical tests: One sample rank test and Wilcoxon signed rank test for two independent groups of samples. The results obtained
with One sample rank test could not give statistical justification for the selection of linear vs. quadratic regression models because slight
differences between the error (presented through the relative residuals) were obtained. Estimation of the significance of the differences in the
RR was achieved using Wilcoxon signed rank test, where linear and quadratic regression models were treated as two independent groups.
The application of this simple non-parametric statistical test provides statistical confirmation of the choice of an adequate regression model.
Keywords: regression model, bioanalytical method, non-parametric statistical test, validation
Introduction
The quality of the bioanalytical data is highly dependent on the appropriateness of the regression model applied for quantitative analysis. The selection of an adequate
regression model is the basis for accurate and reproducible
quantification over the whole concentration range, whereas the inappropriate regression model presents a source of
error and imprecision of the bioanalytical method (Rozet et
al., 2011). The Ordinary Least Squares (OLS), which belong to the group of linear regression models is the most
commonly used regression model for narrow range of concentration, where homoscedasticity of the data is presupposed (constant variance over the whole concentration
range) (Singtoroj et al., 2006; Peters et al., 2007)
Most bioanalytical methods for estimation of drug concentration in all pharmacokinetic phases, usually apply wide
* phone: +389 2 3126032; fax: + 389 2 3123054
email: natalijan@ff.ukim.edu.mk
concentration range. When the concentration range is broad,
heteroscedasticity of the data is expected (variance increases
with increasing concentration). The application of the OLS
model on heteroscedastic data will generate inaccurate analytical results, especially in low concentrations. One way to
handle heteroscedastic data is to apply weighted least square
(WLS) regression on OLS. The principle of weighting is to
give more importance to the data points with low variance
and less importance to data points of high variance. The selection of adequate WLS model will balance the regression line to generate an evently distributed error throughout the calibration range (Kimanani E.K, 1998; Almeida et
al., 2002; Singtoroj et al., 2006; Tellinghuisen, 2008). Most
commonly used weights in bioassays are 1/x, 1/x2, 1/y and
1/y2. Another approach to linearise non-linear data and reducing heteroscedasticity is the use of quadratic regression
model. This model is the simplest form of polynominal regression and the same as with the linear regression, could be
weighted or not (Singtoroj et al., 2006, Hubert et al., 2007).
Considering the drug regulatory agencies request “The
20
Natalija Nakov, Jasmina Tonic-Ribarska, Aneta Dimitrovska, Rumenka Petkovska
simplest model that adequately describes the concentration-response relationship should be used” (EMA 2011,
FDA 2001), the question is how to select the adequate regression model. The statistic criteria such as coefficient of determination (R2) is only informative and not relevant, since different regression models give similar values for R2 and in addition acceptable R2 values may be obtained despite the impaired accuracy (Hartmann et al., 1998; Castillo & Castells,
2001; De Souza & Junqueira, 2005; Stockl et al., 2009). The
approach based on comparison of the sum of relative residuals (SRR) obtained for each regression model is reported by
several authors (Lang & Bolton, 1991; Wieling et al., 1996;
Almeida et al., 2002). According this approach the regression model with lowest SRR should be chosen as adequate.
Singtoroj et al (Singtoroj et al., 2006) proposed a strategy
based on using non-parametric test of ranks for evaluation of
regression models in terms of SRR obtained from calibration
curve fit and calibration curve predictability.
An issue that arises is whether these approaches enable selection of the simplest regression model, especially
in cases when slight difference between the SRR are obtained. The objective of our work is to present a statistical approach for selection of an adequate regression model
during the bioanalytical method validation. The proposed
approach is based on two non-parametric statistical tests:
One sample rank test and Wilcoxon signed rank test for
two independent groups of samples.
Experimental
Sample preparation and chromatographic conditions
The data that were statistically evaluated were derived
from the validation of the HPLC-MS/MS method for ibuprofen (IBP) quantification in human plasma. Briefly, the
calibration standards (CS) and quality control (QC) samples were prepared by spiking 50 µl working standard solutions of rac-IBP with 950 µl blank human plasma. The plasma samples, containing ketoprofen as an internal standard
(IS), were extracted using LLE procedure. The analysis was
conducted on TSQ Quantum Discovery Max triple quadrupole mass spectrometer (Thermo Scientific, USA). Chromatographic separation was achieved on Lux Cellulose 3
chromatographic column (Phenomenex, 250 x 4.6 mm) using 0.1 % (v/v) acetic acid in mixture of methanol/water
(90:10, v/v) as a mobile phase. Analyses were conducted
at a flow rate of 0.6 mL/min and the injection volume was
10 µl. The mass spectrometer was operated in the selected
reaction monitoring (SRM) mode using negative electrospay ionization. Ibuprofen and ketoprofen were quantified
in selected reaction monitoring (SRM) using the transition
of m/z 205.1→161.3 and m/z 253.2→209.2, respectively.
Data analysis
Six-point calibration curve was constructed from the
peak area ratio (peak area IBP / peak area IS) vs. concen-
tration in a range from 0.1 - 50 mg/L, over three days. QC
samples at concentrations of 0.1mg/L (LLOQ); 0.2 mg/L
(LQC), 20mg/L (MQC) and 40 mg/L (HQC) in five replicates for each QC level, were analyzed along with the
calibration curves. Five linear and five quadratic regression models were evaluated during the selection of the adequate curve fit.
The CS and QC data were fitted on OLS, weighted linear (1/x, 1/x2, 1/y and 1/y2) and on quadratic models
(non-weighted and weighted) using calibration curve options in MS data system (XcaliburTM Data system, Thermo Scientific, USA). The relative residuals (RR) were calculated based on back-calculated concentration obtained
from each regression model and nominal concentration
(Equation 1).
Eq. 1
The SRR was computed from the average RR (%)
obtained from each calibration level and every QC level
(Equation 2).
Eq. 2
The test of homoscedasticity was carried out using
F-test and the obtained experimental value was compared
with tabled one, for level of significance 0.05.
The non-parametric statistical tests (One sample rank
test and Wilcoxon signed rank test for two independent
groups of samples) were calculated using Windows Excel
(Microsoft Corporation).
Results and discussion
The range of the calibration curve for the bioanalytical methods should be established to allow adequate description of the pharmacokinetics of the analyte of interest
(EMA 2011). Therefore before defining the concentration
range, it is essential to have information about the expected
concentrations of the analyte in biological matrix. Considering that different dosage strength (200 mg, 400 mg, 800
mg etc) of IBP are available on the market and the information obtained from the literature data (Canaparo et al.,
2000; Bonato et al. 2003; Szeitz et al., 2010), a 500-fold
concentration range was required (0.1 - 50 mg/L).
The data of CS and QC samples obtained during the validation process were fitted to the OLS (linear
non-weighted) regression model, as this model is the starting point for the selection of the adequate calibration curve.
Given the wide concentration range, heteroscedasticity of
the data was expected. In order to confirm this expectation,
a test for homoscedasticity was carried out. The F-test revealed that the variance is not evenly distributed over the
Maced. pharm. bull., 60 (1) 19 - 25 (2014)
Statistical approach for selection of regression model during validation of bioanalytical method
21
Fig. 1 Residuals of the calibration standards plotted against concentration
whole concentration range, since the experimental F-value
(F=75.4) was significantly higher than the tabled one (Ftab
=9.28). The heteroscedasticity of the data was further confirmed from the RR of the CS samples obtained from three
days calibration curve (Fig.1). The RR obtained from the
lowest concentration was 930% compared with 5% RR for
the highest concentration. The RR data clearly showed that
fitting the linear non-weighted model generates inaccurate
results especially in the lower concentration range, indicating the need of regression models different from the linear non-weighted. Therefore four linear WLS and five quadratic models (one non-weight and four weighted) were further constructed on the same data set.
The calculated regression models were evaluated according the traditional approach based on the SRR
(Almeida et al., 2002) with slight modification referred to
the calculation of SRR. In our investigation the SRR calculation was based on errors obtained from the CS and the
QC samples, whereas in the literature data the SRR was
based on the errors obtained just from the CS. The SRR
generated from each regression model is present in Table
1. The SRR obtained using quadratic models (non-weight
and weighted) were lower than the SRR obtained from the
linear models. In addition all quadratic models generate
same percent of error (presented through SRR), which is
not the case for linear models where large difference between the errors were observed. The OLS and weighted
1/x and 1/y linear regression models generate large error,
whereas 1/x2 and 1/y2 weighted linear models gave similar error as the corresponding quadratic weighted models.
Considering that quadratic models are more complex than
weighted linear, additional investigation should be carried
out before selecting the quadratic models as an adequate
regression model.
Statistical tests were applied for evaluation of the significance of the difference between the errors obtained with
quadratic and linear 1/x2 and 1/y2 models. The use of statistical approach for the selection of the adequate regression
model is important, since the selection of more complex
regression model should be justified. The proposed statisti-
Table 1. Data obtained using traditional SRR approach
Regression model
Linear non-weighted (OLS)
Linear 1/x
Linear 1/x2
Linear 1/y
Linear 1/y2
Quadratic
non-weighted
Quadratic 1/x
Quadratic 1/x2
Quadratic 1/y
Quadratic 1/y2
Макед. фарм. билт., 60 (1) 19 - 25 (2014)
SRR (%) for
CS
SRR (%) for
QC samples
Total SRR (%)
1097.92
78.08
31.14
86.07
40.78
1406.64
65.88
18.77
80.40
22.73
2504.56
143.96
49.91
166.47
63.50
26.52
21.23
47.75
7.36
6.88
6.64
6.70
40.74
40.74
38.45
41.17
48.11
47.62
45.09
47.87
0.1 mg/L
RR (%) Rank
922.55
10
24.51
8
1.31
5
32.35
9
2.29
6
15.13
7
0.98
3.5
0.65
1.5
0.98
3.5
0.65
1.5
0.635 mg/L
RR (%) Rank
147.40
10
11.65
9
7.30
6
10.50
8
9.61
7
6.30
5
0.89
4
0.26
1
0.58
3
0.37
2
6.35 mg/L
RR (%) Rank
8.52
6
17.99
9
9.73
7
18.83
10
11.04
8
2.87
5
2.71
4
1.93
1
2.56
3
2.05
2
12.5 mg/L
RR (%) Rank
9.22
8
10.78
9
2.14
6
11.43
10
3.85
7
1.55
3
1.33
1
1.90
5
1.37
2
1.81
4
1
3
2
6.5
6.5
4.5
4.5
-3.77
-50.94
-10.38
-6.92
17.81
17.81
17.50
17.50
Linear 1/x2
Linear 1/y
Linear 1/y2
Quad. nonweighted
Quad.1/x
2
Quad.1/y
2
Quad.1/y
Quad.1/x
2.50
8
9
3.27
10
Reg.model
Lin. nonweighted -930.19
(OLS)
Linear 1/x -44.34
0.54
0.54
0.00
0.00
0.54
1.63
2.50
6.60
Prec.
(SD%)
Acc.
(%)
LLOQ
Rank
QC sample
4
4
1.5
1.5
4
6
7.5
9
7.5
10
Rank
-8.96
-6.29
-8.33
-8.33
-1.89
-3.30
-12.58
-2.67
-6.76
-452.36
Acc.
(%)
8
4
6.5
6.5
1
3
9
2
5
10
Rank
2.95
1.91
1.91
1.91
2.16
2.16
2.33
0.98
2.37
4.97
Prec.
(SD%)
LQC
9
3
3
3
5.5
5.5
7
1
8
10
Rank
4.54
4.51
4.46
4.48
4.28
4.99
14.05
3.68
12.95
14.80
Acc.
(%)
6
5
3
4
2
7
9
1
8
10
Rank
3.65
3.65
3.65
3.65
3.65
3.22
3.50
3.00
3.47
3.66
Prec.
(SD%)
MQC
7
7
7
7
7
2
4
1
3
10
Rank
8.17
8.15
8.14
8.12
8.14
-4.05
2.83
-6.13
1.83
5.29
Acc.
(%)
10
9
7.5
6
7.5
3
2
5
1
4
Rank
25.4 mg/L
RR (%) Rank
7.64
10
5.41
8
2.77
7
6.08
9
1.31
6
0.30
1
1.14
4
0.36
2
0.93
3
1.22
5
Table 3. One sample rank test - Ranking of the regression models based on the accuracy and precision of the QC samples
Conc.
Reg.model
Linear 1/x
Linear 1/x2
Linear 1/y
Linear 1/y2
Quadratic non-weighted
Quad.1/x
Quad.1/x2
Quad.1/y
Quad.1/y2
Table 2. One sample rank test - ranking of the regression models based on the RR(%) obtained for the CS
2.83
2.83
2.84
2.83
2.85
1.92
1.91
2.33
1.89
2.00
Prec.
(SD%)
HQC
7
7
9
7
10
3
2
5
1
4
Rank
50.8 mg/L
RR (%) Rank
2.59
6
7.72
8
7.90
9
6.87
7
12.69
10
0.08
1
0.31
3
0.83
5
0.22
2
0.59
4
55.5
43.5
44
41.5
39
32.5
49.5
25
41.5
68
Rank
sum
Rank
sum
50
51
40
53
44
22
19.5
15.5
16.5
18.5
22
Maced. pharm. bull., 60 (1) 19 - 25 (2014)
23
Table 4. One sample rank test - Final ranking of the regression models
Regression
model
Rank sum
from CS
Rank sum from
QC samples
Rank sum
Final rank
50
51
40
53
44
22
19.5
15.5
16.5
18.5
68
41.5
25
49.5
32.5
39
41.5
44
43.5
55.5
118
92.5
65
102.5
76.5
61
61
59.5
60
74
10
8
5
9
7
3.5
3.5
1
2
6
Linear 1/x
Linear 1/x2
Linear 1/y
Linear 1/y2
Quad. non-weighted (OLS)
Quad.1/x
Quad.1/x2
Quad.1/y
Quad.1/y2
Table 5. Data obtained using Wilcoxon sign rank test
Concentration
RR
Linear weighted
0.1
1.31
2.29
7.30
9.61
9.73
11.04
2.14
3.85
2.77
1.31
7.90
12.69
3.77
10.38
2.67
3.30
3.68
4.99
6.13
4.05
(mg/L)
0.635
6.35
12.5
25.4
50.8
LLOQ
LQC
MQC
HQC
RR
Quadratic
weighted
0.65
0.65
0.26
0.37
1.93
2.05
1.90
1.81
0.36
1.22
0.83
0.59
17.81
17.50
8.33
8.96
4.46
4.54
8.14
8.17
cal approach was based on nonparametric tests because the
obtained data were independent, their distribution was not
Gaussian and generally these statistical tests are less affected by outlying values.
Non-parametric One sample rank test based on ranking of the regression models in terms of SSR obtained from
calibration curve fit and calibration curve predictability
was reported by Singtorojet al (Singtoroj et al., 2006). The
SRR of the CS obtained from each calibration level over
three days were used for the assessment of the calibration
Макед. фарм. билт., 60 (1) 19 - 25 (2014)
Difference
0.65
1.63
7.04
9.24
7.80
8.99
0.25
2.04
2.41
0.09
7.07
12.10
-14.04
-7.12
-5.66
-5.66
-0.78
0.45
-2.01
-4.12
Ordered absolute
values of
difference
0.09
0.25
0.45
0.65
0.78
1.63
2.01
2.04
2.41
4.12
5.66
5.66
7.04
7.07
7.12
7.80
8.99
9.24
12.10
14.04
W+
W-
Signed
Ranks
1
2
3
4
-5
6
-7
8
9
-10
-11.5
-11.5
13
14
-15
16
17
18
19
-20
130
80
curve fit and the calibration curve predictability was evaluated through the accuracy and precision obtained from
four independent QC levels. Afterwards, the investigated
regression models were ranked according SRR obtained
from the CS (Table 2) and QC samples (Table 3). The final rank was obtained as a sum of ranks of the calibration
curve fit and their predictability (Table 4). The OLS, 1/x
and 1/y weighted linear regression models did not meet the
acceptance criteria of ±15 % and ±20 % for LLOQ (Table
2 and Table 3) given by the EMA guide for bioanalytical
24
method validation (EMA 2011) and consequently were not
included in the further evaluation. According this statistical
test, the selection of the regression model is based on the final rank, so the quadratic 1/x2 model ranked with 1 should
be selected as an adequate (Table 4).
The data shown in Table 2 and Table 3 indicated that
weighted 1/x2 and 1/y2 linear models also fulfill the EMA
criteria of ±15 % and ±20 % and gave similar SRR compared to the quadratic models, using the traditional approach. However these weighted linear models had final
rank 5 and 7 respectively, while the corresponding quadratic models (1/x2 and 1/y2) were ranked with 1 and 6, respectively (Table 4). This evaluation indicated that One sample rank test could not give justification for the selection
of the weighted quadratic over the weighted linear regression models. In order to evaluate the significance of the differences between the errors obtained using weighted linear and quadratic regression models, non-parametric Wilcoxon sing rank test for two independent groups of samples was applied. The Wilcoxon signed rank test was performed using data for RR obtained by linear 1/x2 and 1/y2
models as first group and quadratic 1/x2 and 1/y2 models as
a second group of samples. This test analyses not just the
differences between the RR, but it also takes into account
the magnitude of the observed differences. The data evaluated in the two matched groups were the average RR obtained from each calibration level during three days and the
average RR obtained from the replicates of four QC sample levels. Afterwards the differences between the RR were
computed, ranked and depending on the observed differences sign (“+”or “-”) was attached on each rank (Table 5).
The null hypothesis (H0) was that there is no difference between the RR generated with linear and quadratic models
versus H1 hypotheses: there is a difference between the RR
obtained with the investigated regression models. The test
statistic for the Wilcoxon signed rank test is W, defined as
the smaller of W+ (sum of positive ranks) and W- (sum of
negative ranks). The W+ was found to be 130 and the Wwas 80. The critical value of W for n=20 with α=0.05 is 52.
The decision rule is reject H0 if W ≤ 52. Given that 80 > 52,
the null hypothesis should be accepted.
The results derived using Wilcoxon signed rank test
showed that although quadratic models generated smaller error than the weighted 1/x2 or 1/y2 linear models, the
difference between the errors were not statistically significant. Considering the request of the regulatory agencies that the simplest model that adequately describes data
should be chosen, the proposed statistical approach gave
justification for the selection of linear 1/x2 model as an
adequate regression model for the IBP calibration curve.
Conclusion
The present investigation showed that during the selection of a regression model for bioanalytical assays with
broad concentration range, statistical approach based on
non-parametric tests should be used. The results obtained
within this paper showed that One sample rank test could
not give statistical justification for the selection of linear
vs. quadratic regression models because slight differences between the SRR were obtained. The proposed statistical approach, based on Wilcoxon signed rank test wherein linear and quadratic regression models were evaluate as
two separate groups, allowed estimation whether the differences in errors are statistically significant. The application of this simple non-parametric statistical test provides
statistical justification of the choice of an adequate regression model.
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Резиме
Избор на соодветен модел на регресија при валидацијата на
биоаналитички метод
Наталија Наков*, ЈасминаТониќ-Рибарска, Анета Димитровска,
Руменка Петковска
Институт за применета хемија и фармацевтски анализи, Фармацевтски факултет,
Универзитет „Св. Кирил и Методиј“, Водњанска 17, 1000 Скопје, Република Македонија
*
Клучни зборови: модел на регресија, биоаналитички метод, непараметарски статистички тестoви, валидација.
Добивањето на точни и репродуцибилни податоци со биоаналитичкиот метод зависи од изборот на соодветен
модел на регресија. Во биоаналитичките методи подрачјето на калибрационата крива најчесто е широк, поради што
се очекува хетеросцедентност на добиените податоци. За евалуација на неколку линеарни и квадратни регресиони
модели при изборот на соодветен модел на регресија беа користени непараметарски статистички тестови: Тест
на рангови и Wilcoxon тест на сума на рангови за два независни примерока. Резултатите покажаа дека Тестот на
рангови не може да даде статистичко оправдување за изборот на линеарен наспроти квадратен модел на регресија,
поради малите разлики во грешките (претставени преку сумата на релативните резудуи) добиени при користење
на соодветните модели на регресија. Статистичка проценка на значајноста меѓу грешките добиени од различните
регресиони модели е постигната со примена на Wilcoxon тест на рангови, според кој линеарните и квадратните
регресиони модели со фактори на надоместок беа третирани како две независни групи на примероци. Примената
на овој едноставен непараметарски тест овозможува добивање на статистичка оправданост за изборот на соодветен
модел на регресија.
