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CONTENTS
ARTICLES Page
A Study on the Stability of Different Frusemide Liquid Dosage Formulas: 1
Oral Solution, Syrup, Elixir, Suspension and Emulsion
Fatima J. Jawad
Cytotoxic Assay of Nigella sativa Leaf Callus Extract (Thymol) on Hep-2 Cell Line 63
Using ELISA Assay
Zaynab S. Abdel Gany and Mayasaa F. Mahdi
*
Department of pharmaceutics, College of Pharmacy, University of Baghdad. , Baghdad , Iraq
Abstract
The present study aim at preparing frusemide in liquid form suitable for oral use. This is
achieved through preparing different liquid forms of frusemide. The frusemide liquid is prepared in the
following forms: oral solution, syrup and elixir with intensity of 1, 0.4 and 0.8% weight /volume
respectively and in combination with potassium carbonate, polysorbate 80, alcohol and phosphate
buffer solution of pH8 to dissolve the frusemide in the above mentioned forms. The different forms of
the prepared medicine have been stored in glass bottles that can provide protection against light and at
40, 50, 600C for four months. Besides the pH has been checked to decide the period of validity. The
results show that the expiration date of frusemide have lasted for 1.8, 1.07 and 1.22 years respectively
for the oral solution, the syrup and the elixir. The suspensions of frusemide are formulated in
combination with the following: polyvinyl pyrolidine, xanthan gum, the combination of (xanthan gum
and sodium carboxymethyl cellulose), the combination of (xanthan: methyl cellulose) and chitosan.
The formulas which give suitable release of the drug are chosen for assessment according to the
following considerations: The rat of sedimentation and apparent zero order degradation constant at
250C. In conclusion, it is found the best formula is that which includes poly vinylpyrolidine, tween20,
glycerol, sorbitol, cocoa syrup and parabens at pH7. the fluidity of this chosen formula is psendoplastic
type and its validity has lasted for about three years. The emulsion of frusemide is also prepared
extemporaneously by using the commercial frusemide tablets in combination with acacia and olive oil.
This should be consumed within 45 days of the date of production.
Key word: frusemide, elixir, suspension, emulsion.
الخالصة
حؼش.ٯخ٪٠ِٛخ اٜغبئٛ اَٙ االش٘بٜخز٠ ث٦ٰو٤ رظٙ خال٢ٟ بٯذ٠ٰع٩ِشٜٛ ٙ٪ٔجٟ ٭٪٠ُ ٚذساعخ رحؼٰش عبئٛذٍ ا٨ر
سثبد٪ٮ عٛ٪جٛا٩ ٝ٪ٰربع٪جٛبد ا٣٪ن ٗبسثٟ زوبٓتٛ ا٬ٜ هٞحغ/١ص٩ % 0.8 ، 0.4 ،1 ح٪ٔ٘غٰش ثٛا٩ ششاة٩ ٭٪٠ُ ٙ٪ٜح٠ٗ بٯذ٠ٰع٩ِشٛا
ِخٜخز٠ٛ ا٦ٛحؼش ثبش٘ب٠ٛاء ا٩ذٛ ا١ خض.٥سح اهال٪ٗز٠ٛ اٙبٯذ ُٮ االش٘ب٠ٰع٩ِشٛ الراثخ ا8 ٞٓذاسئخ سٛعِبد ا٪ِٛ اٙ٪ٜحٟ٩ ٙ٪٘حٛا٩ 80
8 د٩ٮ ثحذ٤ٰع٩ٰذس٨ٛن ػجؾ االط اٟ ش٨ذح أسثوخ أش٠ٛ ٯخ٪ئٟ دسعخ60 ،50 ،40 ء ثذسعبد حشاسح٪ؼٜٛ ؼبدحٟ ٮ صعبعٰخ٣ب٤ٓ ُٮ
٭٪٠ِٛ اٙ٪ٜح٠ٛخ ُٮ ا٤ ع1.22 ، 1.07 ، 1.8 ذ٣بٯذ ٗب٠ٰع٩ِشٛذح طالحٰبد اٟ ١زبئظ ثأ٤ٛشد ا٨ اك.ظالحٰخٜٛ ٰخ٤ٟضِٛزشح اٛزحذٯذ اٛ
ٝ٪دٯ٪ظٛا٩ ١ضب٣ضاٛي ا٠َ (طٜإرٟ٩ ١ضب٣ضاٛي ا٠ط٩ ٢ذٯٛ٩ٮ ثبٯشٛ٪ن ثٟ بٯذ٠ٰع٩ِشٛٔبد اٜوٟ ٰن٤ رظٞ ص.زوبٓتٛ٘غٰش ثبٛا٩ ششاةٛا٩
٢ٟ ٰٰٞٔزٜٛ ب٧ اخزٰبسٞاء ر٩ذٜٛ بعت٤ٟ زٮ اهـذ رحشسٛزشا ٰٗت اٛ ا.١عب٪ٰ٘زٛا٩ )ص٪ٰٜٜ عٰٚض٠ٛ ا:١ضب٣ضاٛي ا٠َ (طٜإرٟ٩ )ص٪ٰٜٜ عٰٚضٟ
ٮ٧ رشٰٗجخ٢ احغ١عذ ثب٩ ٓذ٩ ٯخ٪ئٟ 25 ـٛحشٰٗخ ثذسعخ حشاسح اٛشٯخ ا٧لبٛظِش اٛشرجخ ا٠ٛ ِٖ٘زٛصبثذ ا٩ زشعتٛ ٰٓبط عشهخ اٙخال
.7 ٮ٤ٰع٩ٰذس٧ ذ اط٤بد ه٤ٰجشاثٛا٩ ٩٘بٗبٛششاة ا٩ ٙ٪سثز٪ع٩ ٙ٩ٰغشٜٗ٩ 20 ٢ٯ٪ر٩ ٢ذٯٛ٩ ثبثشٚ٤ُٰ ٮٛ٪ ث٬ٜ٭ ه٪ذ رحز٣زٮ ٗبٛا
ًٰب٣بٯذ ا٠ع٩ِشٛت اٜغزحٟ حؼش.اد٪٤د صالس ع٩ذ ثحذ٣ب ٗب٨ب طالحٰبرٟثالعزٰ٘ٮ ا٩ٕ عٰذ٪ٜخزبسح ع٠ٛزشٰٗجخ اٛ ا٥ز٧ ١ عشٯبٚ٘ش٩
.٥ رحؼٰش٢ٟ ٝ٪ ٯ45 ٙ خال٦ٛ٩ رذاٞ ٯز١ ا٬ٜ ه١٪ضٯزٛصٯذ ا٩ وشثٮٛي ا٠ظٛن اٟ زغبسٯخٛبٯذ ا٠ٰع٩ِشٛ آشاص ا٢ٟ
Introduction
Frusemide which belongs to the suppositories to increase the drug liberation
group of loop diuretic is very effective with the use of non-ionic surfactants (solutol of
indraining all kinds of oedemas (of cardiac, HS 15, cremophor RH60 and montanox
hepatic or renal origin), in mild or moderate 60DF).(3) Frusemide adhesive micro-spheres in
hypertension or used in greater doses in acute hard gelatin capsules, frusemide granules with
and chronic renal failure, oliguria. (1) dika fat with maize starch and microcapsules
Commerrically available as tablets (20, 40, 80 of frusemide with Acrycoat E30 acrylic
and 500mg) and injection (10 and 20 mg/ml) polymer were prepared and evaluated in man
and frusemide oral solution which mentioned resulting in sustained release. (4) To the patients
in USP. (2) Many studies concered frusemide who have difficulty in swallowing the oral
to prepare it in defferent pharmaceutical liquid dosage forms (syrup, elixir, suspension
dosage forms as frusemide containing rectal and emulsion) and rectal are offen supplied.
1
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
2
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
previous mixture. Sorbitol 32.4ml and 5ml Table (2): Different formulas of frusemide
dispersion of sodium carboxy methyl cellulose prepared as suspension (A, B, C, D and E).
(1% w/v) were measured in graduated cylinder and emulsion (F).
and added to resulting product with stirring. formulas
After the product was filtered by cotton the Matarials
A B C D E F
cherry flavor was added. The volume was Frusemide (gm) 2.5 2.5 2.5 2.5 2.5 -
completed to 100ml by phosphate buffer 8.