Макед. фарм. билт., 60 (1) 19 - 25 (2014)
ISSN 1409 - 8695
UDC: 582.949.2-035.66(497.7)
582.949.2-035.66(496.5)
582.949.2-035.66(495)
Volatile aroma сompounds in infusions of stems and rosette
leaves of Sideritis raeseri Boiss. & Heldr. from R. Maсedonia,
Albania and Greeсe
Bujar Qazimi*, Gjoshe Stefkov, Marija Karapandzova, Ivana Cvetkovikj,
Svetlana Kulevanova
Institute of Pharmacognosy, Faculty of Pharmacy, University SS Cyril and Methodius, Skopje, Republic of Macedonia
Received: April 2014; Accepted: June 2014
Abstract
The composition of the volatile aroma components was defined in the infusion prepared by stems of Sideritis raeseri (SR-S) from
R. Macedonia, Albania and Greeсe and in the infusion of rosette leaves of S. raeseri (SR-R) from R. Macedonia. Analysis were made by
gas chromatography (GC/FID/MS) equipped with a headspace (HS) sampler. Twenty components (12 monoterpenes representing 76.7094.84% and 8 sesquiterpenes representing 5.16-18.55% of the entire volatiles) were identified as aroma components in SR-S samples. The
predominant components in all samples were β-pinene, α-pinene and trans-сaryophyllene. The high abundance of limonene and δ-3-сarene
were characteristic for the samples from Greeсe and 1,8-сineole was for the samples from R. Maсedonia and Albania. In the infusions of
SR-R, ten components were identified, 5 monoterpenes (73.20-83.89%) and 5 sesquiterpenes (16.10-26.80%). Prevailing components in
all tested samples of SR-R were β-pinene, α-pinene, 1,8-cineole and α-epi-murolol. There was almost no difference in the chemical profiles of the aroma compounds between SR-S and SR-R. Infusion of rosette leaves exhibit very similar aroma compounds profile with the
infusion of stems of S. raeseri.
Keywords: Sideritis raeseri, infusion, volatile aroma components, Headspace, GC/MS.
Introduction
Sideritis raeseri Boiss. & Heldr. is endemic to the Balkan Peninsula and is reported to grow in Greece, R. Macedonia and Albania. Mountain tea is a traditional beverage
in the Balkan countries, prepared as a refreshing herbal tea.
Aerial parts of this plant are widely utilized in Mediterranean folk medicine in the form of a decoction or infusion.
This herb is often used to treat the common cold, to alleviate sinus congestion, pains and virus infections, including
influenza (Heywood, 1972; Obon de Castro, 1994; Gabri-
* bqazimi2003@yahoo.com
eli et al., 2005; Bojović et al., 2011). Rosette leaves traditionally is not used.
Various extracts and essential oils of S. raeseri have
shown different types of activities as: antioxidant (Gabrieli
et al., 2005; Koleva et al., 2003; Pljevljakusić et al., 2011;
Karapandzova et al., 2013; Petreska et al., 2011b), antibacterial (Kostadinova et al., 2008; Menković et al., 2010),
spasmolytic (Branković et al., 2011), hypotensive, vasorelaxant and cardiodepressant (Kitić et al., 2012). These activities are due to the presence of flavonoids, phenolic acids, terpenes, iridoids, coumarins, lignans, sterols, phenylpropanoids (Kostadinova et al., 2008; Pljevljakusić et al.,
2011; Karapandzova et al., 2013; Janeska et al., 2007; Qazimi et al., 2010; Fraga B.M., 2012; Petreska et al., 2011a;
28
Bujar Qazimi*, Gjoshe Stefkov, Marija Karapandzova, Ivana Cvetkovikj, Svetlana Kulevanova
Petreska et al., 2011b; Alipieva et al., 2010), and a complex
mineral composition (Pljevljakusić et al., 2011; Spaseska
et al., 2011; Karapandzova et al., 2013).
However, the specific and particular aroma is maybe
the most important reason for the wide use of the tea by the
Balkan peoples. Although numerous data have been published about the chemical composition of the essential oils
(Kostadinova et al., 2008; Pljevljakusić et al., 2011; Petreska et al., 2011b; Menković et al., 2013; Tzakou, 2002;
Galati et al., 1996) and aroma сomponents in the fresh and
dried aerial parts of S. raeseri (Qazimi et al., 2014), there
are no data about the volatile aroma compounds in the infusion of S. raeseri. To analyze these compounds a refined
method of headspace sampling hyphenated with GC/FID/
MS analysis can be utilized (Watson, 2005).
Taking into account all the considerations mentioned
above, the aim of this work was the determination of the
volatile aroma compounds in the infusion of dried stems
and infusion of rosette leaves of mountain tea (S. raeseri)
using a headspace (HS) method with GC/FID/MS.
Material and methods
Plant material
The aerial parts (stems) (SR-S) of the flowering plants
(20–25 cm from the top) of S. raeseri were collected in different localities in National Park Galiсhiсa (R. Maсedonia),
Albania and Greeсe and rosette leaves (SR-R) of S. raeseri were collected in different loсalities in National Park
Galiсhiсa (R. Maсedonia) during the summer of 2012 (Table 1). The plant material was air dried, packed in paper
bags and kept in a dark and cold place until analysis. Plant
identity was verified and voucher specimens were deposited at the Institute of Pharmacognosy, Faculty of Pharmacy,
Skopje, R. Macedonia.
Preparation of the infusion
Infusions were made from 1 g plant material covered
with 100 ml boiling water and left for 5 minutes in a closed
vessel, stirring and filtered.
GC and GC-MS analyses
5 ml of infusion of the stems or infusion of the rosette leaves was put in sealed vials, warmth for 5 minutes
and the gas phase (highly volatile compounds) was investigated on Agilent 7890А Gas Chromatography system
equipped with flame ionization detector (FID) and Agilent 5975C Mass Quadrupole detector as well as capillary
flow technology which enable simultaneous analysis of the
sample on both detectors. HP-5ms (30 m x 0.25 mm, film
thickness 0.25 µm) capillary column was used. Operating
conditions were as follows: oven temperature 60 °C, 20
°C/min to 280 °C; helium as carrier gas at a flow rate of
1mL/min; injector temperature 260 °C and FID temperature 270 °C. 1000 µL of gas phase was injected at split ratio 1:1. The mass spectrometry conditions were: ionization
voltage 70 eV, ion source temperature 230 °C, transfer line
temperature 280 °C and mass range from 50-500 Da. The
MS was operated in scan mode.
Head Space method
Incubation temperature was 80 ˚C, incubation time 5
min, syringe temperature 85 ˚C and agitator speed 500 rpm.
Identification and quantification of the components
Identification of the components was made by comparing mass spectra of components with those from Nist,
Wiley and Adams mass spectra libraries, by AMDIS (Automated Mass Spectral Deconvolution and Identification
Table 1. Specimens of S. raeseri
Species
Locality
R.M., Galichica, Baba
R.M., Galichica, Kazani
R.M., Galichica, Korito
R.G., Lefkada
S. raeseri-stems
R.A., Tepelena
R.A., Gramoz
R.G., Parga
R.M., Galichica, Baba
S. raeseri-rosette
R.M., Galichica, Kazani
leaves
R.M., Galichica, Korito
R.M. – R. Macedonia, R.G. – R. Greece, R.A. – R. Albania
Voucher
specimen
S1/12
S2/12
S3/12
S4/12
S5/12
S6/12
S7/12
S1/R-12
S2/R-12
S3/R-12
Symbol
SR-S (1)
SR-S (2)
SR-S (3)
SR-S (4)
SR-S (5)
SR-S (6)
SR-S (7)
SR-R (1)
SR-R (2)
SR-R (3)
Maced. pharm. bull., 60 (1) 27 - 33 (2014)
29
Volatile aroma сompounds in infusions of stems and rosette leaves of Sideritis raeseri Boiss. & Heldr. from R. Maсedonia...
System) and by comparing literature and estimated Kovat′s
(retention) indices that were determined using mixture of
homologous series of normal alkanes from C9 to C25 in hexane, under the same above mentioned conditions.
The percentage ratio of the components was computed by the normalization method of the GC/FID peak areas
and average values were taken into further consideration.
Results and disсussion
The chemical composition of aroma compounds in the infusion of stems of S. raeseri (SR-S)
Total of 20 individual components were identified
in the infusion of stems of S. raeseri (SR-S), collected
from three different localities in R. Macedonia, two localities in Albania and two localities in Greece, representing 92.25-100% of the total content (Table 2). Data analysis of the chemical composition revealed four different
classes of components: monoterpene hydrocarbons (MH)
58.32-94.84%, oxygen containing monoterpenes (OM) up
to 29.93%, sesquiterpene hydrocarbons (SH) 3.66-15.55%
and oxygen containing sesquiterpenes (OS) up to 8.17%
(Fig. 1).
The most abundant components in all SR-S samples
were monoterpenes β-pinene (26.83-63.13%) and α-pinene
(16.82-28.16%), followed by sesquiterpenes trans-caryophyllene (2.36-7.07%), α-copaene (1.08-4.95%) and α-epimurolol (1.14-4.88%). It is important to stress the presence
of 1,8-сineole (3.49-9.35%), found only in the samples
from R. Maсedonia and Albania. The higher abundance of
limonene and δ-3-сarene (3.67-18.7% and 1.06-10.63%,
respeсtively) were specific for the samples from Greeсe.
The chemical composition of aroma compounds in the infusion of rosette leaves of S. raeseri (SR-R):
Total of 10 individual components were identified in
the infusion of rosette leaves samples of S. raeseri (SRR), collected from three different localities in R. Macedo-
Table 2. Chemical composition of volatile aroma compounds in the infusion of stems of S. raeseri (SR-S) (%)
No. Components
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
α-Thujene
α-Pinene
Sabinene
β-Pinene
δ-3-Carene
Limonene
1,8- Cineole
α-Campholenal
trans-Pinocarveol
Pinocarvone
Myrtenal
Bornyl acetate
α-Copaene
trans-Caryophyllene
Bicyclogermacrene
α- Muurolene
Caryophyllene oxide
Globulol
α-epi-Murolol
Valeranone
KIL
KIE
924
932
969
974
1008
1024
1033
1122
1135
1160
1195
1284
1374
1417
1500
1500
1582
1590
1640
1674
933.7
937.5
966.4
973.8
989.7
1005.9
1017.2
1102.0
1118.0
1159.3
1195.2
1260.9
1345.9
1387.3
1501.7
1468.0
1552.6
1613.4
-
Total (%)
SR-S
(1)
(2)
(3)
(4)
(5)
(6)
(7)
16.82
41.50
5.49
2.89
7.15
5.37
6.25
2.78
4.75
2.12
4.88
-
22.66
58.74
9.55
4.95
4.1
-
23.43
3.30
48.13
1.27
6.26
1.93
3.24
2.55
1.72
1.61
2.18
4.38
-
2.41
18.13
26.83
10.63
18.70
4.46
7.07
4.02
3.00
-
28.16
62.73
3.49
4.20
1.42
26.15
60.14
3.84
1.24
1.54
1.08
1.08
2.58
1.21
1.14
-
26.98
63.13
1.06
3.67
2.80
2.36
-
100
100
100
95.25
100
100
100
SR-S (1)-(7) = infusions of stems of S. raeseri from Galichica, Baba, R.M., Galichica, Kazani, R.M., Galichica, Korito, R.G., Lefkada, R.A., Tepelena, R.A., Gramoz, R.G., Parga, respectively), (-)- not found; KIL- Kovats (retention) index- literature data (Adams, 2007),
KIE – Kovat’s (retention) index experimentally determined (AMDIS).
Макед. фарм. билт., 60 (1) 27 - 33 (2014)
30
Fig.1. The content of different сlasses of volatile aroma components in the infusion of stems of S. raeseri: MH-monoterpene hydrocarbons, OM-oxygen containing monoterpenes, SH-sesquiterpene hydrocarbons, OS-oxygen containing
sesquiterpenes.
Table 3. Chemical composition of volatile aroma compounds in the infusion of rosette leaves of S. raeseri (SR-R) (%)
No.
1
2
3
4
5
6
7
8
9
10
Components
KIL
KIE
α-Pinene
Sabinene
β-Pinene
1,8- Cineole
Bornyl acetate
α-Copaene
Globulol
932
969
974
1033
1284
1374
1590
1640
1652
1674
937.5
966.4
973.8
1260.9
1345.9
1613.4
1621.8
-
α-epi-Murrolol
α-Cadinol
Valeranone
Total (%)
SR-R
(1)
(2)
(3)
18.11
5.78
40.85
8.46
6.26
6.92
13.62
-
17.34
4.48
47.82
10.90
3.35
2.95
7.91
5.24
19.75
53.43
9.91
16.91
-
100
99.99
100
SR-R (1)-(3) = infusions of rosette leaves of S. raeseri from R.M., Galichica, Baba, R.M., Galichica, Kazani and R.M., Galichica, Korito, respectively; (-)- not found; KIL- Kovats (retention) index- literature data (Adams, 2007), KIE – Kovat’s (retention) index experimentally determined (AMDIS).
nia, representing 99.99-100% of the total content (Table 3).
Data analysis of the chemical composition revealed four
different classes of components: MH (64.74-73.18%), OM
(8.46-14.25%), SH (2.95-6.26%) and OS (13.15-20.54%)
(Fig. 2).
The prevailing components in all SR-R samples were
monoterpenes β-pinene (40.85-53.43%), α-pinene (17.3419.75%) and 1,8-сineole (8.46-10.90%), followed by sesquiterpene α-epi-murolol (13.62-16.91%).
The number of components found in SR-S was larger than in SR-R. Also, the share of the classes of components in SR-S (MH>OM>SH>OS) differed from SR-R
(MH>OS>OM>SH). Infusion of rosette leaves exhibit
only smaller differenсes of aroma compounds profile with
the infusion of stems of S. raeseri. The Fig. 3 shows the
ratio between 5 major aroma volatile components in infusions of stems and rosette leaves, where small variations
observed in the content of the dominant components.
Maced. pharm. bull., 60 (1) 27 - 33 (2014)
31
Fig.2. The content of different сlasses of volatile aroma components in the infusion of rosette leaves of S. raeseri: MHmonoterpene hydrocarbons, OM-oxygen containing monoterpenes, SH-sesquiterpene hydrocarbons, OS-oxygen
containing sesquiterpenes.
Fig.3. Main volatile aroma compounds in the infusion of stems (SR-S) and rosette leaves (SR-R) of S. raeseri ((1)-(3) –
samples collected from National park Galichica, R.M, from localities Baba, Kazani and Korito, respectively).
As there are no data for the volatile aroma compounds
in the infusion of stems and rosette leaves of S. raeseri analyzed by HS-GC/FID/MS, most of our findings were compared with the aroma сompounds in the fresh and dried
stems of S. raeseri and essential oil composition. Similar
results were obtained in our previous work where we analyzed the aroma сompounds in the dried aerial parts of S.
raeseri from R. Maсedonia, Albania and Greeсe, and in the
fresh aerial parts of S. raeseri from R. Macedonia, by HSМакед. фарм. билт., 60 (1) 27 - 33 (2014)
GC/MS-FID. The monoterpene hydrocarbons β-pinene
(21.9-48.8% and 37.9-40.7%), α-pinene (17.5-41.0% and
21.5-29.1%), and limonene (1.2-15.7% and 5.1-7.1%), followed by the sesquiterpene hydrocarbons α-copaene (0.99.8% and 3.7-8.2%) and trans-caryophyllene (1.9-6.5%
and 1.4-4.5%, respeсtively) were the major identified
сomponents (Qazimi et al., 2014). These components were
identified as major components in the infusions of stems
and rosette leaves as well. The infusions were also charac-
32
terized with high presence of 1,8-сineole.
Pljevljakusic et al. analyzed the chemical composition
of the essential oils from cultivated S. raeseri aerial parts
at four different developmental phases using GC-MS and
revealed that the sesquiterpene fraction was the predominant terpenoid group in all samples (58.2-67.8%). The sesquiterpene hydrocarbon bicyclogermacrene was the main
constituent of all analyzed oils (35.8-42.5%), followed
by spathulenon and β-caryophyllene (5.0-15.0% and 0.19.0%, respectively); the major monoterpene hydrocarbon
was cis-β-ocimene (3.0-4.9%) (Pljevljakusic et al, 2011).
Compared to these data, in the HS profile of our samples,
bicyclogermacrene exceeded only in the sample SR-S (8)
from Lefkada, Greece (4%).
Kostadinova et al. reported the sesquiterpenes germacron and elemol acetate (25.0 and 15.9%, respectively) as dominant components of the essential oil of S. raeseri from Galichica Mtn. (Kostadinova et al., 2008). These
compounds were not identified in our tested samples.
Conсlussion
Speсifiс and partiсular aroma of S. raeseri is one of
the reasons for the wide use by the Balkan’s people. The
сhemiсal composition of the volatile aroma components
was defined in the infusion prepared by stems of S. raeseri (SR-S) from R. Macedonia, Albania and Greeсe and in
the infusion of rosette leaves of S. raeseri (SR-R) from R.
Macedonia. Analysis were made by gas chromatography
(GC/FID/MS) equipped with a headspace (HS) sampler. In
SR-S and SR-R samples were identified 20 and 10 components, respectively. The components belonged to several
classes of components such as monoterpene hydrocarbons,
oxygen containing monoterpenes, sesquiterpene hydrocarbons and oxygen containing sesquiterpenes. Infusion of the
rosette leaves exhibit very similar aroma compounds profile with the infusion of the stems, comprising β-pinene,
α-pinene, 1,8-сineole, α-copaene and α-epi-murolol as predominate components and accordingly can be consider as
a additional plant material source of this endemic aromatic plant. For complete assessment additional phytochemical analysis are require.
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Резиме
Испарливи арома компоненти во инфузи од цветни
изданоци и розетни листови на Sideritis raeseri Boiss. & Heldr.
од Р. Македонија, Албанија и Грција
Бујар Ќазими, Ѓоше Стефков, Марија Карапанџова, Ивана Цветковиќ,
Светлана Кулеванова
Институт за Фармакогнозија, Фармацевтски Факултет, Универзитет Св Кирил и Методиј, Скопје, Република
Македонија
Клучни зборови: Sideritis raeseri, инфуз, арома испарливи компоненти, хедспејс, GC/MS
Составот на испарливи арома компоненти е испитуван во инфузи подготвени од цветни изданоци на Sideritis raeseri (SR-S) од Р. Македонија, Албанија и Грција и во инфузи на розетни листови на S. raeseri (SR-R) од Р.
Македонија. Анализите се направени со помош на гасна хроматографија (GC/FID/MS) опременa со хедспејс (HS)
семплер. Дваесет компонентите (12 монотерпени 76,70-94,84% и 8 сесквитерпени 5,16-18,55%) се идентификувани
како арома компоненти во SR-S примероци. Доминантни компоненти во сите примероци беа β-pinene, α-pinene и
trans-кариофилен. Висока застапеност на лимонен и δ-3-карен се карактеристични за примероците од Грција и
1,8-цинеол за примероците од Р. Македонија и Албанија. Во инфузи на SR-R, идентификувани се десет компоненти,
5 монотерпени (73,20-83,89%) и 5 сесквитерпени (16,10-26,80%). Доминантни компоненти во сите испитувани
примероци на SR-R се β-pinene, α-pinene, 1,8-цинеол и α-epi-муролол. Речиси не постои разлика на хемиските
профили на арома компоненти меѓу SR-S и SR-R. Инфузи од розетни листови покажуваат многу слични профили на
арома компоненти со инфузи од цветни изданоци на S. raeseri.
Макед. фарм. билт., 60 (1) 27 - 33 (2014)
ISSN 1409 - 8695
UDC: 615.277:547.875.51
Synergy of novel coumarin derivatives and tamoxifen in
blocking growth and inducing apoptosis of breast cancer cells
Lulzime Ballazhi1,2, Faik Imeri3, Aleksandar Dimovski1, Ahmed Jashari4,
Emil Popovski5, Pranvera Breznica-Selmani1,6, Bozhana Mikhova7, Gerald Dräger8,
Edita Alili-Idrizi2, Kristina Mladenovska1*
Faculty of Pharmacy, University “Ss Cyril and Methodius”, Mother Theresa 47, 1000 Skopje, Macedonia
2
Faculty of Medical Science, State University of Tetovo, Pashe Deralla bb, 1200 Tetovo, Macedonia
3
Institute of Pharmacology, University of Bern, Friedbühlstrasse 49, CH-3010 Bern, Switzerland
4
Faculty of Natural Sciences & Mathematics, State University of Tetovo, Pashe Deralla bb, 1200 Tetovo, Macedonia
5
Institute of Chemistry, Faculty of Natural Sciences & Mathematics, University “Ss. Cyril and Methodius”,
PO Box 162, 1000 Skopje, Macedonia
6
Faculty of Medicine, University “Hasan Pristina”, Mother Theresa 10 000, Pristina, Kosovo
7
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences,
Acad. G. Bonchev 9, 1113 Sofia, Bulgaria
8
Institute of Organic Chemistry, Leibniz Universität Hannover, D-30167, Hannover, Germany
1
Received: October 2014; Accepted: November 2014
Abstract
Possible synergistic effect of tamoxifen (2 μM) and hydrazinyldiene-chroman-2,4-diones (10-100 μM) was examined with an aim to
create more effective treatment for ER+ breast cancer. Anti-breast cancer effect has been evaluated on the proliferation of MCF-7 breast adenocarcinoma cells using MTT and alamarBlue assays. Cell viability was evaluated after 48h-treatment and the ICs50 of the coumarin derivatives were determined. The apoptotic effect was evaluated by detection of PARP cleavage and reduced activity of the survival kinase Akt.