Frusemide tablet 5
finally the pH was adjusted by using pH- (80mg)
meter.(8)
PVP (gm) 10
Xanthan gum 0.5 0.5 0.5
Frusemide elixir (formula III)
(gm)
0.8gm of frusemide was dissolved in
Sodium carboxy 0.5
10ml sorbitol plus 40ml ethanol. Then citric
methyl cellulose
acid 0.1 gm and sodium saccharin 0.08gm
(gm)
were mixed in 20 ml purified water puls 5 ml
Methyl cellulose 0.25
tween 20. Then the aqueous solution was
added to the alcoholic solution to maintain the (gm)
highest possible alcoholic strength at all times Chitosan (gm) 1.5
so that the minimal separation occurs when the Acacia (gm) 6
two solutions were completely mixed, the Olive oil (ml) 18
cherry flavor was added. Then the volume was Tween 20 (ml) 1
completed to 100ml by phosphate buffer 8. Glycerol (ml) 10
The elixir was permitted to stand for a few Sorbitol (ml) 5
hours to ensure saturation of alcoholic solvent. Cocoa syrup (ml) 20
The product was filtered by using talc as filter Methyl + propyl 0.18 +0.03
aid to prevent cloudy appearance. Finally the paraben (gm)
pH was adjusted by using pH-meter. (6) purified water 100 90
Phosphate buffer pH8 was prepared by mixing Qs(ml)
50ml of a solution of 0.2 M potassium
dihydrogen orthophosphate with 46.8ml of Dissolution rate measurement
0.2M sodium hyproxide then diluted to 200ml The dissolution medium was 900ml
with water.(2) of phosphate buffer 6.8. The temperature of
study was 370C and the rotating velocity was
Formulation of frusemide suspension 100 rev. min-1. 5 ml of each formulas A, B, C,
Table (2) shows 6 formulas of D and E was transferred to the jar bottom using
frusemide suspension prepared by the a syringe. At appropriate intervals samples of
following method: frusemide, methyl plus 5ml were taken from the jar and analyzed for
propylparaben, sorbitol and glycerol were total content of frusemide by UV-
Levigated in the mortar with tween20 and part spectrophotometer. Detection was done at
of prepared dispersions of suspending agents 330nm. 5ml of fresh phosphate buffer was
(PVP, xanthan gum, sodium carboxy methyl added to the jar with each time intervals to
cellulose, methylcellulose and chitosan) in keep the volume constant.(10)
different concentration as summarized in table
(2). The remaining amounts of the dispersions Sedimentation volume
were added in divided portions to the mixture. 100 ml of each formulas (A, B, C, D and
The mortar was rinsed several times with E) was transferred to the stoppered graduated
purified water and the rinsed volume of cylinder. The suspension were shaken
dispersion was added to cylinder, cocoa syrup vigorously to ensure uniformly then left
was added before the volume was completed to undistributed. The sedimentation volume was
100ml by adding purified water. measured at selected time intervals during
storage without agitation for a period of
(5)
Comparison studies of formulas A, B, C, D 8weeks and was calculated in terms of the ratio
and E of ultimate settled height (Vu) to the original
The following parameters were used to height (Vo).(11)
compare the prepared formulas A, B, C, D
and E. Extemporaneous preparation of frusemide
emulsion (formula F)
Acacia was triturated in mortar to be in
powder form. 12ml water was added to get
primary emulsion. 18ml olive oil was added
3
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
4
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
1.998
K40(x10-3) K50(x10-3) K60(x10-3)
1.996 Formulas
1.994 month-1 month-1 month-1
1.992
1.99
1.988
I 5.9 7.1 8.0
1.986
1.984
II 10.7 26 35
0 1 2 3 4 5
Time (months)
III 10.1 15 20
Figure (1): Degradation curve of frusemide
oral solution (formula I) at 40, 50, 60 0C
40 C 50 C 60 C 9
8
2.002
7
)
2
-1
(mounth
6
log % remaining
1.998
1.996 5
1.994 4
-3
1.992 3
kx 10
1.99
2
1.988
1
1.986
0
1.984
0 1 2 3 4 5 2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4
Figure (2): Degradation curve of frusemide Figure (4): Arrhenious plot for estimation of
syrup (formula II) at 40, 50, 60 0C the expiration date of formula I at 25 0C.
2.005
2
1.995 35
Log% remining
1.99 30
1.985 40
kx10-3(month-1)
25
1.98
50 20
1.975
1.97 15
60
1.965 10
1.96 5
1.955 0
0 1 2 3 4 5 2.9 3 3.1 3.2 3.3 3.4
Time (month)
3 -1
1/Tx10 (kelvin )
Figure (3): Degradation curve of frusemide Figure (5): Arrhenious plot for estimation
elixir (formula III) at 40, 50, 60 0C
of the expiration date of formula II at 25 0C
Table (3) summarized the degradation rate
constant of formulas I, II and III. Arrhenious
plots were constructed to predict the
degradation rate constant of frusemide at 250C
as shown in figures (4, 5 and 6). The results
indicate no significant differences (P>0.05)
between K250C for formulas (I, II and III). The
expiration data of frusemide was calculated
according to first order reaction equation:
0.104
t10% -----------------2
K 250 C
5
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
20 120
18
%dissolved of frusemide
16 100
)
14
-1
80
kx10 3 (month
12 A
10 60
8 B
6 40
4 C
2 20
0
0 Pol
2.95 3 3.05 3.1 3.15 3.2 3.25 3.3 3.35 3.4 y.
0 10 20 30 40 50 60
(B)
Pol
1/Tx10 3(kelvin-1) Time min
y.
(B)
Figure (6): Arrhenions plot for estimation of Figure (7) : Dissolution rate profile of
the expiration date of formula III at 25 0C frusemide formula (A, B and C)
120
The expiration dates were found to be equal to
% dissolved of frusemide
1.8, 1.07 and 1.22 years for formulas I, II and 100
III respectively as shown in table (4). 80
D
60 E
Table (4): Degradation rate constants at
F
250C and the corresponding expiration 40
dates of the prepared formulas. 20
0
K25(x10-3) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Formulas no. t 10% (years)
month-1 time (min)
II 8.09 1.07
1.2
1
Sedmentation volume
6
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
wetting and viscosity enhancing agents used. Then, the expiration date of frusemide
As a result, a high sediment volume observed suspension formula (A and D) which showed
in formula A could be a result of the increase the best release and sedimentation volume than
in the viscosity of the PVP dispersion in other formulas was calculated according to
alkaline media which may be caused by the apparent-zero order reaction equation
expansion resulting from electrostatic
repulsion between negatively charged 0.10[ A0 ]
carboxylate group. (17, 18) In formula D the t10% ----------- (6)
increase in viscosity due to strong cross- KO
linking between the nonionic gum
(methylcellulose) and anionic gum (xanthan
2.5
gum).(19) The presence of glycerol, sorbitol and
cocoa syrup adds to the viscosity of suspension 2
Log % remaining
especially cocoa syrup (chocolate syrup) which
40
is a suspension of cocoa powder in vehicle 1.5
containing liquid glucose, glycerine, vanillin 50
1
and sucrose. It is effective because of its high
viscosity and enhance the palatability by 0.5
60
d [c ]
k[c] -------- (4)
dt
16
is applied. 4
7
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Frusemide oral liquid dosage forms
600
FEBIGER Philadelphia. London 4th ed.,
-1
Shear rate (sec.
500
1993-286.
400 13- Qasem, J. G, Bummer, P.M and Digenis,
300 G. A, Kinetics of pactitaxel, Pharm
200 SciTech, 4(2), 2003,21.
100 14- Cohen, N., Golik, A., Effect of frusemide
0 oral solution versus frusemide tablet, J-
0 200 400 600 800 1000 1200 Miner Electrolyte Metab, 1996; 22(4); PP
-1
Shear stress (dyne cm ) 248-52.
15- Shihad, A.R., Mustafa, R.M., Effect of
Figure (12): Rheogram of the selected
polyethylene Glycol, Sodium lauryl sulfat
frusemide suspension formula (A)
and polysorbate- 80 on the solubility of
frusemide, International Journal of
pharmaceutics, 1979, 4, P 13-20.
References: 16- Ghebre, S., Isac, J., Solid pharmaceutical
1- Martindale, ―The extra pharmacopoeia‖ dispersion, united state patents, Oncology
31st ed., Royal pharmaceutical Society; out sourcing partner in development,
1996; P 871-874. 2004, (13), P 1-5.
2- British Pharmacopeia vol.2; 1993; P 784. 17- Eczacilik, B., Studies on furazolidone
3- Szilvia, B., Geza, R.J., Eszter, D., George, suspension formulation, Acta
F. and Istvan, In vivo and in vitro study in pharmaceutic Turcica, 1992. 13, 97-103.
rats of rectal suppositories containing 18- Kassem, M. A., Matha, A. G., Rheologic
Frusemide, European Jaurnal of studies on dispersion of
pharmaceutics and Biopharmaceutics, polyvinylpyrrolidone, pharmaceutical acta
May 2002, 53, Issue 3, P 311-315. helvetiae, 1970, 18-26.
4- Akiyama, Y., Nagahara, N. E., Kitano, M., 19- Walker, C. V., Wells, J. I., Rheological
Iwasa, S., Yamamoto, I., Azuma, J., synergism between ionic-nonionic gums,
Evaluation of oral mucoadhesive International Journal of pharmaceutics,
microspheres in man on the basis of the 1982, 11, P 309-322.
pharmacokinetics of frusemide, J-pharm- 20- Chesney, R. M, Patters, A., Acacia gum in
pharmacol; 1998; 50(2); 159-66. chronic renal failure; J-therapy, 2006,
5- Aulton, M.E., Pharmaceutics: The science 3(2), P 183-185.
of Dosage Form Design, 2nd ed., 2002, PP
242, 91-93.
6- Ansel, H.C., Allen, Jr.L., Popovich, N.G.,
pharmaceutical Dosage Forms and Drug
Delivery System, 2005, seventh ed., PP
338-342, 360-365.
8
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
Abstract
Anabolic androgenic steroids (AAS) are man-made derivatives of the male sex hormone
testosterone, originally designed for therapeutic uses to provide higher anabolic potency with lower
androgenic effects. Increasing numbers of young athletes are using these agents illicitly to enhance
physical fitness, appearance, and performance despite their numerous side effects and worldwide
banning. Today, their use remains one of the main health problems in sports because of their
availability and low price. The present study focuses on investigating the adverse effects of anabolic
androgenic steroid abuse on sex hormones, liver and renal function tests, fasting glucose levels and
lipid metabolism in Iraqi male recreational bodybuilders. We have recruited fifteen male bodybuilders
(age 19-32 years) and an equal number of healthy non-obese, non-AAS-using sedentary controls.