The results demonstrated dose-dependent activity, with a percent of growth inhibition after combination treatment being significantly higher (53% to 79%, 10 μM and 100 μM, respectively) than the one in the cell lines treated with tamoxifen (29% to 37%) and the synthesized coumarin derivatives alone (11% to 68%, 10 μM and 100 μM, respectively). The ICs50 of the synthesized compounds significantly decreased in synergy with tamoxifen (33% to 51%). Coumarin derivative having thiazole moiety with additional methyl groups attached
to the carbons at positions 5 and 4 in the thiazole ring showed to be the most potent, with IC50 20 µM when administered alone and 10 µM
in synergy with tamoxifen. The levels of phospho-Thr308 Akt were down-regulated by the combination treatment, pointing to tyrosine kinase phosphorylation inhibition. In conclusion, the novel coumarin derivatives enhance the activity of tamoxifen and this combination may
be suitable for prevention of ER+ breast cancer or development of related compounds. Further studies are needed to elucidate precisely the
type of receptor involved in the activity and the mechanism of action.
Keywords: hydrazinyldiene-chroman-2,4-diones, tamoxifen, breast cancer, MCF-7 cells, antiproliferative effect
* tel: 00389 2 3126 032; fax: 00389 2 3123 054
e-mail: krml@ff.ukim.edu.mk
36
L. Ballazhi, F. Imeri, A. Dimovski, A. Jashari, E. Popovski, P. Breznica-Selmani, B. Mikhova, G. Dräger, E. Alili-Idrizi, K. Mladenovska
Introduction
The balance between proliferation and cell death,
mainly apoptosis, is crucial in determining the overall
growth and regression of tumor. For this reason, traditional cytotoxic agents, such as DNA intercalating agents,
DNA cross-linking agents, topoisomerase inhibitors, cytoskeleton disrupting agents and antimetabolites have usually been targeted to damage the aberrantly dividing cancer
cells by interrupting the cell division process (Hengartner,
2000; Johnstone et al., 2002; Mehlen and Puisieux, 2006).
However, rapidly growing nonmalignant cells are also significantly affected, which in turn, limits efficacy and probably increases the risk of drug resistance, particularly when
toxicities lead to less than optimal drug dosing. Drug resistance, the signal transductions within the process of oncogenesis, cancer cell survival and metastasis are complex,
and for survival, cancer cells often rely on redundant signaling pathways when the original pathway is blocked.
Therefore, to achieve the best outcome for the cancer patients, often several anti-cancer agents that inhibit multiple
targets or redundant pathways are combined in the treatment regimen (Li et al., 2014; LoRusso et al., 2012). Treatment combinations, including combination of nonspecific small molecule chemotherapeutic agents have been successfully applied to various cancer types, including (metastatic) breast cancer (Lee and Nan, 2012).
Breast cancer is still the most frequent malignancy
cancer in women and principal cause of cancer death for
females worldwide (Jemal et al., 2009; Ferlay et al., 2010).
It is estimated that app. 80% of the breast tumors are estrogen receptor positive (ER+) and their growth is stimulated by estrogens (Hertz et al., 2009). Despite tremendous
progress in their treatment during past decades, severe toxic side-effects and de novo and acquired resistance to the
existing therapies present a major clinical problem urging
for design and development of novel drugs and treatment
combinations.
The coumarin (benzopyran-2-one) ring system, present in natural products, has intrigued medicinal chemists
for decades to explore the natural coumarins or their synthetic analogs for their applicability as anticancer drugs
due to the antiproliferative and cytotoxic effect on malignant cells and usefulness in prevention of recurrence and
metastases of malignant cells (Sadiu et al., 2012; Goel et
al., 2009; Jacquot et al., 2001). Of particular interest in
breast cancer chemotherapy, some coumarins and their active metabolite 7-hydroxycoumarin analogs have shown
sulfatase and aromatase inhibitory activities. Coumarin
based selective estrogen receptor modulators and coumarin-estrogen conjugates have also been described as potential anti-breast cancer agents (Musa et al., 2008).
In search for novel coumarin based antibreast cancer drugs, we have synthesized compounds that combine
the coumarin core and five membered heterocycles (isoxazoles and thiazoles) in hydrazinylidiene-chroman-2,4-di-
ones (Jashari et al., 2013; 2014; Ballazhi et al., accepted
for publication). Izoxazole and thiazole substituents were
chosen as important pharmacophores in agents with antiproliferative and tumor vascular-disrupting activity (Gulsory and Guzeldemirci, 2007; Poma et al., 2007). Eight
compounds were synthesized, showing to have antiproliferative effects and induce apoptosis on breast cancer cells
MCF7 and MDA-MB-231 (Jashari et al., 2014) and bone
and lung metastatic cell lines from breast cancer, SCP1833
and SCP4175, respectively (Ballazhi et al., accepted for
publication). Three of the eight compounds tested, having
thiazole moiety, without or with additional methyl group(s)
attached to the carbon(s) at the position(s) 5 and/or 4 in the
thiazole ring showed to be the most potent, requiring further evaluation.
Based on these findings, the aim of this study was to test
their antiproliferative effects on MCF-7 cell lines in synergy
with tamoxifen (Tam). Tam induces growth arrest and apoptosis in breast cancer cells through the inhibition of estrogen binding to the ERs, improving the clinical outcome of
patients with both early and advanced breast cancer. Also,
at high concentrations (≥10 μM), it has been shown to mediate apoptosis in ER-negative cancer cells (Higgins et al.,
2011; Fisher et al., 2005; Gelmann, 1996; Sutherland et al.,
1982). However, about half of patients with advanced ER+
disease immediately fail to respond to Tam, and the disease
progresses to a resistant phenotype. Mechanisms by which
breast cancer cells become resistant may include changes in
the expression of ERα or ERβ, alterations in co-regulatory
proteins, and the influences of cellular kinase signal transduction pathways (Kruh, 2003; Dorsses et al., 2001). Significantly higher concentration-dependent reduction in cell viability and increase in apoptosis, confirmed by PARP cleavage and reduced activity of the survival kinase Akt, was observed with combination of Tam and coumarin derivatives
in comparison with the compounds alone.
Materials and Methods
Cell culturing
The human breast cancer cell line MCF-7 (MCF-7 from
American Type Culture Collection) was maintained in Dulbecco’s modified eagle’s medium (DMEM, Invitrogen,
Switzerland) containing 10 % fetal bovine serum (FBS, Invitrogen, Switzerland) supplemented with 300 µg mL-1 glutamax (Invitrogen, Switzerland), 100 µg mL-1 benzylpenicillin (Invitrogen, Switzerland) and 100 units mL- streptomycin (Invitrogen, Switzerland). MCF-7 cells were incubated at 37 oC in an atmosphere containing 5% CO2. For experiments, cells from exponentially growing culture were used.
MTT assay
Cytotoxic effects of Tam (T5648 Sigma, USA), 4-hydroxycoumarin (4-HC, as a reference, Merck KGaA, GerMaced. pharm. bull., 60 (1) 35 - 44 (2014)
Synergy of novel coumarin derivatives and tamoxifen in blocking growth and inducing apoptosis of breast cancer cells
R2
S
X
O
NH
R1
X
R1
R2
1c
C
H
H
1d
C
H
CH3
1e
C
CH3
CH3
N
N
O
Compound
37
O
Fig. 1. Structure of the novel hydrazinylidiene-chroman-2,4-diones obtained after reaction of the coumarin with the
corrsponding salts of the heterocyclic amines (Jashari et al., 2013; 2014; Ballazhi et al., accepted for publication).
many) and the three coumarin derivatives synthesized in
our laboratory (1c, 1d, 1e; Fig. 1) on MCF-7 cells were determined by 3[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT, Sigma-Aldrich, USA) assay.
In short, 100 µL of the growth medium DMEM was
poured in each well of a 96-well plate and seeded with
5000 MCF-7 cells per well. Cells were allowed to attach
overnight and then 4-HC, 1c, 1d and 1e in increasing concentrations (10, 20, 50, 100 µM) and Tam (2µM) were added to respective wells. After 48 hours of incubation at 37
o
C, 5% CO2 and relative humidity 95%, 20 µL of MTT reagent (5 mg mL-1) was added to each well. After additional
incubation within 4 hours, 100 µL of MTT-solvent (4 mM
HCl, 0.1% Nonidet P-40 (in isopropanol), AppliChem,
Germany) was added to each well to solubilize the MTT
crystals. The plates were covered with tinfoil and the cells
were agitated on orbital shaker for 15 min. Then, the absorbance was read at 570 nm in a microplate reader (SpectraMax M2 Fluorimeter, BucherBiotec Inc, USA). All experiments were performed at least 3 times, with 4 wells for
each concentration of the tested agents. The control cells
were grown under the same conditions without adding the
test compounds. Cell survival was calculated using the following formula:
%of cell viability
mean experimental absorbance
⋅100%
mean control absorbance
AlamarBlue assay
AlamarBlue assay was performed in 96-well microtiter plates. In each well, 100 µL of the culture medium with
4000 cells was added. After 24 hours, the medium was removed from all wells and replaced by fresh medium (150
µL) containing the compounds. After 48 hours of incubation, the plates were inspected under an inverted microscope to assure growth of the controls and sterile conditions. Afterwards, alamarBlue reagent® (10 μL) (Invitrogen, Basel, Switzerland) was added to each well and the
plates were incubated for another 2 to 3 hours. Then, the
plates were read with a SpectraMax microplate reader
(SpectraMax M2 Fluorimeter, BucherBiotec Inc, USA) at
Макед. фарм. билт., 60 (1) 35 - 44 (2014)
excitation wavelength of 540 nm and emission wavelength
of 570 nm. The percent of viability was expressed as fluorescence counts in the presence of test compound as a percentage of that in the vehicle control.
Western blot analysis
The procedure for Western blot analysis has been already described (Doll et al., 2005; Jashari et al., 2014).
Stimulated cells were homogenized in lysis buffer and centrifuged for 10 minutes at 14000×g. The supernatant was
taken for protein determination. 30 μg of protein were separated on SDS-PAGE, transferred to nitrocellulose membrane and Western blot analysis was performed using antibodies as indicated in the figure legend (Figure 3).
Statistical analysis
The 50% inhibitory concentration (IC50) was determined as the anticancer drug concentration causing 50%
reduction in cell viability and calculated from the viability
curves by linear interpolation between the values immediately above and below the 50% inhibition using the Bliss’s
software (Bliss Co, Castro Valley, CA 94552, USA).
The results were presented as mean ± SD and the statistical analysis was performed using one-way analysis of
variance (ANOVA) followed by a Bonferroni’s post hoc
test for multiple comparisons (GraphPad InStat version
3.00 for Windows NT, GraphPad Software, San Diego,
CA, USA).
Results
Cytotoxic and anti-proliferative effects of Tam, 4-HC and
coumarin derivatives on breast cancer cells
The effects of Tam, 4-HC and coumarin derivatives
(1c, 1d and 1e) on cell viability and proliferation, alone or
in combination, evaluated by MTT and alamarBlue assays,
are presented in Fig. 2A and 2B, respectively. Differences
in cell viability and proliferation were observed when data
obtained from the two assays were compared, with the per-
38
120
A
MTT-Absorbance (% of Control)
110
Co
Tam
100
4-HC+Tam
90
1c+Tam
80
1d+Tam
*
70
60
50
#
40
##
30
##
20
#
#
##
*
**
##
##
1e+Tam
*
*
**
4-HC
**
**
1c
** **
1d
1e
##
10
0
-
2 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100 10 20 50 100
µM
Fig 2.
2. Effects
Effects of
of Tam,
Tam, 4-HC
Fig
4-HC and
and coumarin
coumarin derivatives,
derivatives, alone
alone or
or in
in combination,
combination, on
onviability
viabilityof
ofMCF-7
MCF-7breast
breastcancer cells,
measured
by measured
MTT (A) by
andMTT
alamarBlue
assays. The
ofpercentage
viable cellsofwas
determined
cancer
cells,
(A) and(B)
alamarBlue
(B)percentage
assays. The
viable
cells wasas a ratio of treated cells to untreated
expressed
as mean
± SEM
triplicate as
experiments.
*p of
<0.05 considered
determined
as a ratiocontrols.
of treatedResults
cells toare
untreated
controls.
Results
areofexpressed
mean ± SEM
#
## the unstimulated
triplicate
experiments.
<0.05
considered
statistically
significant
compared
statistically
significant *p
when
compared
to the
unstimulated
control when
values;
p<0.05, to
p<0.01 considered statistical#
##
p<0.05,
p<0.01
considered
statistically
significant when compared to the Tamcontrol
values;when
ly significant
compared
to the
Tam-stimulated
values.
stimulated values.
cent of growth inhibition being higher when MTT assay
was applied.
The results showed that only synthesized coumarin derivatives, but not 4-HC induced anti-proliferative effect in
MCF-7 cell lines. Even when 4-HC was administered with
Tam, the number of viable cells remained high, from app.
67% (100 µM, MTT assay) to 100% (10 µM, alamarBlue
assay). 4-HC alone induced no apoptosis when concentrations equal to or lower than 20 µM were applied.
The percentage of growth inhibition after treatment
with the corresponding hydrazinylidiene-chroman-2,4-diones was dramatically higher than the one in the untreated control cells, especially when concentrations higher
than 10 µM were applied in synergy with Tam. Namely,
Maced. pharm. bull., 60 (1) 35 - 44 (2014)
10
as MTT and alamarBlue assays showed, when the synthesized agents were administered in combination with Tam,
the percent of growth inhibition ranged from app. 51% to
app. 79% (MTT assay) and from app. 12% to 65% (alamarBlue assay), with increase in the concentration of the
agents from 10 to 100 µM. Administration of Tam alone
decreased the number of viable cells to lower extent, from
37% (MTT assay) to 29% (alamarBlue assay), expressed
as a percent from control (100%). Similarly, when synthesized compounds were administered alone, with the lowest concentration, the percent of viable cells was higher in
comparison with Tam alone, from 89% to 83% (MTT and
alamarBlue assays) vs. app. 63% (MTT assay) and 71%
(alamarBlue assay) for Tam, respectively. With increase in
the concentration of the synthesized coumarin derivatives,
from 50 to 100 µM, when they solely administered, the percent of viable cells was lower than the one when only Tam
was administered, app. 32% and 45% (1d, 100 µM, MTT
and alamarBlue assay, respectively). Synergy of the syn-
39
thesized agents with Tam resulted in average 30.3±7.6%
(MTT assay) to 21.0±3.5% (alamarBlue assay) higher decrease in viable cells in comparison with the coumarin derivatives alone, when administered in concentration of
100 µM. When concentration of 10 µM was applied, decrease in non-apoptotic cells was 43.3±2.5% (MTT assay)
to 11.1±4.7% higher in comparison with the coumarin derivatives alone.
As Table 1 shows, the IC50 of the synthesized compounds significantly decreased when they administered in
synergy with Tam, from 33% (1c, alamarBlue assay) to
51% (1c, MTT assay). The coumarin derivative having thiazole moiety, with additional methyl groups attached to the
carbons at the positions 5 and 4 in the thiazole ring (1e)
showed to be the most potent, with IC50 around 20 µM and
10 µM when administered alone and in synergy with Tam,
respectively (Table 1).
Table 1. Cytotoxicity of 4-hydroxycoumarin and its synthesized derivatives against MCF-7 breast cancer cell lines determined by MTT and alamarBlue assaysa
Coumarin
derivatives
4-HC
1c
1d
1e
Compound
> 100
19.76±3.95
19.64±5.40
19.94±1.90
lC50 (µM)
MTT assay
Compound+Tam (2 µM)
> 100
9.72±3.91
10.05±4.58
9.80±3.45
lC50 (µM)
AlamarBlue assay
Compound
Compound+Tam (2 µM)
> 100
> 100
29.60±2.19
19.76±5.52
29.24±1.60
16.60±7.87
19.91±5.98
10.33±2.00
Values are means ± SD from at least three independent experiments
a
CF-7 cells with the
Fig. 3. Western blot analysis of cleeaved PARP and phospho-Akt after incubation of MC
Fig. 3. Western
blot(Co),
analysis
cleaved
PARP and
phospho-Akt
after for
incubation
of MCF-7
the vehicle
(Co), Tam
vehicle
Tamofand
Tam
m+novel
coumarin
derivatives
48 h. Western
blotcells
a withwas
analysis
performed
and by
Tam+novel
coumarin
derivatives
48 h.
Western
blot analysis was performed
using antibodies
against
t PARP for
(upper
panels),
phospho-Thr308-Akt
(middle panels)
p by using
and antibodies
the house- against
PARP
(upperprotein
panels),GAPDH
phospho-Thr308-Akt
panels)
and the
house-keeping
protein
GAPDH (lower panels).
keeping
(llower panels).(middle
The data
represent
three
identical experi
iments.
The data represent three identical experiments.
Макед. фарм. билт., 60 (1) 35 - 44 (2014)
40
Apoptotic effects of Tam and coumarin derivatives on
breast cancer cells
We investigated whether the combination of Tam and
4-HC or coumarin derivatives synergistically affected
apoptosis of MCF-7 cells. PARP (poly ADP ribose polymerase) cleavage which is a marker of apoptosis was determined by Western blot analysis. Fig. 3 shows that in MCF7 cells, all concentrations of 1c, 1d and 1e in combination
with Tam led to a reduced protein expression of 116 kDa
full length PARP with a concentration-dependent appearance of a cleavage product at 86 kDa. In parallel, the levels of phospho- Thr308 Akt were downregulated by combination of Tam and 1c, 1d and 1e when compared to control and to combination of Tam and 4-HC treated cells. Akt
is a well known protein kinase involved in cell growth and
survival and reduced activity often correlates with reduced
proliferation and increased apoptosis. The level of phospho-Thr308 Akt is well accepted to reflect Akt activity.
Discussion
Tam, a selective ER modulator is a partial agonist of
ER, blocking the proliferative effects of estrogen via this
receptor. Since its introduction in cancer therapy, it has become the standard treatment option for hormone-responsive
breast cancer patients (Bush, 2007; Goldhirsch et al., 2009;
Fisher et al., 1998; 2005). It was shown that the Tam effect
can be reversed by addition of 17β-estradiol and that Tam
has no effect on the cell cycle kinetics of the receptor-negative MDA-MB-231 cells, confirming that the antiestrogen
effect is mediated through the ER (Osborne et al., 1982).
This was also confirmed in the study of Gelmann (1996)
and Mandlekar and Kong (2001), in which more apoptosis
was induced in ER+ MCF-7 breast cancer cells compared
to the MDA-MB-231 cells. Several hormone receptor-independent effects have been also described for Tam, leading
to apoptosis when higher concentrations are applied (Fisher
and Redmond, 1992; Mandlekar and Kong, 2001).
Despite the relative safety and significant anti-neoplastic activities of Tam, most initially responsive breast
tumors develop resistance to this drug. Even though an improved understanding, resistance to anti-estrogen therapy
remains a significant clinical problem. However, combination therapies of Tam with other drugs that aimed at the
signaling pathways underlying the development of resistance may be a potential mean of delaying the arrival resistance.
One of the new treatment strategies for improving clinical outcome in breast cancer involves development of ER
antagonists (Musa et al., 2008). Estrogens are formed exclusively in peripheral tissues, and there are two pathways
associated with their synthesis in such tissues, the aromatase and sulfatase pathways. The aromatase pathway involves the conversion of androgen precursor, androstenedione, to estrone by the aromatase (AR) enzyme complex,
while the estrone sulfatase pathways (E1-STS) involves
the conversion of estrone formed via the aromatase route
to estrone sulfate (E1S) by the sulfotransferase enzymes
(Woo et al., 1998; Suzuki et al., 2003; Yoshimura et al.,
2004; Utsumi et al., 1999; Miyoshi et al., 2003). The E1STS pathway is considered the major source leading to estrogen formation, causing low response rate in ER+ breast
tumor patients to highly potent AR inhibitor (Jonat et al.,
1996; Santner et al., 1984; Yamamoto et al., 1993). Furthermore, studies have shown that endocrine therapy involving the inhibition of enzymes within the steroid biosynthetic cascade may be one route to controlling the disease. This approach has led to the development of novel
coumarins as STS (Purohit et al., 2003) as well as AR inhibitors (Chen et al., 2004).