Serum hormones (luteinizing hormone (LH), follicle stimulating hormone (FSH), total testosterone,
and prolactin), liver function indices (serum alanine aminotransferase (ALT), aspartate
aminotransferase(AST), alkaline phosphatase(ALP), total and direct bilirubin), renal function
parameters (serum creatinine and urea), lipid profile and serum glucose levels were measured. Abuse
of AAS was associated with significant decreases (p< 0.005) in serum levels of LH (66.9%), FSH (49.8
%) and testosterone (63.7%) together with significant increases (p< 0.05) in prolactin concentrations
(49.8%) in AAS-using bodybuilders compared to sedentary controls. Anabolic androgenic steroids-
using athletes had significantly higher (p< 0.05) circulating levels of total bilirubin (116.3%), direct
bilirubin (127.6%), aspartate (1752.9%) and alanine (263.1 %) transaminases than those of sedentary
control subjects. Serum alkaline phosphatase levels were not significantly different (p> 0.05) between
the two groups. Concerning renal function, AAS-using athletes had significantly higher serum
concentrations of creatinine (28.6%) and urea (21.3%) than sedentary controls. Meanwhile, AAS abuse
was accompanied by atherogenic lipid profile. Anabolic androgenic steroids -using athletes had
significantly higher (p< 0.05) serum levels of triglycerides (TG) (45.6%), low density lipoprotein-
cholesterol (LDL-C) (26.0%) and very low density lipoprotein-cholesterol(VLDL-C) (45.6%) together
with significantly lower serum concentrations of high density lipoprotein-cholesterol (HDL-C)
(31.3%) than sedentary controls. Serum total cholesterol (TC) and fasting glucose concentrations were
not significantly different (p> 0.05) between the two groups. The results presented in the study confirm
that abuse of AAS induces unfavorable body functions and undesirable side effects. Therefore, efforts
should be sought against use of these compounds outside the therapeutic frame.
Key words: anabolic steroids, athletes, bodybuilding, exercise.
الخالصة
٩ ة٪ٰ ه٬ٜت هٌٜزٜٛ ًوذ خظٰظب٤ُ) طTestosterone( ٍ ثـ٩وش٠ٛزٗش٭ اٛ ا١٪ٟش٨ٛشزٔبد اٟ ٮ٧ بئٰخ٤جٛٯذاد ا٩غزٰشٛا
شٗجبد٠ٛ ا٥ز٧ ٚضٟ روبؿٮ٬ٜ ه١٪ٜ ٯٔج٢زٯٛ ا٢ٰٰشٯبػَٛ أهذاد ا١ذساعبد أٛ رزٗش ا.ائٮ٩غزحؼش د٠ٗ ) Testosterone( ـٛئ ا٩غبٟ
ػن٩ ٢ٟ ًٞشٛ ا٬ٜ ه٩ جٰخ٣غبٛب ا٨ أهشاػٙ٪ز٘شسح ح٠ٛزحزٯشاد اٛ ا٢ٟ ًٞشٛ ا٬ٜ ه٢ٰ٤غٛس ا٩ش٠ف ث٪حٜٟ رظشٯح ؿجٮ ُٮ رضاٯذ١٩ثذ
٥ز٨ٛ خبؿئٛ اٝ ٯوزجش االعزخذا.ب٨ ٯزبعش ث٩اد أ٪٠ٛ ا٥ز٧ ٬ ٯزوبؿ٢ٟ ٬ٜخ هٟثبد طبس٪ٰٔخ هٟ٪٘حٛبد ا٨غٛ ا٩ جٰخ٠ٛ٩بد اال٠ل٤٠ٛا
٥ز٧ رأصٰشاد روبؿٮٰٰٞٔزٛ ذ٠٠ ط٩ ذساعخٛ ا٥ز٧ اُعشٯذ.حذٯشٛوظش اٛشٯبػخ ُٮ اٛ اٚٗشبٟ ٞ٧ أ٢ٟ ٢ٰٰشٯبػٛ اٚ ٓج٢ٟ شٗجبد٠ٛا
غ٘ش ُٮٛ ا٫٪غزٟ ٩ ٝ٪شحٛصٯن ا٪ ر٫٪غزٟ ٩ ٬ٜ٘ٛ ا٩ ٘جذٛكبئَ ا٩ ٩ ٝذٛ اٚظٟ بد ُٮ٣٪ٟش٨ٛ ا٫٪غزٟ ٬ٜبئٰخ ه٤جٛٯذاد ا٩غزٰشٛا
٩ بئٰخ٤جٛٯذاد ا٩غزٰشٛ ا١٪) سٯبػٰبً ٯزوبؿ15( ٬ٜذساعخ هٛ ا٥ز٧ ذٜ٠ اشز.ٝ األعغبٙب٠ٗ بسعٮ سٯبػخ٠ٟ ٢ٟ هخ٪٠غ٠ٛ ٝذٛ اٚظٟ
٫٪غزٟ ٰٓبط٬ٜ هٰٰٞٔزٛذد ؿشّ ا٠ ئهز.بئٰخ٤جٛٯذاد ا٩غزٰشٛ ا١٪ ال ٯزوبؿ٩ ٝ األعغبٙب٠ٗ سٯبػخ١٪بسع٠هبً ال ٯ٪زـٟ )15(
Total and direct bilirubin, ( ٘جذٛكبئَ ا٩ ٩ )LH, FSH, Testosterone, and prolactin( ٝذٛ اٚظٟ بد ُٮ٣٪ٟش٨ٛا
ُٮٝ٪شحٛصٯن ا٪ ر٫٪غزٟ ٩ )S creatinine and S urea( ٬ٜ٘ٛكبئَ ا٩ ٩ )SAST, SALT, and alkaline phosphatase
٩بد رأصٰشاً ر٣جٰبٛ اٰٜٚش رح٨ أك.ٝذٛغ٘ش ُٮ اٛ رشٰٗض ا٬ٛ) ثبإلػبُخ ئTC, TG, HDL-C, LDL-C, and VLDL-C( ٝذٛ اٚظٟ
. ٝذٛ اٚظٟ بد ُٮ٣٪ٟش٨ٛ رشٰٗض ا٬ٜبئٰخ ه٤جٛٯذاد ا٩غزٰشٛزوبؿٮ اٛ اػحخ٩ ٯخ٪٤وٟ خ٠ٰٓ
9
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
ٯخ٪٤وٟ سح٪) ثظprolactin( ٫٪غزٟ ئسرِن٩ )LH, FSH, and total testosterone( ـٛ٭ ُٮ رشاٰٗض ا٪٤وٟ خِبع٣حق ئ٪ٛ حٰش
Total and direct bilirubin, SAST ( ـٛٯبد ا٪غزٟ اػحخ ُٮ٩ ٯخ٪٤وٟ ن صٯبدحٟ ٖٛ رشاُْ ر،بئٰخ٤جٛٯذاد ا٩غزٰشٛزوبؿٮ اٟ ٫ذٛ
ًذساعخ أٯؼبٛشد ا٨ أك.٢ٰهز٪٠غ٠ٛ ا٢ٰ) ثalkaline phosphatase( ـٛ ا٫٪غزٟ ٭ ُٮ٪٤وٟ ّد أ٭ ُش٪ع٩ ٝن هذٟ )and SALT
٫٪غزٟ ٭ ُٮ٪٤وٟ ئسرِبم٬ٛبئٰخ ثبإلػبُخ ئ٤جٛٯذاد ا٩غزٰشٛزوبؿٮ اٟ ٫ذٛ ٝذٛ اٚظٟ ) ُٮcreatinine and urea( ـٛ ا٫٪غزٟ ئسرِبم
٭ ُٮ٪٤وٟ ّالحلخ أ٭ ُشٟ ٞ ٯزٞٛ .ٝذٛ اٚظٟ ) ُٮHDL-C( ٫٪غزٟ ٭ ُٮ٪٤وٟ خِبع٣ ا٩ )TG, LDL-C, and VLDL-C( ـٛا
ٝ ئعزخذا١زبط أ٤ب اعز٤٤٘٠ ٯ،ذساعخٛ ا٥ز٧ ب٨زٮ أُشصرٛزبئظ ا٤ٛء ا٪ ُٮ ػ.٢ٰهز٪٠غ٠ٛ ا٢ٰ ثٝذٛ اٚظٟ غ٘ش ُٮٛ ا٩ ) TC( ٫٪غزٟ
ٙ ُٮ ثز٢٠٘ب ٯ٤اعج٩ .١غب٣ اإلٞكبئَ عغ٩ ٬ٜجبً هٜؼبهِبد رإصش عٟ ٙ٪ حظ٬ٛوالط ٯإد٭ ئٛبئٰخ خبسط ئؿبس ا٤جٛٯذاد ا٩غزٰشٛا
.والعٮٛشٗجبد خبسط االؿبس ا٠ٛ ا٥ز٧ ٝن اعزخذا٤ٟ ٩ أٰٜٚٔ رٚ أع٢ٟ د٪٨غٛ ا٫ٓظبس
Introduction
Self-administration of large doses of of use, and whether other supplements and
anabolic androgenic steroids ( AAS ) by drugs being used. Exclusion criteria included
athletes to obtain a well-shaped body and to smoking, alcohol consumption, presence of
increase muscular strength has been chronic medical conditions (diabetes mellitus,
increasingly noticeable since the 1950s.1,2 liver or kidney disorders) and the use of
Anabolic androgenic steroids are widely used growth hormone. Anabolic steroid abusers
by professional and recreational athletes as were selected if they were currently using
well as nonathletes.3 Abuse of AAS is not AAS. Table (1) summarizes AAS used with
limited to male adults but also reported in their doses and duration of use prior to sample
female adults as well as adolescents of both withdrawal. . All of the participants took
sexes.1 Every tissue in the body, including the androgens in cycles and none was taking AAS
brain, has androgen receptors; therefore, AAS in a continuous pattern. Cycles were 4-8 weeks
exert systemic as well as psychological in duration separated by suspension periods of
effects.4 Anabolic androgenic steroids have 4-12 weeks. A control group of healthy
been linked with a wide range of unwanted sedentary males (n=15) with a mean age of
adverse effects. These effects may range from 22.1 ± 3.65 years were recruited from the
physically unattractive, such as acne and community and historically had not ever used
gynecomastia in males, to serious and life anabolic steroids. Unfortunately, absence of
threatening, such as cardiovascular diseases bodybuilding controls was evident because, as
and hepatic carcinoma. Most effects are was found, persons who continue to exercise
reversible upon withdrawal.2,5 Because of their regularly use AAS routinely. The two groups
widespread use, many side effects may turn of volunteers were comparable with respect to
out to be significant risk factors when their age and height. However, the study group
considering public health. Increased risk of taking AAS had significantly greater mean
violent death was reported among AAS weight and BMI.
abusers from impulsive, aggressive behavior,
or depressive symptoms.6 Anabolic androgenic Sample collection
steroids have been taken in cycles. Subjects' weight and height were measured
Traditionally, AAS users combine two or more using a balance beam and a vertical ruler.
different drugs, mixing oral and injectable Participants were asked to fast for 12 hours
AAS. They begin a cycle with a low dose of and avoid heavy physical exercise before
AAS and slowly increase the dose and then the attending for sample collection. One blood
dose is tapered to zero.3 Doses taken by sample was collected from each volunteers by
abusers can be 10-100 times higher than those venipuncture between 08:00-10:00 AM. A total
used for medical purposes. The aim of the of 15 ml blood was obtained and placed in
present study was to evaluate the changes in EDTA-free tubes to be centrifuged for 5-10
serum sex hormones, liver and renal function minutes at 3000 rpm. Serum was then divided
indices, glucose level and lipid metabolism in into several 1.5 mL Eppendorf tubes and
Iraqi male anabolic androgenic steroid abusers. stored at (-20oC) until time for the assay.