Considering all above mentioned, MCF-7 breast cancer
cell were used to evaluate the synergy effect of Tam and the
coumarin derivatives synthesized in our laboratory. MCF7 cells were used because they possess several ideal characteristics particular to the mammary epithelium. These include the ability for MCF-7 cells to process estrogen, in
the form of estradiol, via ER in the cell cytoplasm, which
in turn regulates the cell proliferation and survival and the
down-regulation of genes with anti-proliferative or proapoptotic activity (Huwiler et al., 1995). This makes the
MCF-7 cell line an ER+ control cell line. In addition, treatment of MCF-7 cancer cells with anti-estrogens can modulate insulin-like growth factor finding proteins, which ultimately have the effect of a reduction in cell growth (Levenson and Jordan, 1997). It was also demonstrated that Tam,
at pharmacological concentrations achievable in tumor tissue of Tam-treated breast cancer patients, can induce rapid
death of MCF-7 cells associated with release of mitochondrial cytochrome c, a decrease of mitochondrial membrane
potential and an increase in production of reactive oxygen
species (Kallio et al., 2005). This suggests that disruption
of mitochondrial function but not immediate activation of
caspase-9 or cleavage of poly (ADP-ribose) polymerase has
a primary role in the acute death response of the MCF-7
cells. Accordingly, an inhibitor of mitochondrial permeability transition but not the caspase inhibitor could be able to
protect MCF-7 cells against Tam.
In this study, we have demonstrated that the antiestrogen Tam, used in concentration of 2 µM inhibits proliferation of ER+ MCF-7 cells. These results, are similar to those reported by Lippman et al. (1976) and Sutherland et al. (1982) who found that, at concentrations of Tam
observed in women treated for breast cancer (<1.0 µM),
the antiestrogen slowed proliferation but was not lethal to
MCF-7 cells in short-term culture. Literature data suggest
that Tam inhibits cell proliferation by invoking a transition
delay in early to mid-G1 phase of the cell cycle (Osborne
et al., 1983).
In our previous studies, we have shown that three of
the eight newly synthesized coumarin derivatives, having
thiazole moiety without or with additional methyl group(s)
Maced. pharm. bull., 60 (1) 35 - 44 (2014)
attached to the carbon(s) at the position(s) 5 and/or 4 in
the thiazole ring were effective in inhibiting the growth
of different cancer cells, including MCF-7 breast cancer
cells, in a dose- and time-dependent manner (Jashari et
al., 2014). Similar results were obtained when estrogenic
activity on MCF-7 breast carcinoma cells of compounds
prepared by addition of 2-aminothiophenol to substituted
4-HC derivatives was evaluated. Among the compounds
tested,
6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b]
[1,4]benzothiazin-6-one and 6,12-dihydro-3-hydroxy1-benzpyrano[3,4-b][1,4]benzothiazin-6-one exhibited an
ER-dependent proliferation and a high binding affinity to
ER, but a moderate capacity to activate the transcription of
a reporter gene (Jacquot et al., 2001). The pyrone-substituted coumarin, namely warfarin, isoflavone genistein and
6,7-dihydroxycoumarin known as esculetin were also examined, under similar assay procedures, using the MCF-7
cell lines. Proliferation assays yielded that the MCF-7 cells
tested were quite sensitive to the effects of all three compounds, with potency of growth inhibition by genistein being greater than the one of esculetin which, in turn was
greater than that of warfarin. Genistein has exceptionally
interesting, multidirectional therapeutic properties and the
biological activity of this substance, as of all isoflavones, is
conditioned by the location of the phenyl ring near the third
carbon of the benzo-γ-pyrone. It has been shown that inhibition of cancer cell proliferation was attributed to inhibition of several key enzymes, especially tyrosine kinase,
which plays a critical role in cell proliferation and transformation, and is also associated with oncogene expression in
breast cancer. The investigation of how esculetin, genistein
and warfarin affect the signal transduction cascade and cell
cycle progression in MCF-7 cells confirmed inhibition of
tyrosine phosphorylation, with warfarin being a less potent inhibitor of cell proliferation and metabolic activity
than esculetin and genistein, partly due to the fact that it
shows no significant tyrosine kinase phosphorylation inhibition in comparison to the other compounds (Lacy and
O’Kenendy, 2004). In addition, in vitro studies showed that
genistein also exhibited a synergistic additive effect when
cancer cells were exposed to a combination treatment with
Tam, confirming to have both estrogenic and anti-estrogenic effects (Tanos et al., 2002; Luczkiewicz et al., 2003).
All these findings implicated that the effects of Tam
may be enhanced in combination with our coumarin derivatives, which was confirmed by a significant additive cytotoxic effect in the MCF-7 cell lines. Non-significant differences in cell viability and proliferation, observed when
the data obtained from the two assays were compared, can
be explained by the different sensitivity to the effects of the
compounds on cellular metabolism of the two assays. The
MTT assay is based on the reduction of tetrazolium salt
such as MTT into formazan crystals by living cells, which
determines mitochondrial activity. It relies on the activity
of just one group of mitochondrial enzymes to predict adverse effects on metabolism, and in doing so may underМакед. фарм. билт., 60 (1) 35 - 44 (2014)
41
estimate metabolic effects, which was confirmed for interferons in the past (Jabbar et al., 1989). Upon entering the
cells, the active ingredient of alamarBlue®, resazurin, is
continuously reduced to resofurin which is further reduced
to hydroresorufin. It is still unknown how this reduction
occurs, intracellularly via enzyme activity or in the medium as a chemical reaction, although the reduced fluorescent form of alamarBlue was found in the cytoplasm of living and cells nucleus of dead cells (O’Brien et al., 2000).
Our data are in contradiction with the previous study (Jabbar et al., 1989) and also with the results of Hamid et al.
(2004) who reported slightly higher sensitivity for alamarBlue assay than the MTT assay for most of the tested drugs, out of 117 in total. However, it must be emphasizes that the results of Hamid et al. (2004) were obtained
from tests in human hepatoma cell line HepG2, at a single
point screen. When the drugs were re-tested in both assays
for reconfirmation of cytotoxicity and determination of the
IC50 values, except for daunorubicin, the IC50 values were
comparable in both assays, pointing that both assays provide useful information to identify in vitro cytotoxic drugs
at early stages of drug candidate selection. There is also
possibility different results to be caused by different level of induction and/or inhibition of the metabolic enzymes
responsible for transformation of cell toxicity end points,
as was demonstrated using dicumarol (Hamid et al., 2004).
This could be one of the reasons why combination treatment of Tam and 4-HC results in higher cell viability in
comparison with Tam alone (Fig 2A and B). Namely, there
are literature data that report on inhibition of microsomal mixed function oxidase by 4-HC (Deckert et al., 1972)
and this could decrease Tam activity knowing that its antiestrogenic effect has been attributed to its metabolism to
an active 4-hydroxy derivative and the avid binding of the
active metabolite to the estrogen receptor. Biotransformation by P450 forms are expressed extrahepatically, in the
breast and endometrium, and may be particularly important in determining tissue-specific effects of Tam (Crewe
et al., 2002).
In addition, Tam has different partial agonist-antagonist activities in different tissues and the differences may
be related in part to the milieu of ER coactivators and corepressors in these tissues. If a cell or tissue requires only
activating factor 1 to interact with transcription factors at
the promoter, Tam is agonistic. However, if a cell type requires activating factors 1 and 2 of the estrogen receptor
to be functioning concurrently, Tam is antagonistic (Shou
et al., 2004). There is a probability that the newly synthesized coumarine derivatives and 4-HC at higher concentrations (50-100 μM) prevent activation of ER and co-actovators (such as A1B1) in MCF-7 cells, reduce the recruitment
of co-activator complexes and enhance recruitment of corepressor complexes to Tam-bound Er on gene promoters, thus potentiating Tam’s estrogen antagonist effects on
gene expression and tumor growth. However, this should
be confirmed in further studies in which MCF-7 breast can-
42
cer cells, which express high levels of A1B1, will be treated with Tam alone and in combination with these coumarine derivatives. In addition, effects of these newly synthesized coumarine derivatives on mitochondrial permeability transition as well as enzyme induction/inhibition effects
should be evaluated.
Our data demonstrated that Tam and 4-HC derivatives
inhibited MCF-7cells proliferation by inducing apoptosis.
The enhanced apoptosis may account for the synergistic inhibition of the combination treatment. The levels of phospho- Thr308 Akt were down-regulated by combination of
Tam and the coumarin derivatives, pointing to tyrosine kinase phosphorylation inhibition.
Conclusion
The results of this study indicate that the combination
of Tam and hydrazinyldiene-chroman-2,4-diones having
thiazole moiety without or with additional methyl group(s)
attached to the carbon(s) at the position(s) 5 and/or 4 in the
thiazole ring can effectively inhibit cell proliferation and
induce apoptotic pathway. They may be considered as new
candidates that could contribute to the development of a
large chemical library or related compounds by a combinatorial synthesis approach. Further studies are needed to
elucidate with precision the type of receptor involved in
the activity and their mechanism of action, including binding mode.
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44
Резиме
Синергистички ефект на нови кумарински деривати и
тамоксифен во блокирање на растот и предизвикување
апоптоза на клетки од рак на дојка
Лулзиме Балажи1,2, Фаик Имери3, Александар Димовски1, Ахмед Јашари4, Емил
Поповски5, Пранвера Брезница-Селмани6, Божана Микхова7, Гералд Дрегер8,
Едита Алили Идризи2, Кристина Младеновска1*
Фармацевтски факултет, Универзитет “Св. Кирил и Методиј”, бул. Мајка Тереза 47, 1000 Скопје, Македонија
Факултет за медицински науки, Државен универзитет во Тетово, ул. Паше Дерала бб, 1200, Тетово, Македонија
3
Институт за фармакологија, Универзитет во Берн, ул. Фридбул 49, CH-3010 Берн, Швајцарија
4
Факултет за природни науки и математика, Државен универзитет во Тетово, ул. Паше Дерала бб,
1200, Тетово, Македонија
5
Институт за хемија, Факултет за природни науки и математика, Универзитет “Св. Кирил и Методиј”,
PO 162, 1000 Скопје, Македонија
Медицински факултет, Универзитет “Хасан Приштина”, ул. Мајка Тереза 10 000, Приштина, Косово
6
Институт за органска хемија со центар за фитохемија, Бугарска академија за науки,
Акад. Г. Бончев 9, 1113 Софија, Бугарија
7
Институт за органска хемија, Лајбниц Универзитет во Хановер, D-30167, Хановер, Германија
1
2
Клучни зборови:
хидразинилдиен-хроман-2,4-диони, тамоксифен, рак на дојка, MCF-7 клетки, антипролиферативен ефект.
Во овој труд е испитуван потенцијалниот синергистички ефект на тамоксифен (2 μM) и хидразинилдиен-хроман2,4-диони (10-100 μM) со цел да се дизјанира поефективен третман за ER+ клетки на рак на дојка. Антиканцер
ефектите беа оценувани во однос на пролиферацијата на MCF-7 аденокарциномните клетки на дојка со користење на
MTT и аламарСино есеите. Виталноста на клетките беше следена после третман од 48 часа, после што беа одредувани
IC50 на кумаринските деривати. Апоптотичниот ефект беше оценет со детекција на раскинувањето на поли ADP
рибоза полимераза и преку намалувањето на активноста на киназа на преживување Akt. Резултатите покажаа дознозависна активност, при што процентот на инхибиција на клеточен раст после комбинираниот третман беше значајно
повисок (53% до 79%, 10 μM и 100 μM, соодветно) од соодветниот процент во клеточните линии третирани само со
тамоксифен (29% до 37%) и синтетизираните кумарински деривати (11% до 68%, 10 μM и 100 μM, соодветно). IC50
на синтетизираните соединенија значајно се намалија во синергија со тамоксифенот (33% до 51%). Кумаринскиот
дериват со тиазолна структура и дополнителни метил групи на јаглеродите во позиција 5 и 4 во тиазолното јадро
прикажа најголема потентност, со IC50 20 µM, применет сам, и 10 µM, во синергија со тамоксифен. Со комбинираниот
третман, нивоата на фосфо-Thr308 Akt беа надолно регулирани, упатувајќи на инхибиција на фосфорилацијата
на тирозин киназа. Како заклучок, новите кумарински деривати ја зголемуваат активноста на тамоксифен и овој
комбиниран третман може да биде соодветен за превенција на ER+ рак на дојка или за развој на сродни соединија
со комбинаториска синтеза. Потребни се понатамошни студии со кои прецизно ќе се оцени типот на рецепторите
одговорен за активноста и механизмот на дејство.
Maced. pharm. bull., 60 (1) 35 - 44 (2014)
ISSN 1409 - 8695
UDC: 582.661.15-113.55
Short Communication
Chemical composition of Chenopodium botrys L.
(Chenopodiaceae) essential oil
Ljubica Adji Andov*, Marija Karapandzova, Ivana Cvetkovikj, Gjose Stefkov,
Svetlana Kulevanova
Institute of Pharmacognosy, Faculty of Pharmacy, University”Ss. Cyril and Methodius”,Majka Tereza str. 47, 1000 Skopje,
R. Macedonia
Received: June 2014; Accepted: August 2014
Abstract
Chemical composition of essential oil isolated from aerial parts of Chenopodium botrys L. (Chenopodiaceae) collected from five different locations in the Republic of Macedonia was analysed by GC/FID/MS. Seventy five compounds were identified representing 90.0291.24% of the oil. The analysis has shown that the oils were rich in sesquiterpenе components (83.18-87.54%) comprising elemol acetat (9.88%-21.98%), seline-11-en-4α-ol (9.81%-13.5%), selina-3,11-dien-6α-ol (6.42%-9.71%) and elemol (5.57%-9.49%) as major oxygen containing sesquiterpenes, followed by lower content of α-eudesmol acetat (3.24%-4.11%), α-chenopodiol (2.42%-5.43%), botrydiol
(1.87-5.73%) and α-chenopodiol-6-acetat (1.9%-4.73%).
Keywords: Chenopodium botrys, essential oil composition, gas-chromatography/mass spectrometry (GC/MS), sesquiterpenes
Introduction
Chenopodiaceae is a large family consisting of approximately 102 genera and 1400 species (Kokanova-Nedialkova et al., 2009). The typical genus Chenopodium
comprises numerous species of perennial and annual plants
known as goosefoots, which occur anywhere in the world.
According to Fuentes-Bazan et al. (2012) the species of
Chenopodium could grow as herbaceous plant or as shrubs
and small trees and they are mainly non-aromatic but could
be fetid. Only few species produce essential oil (glandular
goosefoots) usually with characteristic chemical composition. Thus, for South America (Argentina) C. ambrosioides L., C. multifidum L., C. pumilio R. Br., and few other
species were claimed as aromatic (Bonzani et al., 2003). In
Europe, the most interesting is C. botrys L. (syn. Dyspha-
* *bube.petrovska@yahoo.com
nia botrys (L.) Mosyakin and Clemants, known as Jerusalem Oak Goosefoot also called Feathered Geranium, native
to the Mediterranean region. As native or introduced plant
it also grows in the territory of Asia, India, Himalayas,
Northern Europe, Turkey, Cyprus, Africa, Australia and
North and South America (Seidemann, 2005). Chenopodium species have been used in folk medicine worldwide
for treatment of different ailments. It is well known that C.
album improves the appetite and act as anthelmintic, laxative, diuretic and tonic. In South America C. ambrosioides is used against intestinal parasites from immemorial
time. The plant is also known as carminative, diaphoretic and emmenagogue and as a remedy aginst cough, pulmonary obstruction and amenorrhea (Yadav et al., 2007).
In Europe, C. botrys has been used for treatment of catarrh
and humoral asthma and is known as a good substitute for
C. ambrosioides (Yadav et al., 2007).
46
Ljubica Adji Andov, Marija Karapandzova, Ivana Cvetkovikj, Gjose Stefkov, Svetlana Kulevanova
C. botrys possesses glandular trichomes that produce
essential oil with intense characteristic odour reminiscent to
the frankincense scent. Essential oil of this plant has been
studied and presence of some oxygen-containing sesquiterpenes was correlated with pronounced antibacterial and antifungal activity (Kokanova-Nedialkova et al., 2009; Yadav
et al., 2007; Tzakou et al., 2007). Iranian researchers in essential oil of C. botrys showed presence of 2,3-dehydro-4oxo-β-ionon, (+)-7-epi-amiteol, elemol, α-cadinol and taucadinol as main components (Mahboubi et al., 2011). In
the essential oil of Greek C. botrys, Tzakou et al. (2007)
have identified the sesquiterpenes: elemol acetate, elemol,
botrydiol, α-chenopodiol, β-eudesmol and selina-3, 11-dien6α-ol as major components. For C. botrys from Saudi Arabia, El-Sayed et al. (1989) showed presence of oxygencontaining sesquiterpenes, primarily α and β-eudesmol
while Chalabian et al. (2006) identified α-eudesmol, epi-αmuurolol and cubenol as major constituents.
In the flora of the Republic of Macedonia (RM), 15
species of the genus Chenopodium occur including C.
botrys, C. multifidum and C. ambrosioides as aromatic
plants and 12 other species without glands (non-aromatic) such as C. bonus-henricus, C. hybridum, C. glaucum,
C. murale, C. album etc. (Miceveski, 1995). Dried herbal parts of C. botrys are used from local people for preparing infusions or liquid extracts with diuretic, antispasmodic, carminative and antidiarrheal properties (Maksimovic
et al., 2005). The herb of the plant has not been issue of
chemical characterisation so far. Therefore, the aim of this
study is determination of the chemical composition of the
essential oil of wild samples of C. botrys collected from
different localities of the Republic of Macedonia.
Materials and methods
Plant material
The aerial flowering parts of C. botrys were collected
in the period from July to September in 2013 at five localities in RM: Kozuf, Pretor, Strumica, Zletovo and Radovish. Botanical identification of the species was performed
at the Department of Pharmaceutical Botany, Faculty of
Pharmacy, University Ss. Cyril and Methodius, Skopje,
RM. Voucher specimens (CB-K-1/13; CB-P-1/13, CB-S1/13, CB-Z-1/13 and CB-R-1/13 for samples from Kozuf, Pretor, Strumica, Zletovo and Radovish, respectively)
were deposited at the herbarium of the Faculty of Pharmacy, University Ss. Cyril and Methodius, Skopje, RM.
Collected plant material was air-dried and preserved
in paper bags until analysis was performed, when it was
minced and homogenised appropriately.
Isolation of the essential oil
The essential oil was isolated with steam distillation
using all-glass Clevenger type apparatus according to the
European pharmacopoeia method (Ph. Eur. 7; Method
2.8.12.). Briefly, 20 g of dry plant material was measured
and transferred in a balloon of Clevenger distillation apparatus, in which 500 ml of distilled water and 0.5 ml of xylene were added and distillized for 2 hours. The obtained
oil in xylene was treated with anhydrous sodium sulfate in
order to be purified and dried.
GC/FID/MS analysis of essential oil
Essential oil samples were analysed on Agilent 7890А
Gas Chromatography system equipped with FID detector and Agilent 5975C Mass Quadrupole detector as well
as capillary flow technology which enables simultaneous
analysis of the samples on both detectors. For that purpose,
HP-5ms capillary column (30 m x 0.25 mm, film thickness
0.25 µm) was used. Operating conditions were as follows:
the oven was heated to 60 °C with a gradual increasement
in temperature of 3°C/min to 240°C, which was maintained 1 min and then at rate of 10 °C/min the temperature
was rised to 280 °C and held 1 min; helium as carrier gas
at a flow rate of 1ml/min; injector temperature 220 °C and
that of the FID 270 °C. 1µl of each sample was injected
at split ratio 1:1. The mass spectrometry conditions were:
ionization voltage, 70 eV, ion source temperature 230 °C,
transfer line temperature 280 °C and mass range from 50 500 Da. The MS was operated in scan mode. For GC/FID/
MS analysis, the essential oil was dissolved in xylene to
obtain 1 µl/ml oil solution.
Identification of the components present in essential
oils was made by comparison of obtained mass spectra
with those from Nist, Wiley and Adams mass spectra libraries, using AMDIS (Automated Mass Spectral Deconvolution and Identification System) as well as by literature
and estimated Kovat′s (retention) indices that were determined using mixture of homologous series of normal alkanes from C9 to C25 in hexane, under the same above mentioned conditions.
The percentage ratio of essential oils components was
computed by the normalisation method of the GC/FID
peak areas without any correction factors.
Results and discussion
The results of the analysis of the chemical composition
of essential oils isolated from samples of C. botrys collected from five different locations in RM are presented in Table 1. Using GC/FID/MS, 75 components were identified
representing 90.02 to 91.24% of the total oil content.
Generally, the identified components belonged to four
classes of compounds: monoterpene hydrocarbons (MH),
oxygen-containing monoterpenes (OM), sesquiterpene hydrocarbons (SH) and oxygen-containing sesquiterpenes
(OS). Predominant components were total sesquiterpenes
(83.18 to 87.54%) compared to minor quantity of the total monoterpene (2.70 to 6.95%). OS were represented in
higher percentage (from 71.38 to 81.81%), followed by a
Maced. pharm. bull., 60 (1) 45 - 51 (2014)
47
Chemical composition of Chenopodium botrys L. (Chenopodiaceae) essential oil
Table 1. The chemical composition of the essential oil of Chenopodium botrys (%)
No
KIL
KIE
Components
Kozuf
Pretor
Strumica
Zletovo
Radovis
0.09
tr
0.08
0.05
0.44
0.37
1.