Laboratory measurements
Material,Subjects and Methods Serum concentrations of LH, FSH, prolactin
Participants and total testosterone were determined by
Fifteen non-obese male bodybuilders aged immunofluorometric assays on a mini VIDAS
19-32 years (mean 23.27 ± 3.73) were analyzer 7,8 . Liver and renal function indices
recruited at local gyms in Baghdad city . were measured by colorimetric methods using
Bodybuilders were interviewed concerning the commercially available kits9,10,11,12,13,14,15.
their health (current diseases and family Serum total cholesterol, triglyceride, high-
diseases), consumption of high protein diet, density lipoprotein cholesterol and fasting
regular exercise, lifetime steroid abuse, pattern glucose concentrations were determined by
10
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
Table 1 : Dose and duration of AAS use for fifteen body builders
Total
Duration of
Subject Route of Dose dose
AAS used prior
no, administration (mg/kg) received
sampling(wk)
(mg)
1 Methandrostenolone O 175 6 1350
Nandrolone decanoate P 50
2 Methandrostenolone O 140 4 660
Nandrolone decanoate P 25
3 Methandrostenolone O 140 4 760
Nandrolone decanoate P 50
4 Methandrostenolone O 245 4 980
5 Methandrostenolone O 210 4 840
6 Testodterone proponate P 50 3 450
Nandrolone decanoate P 100
7 Mthenolone O 20 3 660
Oxymetholone O 150
Nandrolone decanoate P 50
8 Methandrostenolone O 175 6 1350
Nandrolone decanoate P 50
9 Methandrostenolone O 140 4 760
Nandrolone decanoate P 50
10 Methandrostenolone O 175 6 1650
Nandrolone decanoate P 100
11 Methandrostenolone O 245 4 2780
Sustanon P 250
Nandrolone decanoate P 200
12 Methandrostenolone O 175 4 1800
Nandrolone decanoate P 25
Sustanon P 250
13 Methandrostenolone O 175 4 1100
Nandrolone decanoate P 100
14 Methandrostenolone O 245 6 2670
Nandrolone decanoate P 200
15 Methandrostenolone O 245 6 2670
Nandrolone decanoate p 200
11
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
Sedentary AAS-using BB
Controls N=15
Table 3: Effects of anabolic androgenic Variable
N=15 (mean ± SD)
steroids on liver function tests in AAS-using (mean ± SD)
bodybuilders compared to sedentary controls.
Cr
0.84 ± 0.19 1.08 ± 0.21**
(mg/dL)
.Variable Sedentary AAS-using BB
Controls N=15 Urea
N=15 (mean ± SD) 38.07 ± 8.58 46.18 ± 14.37*
(mg/dL)
(mean ± SD)
Values expressed as mean + SD
Total
The P- values refer to the differences from the
bilirubin 0.49 ± 0.63 1.06 ± 0.74*
control group.
(mg/dL)
* : P < 0.05 significant difference between the
Direct
two groups.
bilirubin 0.29 ± 0.41 0.66 ± 0.47*
** : P <0.005 highly significant difference
(mg/dL)
between the two groups.
ALP N = no. of subjects.
80.20 ± 20.26 93.20 ± 37.19 ns
(U/L)
AST
1.21 ± 4.69 22.42 ± 27.02**
(U/L) Lipid profile and fasting serum glucose
ALT Circulating levels of HDL-C were
4.69 ± 6.52 17.03 ± 17.02* significantly lower (p< 0.005) (31.3%) in
(U/L)
Values expressed as mean + SD AAS-abusing bodybuilders than sedentary
The P- values refer to the differences from the controls. Table (5) indicates that AAS-using
control group. bodybuilders had significantly higher (p< 0.05)
* : P < 0.05 significant difference between the serum levels of triglycerides (45.6%), LDL-C
two groups. (26.0%) and VLDL-C (45.6%) than sedentary
** : P <0.005 highly significant difference controls. Serum total cholesterol and glucose
between the two groups. concentrations were not significantly different
ns
: non significant (P > 0.05 ) significant
(p> 0.05) between AAS-using bodybuilders
difference between the two groups and sedentary control subjects.
N = no. of subjects.
12
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
Table 5: Effects of anabolic androgenic adverse effects which affect almost all organs
steroids on lipid profile and serum glucose and systems of the human body. Anabolic
in AAS-using bodybuilders compared to androgenic steroids-using bodybuilders had
sedentary controls significantly lower (p< 0.005) serum levels of
LH, FSH and total testosterone than sedentary
Variable Sedentary AAS-using BB controls (table 2). The results were consistent
Controls N=15 with those reported by Holma et al21 who
N=15 (mean ± SD) observed reduced serum levels of LH, FSH
(mean ± SD) and testosterone in athletics during a course of
Cholesterol 153.80 ± 21.62 171.20 ± 37.19 ns oral intake of methandrostenolone (15
(mg/dL) mg/day). Exogenously administered anabolic
TG 74.93 ± 42.84 109.13 ± 57.50* androgenic steroids exert a negative feedback
(mg/dL) on the secretion of gonadotrophins, mostly
HDL-C 44.60 ± 7.15 30.67 ± 7.64** attributed to a direct effect on the
(mg/dL) hypothalamus to decrease secretion of GnRH.
LDL-C 94.21 ± 21.13 118.71 ± 34.76* This in turn causes a corresponding decrease in
(mg/dL) secretion of both LH and FSH and eventually
biosynthesis and release of testosterone from
VLDL-C 14.99 ± 8.57 21.83 ± 11.50*
the testes.22 In addition, anabolic androgenic
(mg/dL)
steroids may produce local suppressive effects
FSG 83.20 ± 17.63 89.93 ± 9.16ns
on the testes and on adrenal androgen
(mg/dL)
production.23 Serum prolactin levels in AAS-
Values expressed as mean + SD using bodybuilders were significantly higher
The P- values refer to the differences from the than those in sedentary controls (p < 0.05)
control group. (table 2). Data reported by Stoffel-Wagner et
*: P < 0.05 significant difference between the al24 and Leibenluft et al25 were consistent with
two groups. the interpretation that testosterone and/or its
**: P <0.005 highly significant difference metabolites facilitate the secretion of prolactin.
between the two groups. Estrogen is known to stimulate prolactin
ns
non significant (P > 0.05 ) significant release from the anterior pituitary.26 Non-
difference between the two groups aromatizable AAS (stanozolol and
N = no. of subjects. methandrostenolone) were reported to activate
estrogen receptors through interaction of either
Adverse effects the parent compound or its metabolites
Participants were asked questions about indicating a possible mechanism for increased
unusual adverse effects that would be felt prolactin secretion.27 The available data in the
during an AAS cycle and the most common corresponding literature on the influence of
reported side effects were aggression, changes exogenously administered androgens on
in libido, acne formation, headaches, and prolactin serum level were found controversial.