2.
924
939
932.0 α-Thujene
932.4 α-Pinene
0.20
0.10
3.
946
951.8 Camphene
0.31
0.22
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
969
991
1002
1007
1014
1022
1024
1032
1054
1065
1083
1118
1136
975.7
987.9
1007.5
1013.1
1019.0
1026.4
1028.5
1037.0
1060.2
1067.1
1089.7
1123.4
1142.5
3.23
0.98
0.24
0.05
0.20
0.31
-
0.08
4.32
0.05
0.15
0.10
0.03
0.38
0.08
0.03
0.03
0.26
0.32
0.05
2.58
1.40
0.03
0.17
0.16
0.27
-
2.95
1.94
tr
0.10
0.19
0.19
-
1.81
1.42
0.04
0.10
0.04
0.16
0.30
-
17.
1154
1158.5 β-Pinene oxide
-
0.11
-
-
-
18.
19.
20.
21.
22.
23.
24.
25.
26.
1165
1174
1186
1279
1284
1294
1335
1346
1346
1170.4
1181.0
1195.0
1238.5
1290.4
1303.1
1342.4
1342.7
1355.1
0.06
0.12
0.14
0.05
0.08
0.03
0.07
0.11
0.02
0.01
0.35
0.05
0.05
0.08
0.05
0.06
0.12
tr
tr
tr
-
0.04
0.05
0.14
-
27.
1374
1382.0 α-Copaene
-
0.09
0.06
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
1389
1409
1417
1430
1434
1437
1451
1452
1465
1478
1485
1491
1498
1500
1396.5
1415.9
1425.9
1435.6
1438.4
1443.8
1452.3
1460.4
1469.2
1481.1
1493.5
1496.7
1501.1
1505.9
4.21
0.17
0.38
0.06
0.09
0.67
0.96
0.13
1.87
-
2.88
0.07
0.24
0.04
0.25
0.04
0.12
0.09
0.38
0.62
0.63
tr
1.63
0.49
1.36
0.05
0.23
0.06
0.03
0.06
0.15
0.31
0.28
tr
0.78
0.35
0.99
tr
0.12
0.05
0.11
0.19
0.18
0.86
0.47
0.82
tr
0.12
0.11
0.12
0.56
0.25
Sabinene
Myrcene
α-Phellandrene
D3-carene
α-Terpinene
o-cymonene
Limonene
β-Z-ocimene
γ-Terpinene
cis-Sabinene hydrate
Fenchone
cis-p-Menth-2-en-1-ol
trans-p-Menth-2-en-1-ol
Borneol
Terpinene-4-ol
α-Terpineol
3Z-hexenyl valerate
Bornyl acetate
p-Menth-1-en-9-ol
δ-Elemene
α-Terpenil acetat
α-Cubebene
β-Elemene
α-Gurjunene
trans-E-Caryophyllene
β-Copaene
γ-Elemene
α-Guaiene
trans-Muurola-3.5-diene
α-Hummulene
cis- Muurola-4(14),5-diene
γ-Muurolene
β-Selinene
10,11-epoxy-Calamenene
α-Selinene
α-Muurolene
Макед. фарм. билт., 60 (1) 45 - 51 (2014)
48
No
KIL
KIE
42.
1508
43.
Components
Kozuf
Pretor
Strumica
Zletovo
Radovis
1511.1 Germacrene A
0.28
0.19
0.16
0.25
0.31
1513
1519.4 γ-Cadinene
0.23
1.20
0.86
0.83
0.43
44.
1522
1528.3 δ-Cadinene
0.63
1.83
1.08
1.51
0.66
45.
1533
1538.3 trans-Cadina-1,4-diene
-
0.19
0.11
0.32
0.16
46.
1537
1543.7 α-Cadinene
0.32
0.43
0.22
0.49
0.21
47.
1545
1547.1 Selina-3,7(11)-diene
0.21
0.19
0.07
-
-
48.
1548
1553.6 Elemol
8.14
9.49
6.31
5.57
5.82
49.
1564
1570.8 β-Calacroene
-
0.07
-
-
-
50.
1567
1576.2 Palustrol
0.99
0.17
0.17
-
0.76
51.
1574
1583.5 α-Cederene epoxide
-
2.64
2.04
2.43
-
52.
1582
1591.4 Cariophyllene oxide
0.42
0.28
0.39
0.44
0.82
53.
1592
1599.4 Viridiflorol
0.23
0.54
0.46
0.77
1.44
54.
1600
1602.5 Guaiol
0.29
-
0.55
0.57
0.92
55.
1609
1611.4 Ledol
0.57
0.84
0.92
0.95
1.45
56.
1622
1637.7 γ-Eudesmol, 10-epi
tr
0.46
-
-
tr
57.
1630
1641.8 γ-Eudesmol
2.40
1.71
2.66
2.67
1.57
58.
1642
1647.3 Selina-3,11-dien-6α-ol
8.94
6.42
8.98
8.78
6.57
59.
1649
1656.8 β-Eudesmol
2.52
-
2.96
2.52
2.41
60.
1653
1661.9 α-Cadinol
-
12.11
tr
tr
tr
61.
1658
1662.9 Selin-11-en-4α-ol
11.90
tr
11.96
13.51
9.81
62.
1668
1676.1 Caryophyllene , 14-hydroxy-9-epi-(E)
1.32
-
1.68
1.55
1.33
63.
1680
1678.7 Elemol acetat
15.41
21.98
12.50
10.79
9.88
64.
1689
1694.1 Botrydiol
2.41
1.21
2.82
1.87
5.73
65.
1700
1702.6 Eudesm-7(11)-en-4-ol
2.26
1.51
2.41
2.84
3.95
66.
1725
1727.0 Guaiol acetat
0.89
0.62
1.15
2.24
9.92
67.
1766
1761.6 β-Costol
1.06
0.71
1.05
1.63
1.89
68.
1777
1775.6 α-Santalol acetate
1.83
-
1.67
1.54
-
69.
1783
1780.8 γ-Eudesmol acetat
0.51
0.41
0.63
0.59
0.92
70.
1794
1791.8 α-Eudesmol acetat
3.51
3.61
3.34
3.88
3.75
71.
1811
1814.9 β-Chenopodiol
1.61
1.20
2.23
2.24
1.85
72.
1855
1859.8 α-Chenopodiol
3.10
2.42
5.43
4.41
3.91
73.
1890
1890.7 β-Chenopodiol-6-acetat
0.91
0.64
1.55
1.66
1.45
74.
1938
1945.4 4α-Acetoxy-eudesman-11- ol
0.42
0.51
0.96
0.83
0.93
75.
1960
1963.9 α-Chenopodiol-6-acetat
2.20
1.90
4.32
4.70
4.73
90.02
90.13
90.29
91.24
90.08
Total
KIL – Retention index - literature data (Adams, 2007); KIE – Retention index experimentally determined with reference to a
homologous series of n-alkanes on HP-5ms column (AMDIS), (-) – not found, tr – traces < 0.02;
Maced. pharm. bull., 60 (1) 45 - 51 (2014)
49
Fig.1. The abundance (%) of different classes of components in the essential oil composition of Chenopodium botrys (1Kozuf, 2 - Pretor, 3 - Strumica, 4 - Zletovo and 5 - Radovis), OS - oxygen containing sesquiterpene; SH - sesquiterpene hydrocarbons; OM - oxygen containing monoterpenes; MH - monoterpene hydrocarbons.
Fig.2. The abundance (%) of main sesquiterpene components in the essential oil of Chenopodium botrys (1- elemol;
2 - selina-3,11-dien-6α-ol; 3 - selin-11-en-4α-ol; 4 - elemol acetat; 5 - α-eudesmol acetat; 6 - α-chenopodiol; 7 α-chenopodiol-6-acetat).
lower content of SH (3.75 to 11.8%). MH were present in a
small percentage (1.93 to 5.54%) as well as OM (from 0.38
to 1.41%) (Fig. 1).
Elemol acetate (9.88%-21.98%), saline-11-en-4α-ol
(9,81%-13,5%), selina-3,11-diene-6α-ol (6.42%-9.71%)
Макед. фарм. билт., 60 (1) 45 - 51 (2014)
and elemol (5.57%-9.49%) were identified as major
OS, followed by a lower content of α-eudesmol acetate
(3,24%-4,11%), α-henopodiol (2.42%-5.43%), botrydiol
(1.87-5.73%) and α-chenopodiol-6-acetate (1.9%-4.73%)
(Table 1, Fig. 2). Traces of α-cadinol were identified in
50
all essential oil samples except in the sample from Pretor, which contained 12.11% of this component. From the
fraction of MH only myrcene (0.73-4.32%) and sabinene
(0.08-3.23%) were more present. The essential oil composition was almost constant as the main components were
presented in similar percentages in all investigated samples. Difference was noticed only in the sample originating from Pretor, as in this oil elemol acetate was present in
higher amount compared to all other oils, while selina-11en-4α-ol was absent (Fig 2).
The present findings were in correlation with the results obtained in the studies conducted in Greece and North
America, showing predominant oxygen-containing sesquiterpenes with elemol acetate (16.3%), elemol (14.1%),
botrydiol (11.1%), α-chenopodiol (9.5%), β-eudesmol
(7.0%) and selina-3,11-dien-6α-ol (6.1%) as major components for the samples originated from Greece (Tzakou
et al., 2007) and α and β-chenopodiol (36%), eudesma3,11-dien-6α-ol (9.4%), botrydiol (9.0%), elemol (6.5%),
elemol acetate (5.5%), γ-eudesmol (5.4%) and α and
β-eudesmol (3.7%) as major components in samples originated from North America (Bedrossian et al., 2001). Studies conducted in Iran and Saudi Arabia also showed high
content of sesquiterpenes and very low presence of monoterpenes, but with different composition of the predominant
components in the oil. In some samples, the most important were 2,3-dehydro-4-oxo-α-ionon (22.4%), (+)-7-epiamiteol (11.5%), elemol (7.4%), α-cadinol (7.0%) and taucadinol (7.0%) (Mahboubi et al., 2011), but in other predominant were γ-terpineol (52.8%), p-cymene (19.0%) and
iso-ascaridol (7.0%) (Morteza-Semnani and Babanezhad,
2007), or camphor (16.5-25.7%), elemol (14.3-13.4%) and
α-cadinol (8.2-11.6%) (Feizbakhsh et al., 2003). Recently,
Mokhtari-Karchegani et al. (2014) published different essential oil composition of three ecotypes of C. botrys from
Iran characterised mainly by monoterpene compounds
such as α-pinene, camphene, β-myrcene and 1,8-cineole. One ecotype additionally contained high amounts of
β-pinene and camphor.
Evaluation of the biological activity of the essential oil
of C. botrys was most likely related to the high content
of ОS with significant antimicrobial activity (Maksimovic et al., 2005; Mahboubi et al., 2011; Tzakou et al., 2007).
These findings rationalize further investigations for determination of any antimicrobial activity of essential oil of
C. botrys from RM. Positive results of this activity could
be used in assessment of the antimicrobial potential of this
raw material in order to use it in the pharmaceutical and
cosmetic industry as well as in food production.
Conclusion
GC/FID/MS analysis of the essential oil composition
of Chenopodium botrys collected at five different locations
in the Republic of Macedonia showed sesquiterpene profile of the oil with major components belonging to the frac-
tion of oxygen-containing sesquiterpenes. The most abundant components were elemol acetate, saline-11-en-4α-ol,
selina-3, 11-diene-6α-ol and elemol, followed by lower
content α-eudesmol acetate, α-henopodiol, botrydiol and
α-chenopodiol-6-acetate.
References
Adams, R., 2007. Identification of essential oil components by
gas chromatography/mass spectrometry. 4th Ed. Illinois:
Allured Publishing Corporation IL, USA, pp 9-31.
Bedrossian, A.G., Beauchamp, P.S., Bernichi, B., Dev, V.,
Kitaw, K.Z., Rechtshaffen, H., Bottini, A.T., Hope, H. 2001.
Analysis of North American Chenopodium botrys Essential
Oil Isolation and Structure of Two New Sesquiterpene
Alcohols, Journal of Essential Oil Research 13(6), 393-400.
Bonzani, N.E., Barboza, G.E., Bugatti, M.A., Ariza Espinar, L.
2003. Morpho-histological studies in the aromatic species
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Chalabian, F., Azam, M., Kambiz, L., Saldouzi, S. 2006.
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composition and antimicrobial activity of the essential
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Strazbourg, 2010.
Feizbakhsh, A., Sedaghat, S., Tehrani, M.S., Rustaiyan, A.,
Masoudi, S. 2003. Chemical Composition of the Essential
Oils of Chenopodium botrys L. from Two Different Locations
in Iran, Journal of Essential Oil Research 15(3), 193-194.
Fuentes-Bazan, S., Uotila, P., Borsch, T. 2012. A novel
phylogeny-based generic classification for Chenopodium
sensu lato, and a tribal rearrangement of Chenopodioideae
(Chenopodiaceae). Willdenowia 42 (1), 5-24.
Kokanova-Nedialkova, Z., Nedialkov, P.T., Nikolov, S.D.
2009. The genus chenopodium: Phytochemistry,
ethnopharmacology and pharmacology, Pharmacognosy
Review 3(6), 280-306.
Mahboubi, M., Bidgoli, F. G., Farzin, N. 2011. Chemical
composition and antimicrobial activity of Chenopodium
botrys L. essential oil, Journal of Essential Oil Bearing
Plants 14(4), 498-503.
Maksimović, Z. A., Đorđević, S., Mraović, M. 2005.
Antimicrobial activity of Chenopodium botrys essential oil,
Fitoterapia 76(1), 112-114.
Micevski, K. 1995. Flora of the Republic of Macedonia. MANU,
Skopje, 1(3), pp. 401-772.
Morteza-Semnani, K., Babanezhad, E. 2007. Essential oil
composition of Chenopodium borys L. from Iran, Journal of
Essential Oil-Bearing Plants 10(4), 314-317.
Mokhtari-Karchegani, N., Amjad, L., Ranjbar, M. 2014.
Comparative Analysis of Chemical Composition of three
Ecotypes of Chenopodium Botrys L. in Iran. Adv. Environ.
Biol. 8(24), 43-47.
Seidemann, J. World Spice Plants: Economic Usage, Botany,
Taxonomy. Berlin Heidelberg: Springer 2005, VIII, 592 p. 93.
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Tzakou, O., Pizzimenti, A., Pizzimenti, F. C., Sdrafkakis, V.,
Galati, E. M. 2007. Composition and antimicrobial activity
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of Essential Oil Research 19(3), 292-294.
51
Yadav, N., Vasudeva, N., Singh, S., and Sharma, S. K. (2007).
Medicinal properties of genus Chenopodium Linn, Natural
Product Radiance 6(2), 131-134.
Резиме
Хемиски состав на етерично масло од Chenopodium botrys L.
(Chenopodiaceae)
Љубица Аџи-Андов*, Марија Карапанџова, Ивана Цветковиќ, Ѓоше Стефков,
Светлана Кулеванова
Институт за фармакогнозија, Фармацевтски факултет, Универзитет „Св. Кирил и Методиј“, Мајка Тереза 47,
1000 Скопје, Република Македонија
Клучни зборови:Chenopodium botrys, состав на етерично масло, гасна хроматографија/масена спектрометрија (GC/MS),
сесквитерпени
Хемискиот состав на етеричното масло изолирано од надземните делови на Chenopodium botrys L. (Chenopodiaceae) собран на 5 различни локалитети во Република Македонија е испитуван со помош на гасна хроматографија во
спрега со масена спектрометрија (GC/FID/MS). Идентификувани се 75 компоненти кои претставуваат 90,02-91,24%
од целокупното масло. Анализата покажа дека во маслото преовладуваат соединенија од групата на сесквитерпени
(83,18-87,54%) со елемол ацетат (9,88%-21,98%), селин-11-ен-4α-ол (9,81%-13,5%), селина-3,11-диен-6α-ол (6,42%9,71%) и елемол (5,57%-9,49%) како доминантни сесквитерпенски соединенија со кислород, следени со помал
процент на α-еудезмол ацетат (3,24%-4,11%), α-хеноподиол (2,42%-5,43%), ботридиол (1,87-5,73%) и α-хеноподиол6-ацетат (1,9%-4,73%).
Макед. фарм. билт., 60 (1) 45 - 51 (2014)
ISSN 1409 - 8695
UDC: 615.225.2.036.8
Effects of Valsartan vs Amlodipin on renal function in salt
loaded spontaneously hypertensive rats
Kalina Gjorgjievska1*, Dimce Zafirov1, Maja Jurhar Pavlova2, Svetlana Cekovska3
Institute of Preclinical and Clinical Pharmacology with Toxicology, Medical Faculty, Skopje
2
Institute of Microbiology and Parasitology, Medical Faculty, Skopje
3
Institute of Medical and Experimental Biochemistry, Medical Faculty, Skopje
1
Received: April 2014; Accepted: June 2014
Abstract
The goal of this study was to compare the effects of valsartan and amlodipin on the systolic blood pressure and parameters specific
to the renal function in salt loaded spontaneously hypertensive rats (SHR). 32 male SHR were used at age of 20 weeks and body weight
ranging between 265-300 g. From 8 weeks of age tab water was replaced with a solution of NaCl (1%) given ad libitum. Rats were divided
into 2 groups: valsartan treated group SHRVAL (n=16) in which valsartan was given at a dose of 10 mg/kg b. w. and amlodipine treated
group SHRAMLO (n=16) in which amlodipine was given at a dose of 5 mg/kg b. w. For a period of 12 weeks we have evaluated the
effect of the investigated drugs on systolic blood pressure, body weight and renal function tests. In salt loaded rats amlodipine was more
effective in reducing the systolic blood pressure in contrast to valsartan who had more pronounced effect on renal parameters most evident
in proteinuria. Since both treatment groups have different mechanism of action a combination therapy may be beneficial in improving renal
function in SHR rats.
Key words: Salt loading, SHR rats, renal function, valsartan, amlodipin.
Introduction
In patients with kidney disease antihypertensive
drugs are given in order to reverse the pathological
processes in the kidneys and to prevent the development of
nephrosclerosis. Pharmacological inhibition of the renninangiotensin-aldosteron system (RAAS) with angiotensin
–converting enzyme inhibitors (ACEi) and angiotensin
receptor blockers (ARB) has attenuated the decline of
renal function associated with diabetic (Lewis et al., 1993;
Parving et al., 2004; Ravera et al., 2007; Ravid et al., 1993)
* kmilosevska@yahoo.com
and non-diabetic (GISEN study group, 1997; Kunz et al.,
2008) forms of chronic renal disease (CRD) and as such
became a standard in preserving renal function in patients
with CRD. Whether the protective effects on the kidney
function can be restricted only to the RAAS inhibitors
or this effect can be extended to other forms of therapy
such as calcium channel blockers (CCB) is not clearly
defined. CCBs are heterogeneous class of drugs used in
treatment of coronary heath disease and hypertension.
Amlodipine, a third-generation dihydropyridine CCB is
characterized with high vascular selectivity and relatively
low negative inotropic effects. In animal experimental
studies amlodipine was able to prevent the development
of hypertension-related left ventricular hypertrophy and
54
Kalina Gjorgjievska, Dimce Zafirov, Maja Jurhar Pavlova, Svetlana Cekovska
dysfunction (Tomassoni et al., 2003; Watanabe et al.,
1998). Controversy exists regarding the effects of CCB on
progressive renal injury since both positive (Bezerra et al.,
2005; Kanno et al., 1994) and negative (Dworkin et al.,
1996) results have been published.
The goal of this study was to compare the effects of
valsartan and amlodipin on the systolic blood pressure
and parameters specific to the renal function in salt loaded
spontaneously hypertensive rats (SHR) rats.
Materials and methods
In this study 32 male SHR rats were used. These
animals were 5 generation of rats originating from Charles
River Laboratories Inc. breaded at the animalia of the
Institute of Preclinical and Clinical Pharmacology and
Toxicology, Med. Fac. SK.
In order to minimize the effects of the inter-individual
differences, all animals were at age of 20 weeks and body
weight ranging between 265-300 g. Rats were fed with
standard rats chow and housed in cages in groups of 5-6
in a temperature controlled environment (~ 22 oC) and
12-h light/dark cycle. From age of 8 weeks tab water was
replaced with a solution of NaCl (1%) given ad libitum.
The experimental protocol met the national guidelines on
proper care and use of animals in laboratory research.
SHR rats were divided into 2 groups: valsartan treated
group SHRVAL (n=16) in which valsartan was given at
a dose of 10 mg/kg b. w. and amlodipine treated group
SHRAMLO (n=16) in which amlodipine was given at a
dose of 5 mg/kg b. w. Drugs were given daily, for a period
of 12 weeks, in the morning, by gavage.