premature hair loss as summarized in table 6. Serum total and direct bilirubin levels in AAS-
using bodybuilders were significantly higher
Table 6: Adverse effects reported by AAS- (p< 0.05) than those in sedentary controls
using bodybuilders. (table 3). Androgens can selectively interfere
with bile excretion by the liver. Canalicular
Adverse effect No. of bile plugs were observed after treatment with
Subjects % methyltestosterone, oxymetholone, mestranol,
N=15 and norethandrolone.28 Cases of cholestatic
Unusual aggression 8 53 jaundice have been recorded in patients
Changes in libido 6 40 therapeutically using or athletes abusing AAS
Acne 3 20 (especially 17 alkylated agents).29,30 Serum
Headaches 4 27 AST and ALT levels in AAS-using
bodybuilders were significantly higher (p<
Hair loss 2 13
0.005 and p< 0.05, respectively) than those in
sedentary controls (table 3). Canalicular
Discussion cholestasis is characterized by mild
Subjects of this study use independently hepatocellular injury and release of
anabolic androgenic steroids mainly to transaminases leading to mild elevations in
enhance external physique. Besides being an serum levels of these enzymes.29 However,
unethical practice, abuse of AAS has been since sustained weightlifting alone can result
associated with several health risks and various in mild elevations in serum transaminase
13
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
activities31,32, the increase in serum levels was found between the two studied
transaminases may be attributed to mild groups (p > 0.05) (table 5). Our results confirm
hepatocellular damage, muscle injury, or both. those reported by many studies40,42 . Anabolic
Urhausen et al33reported that serum androgenic steroids effects on plasma lipids
transaminase levels were significantly higher have been reported to be unpredictable and
(p< 0.001) in anabolic androgenic steroid- depend on the dose, route of administration,
abusing athletes than bodybuilders who and type of AAS (aromatizable or not).46 Low
stopped using anabolic steroids for at least a dosages have been associated with
year. A non-significant difference in serum hypolipemic response, while high doses have
ALP levels was found between the two studied had opposite effects.47,48 Serum HDL-C levels
groups (p > 0.05) (table 3). These results are in AAS-using bodybuilders were significantly
consistent with those reported by O'Sullivan et lower than those in sedentary controls (p <
al34 who observed no significant difference in 0.005) (table 4). The postulated mechanism to
alkaline phosphatase activities between explain anabolic steroid-induced alteration in
anabolic steroid users and potential or past serum HDL-C levels is an increase in hepatic
users. Anabolic androgenic steroids can induce triglyceride lipase activity, an enzyme
cholestasis without elevating alkaline responsible for catabolizing HDL with its
phosphatase levels. ALP activity is usually less phospholipase activity.49 In addition,
than threefold elevated and often is normal.35 apolipoprotein A-1, a major component of
Anabolic androgenic steroids -using athletes HDL particle, was reported to be decreased by
had significantly higher serum creatinine (p< AAS.42,45 The results obtained in the present
0.005) and urea (p< 0.05) levels than sedentary study showed absolute consistency with the
controls (table 4). Studies in rat models available data. A non-significant difference in
provide evidence that, compared with females, serum glucose levels was found between the
aging males exhibit decreased glomerular two studied groups (p > 0.05) (table 5). The
filtration rate and develop glomerular injury, influence of testosterone and anabolic steroids
glomerulosclerosis and proteinuria.36 In on glucose metabolism was found
addition, cases of acute renal failure had been controversial. Results of the present study
reported in clinical patients or bodybuilders agree with those reported by Friedl et al50 who
administering anabolic steroids.29,37 However, observed no alterations in fasting serum
in the present study, we cannot ignore other glucose in normal men treated with
factors that may have participated in testosterone enanthate or nandrolone decanoate
deteriorating renal function parameters in for 6 weeks. On the other hand, Cohen and
anabolic androgenic steroid-using athletes e.g. Hickman51 concluded that power lifters taking
consumption of high protein diet. Serum high dose (mean 200 mg/day) of anabolic
concentrations of triglycerides, VLDL-C and androgenic steroids had diminished glucose
LDL-C were significantly higher in AAS- tolerance compared to non-steroid using
useres (p< 0.05) than those in controls (table athletes, obese sedentary men, or non-obese
5). The rise in serum levels was positively sedentary men. Such controversy in the
correlated with the intake of AAS. Anabolic corresponding literature may be explained to
androgenic steroids can elevate serum levels of be due to differences in doses used. Higher
triglycerides by 40-50% in bodybuilders and AAS doses reduce insulin sensitivity and
other power-training athletes.38 Kiraly39in 1988 impair glucose tolerance.46 Although AAS
reported similar results while studying the doses used by subjects in the present study
effects of large doses of testosterone and other were considered to be high; they were much
anabolic androgenic steroids on serum lipids smaller than those used by athletes in Cohen
during a 12 week strength-training period. and Hickman51 study. The most common side
Elevated serum triglyceride (p< 0.05) levels effects reported by our subjects were unusual
were found with decreased serum HDL-C (p< aggression (53%), changes in libido (40%),
0.005). Serum LDL-C levels were significantly acne formation (20%), headaches (27%) and
higher (p< 0.05) during steroid intake in premature hair loss (13%) (table 6). Perry et
studies reported by Fröhlich et al40 and Palatini al52 reported that anabolic steroid using
et al41. Anabolic androgenic steroids -users weightlifters were more aggressive than
had elevated levels of apolipoprotein B, a nonusers according to different psychiatric
component of both LDL and VLDL.42,43 scores. Changes in libido appear to be the most
Conversely, circulating levels of LDL-C and common adverse effect reported in a group of
VLDL-C were not significantly different while present and past AAS users (approximately
using AAS in studies reported by Sader et al44 61%).34 Reports do indicate that toward the
and Singh et al45, respectively. A non- end of AAS cycle, some males may experience
significant difference in serum total cholesterol loss of libido.53 Acne was also found very
14
Iraqi J Pharm Sci , Vol.17 (2) ,2008 Abuse of Androgen
common side effect among anabolic steroid Bell ET. 4TH edition , Churchill
users as reported by O'Sullivan et al.34 Livingston. New York . 1976 , 293- 332 .
Increases in acne formation is related to 9. Kaplan AL, Pesce AJ ( eds) . Bilirubin .In
stimulation of sebaceous glands resulting in a : Clinical Chemistry : Theory, Analysis
more oily skin.34 Premature hair loss does not and Correlation . 3rd edition , The CV
appear to be very common. It is likely that Mosby Co . St Louis . Toronto.
androgenic alopecia as a result of AAS use is Princeton. 1984 ; 1238 – 1241 .
more prevalent in males who are genetically 10. Malloy HT , Evelyn KA . The
predisposed to balding.54 Headaches are also determination of bilirubin with the
not very common among AAS abusers. photoelectric colorimeter . J Biol Chem.
O'Sullivan et al34 reported only 9% of AAS 1937; 112 (2) : 481-491.
using athletes may develop headaches. 11. Reitman S , Frankel S. As cited by
However, the exact mechanism is unknown. BioMerieux kit ( France ) . Am J Clin
Path . 1957; 28: 56 .
Conclusion 12. Litwack G. Biochemistry of Hormones
In conclusion, anabolic androgenic steroid II : Steroid hormones . In : Textbook of
abuse lowered serum concentrations of Biochemistry with clinical correlations.
pituitary gonadotrophins, LH and FSH, and Devlin TM (ed) . 3rd edition , Wiley J
testosterone. Increased levels of prolactin were and Sons. 1992, p 901 – 925.
also manifested. Abuse of anabolic steroids 13. Bessey OA, Lowry OH, Brock M J . A
probably causes cholestasis, however, with method for the rapid determination of
mildly elevated liver enzymes. In addition, alkaline phosphatase with five cubic
effect of anabolic androgenic steroids on renal millimeters of serum . J Biol Chem .1946:
function indices was not well established 164: 321-329.
indicating that other factors, such as high 14. Jaffe MZ. As cited by Thermo Electron
protein diet, may have contributed in elevation Corporation kit ( Finland ). Physiol
of blood urea and creatinine levels. Finally, Chem. 1886: 10 : 391- 400 .
lipid profile was impaired toward evidenced 15. Fawcett JK, Scott JE. A rapid and precise
dyslipidaemia. method for the determination of urea . J
Clin Path . 1960; 3: 156-159.
16. Richmond W. Preparation and properties
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2. Rogol AD, Yesalis CE. Anabolic- 17. Fossati P, Principe L. Serum triglyceride
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5. NIDA Research Report – Steroid abuse 19. Passey RB , Gillum RL, Fuller JB, et al .
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48. Khaw KT, Barrett-Connor E. Endogenous 52. Perry PJ, Kutcher EC, Lund BC, et al.
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17
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
Abstract
Loratadine is a long acting non-sedating anti-histaminic agent that was developed for the
treatment of seasonal allergic rhinitis, whose anti-histaminic action is more effective than the other
anti-histaminic drugs available commercially. This project was carried out to prepare an acceptable
suspension through studying the release of drug in presence of different types and concentrations of
suspending agents such as polysorbate 40, xanthan gum, sodium carboxymethylcellulose (NaCMC),
aluminum magnesium silicate (veegum) and sodium alginate. The effects of these suspending agents
were studied at pH 1.2 (0.1N HCl) and 37 ْC. The results showed that the release rate of loratadine in
the presence of these suspending agents was dependent on their types and concentrations. The results
showed that loratadine release from the formula prepared from xanthan gum is more than that prepared
from other polymers in the following order: Sodium alginate < NaCMC < veegum < xanthan gum.
However, elegancy of suspension was better on using xanthan gum in a concentration of 0.5%. The
obtained results were utilized to formulate 0.1% suspension of loratadine which is physically stable
with an optimum drug release. The rheology, sedimentation volume, resuspendability and expiration
date were evaluated for the selected formula. The formula that contains loratadine, xanthan gum,
glycerol, sorbitol, methyl paraben, propyl paraben, sodium edetate, raspberry flavor at pH 5.0 appears
to be a promised formula to be present with estimated shelf life of about 3.8 years.
Key word: loratadine, suspension, suspending agent, xanthan gum.
الخالصة
١ ا٩ ٰخ٠ع٪٠َٛ ا٣والط حغبعٰخ االٛ َ اٗزش, وبط٤ٜٛ غجتٟ ًٰش٩ ٙ٪ِو٠ٛ اٚٯ٪ ؿ٢ٰٟغزب٨ٜٛ ؼبدٟ ه ٔبس٢ساربدٯ٪ٜٛا
بحٰخ٤ٛ ا٢ٟ ٙ٪ٔجٟ ْٜوٟ زحؼٰشٛ جحشٛزا ا٧ ئعشاءٞ ر.ُشح رغبسٯب٪ز٠ٛ ا٫ام االخش٪٣ اال٢ٟ ٰخٜ اٗضش ُبه٢ٰٟغزب٨ٜٛ ؼبد٠ٛ ا٦ٛ٪ِوٟ
ي٠ ط,40 سثبد٪ ع٬ٛ٪جٛ ا:ٚضٟ ثوذح رشاٰٗض٩ ٔخٜو٠ٛاد ا٪٠ٛ ا٢ٟ ِخٜخزٟ ام٪٣د ا٪ع٪اء ث٩ذٛ دساعخ رحشس اٙ خال٢ٟ ٰخ٣ظٰذالٛا
زبصٰشادٛزح ا٧ ذ دساعخ٠ ر.ٝ٪دٯ٪ظٛبد ا٤ٰغٛ ا٩ )ٝ٪ٰ٤٠ٛ اال٩ ٝ٪ٰغ٤ٌ٠ٰٛ٘بد اٜ (عٝ ُٰ٘ب,ٝ٪دٯ٪ظٛص ا٪ٰٜٜ عٰٚضٟ ٬ٗغ٪ ٗبسث,١ضب٣ضاٛا
٫٪ رحز٬زٛزشٰٗجخ اٛ ا٢ٟ ٢ساربدٯ٪ٜٛ رحشس ا١ ا٬ٛزبئظ ا٤ٛ اشبسد ا.ْٝ 37 ثذسعخ حشاسح٩ ٠,١ سٯٖ هٰبسٯخ٪ٜٗ٩ٰذس٨ٛغ اٟ حبٙ٪ٜح٠ث
> ٝ٪دٯ٪ظٛبد ا٤ٰغٛ ا:٬ٛزبٛ اٚ٘شٛ ثب٩ ٔخٜو٠ٛاد ا٪٠ٛ ا٢ٟ ٫ام االخش٪٣ اال٬ٜ ه٫٪زٮ رحزٛزشاٰٗت اٛ ا٢ٟ اعشم١ضب٣ضاٛي ا٠ ط٬ٜه
بدحٟ ٬ٜ ه٫٪ رحؼٰش طٰي رشٰٗجٰخ رحز٬ُ زبئظ٤ٛ ا٥ز٧ ٢ٟ االعزِبدحٞ ر.١ضب٣ضاٛي ا٠ ≥ طٝ > ُٰ٘بٝ٪دٯ٪ظٛص ا٪ٰٜٜ عٰٚضٟ ٬ٗغ٪ٗبسث
ْٜو٠ٛ ا١ ٰٓبط عشٯبٙ خال٢ٟ ٰٰٞٔزٜٛ اعزٔشاسٯخٚ اُؼ٩ اء٩ذٜٛ رحشس٢ اهـذ احغ٬زٛظٰي اٛ اخزٰشد ا. ٪ ٠٫١ ثزشٰٗض٢ساربدٯ٪ٜٛا
٬ٜ ه٫٪ رحز٬زٛ ا٬٧ رشٰٗجخ٢ احغ١عذ ثب٩ ٔذٛ . رشٰٗجخٚظالحٰخ الُؼٛ ربسٯخ ا٩ ٦ْ٘ ثوذ رحشٯٜو٠ٛظ ا٣ رغب٩ رشعتٞ حغ٩
ذ االط٤د ه٪زٛخ ا٨٘٣ ٩ ٝ٪دٯ٪ظٛ ا٬بئ٤ اٯذٯزٰذ ص,٢ٰ ثبساثٰٚث٩ ثش,٢ٰ ثبساثٰٚضٟ ,ٙ٪سثٰز٪ ع,ٙ٩ٰغٰشٜٗ ,١ضب٣ضاٛي ا٠ ط, ٢ساربدٯ٪ٜٛا
.اد٪٤ ع٨٫٣ د٩ ثحذ١حؼش ُ٘ب٠ْٛ اٜو٠ٛذح طالحٰخ اٟ بٟ ا. 5 ٬٤ٰع٩ٰذس٨ٛا
Introduction
A coarse suspension is a dispersion of
finely divided, insoluble solid particles (the preferred over the solid dosage form of the
dispersed phase) in a fluid (dispersion medium same drug because of the ease of the
or continuous phase) (1).A conventional swallowing liquids and flexibility in the
suspension may be readily prepared in an administration. The disadvantage of
aqueous solution with small percentage of disagreeable taste of certain drugs (loratadine
hydrophilic polymers (like methyl cellulose, bitter taste) is overcome when the drug is
hydroxypropylcellulose and xanthan gum), as administered as undissolved particles (3). In
well as small percentage of surfactant like general, aqueous suspension gives more
polysorbate 80. The suspending agent was extended effect than aqueous solution (4).
used to achieve homogeneity of redispersed Insoluble or poorly soluble drugs in suitable
suspension while surfactants are used for solvents could be formulated as flavored
wetting and dispersing of insoluble particles suspension as an ideal choice, example;
(2)
.For many patients, the liquid dosage form is nalidixic acid suspension (5) and fluconazole
(6
suspension
1 Corresponding author E-mail : drhaha1971 @ Yahoo. com
Received : 2/ 6/2008
Accepted : 26/10/2008
18
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
19
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
Table (1): Different formulations of paper. Fresh dissolution medium was added to
loratadine as suspension dosage form the jar each time to replace withdrawn
represented as % (w/v) samples. Each sample was suitably diluted and
assayed spectrophotometrically at 278 nm for
Materials Formula number loratadine content.
Measurement of rheograms:
A B C D E
Rheograms was obtained at 25 ْC
Loratadine 0.1 0.1 0.1 0.1 0.1
using Brookfield viscometer.
Xanthan 0.5 0.3 0.3 Sedimentation volume measurement
gum Fifty milliliters of each suspension was
SCMC 0.5 0.5 diluted with distilled water to a volume of 100
Veegum 2 0.5 ml in a stoppered graduated cylinder. The
Polysorbate 1.25 suspensions were shaken vigorously to insure
40 uniformity, and then left undisturbed. The
Methyl 0.2 sedimentation volume was measured every 4
paraben hours for period of 48 hours (17).
Propyl 0.02 Resuspendability of suspension
paraben The efforts required to convert the
Glycerol 5 sedimented system to homogenous suspension
upon shaking the cylinder manually, were
Sorbitol 7
rated with a ranks as follows: resuspendable,
Disodium 0.1
resuspendable with difficulty or not
edetate
resuspendable (18).
Raspberry 0.05 Stability study
flavor The accelerated stability study was done
Final 100 ml in order to determine the expiration date of the
volume selected formula. The suspension was
centrifuged to get the supernatant solution; 5ml
Each formula was prepared as follows: samples of the resultant solution were stored in
Loratadine, methyl paraben, propyl paraben several closed amber glass containers at 35, 45
were levigated in a mortar with glycerol and and 55 ْC for 90 days. Samples were inspected
the prepared dispersion of suspending agent. for change in color, odor, pH, precipitant and
The mixture was triturated with a pestle until a assayed for drug content at suitable time
smooth paste was formed. With continuous intervals (0, 10, 20, 30, 45, 60 and 90) days.
trituration, the paste was diluted with the
remaining amount of the dispersion of the
suspending agent then transferred to graduated Results and Discussion
cylinder. Finally, the required amount of Effect of type and concentration of
disodium EDTA and sorbitol were dissolved in suspending agent on the drug release:
a small portion of water and added to the The effect of various types of
graduated cylinder and raspberry flavor was suspending agents (surfactants and polymers)
added and water to make the final volume. The on the dissolution rate of loratadine was
suspension was shaken and the pH was investigated.
adjusted to 5 with few drops of 5M sodium 120
citrate.
100
% loratadine released
80
In Vitro Evaluation of Suspension
Dissolution rate profile: 60
20
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
% loratadine released
0.5% < 2.0% 1.25% It was seen that
80
increasing polysorbate 40 concentration from
0% to 0.5% and 1.25% (w/v) enhance the drug 60
% loratadine released
hand figures 2, 3 and 4 illustrate the effect of 100
anionic hydrophilic polymers including
xanthan gum, NaCMC and sodium alginate on 80
the dissolution rate of loratadine using
60
different concentrations of these polymers. The
results indicated that the percent of drug 40 0% (w /v)
release were as follows: 0.5% (w /v)
Xanthan gum 0.7% < 0.0% < 0.3% < 0.5% 20 1% (w /v)
NaCMC 0.0% < 1.0% < 0.25% < 0.5% 2% (w /v)
Time (min.)
120
100
Figure (4): Effect of sodium alginate
% loratadine released
20
0.5% (w /v) loratadine, since these polymers behave as
0.7% (w /v) protective colloid by coating the solid
0 hydrophobic particles with multimolecular
0 10 20 30 40 50 60
layer. This imparts hydrophilic character to the
Time (min.)
solid and thus promotes wetting (1), but to
Figure (2): Effect of xanthan gum certain limits, then the dissolution rate of
concentration on the percent of loratadine loratadine decreased by increasing xanthan
released in 0.1 N HCl pH (1.2) at 37 ٥C. gum and NaCMC above 0.5% (w/v), this result
may be attributed to the increase in viscosity of
the prepared formula (21, 22). Moreover the
results showed that the presence of sodium
alginate retarded the dissolution rate of
loratadine at all concentrations used. This
result is related to the formation of higher
viscosity regions due to the hydrated polymer
surrounding drug particles which encounter
high resistance to the dissolution (23). On the
other hand, the effect of veegum on the
dissolution rate of loratadine is shown in
figure(5). 0.0% < 2.0% < 1.5% < 0.5%
21
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
120
prepared suspension; sorbitol and glycerol as a
sweetening agent. They produce pleasant taste,
% loratadine released 100 less viscous suspension and are better than
sucrose in producing structure in vehicle
80
suspension (28). Disodium edetate was involved
60 in the formulation to protect loratadine from
deterioration (29). Raspberry flavor as flavoring
40
0% (w /v)
agent, methyl paraben and propyl paraben
0.50%(w /v)
20 1.5% (w /v)
were added as preservatives. The pH of the
2% (w /v) formula was adjusted to 5 using 5M sodium
0
0 10 20 30 40 50 60
citrate.