During the treatment period we have evaluated the
effect of the investigated drugs on systolic blood pressure
(SBP), body weight and renal function tests. SBP was
measured by a pletismografic method (IITC Life Science
MRBP Blood Pressure System, California, USA) on the
rats tail in 4-week intervals. Body weight was measured
every second week of treatment. Renal functional tests were
performed in 4-week intervals after urine was collected
in metabolic cages in 24 hour intervals for determination
of the following parameters: diuresis (24 hour urine), pH
analysis, proteinuria and urine creatinine. Urine pH was
measured by using test strips (Combina 10 M, Human
GmbH, Germany), urine protein in 24-hour urine samples
were determined turbidimetrically (Biochemical analysator
ChemWell Awareness Technology) with sulfosalyc acid
(Meulemans, 1961).
Creatinine clearance was used as a specific sensitive
marker for detection of altered renal function of the animals.
In order to determine the serum levels of creatinine, blood
was taken by venepuncture from the orbital sinus of the rats.
Blood samples of 400 μl were taken for serum separation
(200 μl). Serum and urine creatinine were determined
fotometrically (Bartels and Bohmer, 1972). Creatinine
clearance (CrCl) was calculated from the creatinine
concentration in the collected urine sample, urine flow rate
and the plasma concentration of creatinine as ClCr (total) =
eliminated Cr in urine (mg/min)/ Cr concentration in serum
(mg/ml) and ClCr/100g b. w. = ClCr (total) x 100/ b.w.
Statistical analysis: Data were expressed as mean ±
SD. Student‘s unpaired and paired t – test and one way
analysis of variance ANOVA were used as appropriate. P
values <0.05 were considered as statistically significant.
*p<0.01 **p<0.001 SHRAMLO vs. SHRVAL group
Fig. 1. Effects of valsartan and amlodipine on body weight in salt loaded SHR rats for a investigative period of 12 weeks.
Maced. pharm. bull., 60 (1) 53 - 59 (2014)
Effects of Valsartan vs Amlodipin on renal function in salt loaded spontaneously hypertensive rats
55
*p<0.001 SHRAMLO vs. SHRVAL group
Fig. 2. Effects of valsartan and amlodipine on SBP in salt loaded SHR rats for a investigative period of 12 weeks.
Results
Body weight: For the investigative period a gradual
decrease of body weight was noticed in both groups. The
decrease of body weight in the SHRVAL group of rats was
less pronounced and by week 12 was 4.47% compared to
baseline values. Decrease of body weight in the SHRAMLO
group by the end of treatment period compared to baseline
values was 9.04% (Fig. 1).
Systolic blood pressure: A gradual decrease of systolic
blood pressure was noticed in the SHRVAL group. Decrease
was more pronounced by week 8 (14.1%) and week 12
(17%) of the study. Amlodipine in SHR salt loaded rats
had greater effect on SBP than valsartan with a decrease
or 18.3% by week 4, followed by 19.3% for week 8 and
21.7% decrease by week 12. For the period of 4-12 weeks
decrease of SBP with amlodipine compared to valsartan
was statistically more significant (p<0.001) (Fig. 2).
Serum Creatinine: A gradual increase of serum
creatinine was noticed in both treated groups. Increase was
less pronounced in the SHRVAL group - 19% by week 8
and 26.5% by week 12. In the SHRAMLO group increase
was 21.1% by week 8 and 35.3% by week 12. Compared to
the SHRAMLO group in the SHRVAL group a significant
*p<0.05 SHRVAL vs. SHRAMLO group
Fig. 3. Effects of valsartan and amlodipine on serum creatinine in salt loaded SHR rats for a investigative period of 12
weeks.
Макед. фарм. билт., 60 (1) 53 - 59 (2014)
56
*p<0.01 **p<0.001 SHRVAL vs. SHRAMLO group
Fig. 4. Effects of valsartan and amlodipine on proteinuria in salt loaded SHR rats for a investigative period of 12 weeks.
Fig. 5. Effects of valsartan and amlodipine on creatinine clearance in salt loaded SHR rats for a investigative period of 12
weeks.
difference in the serum creatinine values was noticed only
for the point of 12 weeks of treatment (p<0.05) (Fig. 3).
Diuresis: In both treated groups a decrease of diuresis
compared to baseline levels was noticed. Decrease in
diuresis was more pronounced in SHRVAL group with a
31% decrease by week 8 and 39% decrease by week 12. In
SHRAMLO group decrease in diuresis was 30% by week
8 and 39% by week 12. For the period of 4-12 weeks the
decrease of diuresis with valsartan compared to amlodipine
was statistically insignificant.
Urine pH: Between the two investigated groups of
animals there was no significant difference in urine pH.
Proteinuria: In the SHRAMLO group a significant
(p<0.001) increase of the proteinuria compared to baseline
values was noticed. Proteinuria was increased to 4.7%
for week 4, 6.2% by week 8 and 9.24% for week 12. In
the SHRVAL group gradual decrease in proteinuria was
noticed mostly evident by week 12 (5%) (Fig. 4).
Creatinine clearance: Creatinine clearance was used
as specific parameter to assess GFR. In both treatment
groups creatinine clearance gradually decreased with
more evident effects at the end of treatment. Between the
two investigated groups during the course of the study no
significant difference in creatinine clearance was noted
(Fig. 5).
Maced. pharm. bull., 60 (1) 53 - 59 (2014)
Discussion
In this study we have decided to additionally aggravate
hypertension and tissue damage by “salt loading” SHR rats
with 1% solution of NaCl ( used as a drinking solution
instead of tab water) from age of 8 to 20 weeks of age.
SHR are often used animal model of human essential
hypertension (Trippodo and Frohlich, 1981). SHR rats
are considered to be a salt-sensitive animal model (Katori
and Maima, 2006; Susic et al., 2009). In this animal model
additional salt loading increases arterial pressure and
also to contributes to the damages to the hypertension
target organs (heart, arteries, kidneys) structurally and
functionally trough pressure-independent mechanisms
(Cavanagh et al., 2010; Limas et al., 1980; Matavelli et al.,
2007; Varagic et al., 2006).
For the investigative period a gradual decrease of body
weight was noticed in both groups. The decrease of body
weight in the SHRVAL group of rats was less pronounced.
SHR rats given salt overload usually have lower body
weight compared to animals that are not salt loaded
(Susic et al., 2010; Varagic et al., 2006) presumably due
to structural and functional cardiovascular and renal injury
(Matavelli et al., 2007; Susic et al., 2010).
In the present study amlodipine has shown to be more
effective in reducing the SBP in SHR rats. Pronounced
hypotensive effect of amlodipine in comparison to valsartan
was evident through the complete course of the study. In
regards to the renal functional tests both amlodipine and
valsartan were effective in increasing serum creatinine
levels. Significant difference in serum creatinine values
in valsartan treated animals in comparison to amlodipine
were shown at the end of treatment (12 week point). Both
investigated groups showed no significant difference
in creatinine clearance. The results of the present study
confirm previous reports on effectives of amlodipine in
reducing SBP in SHR rats. When comparing the effects of
valsartan and amlodipine (Dong et al., 2011) in salt loaded
SHRSP rats, amlodipine was more effective in reducing
blood pressure. In partly nephrectomized SHR rats (Kanno
et al., 1994) amlodipine was able to reduce SBP and also to
suppress urinary protein excression. When given to SHRSP
rats amlodipine in a dose dependent manner reduced SBP
(Kyselovic et al., 2001) and inhibited paradoxical increases
in plasma rennin activity and rennin mRNA in kidneys.
In nephrectomised salt-loaded SHR rats amlodipine
attenuated increases in SBP, urinary protein excretion and
serum creatinine concentration with effects comparable to
enalapril (Saruta et al., 1995). In this study proteinuria was
decreased only in the valsartan treated group.
Although the extent of sustained damage on the
kidneys during hypertension is expected to be proportional
to the degree of the blood pressure exposure, the severity
of renal damage is not determinated by the systemic BP
but to the degree to which the pressure is transmitted to
the renal microvasculature evident in the increase of the
Макед. фарм. билт., 60 (1) 53 - 59 (2014)
57
glomerular pressure. The increase of the glomerular
pressure is ascribed to the greater dilatation of the
afferent as compared to the efferent arteriole. This relative
efferent arteriolar vasoconstriction is attributed to the
tonic vasoconstrictor effects of Angiotensin II (Ang II).
Therefore it is believed that Ang II plays a major role in
the pathogenesis of glomerular hypertension and can be
intrinsic to the glomerular hemodynamic maladaptation
(Griffin and Bidani, 2006). The effects of increased or
disregulated growth factor expression associated with
glomerular hypertrophy are also of great importance.
Increase in locally generated Ang II triggers multiple
downstream pathways that lead to proteinuria and increased
expressions and/or activity of profibrotic mediators such as
TGF-β and plasminogen activator inhibitor-1 (Fogo and
Ichikawa, 1990; Ketteler et al., 1995; Wolf and Neilson,
1993).
The mechanism responsible for valsartan effectiveness
in ameliorating renal function may be due to the attenuation
of both circulating and local rennin-angiotensin system
(Long et al., 2004). It seems that the decrease of SBP
achieved with amlodipin was not as important factor
in preserving renal function since proteinuria was only
decreased in valsartan treated animals. The precise
mechanism by which amlodipine can reverse the
functional renal abnormalities in the essential hypertensive
state, and by which they may attenuate the progression of
hypertensive renal disease, are unknown. The net effect
of calcium antagonists on glomerular hemodinamics is
determent by the balance between the reduction in afferent
arteriolar resistance and the fall in systemic blood pressure
(Robles, 2004). Amlodipine also increases endothelial
nitric oxide (NO) bioavailability, firstly via enhanced NO
formation, and secondly by prolonging the half-life of NO
through antioxidative properties, and suppressing proinflammatory cytokines and free radical generation (Bauer
and Reams, 1989; Berkels et al., 2004), mediated in part
by the prostanoid endothelium-derived factors (Chou et
al., 2002). The calcium antagonism seems to play a role in
the regulation of apoptosis and proliferation of glomerular
cells and may be effective in preventing nephrosclerosis
exacerbated by NO synthesis inhibition (Watanabe et al.,
2000).
Conclusion
In salt loaded rats amlodipine was more effective
in reducing the SBP, but the effects on renal parameters
mostly proteinuria were more evident in the group of
animals treated with valsartan. Since both treatment groups
have different mechanism of action a combination therapy
may be beneficial in improving renal function in SHR rats.
58
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59
Резиме
Ефекти на Васартан и Амлодипин врз реналната функција
кај спонтано хипертензивни стаорци оптеретени со сол
Калина Ѓорѓиевска1*, Димче Зафиров1, Маја Јурхар Павлова2, Светлана Цековска3
Институт за претклиничка и клиничка фармакологија со токсикологија, Медицински Факултет, Скопје
2
Институт за микробиологија со паразитологија, Медицински Факултет, Скопје
3
Институт за медицинска и експериментална биохемија, Медицински Факултет, Скопје
1
Клучни зборови: оптеретување со сол, бубрежна функција, СХР стаорци, валсартан, амлодипин.
Целта на оваа студија е да се компарираат ефектите на васлартан и амлодипин врз систолниот крвен притисок и
параметрите специфични за реналната функција кај спонтано хипертензивни (СХР) стаорци оптеретени со сол. Во
оваа студија беа употребени 32 СХР стаорци на возраст од 20 недели и телесна тежина од 265-300 гр. Кај стаорците
на возраст од 8 недели водата беше заменета со 1% раствор на NaCl даден ad libitum. Стаорците беа поделени во две
групи: валсартан третирана група SHRVAL (n=16) во доза од 10 mg/kg т.т и амлодипин третирана група на стаорци
SHRAMLO (n=16) даден во доза од 5 mg/kg т.т. Во период од 12 недели беше одредуван ефектот на испитуваните
лекови врз систолниот крвен притисок, телесната тежина и параметри специфични за бубрежната функција.
Кај стаорци оптеретени со сол амлодипин се покажа како поефикасен во редукцијата на крвниот притисок
за разлика од валсартан кој покажа поизразени ефекти врз реналните параметри највидливи во намалувањето на
протеинуријата. Бидејќи двата лека делуваат преку различен механизам на дејство, дадени во комбинација би имале
поизразен ефект врз реналната функција кај СХР стаорци.
Макед. фарм. билт., 60 (1) 53 - 59 (2014)
ISSN 1409 - 8695
UDC: 614.26
Professional paper
Quality use of an unlicensed medicine and off label use of a
medicine
Vasilka Nicha1, Maja Simonoska Crcarevska2, Marija Glavas Dodov2,
Renata Slaveska Raichki2*
1
Public Institution in the Health Sector, for the needs of PHI University Clinics, Institution and Urgent Centre,
2
Institute of pharmaceutical technology, Faculty of Pharmacy, University Ss Cyril and Methodius,
Received: May 2014; Accepted: June 2014
Abstract
This paper gives an overview of the concept and process of quality use of unlicensed medicines and off label use of medicines, with
special attention on the professional roles and responsibilities of prescribers and pharmacists. It also focuses on the policy requirements,
sets of guidelines, recommendations, best practices, and other aspects addressed under this topic that represent the state of update
knowledge. As a complex and specific issue, the use of an unlicensed medicine and off label prescribed medicine in different health care
levels is of particular importance for the healthcare settings in the Republic of Macedonia since, the existing regulatory structure requires
adoption and development of a comprehensive strategy relating to this topic in the near future with an aim of encouraging and supporting the development and maintenance of a sound health system with high standards of medication-use policies.
Key words: off label medicine, unlicensed medicine, quality use
The legal foundation of the approval process of a
medicinal product for human use
Marketing authorizations (MA) for medicines and
medical devices for human use are fundamentally granted by a competent regulatory authority in the form
of an official approval (license) for a certain time period, based on a rigorous and comprehensive scientific, clinical, quality and cost- effective evaluation procedure. Globally speaking, several different regulatory approval processes can be distinguished, such as: federal (http://www.
fda.gov) in the USA (regulatory scrutiny from Food and
Drug Administration-FDA), centralized (Regulation (EC)
* tel.: +38923126032; fax: +38923123054.
e-mail address: rera@ff.ukim.edu.mk
No726/2004), decentralized (Directive 2004/27/EC) and/
or mutual recognition procedures (Directive 2001/83/EC)
in the member countries of the European Union (through
the European Medicines Agency-EMA regulatory approval stringent pathways) and national approval processes for
medicines and medical devices coordinated by the competent national authorities.
In our country, as a country with candidate status for
EU membership and for reasons of approximation to the
European legislation, an EU harmonized national MA system was installed by the latest Law on Medicinal Products and Medical Devices, promulgated in 2007 (LMPMD, 2007). Its implementation is more closely defined by
a number of by-laws and rulebooks (https://lekovi.zdravstvo.mk). Some of the by-laws and rulebooks (Rulebook 1-2,
2008) clearly describe the steps of the entire procedure for
MA of medicinal products or medical devices; regulate de-
62
Vasilka Nicha, Maja Simonoska Crcarevska, Marija Glavas Dodov, Renata Slaveska Raichki
tailed requirements and submission of application dossier.
In this context, according to Article 11 from LMPMD, medicinal product can be placed on the Macedonian market
when:
- MA has been issued pursuant to the procedures
prescribed by this Law,
- it is labeled and has patient information leaflet
pursuant to the MA,
- import authorization was issued pursuant to
this Law,
- quality control of medicinal products has been
performed in line with the Law and
- it is used for preclinical or clinical trials providing that notification or authorization of clinical
trial was issued.
Hence, a medicinal product can be placed on the
Macedonian market only if it has a MA, issued by the
Drug Agency as the national competent authority. Further,
it has to be emphasized that the method of medicinal products prescribing must also be defined in the procedure for
MA of medicinal product (Rulebook 3, 2008). To summarize, according to the Macedonian LMPMD, the MA medicines are generally divided into prescription only medicines (POM), over the counter medicines (OTC) and medicines for use only in healthcare institutions.
An application dossier for medicine MA is of substantial importance for the approval process. As an indispensable element of the approval process, an application dossier for medicine MA is a strict and comprehensive document with detailed requirements regarding the proof of
quality, safety, efficacy and cost-effectiveness of medicine obtained from extensive pre-clinical tests and clinical trials (Directive 2001/83/EC). Application dossier also
comprises all data concerning the medicine, including the
name, pharmaceutical dosage form, indication fields, patient population, dose, risk information, etc., structured in
the document known as “The Summary of Product Characteristics”- SmPC (Directive 2001/83/EC ). In this respect,
SmPC for each medicine is a legal document approved as
part of the medicine MA. Actually, it encompasses essential information for healthcare professionals regarding the
use of the medicine, qualitative and quantitative information on the benefits and risks of the medicine for intended
use, information for individualized care and basic pharmaceutical information. The development of the new emerging technologies contributes to the advance knowledge of
the genomic factors so, when this knowledge is clinically
relevant, it should also be presented in the SmPC. All information within SmPC is presented according to a predefined structure (Directive No 2001/83/EC, 2012; http://
eudrasmpc.eudra.org/). It is also worth mentioning that the
enclosed patient information leaflet which is issued with
dispensed medicine, and according to the law, must accurately reflect only the information presented in the SmPC
(Directive No 2001/83/EC, 2012; Rulebook 4, 2009).
Medicines’ approved uses are also indicated on their labels. An MA holder for a certain medicine is obligated to
update the information submitted in support of the marketing authorization application, including information in relation to therapeutic indications, contraindications and adverse reactions. Nevertheless, it is worth to point out that
SmPC is continuously updated throughout the life-cycle of
the medicine, as new evidence and data emerge from postmarketing period and clinical experience.
The existing definitions of off label use of a
medicine and an unlicensed medicine use in the
regulatory context and practice
Prescribing practice of medicines for use outside the
terms of their MA is known as off label (OL) use of a licensed medicines whereas, an unlicensed (UL) medicine
use, in fact, is the use of a medicine that does not has MA.
On the global regulatory scale, the term OL use of a medicine, by wider definition, refers to the use which is outside
the information in SmPC or more precisely, it denotes a
use which is not specified on the product labeling and instructions for use. It is apparent, as a consequence, that this
use of a medicine has not been evaluated nor approved by
the regulatory authorities and therefore has less supporting evidence stipulated by MA process. As a result, it is
not simple to define and regulate this issue. Moreover,
there is a noticeable inconsistency in theliterature data of
how the terms “off‐label”, “unlabeled”, “unproved”, “unlicensed” or unregistered” prescribing and use of medicines
are defined and mentioned in different types of studies
and purposes (Gazarian, 2007). From the pharmaceutical
point of view, for example, in some studies extemporaneously prepared pharmaceutical formulations under the direct supervision of a pharmacist are considered as an unlicensed formulation due to additional pharmaceutical modification of the marketing approved pharmaceutical product
(e.g. pharmacy compounded formulations, such as preparation of a rectal enema from licensed parenteral dosage
forms) whereas others include them in the category of OL
use of medicines (Conroy et al., 2000;). Hence, UL medicines may also include a medicine whose licensed formulation is modified; those that are imported in the country prior a license (MA) has been granted; or those that are chemical entities used for therapeutic purposes. As the legal status of OL use of medicines and use of UL medicines is not
the same it is important to clearly distinguish the meaning of these terms and definitions. Other related terms describing OL use of medicines are as follows: “out-of-label
use,” and “usage outside of labeling”. According to FDA,
the sometimes used term “unapproved use”of medicines
should be avoided because it implies that FDA regulates
prescribing and dispensing activities (ASHP, 1992). In our
country, according to Article 11 from LMPMD: “ …medicinal product, without MA, can be available on the baMaced. pharm. bull., 60 (1) 61 - 69 (2014)
63
Quality use of an unlicensed medicine and off label use of a medicine
Table 1. The terms used to describe the OL use of MA medicines and UL medicine use by EMA, FDA, WHO, and medication use policy in UK
“Off-label” and “offlicense” use of medicine
-an unlicensed medi- -UK licensed medicine made in a phar- cine used outside of
macy under the direct its license
supervision of a pharmacist
Extemporaneously
Prepared Medicines
UK
EMA
WHO
FDA
Unlicensed medicines Specials
do not have:
- a UK MA number
issued by the regulatory authority,
-a European MA issued by the EMA for
use in the UK
-an unlicensed medicine, specially prepared by the holder of a Manufacturers
Special License, to
satisfy an individual
patient’s special need
“Off-label” use of medicine
- intentionally used outside the terms of MA
“Off-label” and “off-license” use of medicine
- use of medicine outside the specifications described in the license (e.g. in terms of formulation, indications, contraindications or age)
“Unlabeled uses” of medicines
-uses that are not included in the indications or dosage regimens listed in the FDA approved
product labeling (not approved labeling reflected in FDA), in:
(1) doses,(2) patient population, (3) indications, (4) routes of administration
sis of compassionate use e.g. for compassionate reasons to
a group of patients with a chronically or seriously debilitating disease or whose disease is considered to be life
threatening and who cannot be treated satisfactorily by
other authorized medicinal product. The medicinal product stipulated in paragraph 2 of this article must either be
subject of a procedure for marketing authorization or must
be undergoing a clinical trial”. Despite the fact that, an unlicensed medicine uses are precisely described and regulated, off label medicinal product are not mentioned and defined in our policy (LMPD, 2007).