Time (min.) In vitro evaluation of suspension:
dissolution rate profile:
Figure (5): Effect of veegum concentration Table (2) and figure (6) showed the
on the percent of loratadine released in 0.1N dissolution rate profiles of the prepared
HCl pH (1.2) loratadine suspensions. The data indicated that
formula A had the highest dissolution rate
This retardation in dissolution of constant as compared with the other formulas,
loratadine as the concentration of veegum since the rate constant of formula A was 24.3 ×
increases may be attributed to adsorption of 10-3 (mg1/3 /min) using Hixson-Crowell root
loratadine on veegum or solid particle films equation, that expresses the dissolution rate of
formed around the drug particles (24). solid particle based on the cube root of the
Formulation of the loratadine suspension: weight of the particles (30), this equation was as
Xanthan gum was used as a suspending follows: Wο1/3 – W1/3 = Kt
agent in concentration of 0.5% to prepare Since: Wο=the original mass of the drug
formula A. This concentration gave the best particles
release as mentioned previously. It is an W = the mass of drug dissolved
effective flocculating agent at relatively low K = the cube root dissolution rate constant
concentration and has excellent suspending T = the time required to dissolve (W) mass
properties to suspend solid (25). The rheological of drug particles
stability of xanthan gum toward pH changes
encountered during transit through the Table (2): The calculated dissolution rate
gastrointestinal tract in addition to large constants of loratadine in the prepared
sedimentation volume presumably by polymer formulas.
bridging phenomena providing reasons for its
use (26). NaCMC was incorporating in formula Formula K × 10-3 (mg1/3 / min)
B in concentration of 0.5% and enhanced the
dissolution rate of loratadine, but its aqueous Formula A 24.3
dispersion has low viscosity and produced Formula B 15.0
sediment layer that was easily redispersed by Formula C 21.3
shaking. Farther more, a combination of
Formula D 21.2
xanthan gum and NaCMC (formula C) resulted
in too viscous suspension which has less Formula E 21.5
flexibility when was pouring. Veegum was
chosen in a concentration of 0.5% (formula D),
since this concentration gave the best release 110
but this suspension had low viscosity. The
100
linear branched chain molecules of veegum
90
% loratadine released
22
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
80 sedimentation
Easily
60 D 0.9
resuspended
40
Resuspendable
E 0.4
20 with difficulty
0
0 10 20 30 40 50
Table (3) shows the sedimentation volume and
-1
Shear rate (S )
Resuspendability of the prepared formulas.
The data indicated that the formulas prepared
Figure (7): Shear rate dependency of the with xanthan gum (formula A, C and D) had
viscosities of the prepared formulas of sedimentation volume almost equal to 1 i.e., no
loratadine suspension at 37 ْC. sedimentation was occurred during the test
period. The obtained results attributed to the
network of flocs formed in the suspension
The results also illustrated that all the which is so loose and fluffy that can extend
prepared formulas exhibited pseudoplastic through out the extra vehicle (31). Xanthan gum
flow which evidenced by shear thinning and is often used as flocculating agent to achieve
increase in shear stress with increased angular non-sedimenting suspension of drugs with no
velocity. This behavior due to the suspending need to other adjuvant(18). However, formula B
agent used, because as a general rule, the had a sedimentation volume equal to 0.6 and
pseudoplastic flow is exhibited by polymer in was easily resuspended by shaking. This low
solution(31). The rheological properties of the sedimentation volume may be due to the type
prepared formulas revealed that formula A was of the suspending agent used, since NaCMC
the best one which offered ease of pouring and may form homogenous network in all
swallowing compared with formula B and E concentrations used (23). On the other hand,
which they are less viscous and could not formula E, which showed sedimentation
obtain the structured suspension with them. during the test period, had low sedimentation
On the other hand, higher viscosity was volume of 0.4 and was resuspendable with
obtained with formula C and D with a difficulty, so it is pharmaceutically
difficulty in the pouring and swallowing from unacceptable.
the bottle orifice. This was a result of using a Stability study:
combination of two polymers (xanthan gum + Formula A was chosen for the stability
NaCMC) and (xanthan gum + veegum) study as the promising formula since it gave
respectively which leads to rheological the optimum physical stability and remarkable
synergism to be occurred due to stronger cross- release profile. The stability study carried at
linking between the two polymers used, where moderate exaggerated temperatures (35, 45
the presence of carboxyl groups on NaCMC and 55 ْC) to predict the expiration date of the
and xanthan gum promote stronger hydrogen promised formula. The degradation of
bonding between them (32). loratadine followed apparent zero-order
Sedimentation volume and resuspendability: kinetics, since the concentration in solution
The sedimentation volume (F) is the depends on the drug solubility. As loratadine
ratio of the ultimate height of the sediment as decomposes in solution, more drug is released
suspension settles in a cylinder (Hμ) to the from the suspended particles so that the
initial height of total suspension (Ho). concentration remains constant (30). The
F = Hμ / Ho resultant solution from centrifuging formula
While the Resuspendability is a quantitative (A) was with no reservoir of loratadine to
test to evaluate the ease of redispersion of a replace that depleted so loratadine degradation
suspension after a long period of standing (30).
23
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
K× 10 (day )
-1
were K0 is the apparent zero-order rate 0.1
-3
constant, [A] is the solubility of loratadine at 0.0749
45 C
2 55 C The expiration date was found to be
equal to 3.8 years. The formula show good
1.995 physical stability, as there was no
1.99
discoloration, precipitation or any other
physical changes after the storage period. The
1.985 pH of the formula was 5.0 for whole the
1.98
period.
Conclusions
1.975
0 10 20 30 40 50 60 70 80 90 100
A stable suspension of loratadine could
Time (days)
be prepared and used efficiently using xanthan
gum as a suspending agent (Formula A), since
Figure (8): Accelerated stability study of it provided an easily pourable suspension with
loratadine in the prepared suspension no sedimentation with expiration date of 3.8
(formula A) at elevated temperatures. years.
24
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Loratadine suspension
M.Sc degree, College of pharmacy, 18. Tempio J.S. and Zatz J.L., Flocculation
University of Baghdad, 1997. effect of xanthan gum in pharmaceutical
6. Laufen H., Yeates R. and Zimmermann suspensions. Pharmaceutical research,
T., Pharmacokinetic optimization of 1980, 69, 1209-1214.
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Drugs. Exp.Clin. Res. 1995, 21(1), 23-28. relation to their solubility parameters. Int.
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cold, antidiarrheal, and liquid theophylline 21. Bosela A.A, Treki M.S., Mahdy M.A. and
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8. Buck M., A guide to pharmaceutical bioavailability of ampicillin. Bull. Pharm.
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25
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Enzymatic antioxidant in chronic gastritis patients
Abstract
Infection of the gastric mucosa with Helicobacter pylori is strongly associated with chronic
gastritis, peptic ulcer and gastric cancer. Helicobacter pylori virulence factors include a variety of
proteins that are involved in its pathogenesis, such as VacA and CagA. Another group of virulence
factors is clearly important for colonization of H.pylori in the gastric mucosa. These include urease,
motility factors (flagellin), and Superoxide dismutase (SOD). Because of this organism's
microaerophilic nature and the increased levels of reactive oxygen in the infected host, we expect that
other factors involved in the response to oxidative stress are likely to be required for virulence.
Superoxide dismutase is a nearly ubiquitous enzyme among organisms that are exposed to toxic
environments. In this study, we measured the SOD in serum of 80 patients complain from chronic
gastritis and infected with H.pylori. 37 patients infected with H.pylori have the CagA gene, and 13
patients are not and also measured the SOD in 30 control groups that not infected with H.pylori. Serum
level of SOD was significantly (p<0.05) higher in patients with chronic gastritis compared to controls.
Also significantly higher (p<0.001) in patients with chronic gastritis infected with H.pylori positive
CagA than patients infected with H.pylori negative CagA.
Key words: chronic gastritis, H.pylori, CagA, SOD
الخالصــة
١عزشؿب٩ وذٯزخ٠ٛٔشحزخ اٛا٩ ٢ٟزض٠ٛوذح ا٠ٛبة ا٨زٛن اٟ ُْ رزشاHelicobacter pylori ٙوذح ثج٘زشٯب ا٠ٜٛ خبؿٮ٠ٌٛشبء اٛئطبثخ ا
٩ CagA ٙزٮ ا٧٩ شاػزٰخ٠ٛزٮ رشبسٕ ُزٮ أحزذاس ُزٮ اٛبد ا٤ٰر٩جشٛ ا٢ٟ ام٪٣ أ٢٠ج٘زشٯب رزؼٛ ا٥ز٨خبطخ ثٛح ا٩ؼشاٛ اٟٚا٪ ه.وذح٠ٛا
ٚزٟ هب٩ سٯض٪ٰزٛزب ا٨٤ٟ وزذ٭٠ٛخزبؿٮ ا٠ٌٛشزبء اٛشاد ُزٮ ا٠غززو٠ٛ ا٢ٯ٪٘خ ُٮ ر٠٨ٟ ١٪٘ح ر٩ؼشاٛ اٟٚا٪ ه٢ٟ ٫هخ أخش٪٠غٟ . VagA
ٞذساعزخ رزٛ ا٥زز٧ ُزٮ.ٝعزؾ عزب٩ ٬زٛززٮ رزوزشع ئٛبد ا٤٘بئٛاعذ ُٮ ا٪ز٭ ٯزٛ اSuperoxide dismutase (SOD) ٙ اٞضٯ٣أ٩ حشٗخٛا
37 ٞ٨٤زٟ H.pylori ٙ ثج٘زشٯزب ا٢ٰظزبثٟ٩ ٢ٟزض٠ٛوزذح ا٠ٛزبة ا٨زٛ ا٢زٟ ١٪٣ا ٯوزب٪ ٣زشٯغ ٗزبٟ 80 ٚظزٟ ُٮSOD ـٛ ا٫٪غزٟ ٰٓبط
. ٢ٰغززٛزززا ا٨ٛ ززخٜٟ ًٰززش حب٢٘ززٛ H.pylori ٙ ا٢ٰظززبثٟ ززشٯغٟ ززشٯغٟ 13٩ CagA ٚززٛ ززخٜٟحبٛ اH.pylori ٙ ا٢ٰظززبثٟ ززشٯغٟ
ًٯزب٪٤وٟ ٯزضدادSOD زـٛ ا٫٪غززٟ ١عزذ أ٩ .ج٘زشٯزبٛ ثب٢ٰظبثٟ ًٰش٩ خ٠ٰٜخ طحٰخ عٛ شخض ثحب30 ٢ٟ ١٪٘غٰـشح رزٛهخ ا٪٠غٟ ذ٣ٗب٩
٢ٟزض٠ٛوذح ا٠ٛبة ا٨زٛ ا٬شػٟ ٯب ُٮ٪٤وٟ ٯضداد٩ غٰـشحٛهخ ا٪٠غ٠خ ث٣ٔبسٟ ٢ٟض٠ٛوذح ا٠ٛبة ا٨زٛ ثب٢ٰظبث٠ٛ ا٬شػ٠ٛ( ُٮ اp<0.001)
ًٰزش٢٘ٛ٩ ج٘زشٯبٛ ثب٢ٰظبث٠ٛ ا٢ٟض٠ٛوذح ا٠ٛبة ا٨زٛ ا٬شػ٠خ ث٣ٔبسٟ (p<0.05) CagA ـٜٛ خٜٟحبٛا٩ H.pylori ٙ ثج٘زشٯب ا٢ٰظبث٠ٛا
.CagA ـٜٛ خٜٟحب
Introduction
The gastric pathogen Helicobacter pylori is The stomach gastritis associated with
a curved, microaerophilic proteobacterium that Helicobacter pylori infection stimulates the
has been implicated as a causal agent of peptic generation of reactive oxygen species (ROS) by
ulcers and a risk factor for adenocarcinoma the inflammatory cells present in the mucosa (7,
(1,2, 3,4,). During the infections, disease 8, 9). An increase in ROS directly correlated
symptoms may or may not occur, though gastric with bacterial load (10). In addition to internally
inflammation is apparently ubiquitous. The generated reactive oxygen species, the
pathogenesis of H.pylori relies on its successful pathogen must also deal with
persistence in surviving a harsh environment, reactive oxygen species that are generated by
including acidity, peristalsis, and attack by phagocytic cells of the host immune response.