To summarize, Table 1 presents the terms explaining
the meaning of UL medicine use and OL use of medicines
described by the regulatory body and medication use policy in UK as an example of EU country.
The most comprehensive definitions of OL medicine
uses in pediatric population are presented in details in Table 2, as an example for highlighting the distinction of several OL categories of medicinal uses (Ufer et al., 2003;
Ufer et al., 2004; Kimland et al., 2007),
Nevertheless, both OL prescribing and use of MA
medicines should be distinguished from improper prescribing, illegal use and medication errors. In line with this, the
practice is experiencing greater challenges since the categories of UL and OL use of licensed medicine are particularly vulnerable to misunderstanding by professionals and
public. These raise many questions and sometimes cause
confusion and concerns that can serve as indication point
for more consideration and attention by all interested parties.
Макед. фарм. билт., 60 (1) 61 - 69 (2014)
Table 2. OL categories use of medicines in pediatric population
Off-label category
Description
Age
Drug not recommended in the
SmPC below a certain age
Weight
Drug not recommended in the
SmPC for children below a certain weight
Absence of pediat- Pediatric use not mentioned at all
ric information
in the SmPC
Lack of pediatric Statement about the lack of evclinical data
idence of efficacy and safety in
pediatric patients in the SmPC
Contraindication
Statement in the SmPC that the
drug is contraindicated in children
Indication
Drug prescribed for indications
outside of those listed in the
SmPC
Route of adminis- Drug administered by a route not
tration
described in the SmPC
64
Statistical data about OL use of licensed
medicines and UL medicine use
The considerable evidences of OL and an UL medicine prescribing practices and the extent of their application in different patient care levels strongly confirm this
widespread practice supported by the overall experiences gained from many studies and surveys (Conroy et
al., 2000; Shah et al., 2007; EMA/794083/2009, 2011). In
fact, an UL and OL use of licensed medicines is not illegal
and the outcomes should not be underestimated particularly when all other options of licensed medicines are unavailable, exhausted, not tolerated, ineffective or unsuitable for
patient’s healthcare needs. For these underlying reasons, in
some instances an UL and OL medicine application may be
the only choice or the best available access to therapy for
vulnerable population such as obstetric (Colvin et al., 2013)
pediatric (‘tJong et al., 2001; Bavdekar and Gogtay, 2005;
Bellis, et al., 2013; Silva et al., 2014), oncologic (Pool and
Dooley, 2004; Soares, 2005), geriatric (Dautzenberg et al.,
2009) and psychiatric (Chouinard, 2006; Hall et al., 2001)
patients as well as patient on palliative care or with terminal disease condition (Pavis and Wilcock, 2001; Culshaw
et al., 2013;). Indeed, OL prescribing is also common for
so-called therapeutic orphan populations and/or rare diseases treatments and special condition (Hampton, 2007; Conroy et al. 2000; Shah et al., 2007). On the other hand,
some older, generic medicinal products have been routinely prescribed as OL for many years (Lindell-Osuagwu et
al., 2009; Rocchi et al., 2010). Global statistical data confirm that in certain patient groups, OL use of medicines has
been proven to be prevalent and in some cases used even
as a common therapeutic strategy, whereas in others it
may be the only treatment option and/or standard of patient health care. For example, the most extensive statistical
data originating from pediatric population studies confirm
that the overall rates of OL and unlicensed medicines prescriptions ranged from 45-60% up to 90% at different care
levels (EMA/794083/2009). Furthermore, based on recent
scientific evidence, the OL medicine use may allow the patients to access innovative and new medicines or in the case
of approved medicines for example, to implement new indications, dose or route of administration (Lindell-Osuagwu et al. 2009; Rocchi et al., 2010; Kimland and Odlind,
2012). Moreover, many new discoveries and experiences
from OL use of medicines and an UL medicine in continuum have not surprisingly contribute to redesign the accepted and “best practice” standards of patient care. These findings emerge much more rapidly than the regulatory authority approves new uses for the marketing approved medicines. It is estimated that around 1,000 specific requests
for OL drug use are made to NHS commissioners in England every year (www.nice.org, 2014). In one study, 57%
of the medicines therapy innovations were discovered by
clinicians, without involvement of pharmaceutical companies or researches (De Monaco et al, 2006). Thus, the im-
portant role of clinicians in the discovery of OL medicines therapies and identification of positive or negative unexpected effects is strongly confirmed. However, a survey
of 150 million OL prescriptions in the United States found
that 73% had little or no scientific support (Gazarian et al.,
2006). By all aspects, the potential of OL and an UL medicines use to expose a patient to harmful consequences is
more pronounced when this use is less supported and justified by solid scientific and medical evidence. Hence, patients might be at risk of receiving harmful or ineffective
treatments. The percentage of an UL and/or OL prescriptions involved in an adverse drug reaction ranged between
23 and 60% in pediatric population (Cuzzolin et al, 2006).
In view of the fact that in many areas of medical practice, the patient care could not proceed without OL prescribing, there is no doubt that this practice and its valuable
role is an integral part of contemporary health care. Therefore, regulatory authorities and professional bodies are expected to ensure clearer understanding and enforce actions
and standards by applying in practice the guiding principles designed to address the quality use of an unlicensed
medicine and OL use of a medicine.
Since, many clinical and ethical concerns and challenges refer to the extent, scope and possible effects and
consequences of such prescribing and use of medicines,
some guidelines for prescribers, clinicians, and pharmacists with regard to the use of an UL medicines and OL
use of a licensed medicines in every level of health care
have been developed and adopted and others will follow
in the near future, with the aim of ensuring and supporting the safe use.
Recognizing this issue as an issue of grave concern,
in our country there is a need of initiating a framework as
a technical support package for dealing with this specific topic.
Basic policy framework for OL use of a licensed
medicine and an UL medicine use
As mentioned above, there are diverse, complex and
justified reasons for OL use of medicines and use of UL
medicines which are not illegal if their prescription,
compounding and use are strictly regulated and evaluated
for appropriateness and safe use (ASHP, 2008; http://www.
gmc- uk.org/ ). Over the years, many potential implications
of OL use of medicines have been arising for example, patient’s insurance costs, regulatory and legislative implication (e.g. risk:benefit ratio; possible redefinition of the evidence-based standard of medication practice, liability for
not applying well-described OL use of medicine), clinical
implication (safety and efficacy of OL use), ethical issues
and economic trends of pharmaceutical companies (Ventola, 2009 ). By all aspects, statistical data about the extent,
possible effects and consequences of the use of the medicines UL and OL strongly confirm the need for national
policy framework that will regulate this issue in order to
Maced. pharm. bull., 60 (1) 61 - 69 (2014)
ensure quality use. This implicates a need to establish: national policies governing OL prescribing and compounding, ethical standards and policy reforms to promote patient and public interests in evidence-based OL prescribing (NHS/Wirral, 2010). A number of initiatives emerge
with the aim to stimulate evidence-based OL medicine
use (Dresser and Frader, 2009; CATAG, 2013). First of
all, regulatory authorities within health system should basically do the following:
- determine the need for UL and OL medicines,
- adopt national policies that will regulate the OL
prescribing,
- regulate certain OL uses,
- promote ethical standards that will regulate the
OL prescribing,
- stimulate policy reforms to promote patient
and public interests in evidence-based OL
prescribing.
Moreover, it is necessary to propose and adopt policy
measures that could decrease risky and ineffective OL prescribing. Systematic monitoring of patient responses to OL
uses with regular information collection about OL uses
and making them publicly available are other aspects
that have to be fulfilled. Monitoring the OL use and record keeping about different patient population by indication and active collecting of safety data should be conducted on regular basis. For example, to continuously monitor OL use of a medicinal product, patient’s medical records, general practice data bases and hospital prescribing
data bases should be created and used (ASHP, 2008). Professional roles and shared responsibilities (clinical and legal liability) of prescribers and pharmacists should also be
clearly defined in order to follow the process of OL medicine use including ethical standards governing OL prescribing. Some countries developed design requests from
secondary or tertiary care (e.g. in the form of a flow chart)
supported by steps, roles and responsibilities of prescribers
when initiation starts in secondary and tertiary health care
(NHS/Wirral, 2010).
Approaches of assessing appropriateness in OL
prescribing and ethical justification
Like any other complex process in health care, medicine prescription process should be based on empirical
knowledge, experience, law regulations and rational steps.
The main objective of each medicine prescribing is to have
a clear therapeutic goal supported by evidence regarding
the safety and effectiveness. Actually, health professionals
with specialized clinical and therapeutics expertise in relevant area, when making prescribing judgment and taking
decisions, should practice evidence based evaluation approach. Medicine prescribing justified by high-quality evidence in one step includes evaluation process based on
the following sources: a new drug monograph, addenda to
Макед. фарм. билт., 60 (1) 61 - 69 (2014)
65
original monographs, therapeutic class reviews, and expedited reviews (ASHP, 2008). Regarding scientific and clinical evidence that can justify and support any medical intervention including prescribing, the proposed hierarchy of
evidence is the following: 1) Large randomized controlled
trials-RTC; 2) Smaller RCT; 3) Cohort studies; 4) Poorly
controlled or uncontrolled studies; 5) Case reports and 6)
Expert’s opinion (Harbour and Miller, 2001). There is no
doubt that all objectives of responsible prescribing are as
relevant for OL prescribing as they are for label prescribing. However, since OL interventions have different objectives mentioned above, it prompts different ethical implications in specific patient populations. For example, in some
circumstances, OL prescribing can be ethically justified on
the basis of evidence that would be considered inadequate
in other contexts (Dresser and Frader, 2009). At the same
time, taking into account that regulatory authorities (c.a.
FDA) cannot approve or disapprove physician and clinicians prescribing practices of MA drugs, OL prescribing is
an area of practice that is not directly regulated by regulatory authorities.The appropriate approach of OL prescribing can be challenging for any prescriber in fact, this is a
major issue of concern.
The common strategy of responsible OL prescribing requires several basic elements (Dresser and Frader, 2009):
- Explicit guidelines to help prescribers assess
the appropriateness of OL prescribing,
- Justifying OL prescribing by high-quality evidence,
- Providing additional information and research
data whenever adequate evidence is lacking
- Informing about the uncertainties and potential
costs associated with OL prescribing
According to the main principle developed for Australian Hospitals the following four categories of OL use are
distinguished, which can be supported by different levels
of evidence and clinical circumstances (CATAG, 2013):
- routine OL use of medicine,
- exceptional/individual OL use of medicine,
- conditional OL use of medicine, with evidence
development,
- research or investigational OL use of medicine.
Proposed routine of OL clinical use of a medicine refers to the use which is based on high quality evidence for
safe, efficacious and cost-effective use. Exceptional/individual OL medicine use denotes patient treatment favorable in individual clinical circumstances where distinct prespecified criteria are met (e.g. serious or rare condition) or
where alternative treatments are not available, or have been
exhausted and/or failed. This OL category use is supported
by low or very low quality evidence but the overall benefit:
harm ratio may be advantageous in this individual use. The
third category, defined as conditional OL use with evi-
66
dence development, covers use support with low to moderate quality evidence in certain types of patients or group
of patients according to an agreed protocol. Therefore, it
is very important to observe the effectiveness and safety outcomes with subsequent record keeping, as it can contribute to systematic development of evidence base about
that drug for the benefit of other patients. For this use the
relevant group of patients is also defined by pre-specified`
criteria (e.g. disease type and severity, age, tried and failed
standard treatment). The fourth OL category use in fact, is
classified as investigational or experimental use because it
is recommended when there is low or very low quality of
evidence, with uncertain benefits and harms that are unknown or which may be significant. However, because of
the potential of clinical benefit for the patient, a research
protocol should be used and reviewed and approved by a
Ethics Committee (CATAG, 2013)
Use is not recommended, when a proposed OL use
does not fit into any of the above the aforementioned four
categories.
It is obvious that determining the appropriateness of
OL prescribing has been associated with difficulties and
it is not easy task to accomplish. While, making efforts
to successfully manage the OL prescribing of a medicine,
many medical organizations and societies have been working to issue guidelines; propose the consensus recommendations to help physicians and clinicians assess appropriateness and decision making in OL prescribing. For example, American Academy of Pediatrics Committee on Drugs
(AAP, 2014) endorses OL prescribing based on sound scientific evidence, expert medical judgment, or published literature and done in line with the best interest of the patient.
In detail, according to ASHP the following principles should guide the OL prescribing and use of medicines
(ASHP, 2008):
1. Whenever possible, OL prescribing should be
based on published evidence,
2. Patient safety should always be priority,
3. The Pharmacy and Therapeutic (P&T) committee should be involved to establish protocols guiding the most frequent OL use,
4. The P&T committee should guide its decisions
based on the scientific evidence,
5. The P&T committee should monitor of OL
uses,
6. The ultimate responsibility for the safety and
efficacy of OL use resides with the prescriber.
1. Prescribers should be familiar with the evidence before considering OL use,
2. Prescribers should be aware of established
protocols for use,
3. When necessary, the prescriber should
consult with a knowledgeable pharmacist,
7. Proper assessment of evidence for OL use
should involve as comprehensive and balanced review as possible,
8. It is strongly recommendable to avoid selective
use of studies to support a decision.
According to MHRA the prescribers have to consider
tree aspects: appropriateness of OL and UL medicine used,
patient information and report suspected adverse reaction.
Thus, the advices for prescriber are to: “Prescribe UL medicine or OL medicine for use”, when:
- Available licensed medicine alternative does
not meet the patient’s needs,
- Sufficient evidence base and/or clinical experience can support safe and effective use:
- both responsibility for prescribing and
for supervising the patient’s care, including monitoring and follow-up can
be taken,
- records of the medicine prescribed are
kept,
- records are kept for all discussed issues
with the patient.
Regarding the “communication to patient”, the best
practice requires:
-
-
-
sufficient information about the proposed treatment, including known serious or common
ADR’s to make available to patients, so as to
allow them to make an informed decision,
to give as much information as patients or carers require or which they may see as relevant,
to explain the reasons for prescribing an OL
medicine or prescribing an unlicensed medicine
where there is little evidence to support its use,
or where the use of a medicine is innovative.
Prescribers are advised to discuss with patients about
potential benefits and harms of OL applications (Eichler,
H., et al, 2009).
Report on suspected adverse reactions is the third aspect of OL and UL use that should be fulfilled by healthcare professionals. They have a responsibility to help monitor the safety of medicines in clinical use through submission of suspected adverse drug reactions to the Institutions
(MHRA 2009).
The ethical justification for OL prescribing by definition, aims to provide the best available therapy for a particular patient and/or patient populations. At least three ethical considerations relevant to OL prescribing can be distinguished (Dresser R., Frader J., 2009):
- evaluation process of the existing evidence for
OL prescribing,
- gathering information and conducting additional research when there is inadequate evidence about an OL use and
- disclosing to patients the reasons about off-label interventions.
Maced. pharm. bull., 60 (1) 61 - 69 (2014)
To summarize, prior to making a decision, it is of paramount importance to consider the overall benefits and
risks of OL medicine use, to inform and communicate to the patient with record keeping and to report on
ADR’s. In line with this, prescribers have been expected
to use their “professional judgments, knowledge and experience” to determine the appropriateness of such prescribing in individual patients or patient population and ultimately to apply evidence-based principles in making clinical decisions about OL or an UL medicines use. However,
in practice, it is obvious that clinicians are often faced with
limited available data or no data at all and/or no explicit
guidance in implementing such judgments and for that reason very often only a small proportion of OL prescribing
may be justified by scientific evidence.
A model strategy on OL use according to NHS in UK
The National Health System (NHS) of UK offers a
more practical and explicit approach with guidelines and
flow charts that support the decision making process of
prescribers regarding the OL medicine prescribing and use.
In this strategy, all the aforementioned principles are included as follows:
- Determining the need for unlicensed and OL
medicines,
- Guidelines regarding alternatives of tablets/
capsules (flow chart),
- Defining roles and responsibilities (clinical responsibility and legal liability) of prescribers
of unlicensed and OL medicines,
- Defining steps (flow chart) and roles and responsibilities of prescribers when initiation
starts in secondary and tertiary care,
- Requests from secondary or tertiary care (flow
chart),
- Patients with professional carers – advice for
prescribers & carers,
- Roles and responsibilities of pharmacists,
- Guidelines for pharmacists & dispensers when
sourcing unlicensed and OL medicines,
- Reporting adverse drug reactions (ADR’s),
- Records keeping.
Roles and responsibilities of pharmacists
regarding OL product
The main professional role and responsibility of a
pharmacist is to ensure that patients receive a medicine
and therapy that is safe, effective, and appropriate for their
medical condition and the circumstances, with minimal
risk and at lower cost. ASHP believes that pharmacists in
organized healthcare settings bear a significant responsibility for ensuring optimal outcomes from all drug theraМакед. фарм. билт., 60 (1) 61 - 69 (2014)
67
py (JCCP, 2014). The so called standard “fit for purpose”
should be achieved by quality and clinically appropriate
medication for the individual patient and all this is equally important for “UL/OL medicines”. Since UL/OL is prescribed by prescriber and supplied by a pharmacist only
when there is no available licensed medicine which fully
meets the patient’s clinical needs, the pharmacist first of
all, should offer guidance to alternatives of pharmaceutical dosage forms as tablets/capsules presented in the flow
chart to help physicians and clinicians in decision making for OL prescription. A pharmacist can supply an unlicensed product only by exception and with the full knowledge of the prescriber. Pharmacists and dispensers when
sourcing unlicensed and OL medicines need to follow certain guidelines in order to understand their legal responsibilities when supplying OL product and minimize the legal
risk of patients and pharmacist. When compounding OL or
UL medicines, pharmaceutical standard of practice should
be applied where possible, or relevant international standards such as those developed by the World Health Organization and the Pharmaceutical Inspection Convention and
Pharmaceutical Inspection Co-operation (PIC/S) as well as
chapters and monographs of the European Pharmacopoeia containing general and specific requirements applicable to medicinal products prepared in pharmacies, in particular about the standards and methods for the control of
the chemical, pharmaceutical and microbiological quality of active substances and excipients, about dosage forms
and containers. However, it is also important to carry out
risk assessment in order to make a distinction between so
called “low risk” preparations (where PIC/S GPP Guide is
recommended as reference) and “high risk” preparations
(where GMP Guide is recommended as reference). Pharmacist should also confirm that the OL product meet the
relevant standard of safety and quality regarding the certificate of analysis or the certificate of conformity. One of the
duties of the pharmacist is to ensure evidence to support
the labeled shelf life of the OL product. Moreover, pharmacist is also responsible to ensure that the OL product is
pharmaceutically appropriate and suitable for the patient
(e.g. to confirm the strength, choice of formulation and excipient details on the certificate, and where applicable, on
the label). Reporting ADR’s reactions of OL product is
the key role of the pharmacist, as well. Record keeping
for all activities of the pharmacists involved in OL medicines is their inevitable obligation. Other recognized duties
of pharmacists are to disseminate medicine information in
areas where decisions are being made about drug therapy
and to communicate with other health care professionals
about recognition, documentation, and prevention of medication errors and educate patients and care givers. American Society of Hospital Pharmacists statement on the use
of medications for unlabeled uses describes Pharmacist’s
Role as the following (ASHP, 1992):
- To fulfill the roles of patient advocate and drug
information specialist,
68
-
-
To develop policies and procedures for evaluating prescriptions and dispensing drugs for UL
uses in their own work settings. Such policies
and procedures might address the documentation of scientific support, adherence to accepted medical practice standards, or a description
of medical necessity,
Develop proactive approaches to promote informed decision making by third-party payers
of health-care services.
Promotion of OL medicines
OL promotion is very different from OL use. On one
hand, it is legal for physicians and clinicians to use OL
medicines; on the other hand, for pharmaceutical companies it is usually not legal to promote their product for off
label use and sale. Thus, bearing in mind that the promotion practice of OL use of medicines by pharmaceutical companies is still prohibited, for example in UK, MA
holders are not allowed to promote ‘OL’ use while, FDA
prohibits the promotion of drugs and devices for OL uses
by companies (Ventola, 2009 ). However, FDA permits the
following sources for Off-label information such as compendia and drug information references, Continuing medical education, Journal articles, medical and graduate education, medical liaisons, sales representatives and web
sites. In our policy, as described in the “Guidelines on
the manner of drugs and medical aids advertising”,
Article4: The advertising for drug should be performed in
compliance with the approved patient information leaflet
and the summary of medical product characteristics. However, nothing is mentioned about the prohibition of OL promotion of medicines. Despite prohibited OL promotion
by pharmaceutical industry companies it is obvious that
independent opinion and practice of leading and influential
academic clinicians and physicians via scientific articles,
congress presentation and other professional activities for
information dissemination can launch OL uses, stimulate
OL prescribing and provide key references for valuable OL
uses. They can also be identified as crucial factors in the
“promotion” of OL medicinal uses.
Conclusion
The main objectives of any contemporary health system are to establish health care policies, health technologies and medicines use policies. Beside the use of licensed
medicines, there is a widespread practice of prescribing
and use of “unlicensed” medicines and “off label” use of licensed medicines which are not illegal if their prescription,
compounding and use are strictly regulated and evaluated
for appropriateness at every heath care level. Health care
practitioners (prescribers, clinicians, pharmacists) have
important roles and responsibilities in the control and validation of this process. In general, they are urged to apply evidence-based principles in decision making process
for OL or UL medicines use; to consider the overall benefits and risks of OL medicine use and to report on ADR’s.
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Hampton, T. 2007. Experts weigh in on promotion, prescription
of off-label drugs. JAMA. 297(7), 683–684.
Harbour, R., Miller, J. A. 2001.New System for Grading
Recommendations in Evidence Based Guidelines,” BMJ
323, 7308, 334-336. http://lekovi.zdravstvo.mk. (Accessed
August 2014)4
http://www.gmc-uk.org/Prescribing_Guidance_2013_50955425.
pdf. Prescribing Medicines and managing medicines and
devices. (Accessed September 2014). http://www.mhra.gov.
uk/home/groups/pl-p/documents/publication/con043810.pdf
(Accessed September 2014).
http://www.nice.org.uk/about/what-we-do/our-programmes/
nice-advice/evidence-summaries- unlicensed-or-off-labelmedicines. (Accessed August 2014). http://www.fda.gov/
Drugs/DevelomentApprovalProcess/ucm090410.htm.
(Accessed March 2014). JCCP, 2014: Pharmacist’s Patient
care Process,
h t t p : / / w w w. a s h p . o r g / D o c L i b r a r y / B e s t P r a c t i c e s /
JCPPCareProcess2014.aspx (Accessed May 2014).‘t Jong,
G.W., Vulto, A.G., de Hoog, M., Schimmel, K.J.M., Tibboel,
D., van den Anker, J.N., 2001. A survey of the use of offlabel and unlicensed drugs in a Dutch Children’s Hospital.
Pediatrics 108(5), 1089–1093.
Kimland, E., Odlind V., 2012. Off-label Drug Use in Pediatric
Patients. Clin. Pharmacol. &Ther. 91,786-801.
Kimland, E., Bergman, U., Lindemalm, S., Böttiger, Y., 2007.
Drug related problems and off-label drug treatment in
children seen at a Drug Information Centre. Eur. J. Pediatr.
166,527-32.
Lindell-Osuagwu, L., Korhonen, MJ., Saano, S., HelinTanninen, M., Naaranlahti T., Kokki H., 2009. Off-label
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and unlicensed drug prescribing in three paediatric wards in
Finland and review of the international literature. Journal of
Clinical Pharmacy and Therapeutics. 34, 277–287.
LMPD, 2007. Law on Medicinal Products and Medical Devices.
Official Gazette of the Republic of Macedonia, No. 106/2007.
MHRA, 2009. Off label and unlicensed use of medicines:
prescribers’ responsibilities. Drug Safety, Update, 2(9) 6-7.
NHS/Wirral, 2010. Medicine Management. Guidance on
Unlicensed and “Off label” medicines. http://mm.wirral.
nhs.uk/document_uploads/guidelines/WirralSpecialsMar11.
pdf
Pavis, H., Wilcock, A. 2001. Prescribing of drug for use
outside their license in palliative care: survey of specialist
in the United Kingdom. BMJ. 323(3), 314-319.
Poole, S.G., Dooley, M.J. 2004. Off-label prescribing in oncology.
Support Care Cancer. 12(5), 302–305.
Regulation (EC) No 726/2004 of the European Parliament and
of the Council, http://eur- lex.europa.eu (Accessed August
2014).
Rocchi, F. Paolucci, P., Ceci, A., Rossi, P. 2010. The European
pediatric legislation: Benefits and perspectives. Italian
Journal of pediatrics,36-56.
Rulebook 1. Rulebook for renewal of the marketing authorization
for medicinal products. Official Gazette of the Republic of
Macedonia, No. 65/08.
Rulebook 2. Rulebook on issuance of marketing authorization
for medicinal products. Official Gazette of the Republic of
Macedonia, No. 29/08.
Rulebook 3. Rulebook on the manner of prescribing and
dispensing medicinal products on a prescription. Official
Gazette of the Republic of Macedonia, No. 153/08.
Rulebook 4. Rulebook on the structure and the contents of
the package leaflet for medicinal product. Official Gazette
of the Republic of Macedonia, No. 29/09.
Shah, S.S., Hall, M., Goodman, D.M., Feuer, P., Sharma, V.,
Fargason, C. Jr., Hyman, D., Jenkins, K., White, M.L., Levy,
F.H., Levin, J.E., Bertoch, D., Slonim, A.D. 2007. Off-label
Drug Use in Hospitalized Children. Arch. Pediatr. Adolesc.
Med. 161(3), 282-290.
Silva, D., Ansotegui, I., Morais-Almeida,M. 2014. Off label
prescribing for allergic diseases in children. WAOJournal.
7(1)4. Published online Feb 14, 2014. doi: 10.1186/19394551-7-4. (Assessed June 2014).
Soares, M., 2005: Off-label indications for oncology drug use
and drug compendia: history and current status. J Oncol
Pract.1(3), 102-105.
Ufer, M., Kimland, E., Bergman, U. 2004. Adverse drug
reactions and off-label prescribing for paediatric outpatients:
a one-year survey of spontaneous reports in Sweden.
Pharmacoepidemiol Drug Saf.13,147-152.
Ufer, M., Rane, A., Karlsson, Å., Kimland, E., Bergman, U. 2003.
Widespread off label prescribing of topical but not systemic
drugs for 350,000 paediatric outpatients in Stockholm. Eur.
J. Clin. Pharmacol. 58,779-783.
Ventola, CL., 2009. Off-label Drug Information: Regulation,
Distribution, Evaluation, and Related Controversies. P&T.
Vol.34 No. 8, 428-449.
70
Резиме
Квалитетна примена на нерегистрирани лекови и
неодобрена употреба на регистрирани лекови
Василка Нича1, Маја Симоноска Црцаревска2, Марија Главаш Додов2,
Рената Славеска Раички2*
1
Јавна установа од областа на здравството за потребите на ЈЗУ Универзитетски Клиники, Завод и ургентен
центар, Мајка Тереза 47, 1000 Скопје, Република Македонија
2
Институт за фармацевтска технологија, Фармацевтски факултет, Универзитет „Св. Кирил и Методиј”,
Мајка Тереза 47, 1000 Скопје, Република Македонија
Клучни зборови: нерегистрирани лекови, неодобрена примена на регистрирани лекови, квалитетна примена
Трудот дава преглед на концептот и процесот за квалитетна примена на нелиценцирани лекови и неодобрена
примена на лиценцирани лекови со посебен акцент на професионалните улоги и одговорности на пропишувачите на
лекови и фармацевтите. Исто така, во фокусот на интерес се ставени регулаторните барања, водичите, препораките,
најдобрите практики и сите аспекти на најнови сознанија од оваа област. Примената на нелиценцираните лекови и
пропишувањето на лекови за неодобрени индикации во различни нивои на здравствената грижа, како комплексно
и специфично прашање, е од посебен интерес за здравствените капацитети во Република Македонија, од причина
што постоечката регулатива во блиска иднина има потреба од усвојување и развој на опсежна стратегија поврзана
со оваа област со цел да се поттикне и поддржи развој и унапредување на здравствен систем со високи стандарди.
Maced. pharm. bull., 60 (1) 61 - 69 (2014)
INSTRUCTIONS FOR AUTHORS
Macedonian Pharmaceutical Bulletin is an official
publication f the Macedonian Pharmaceutical Association.
The journal publishes original scientific papers, short communications, reviews, mini-reviews and professional papers from all fields of pharmacy and corresponding scientific fields of interest for pharmacy (pharmaceutical and
medicinal chemistry, immunology and imunochemistry,
molecular biology, pharmaceutical analyses, drug quality
control, pharmaceutical technology, pharmacoinformatics,
pharmacoeconomics, biopharmacy, pharmacology, applied
botany, pharmacognosy, toxicology, clinical pharmacy,
food and nutrition, physical pharmacy, organical synthesis,
social pharmacy, history of pharmacy etc.). Papers from all
areas of medical and other life sciences are also welcomed.
The Macedonian Pharmaceutical Bulletin, also, publishes and other contributions (recommendations and announcements, reports of meetings, important events and
dates, book reviews, various rubrics).
Types of paper
Original scientific papers (full length manuscripts)
should contain own unpublished results of completed original scientific research.
Short communications also should contain completed
but briefly presented results of original scientific research.
The article should be prepared as described for full length
manuscripts, except for the following: the number of pages should not exceed 10 (including 2 illustrations, figures
or tables). An Abstract should be included as well as a full
reference list.
Reviews and mini-reviews are written at the invitation
of the Editorial Board. “Mini-reviews” of a topic are especially welcome.
They should be surveys of the investigations and
knowledge of several authors in a given research area, the
competency of the authors of the reviews being assured by
their own published results.
Professional papers report on useful practical results
which are not original but help the results of the original
scientific research to be adopted into practical use. Professional papers might be based on the elaborating of theoretical data.
Language
Original scientific papers, short communications, reviews and mini-reviews should be written in good English
(American or British usage is accepted, but not a mixture
of these), while professional papers and all other contributions may be submitted in Macedonian.
Submission declaration
Submission of an article implies that the work described has not been published previously (except in the
form of an abstract or as part of a published lecture or academic thesis), that it is not under consideration for publication elsewhere, that its publication is approved by all authors and tacitly or explicitly by the responsible authorities
where the work was carried out, and that, if accepted, it
will not be published elsewhere in the same form, in English or in any other language.
Policy and ethics
The work described in your article must have been
carried out in accordance with The Code of Ethics of the
World Medical Association (Declaration of Helsinki)
for experiments involving humans http://www.wma.net/
en/30publications/10policies/b3/index.html;
EC Directive 86/609/EEC for animal experiments
http://ec.europa.eu/environment/chemicals/lab_animals/
legislation_en.htm;
Uniform Requirements for manuscripts submitted to
Biomedical journals http://www.icmje.org. This must be
stated at an appropriate point in the article.
Submission
Please submit the manuscript electronically (e-mail
address: magl@ff.ukim.edu.mk) as a single PDF file,
which will be used in the peer-review process. All correspondence, including notification of the Editor’s decision
and requests for revision, takes place by e-mail removing
the need for a paper trail.
72
Referees
Please submit, with the manuscript, the names, addresses and e-mail addresses of 3 potential referees. Note
that the editor retains the sole right to decide whether or not
the suggested reviewers are used.
Papers received by the Editorial Board are sent to referees. The suggestions/comments of the referees and Editorial Board are sent to the author(s) for further action. The
revised article should be returned to the Editorial Board as
soon as possible but in not more than 30 days.
Preparation of manuscripts
Use of wordprocessing software
It is important that the file be saved in the native format of the wordprocessor used. The text should be typed (1
½ spaced) on A4 paper with margins of 3.0 cm on each side
in single-column format, font Times New Roman, Mac C
Times, Macedonian Times and size 11, Keep the layout of
the text as simple as possible. Most formatting codes will
be removed and replaced on processing the article. In particular, do not use the wordprocessor’s options to justify
text or to hyphenate words. However, do use bold face,
italics, subscripts, superscripts etc. When preparing tables,
if you are using a table grid, use only one grid for each individual table and not a grid for each row. If no grid is used,
use tabs, not spaces, to align columns. The electronic text
should be prepared in a way very similar to that of conventional manuscripts. To avoid unnecessary errors you are
strongly advised to use the “spell-check” and “grammarcheck” functions of your wordprocessor.
The pages in the article should be numbered.
Finally, please create PDF file before sending the article. After acceptance, you will be asked to supply the article as wordprocessing document (zip-file).
Appendices
If there is more than one appendix, they should be
identified as A, B, etc. Formulae and equations in appendices should be given separate numbering: Eq. (A.1), Eq.
(A.2), etc.; in a subsequent appendix, Eq. (B.1) and so on.
Similarly for tables and figures: Table A.1; Fig. A.1, etc.
Abbreviations
Define abbreviations that are not standard in this field
in a footnote to be placed on the first page of the article.
Such abbreviations that are unavoidable in the abstract
must be defined at their first mention there, as well as in
the footnote. Ensure consistency of abbreviations throughout the article.
Editor
Units
Follow internationally accepted rules and conventions:
use the international system of units (SI). If other units are
mentioned, please give their equivalent in SI.
The names of substances should be in accordance with
the IUPAC recommendations and rules or Chemical Abstracts practice.
Math formulae
Present simple formulae in the line of normal text
where possible and use the solidus (/) instead of a horizontal line for small fractional terms, e.g., X/Y. In principle,
variables are to be presented in italics.
Footnotes
Footnotes should be used sparingly. Number them
consecutively throughout the article, using superscript Arabic numbers. Many wordprocessors build footnotes into
the text, and this feature may be used. Should this not be
the case, indicate the position of footnotes in the text and
present the footnotes themselves separately at the end of
the article. Do not include footnotes in the Reference list.
Table footnotes
Indicate each footnote in a table with a superscript
lowercase letter.
Figures
Figures (photographs, diagrams and sketches) and
structural formulae should each be given on a separate
sheet (the place to which they belong in the text should be
indicated). The figures should be numbered in Arabic numerals (e.g. Fig. 1). Ensure that each illustration has a caption. Supply all captions separately, not attached to the figure. A caption should comprise a brief title (not on the figure itself) and a description of the illustration. Keep text in
the illustrations themselves to a minimum but explain all
symbols and abbreviations used.
Please submit the pictures in a black and white version.
Tables
The tables should be numbered in Arabic numerals
(e.g. Table 1) and each should be given on a separate sheet
(the place to which they belong in the text should be indicated). Number tables consecutively in accordance with
their appearance in the text. Place footnotes to tables below the table body and indicate them with superscript lowercase letters. Be sparing in the use of tables and ensure
that the data presented in the tables are not duplicated elsewhere in the article.
Maced. pharm. bull., 60 (1) 73 - 76 (2014)
73
Instruction for Authors
Article structure
Manuscript should contain: title, abstract, key words,
introduction, material and methods, results and discussion,
conclusion, acknowledgment (if desired) references and
summary.
Subdivision
Divide your article into clearly defined sections (Abstract, Introduction, Material and methods. etc.). Any section or subsection may be given a brief heading. Each
heading should appear on its own separate line.
Essential title page information
Papers should be preceded by a title page comprising:
the title, the complete name(s) of the authors, and the author’s affiliations.
Title. Concise and informative. Avoid abbreviations
and formulae where possible.
Author names and affiliations. Where the family
name may be ambiguous (e.g., a double name), please indicate this clearly. Present the authors’ affiliation addresses (where the actual work was done) below the names. Indicate all affiliations with a lower-case superscript arabic
number immediately after the author’s name and in front of
the appropriate address. Provide the full postal address of
each affiliation, including the country name of each author.
Corresponding author. Clearly indicate (with *) who
will handle correspondence at all stages of refereeing and
publication, also post-publication. Ensure that telephone and
fax numbers (with country and area code) are provided in addition to the e-mail address and the complete postal address.
Each paper must begin with an Abstract which should
not exceed more than 250 (original scientific and professional papers) or 100 (short communications) words. The
abstract should state briefly the purpose of the research, the
principal results and major conclusions. References should
be avoided, but if essential, then cite the author(s) and
year(s). Also, non-standard or uncommon abbreviations
should be avoided, but if essential they must be defined at
their first mention in the abstract itself. Immediately after
the abstract, provide a list of 3 to 6 keywords arranged in
the order according to their importance.
Introduction
State the objectives of the work and provide an adequate background, avoiding a detailed literature survey or
a summary of the results.
Material and methods
Provide sufficient detail to allow the work to be reproduced. Methods already published should be indicated by a
Макед. фарм. билт., 60 (1) 73 - 76 (2014)
reference: only relevant modifications should be described.
Manuscripts which are related to theoretical studies, instead of Material and methods, should contain a sub-heading and the Theoretical background where the necessary
details for verifying the results obtained should be stated.
Results
Results should be clear and concise.
Discussion
This should explore the significance of the results of
the work, not repeat them. A combined Results and Discussion section is often appropriate. Avoid extensive citations
and discussion of published literature.
Conclusions
The main conclusions of the study may be presented
in a short Conclusions section, which may stand alone or
form a subsection of a Discussion or Results and Discussion section.
Acknowledgements
Collate acknowledgements in a separate section at the
end of the article before the references and do not, therefore, include them on the title page, as a footnote to the title or otherwise. List here those individuals who provided help during the research (e.g., providing language help,
writing assistance or proof reading the article, etc.).
References
Citation in text
Please ensure that every reference cited in the text is
also present in the reference list (and vice versa). Any references cited in the abstract must be given in full. Unpublished results and personal communications are not recommended in the reference list, but may be mentioned in
the text. If these references are included in the reference
list they should follow the standard reference style of the
journal and should include a substitution of the publication
date with either “Unpublished results” or “Personal communication”. Citation of a reference as “in press” implies
that the item has been accepted for publication and a copy
of the title page of the relevant article must be submitted.
Web references
As a minimum, the full URL should be given and the date
when the reference was last accessed. Any further information,
if known (DOI, author names, dates, reference to a source publication, etc.), should also be given. Web references can be listed separately (e.g., after the reference list) under a different
heading if desired, or can be included in the reference list.
74
Reference style
Text: All citations in the text should refer to:
1. Single author: the author’s name (without initials,
unless there is ambiguity) and the year of publication;
2. Two authors: both authors’ names and the year of
publication;
3. Three or more authors: first author’s name followed
by “et al.” and the year of publication.
Citations may be made directly (or parenthetically).
Groups of references should be listed first alphabetically,
then chronologically.
Examples: “as demonstrated (Allan, 1996a, 1996b,
1999; Allan and Jones, 1995). Kramer et al. (2000) have
recently shown....”
List: References should be arranged first alphabetically and then further sorted chronologically if necessary.
More than one reference from the same author(s) in the
same year must be identified by the letters “a”, “b”, “c”,
etc., placed after the year of publication.
Examples:
Reference to a journal publication:
Van der Geer, J., Hanraads, J.A.J., Lupton, R.A., 2000.
The art of writing a scientific article. J. Sci. Commun. 163,
51–59.
Reference to a book:
Strunk Jr., W., White, E.B., 1979. The Elements of
Style, third ed. Macmillan, New York.
Reference to a chapter in an edited book:
Mettam, G.R., Adams, L.B., 1999. How to prepare an
electronic version of your article, in: Jones, B.S., Smith,
R.Z. (Eds.), Introduction to the Electronic Age. E-Publishing Inc., New York, pp. 281–304.
Journal abbreviations source
Journal names should be abbreviated according to
Index Medicus journal abbreviations: http://www.nlm.
nih.gov/tsd/serials/lji.html
List of serial title word abbreviations: http://www.issn.
org/2-22661-LTWA-online.php;
CAS (Chemical Abstracts Service): http://www.cas.
org/sent.html.
Manuscripts written in English should contain a Summary in Macedonian at the end of the paper. The summary should contain: title, author(s) full-name(s), surname(s),
author’s affiliations (institution and address), key words
and abstract. Professional papers written in Macedonian
should contain a summary in English in which the same
data should be included.
Submission checklist
It is hoped that this list will be useful during the final
checking of an article prior to sending it to the journal’s
Editor
Editor for review. Please consult this Guide for Authors for
further details of any item.
Ensure that the following items are present:
One Author designated as corresponding Author:
- E-mail address
- Telephone and fax numbers
- All necessary files have been uploaded
- Keywords
- All figure captions
- All tables (including title, description, footnotes)
- Further considerations: Manuscript has been
“spellchecked” and “grammar-checked”
- References are in the correct format for this journal
- All references mentioned in the Reference list are
cited in the text, and vice versa
- Permission has been obtained for use of copyrighted material from other sources (including the
Web)
After acceptance
Proofs
One set of page proofs (as PDF files) will be sent by
e-mail to the corresponding author. Please list the corrections and return them via e-mail. If, for any reason, this
is not possible, then mark the corrections and any other
comments on a printout of your proof and return by fax,
or scan the pages and e-mail, or by post. Please use this
proof only for checking the typesetting, editing, completeness and correctness of the text, tables and figures. Significant changes to the article as accepted for publication will
not be accepted.
We will do everything possible to get your article published quickly and accurately. Therefore, it is important to
ensure that all of your corrections are sent back to us in one
communication: please check carefully before replying, as
inclusion of any subsequent corrections cannot be guaranteed. Proofreading is solely your responsibility. Note that
Macedonia Pharmaceutical Bulletin may proceed with the
publication of your article if no response is received.
Offprints
The corresponding author, at no cost, will be provided
with a PDF file of the article by e-mail. The PDF file is a
watermarked version of the published article and includes
a cover sheet with the journal cover image.
Additional paper offprints can also be ordered for an
extra charge.
Maced. pharm. bull., 60 (1) 73 - 76 (2014)

волумен 60 (1) 2014 / volume 60 (1)

Transcription

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