phagocytic cells and their released reactive (11, 12). Protection of cells against ROS is
oxygen species (5). Several potential virulence accomplished through the activation of oxygen-
factors derived from H.pylori are considered to scavenging enzymes such as SOD, catalase and
attract or activate neutrophils and mononuclear glutathione peroxidase have been identified
cells., an immunodominant 120-140 KDa (13). Superoxide dismutase is a nearly
antigen termed cytotoxic associated antigen ubiquitous enzyme among organisms that are
(CagA), the CagA positive strain cause more exposed to toxic environ
server inflammations(6).
26
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Enzymatic antioxidant in chronic gastritis patients
The single SOD of Helicobacter pylori, of all subjects in this study, by using the same
encoded by the sodB gene, has been suspected size forceps, from similar topographical sites at
to be a virulence factor for this pathogenic each endoscopy; biopsies were fixed in 10%
microaerophile, but mutations in this gene formal buffer saline for histological
have not been reported previously (14). The examination. Blood samples were taken from
mechanisms for the detoxification of reactive all subjects and the serum were stored at -20 Ċ
oxygen species are of particular interest in until be used.
H.pylori. Despite the fact that this organism is
an obligate aerobe, it is unable to grow in Histology
atmospheric concentrations of oxygen. The biopsy specimens were embedded in
microaerophilic organisms, like H.pylori, are paraffin and stained with haematoxylin – eosin
particularly vulnerable to the detrimental (HandE) and Giemsa stained for H.pylori
effects of oxygen and oxidative stress (15). determination and diagnostic as chronic
Nevertheless, they do possess some of the gastritis. In situ hybridization (ISH) for
enzymatic machinery needed to eliminate or detection of H.pylori / CagA gene. In situ
minimize toxic oxygen-derived products. hybridization (ISH) is a technique makes use
Organisms that grow in toxic environments of the high specificity of complementary
must have mechanisms to handle reactive nucleic acid binding to detect specific DNA or
oxygen species (e.g., superoxide anions, RNA sequence in the cell. For detection of this
peroxides, and hydroxyl radicals) that are by- markers , the biotinylated DNA probe
products of oxygen metabolism (16, 17). Of hybridize to the target sequence (H.pylori /
these genes, only katA (catalase) mutants have CagA mRNA sequence) then a streptavidin-AP
been characterized (18). Although catalase- (streptavidin-alkaline phosphatase) Conjugate
defective mutants are no different from the is applied followed by addition of the substrate
parent in their binding to epithelial cells, this promo-chloro – indolyl – phosphatel / nitro-
enzyme may be important in detoxification of blue tetrazolium (BCIP/NBT) which yield an
reactive oxygen species produced by the host intense blue – black signal appears at the
immune response. Genes encoding an akyl directly specific site of the hybridized probe.
hydroperoxide reductase, a thiol-specific This strepteividin – Ap conjugate like the
peroxidase, and other potential detoxification biotinylated probe provides raid and highly
enzymes were identified, but mutations in sensitive detection method. The use of Biotin –
these genes have not been reported or Labeled DNA probe for H.pylori / CagA (8
characterized (14, 19). Impairment in this g/10015 ML) litter dd H2O . Probe size: 349
important host cell defense mechanism would bp (Maxim Biotech, Inc., U.S.A).
greatly reduce the ability of the gastric to
epithelial cells to tolerate an environment high Scoring
in ROS, such as would be present with the Hybridization /Detection System will
chronic gastritis associated with H.pylori give an intense blue –black color at the
infection. Disturbance of the oxidant- specific sites of the hybridization probe in both
antioxidant balance in the stomach might positive test tissues. A scoring system that
greatly increase the risk of cell death or DNA includes evaluation of the staining percentage
damage, from ROS (20, 21). The aim of this of stained gastric cells was employed for the
study to investigate the relation between the expression of CagA gene of H.pylori.
H.pylori with CagA- positive and CagA - Counting the number of the positive cells in
negative strains and the production of SOD in the gastric tissue which gave a blue-black
patients with chronic gastritis (HP+) and nuclear staining under the light microscope.
healthy control group (HP-). The extent of the ISH signaling the cells of the
examined tissue was determined in 10 fields
Materials and Methods under high power microscope (40X). In each
Eighty subjects (48 male and 32 female; field, the total of examined cell was about 100
mean age 51.7 ), were referred to the cells per field and this gives a total number.
gastrointestinal endoscopy unit at Al-
Yarmook Teaching Hospital, non of whom Measurement of superoxide dimidiate (SOD)
had received non steroidal anti- inflammatory For the quantitative determination of
drugs, within previous three months, superoxide dismutase in whole blood. This
participated in this study. Endoscope fining in produce is suitable for manual use RANDOX,
the patients were as follows: normal mucosa Cat. No. SD 125 Mixed substrates, Buffer,
and no H.pylori infected (30 subjects) and 50 xanthine oxidase and standard. This method
patients with chronic gastritis without ulcer. employs xanthine and xanthine oxidase (XOD)
Biopsy specimens were taken from the antrum to generate superoxide radicals which react
27
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Enzymatic antioxidant in chronic gastritis patients
with 2 (4- iodophonyl) – 3- (4- nitrophenol) -5- Table( 2): The serum level of SOD in
phanyltetrazolium chloride (I.NT) to form a patients infected with H.pylori and CagA
red fermazan dye. The SOD activity is then status.
measured by the degree of inhibition of this
reaction. One unit of SOD is that which causes Mean ± S.E P F
50% inhibition of the rate of reduction of INT Group S.D.
SOD Value Test
under the conditions of the assay. Control 177.70 ±2.91 9.20
Xanthine Uric acid + O2 (Hp-)
I.N.T formazan dye 18.56
O2 + O2 +2 H O2 + H2O Hp+ 44.95 <0.05
CagA- ٬ 284.00 ± 14.22
Statistical analysis
Hp+ 73.84 <0.001
Statistical analysis was performed using ٬٬477.00 ±54.71
CagA+
ANOVA test to determine whether the means
were equal among three groups – i.e. CagA-,
No significant difference p> 0.05
CagA+ and controls, p value of < 0.05 was
٬ Significant at the 0.05 levels
considered statistically significant.
٬٬ Significant at the 0.001 level
Results Hp: H.pylori
The expression of CagA was detected by in
situ hybridization technique. From 50 patients
complaining chronic gastritis and infected with
H.pylori who were tested for CagA, 37 (74%)
were found to be positive CagA and 13 (26%)
patients have CagA – negative (Table 1, Figure
1).The mean level of SOD increased
significantly in patients infected with H.pylori
CagA positive strains p<0.001 when compared
with healthy subjects and patients infected
with H.pylori CagA negative (Table 2,Figure
2).The difference in SOD level between with
H.pylori that have CagA positive or CagA
negative is statistically significant p<0.01.
400
300
200
100
0
control (CagA-) (CagA+)
(HP-) (HP+) (HP+)
Figure (1): Gastric antral biopsy
specimen from stomach infected with Figure (3): The serum level of SOD in
H.pylori that appear curved or round patients infected with H.pylori and
(Giemza stain) (100X). CagA status.
28
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Enzymatic antioxidant in chronic gastritis patients
29
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Enzymatic antioxidant in chronic gastritis patients
8. Suzuki, M.; Nakamura, M.; Miura, S.; 16. Gonzalez-Flecha, B. and Demple, B.:
Tsuchiya, M. and Ishii, H.: Lansoprazole Metabolic sources of hydrogen peroxide
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Helicobacter pylori. J. Clin Gostroenterol. 17. Imlay, J. and Fridovich, I.: Assay of
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9. Yoshida, N.; Yoshikawa, T.; Iinuma S.; Escherichia coli. J Biol Chem.; 1991;266:
Arai M.; Takenaka, S.; Sakamoto K.; 6957–6965.
Miyajima,T.; Nakamura, Y.; Yagi, N.; 18. Odenbreit, S.; Wieland, B. and Haas, R.:
Naito, Y.; Mukai, F. and Kondo. M.: Cloning and genetic characterization of
Rebamipide protects against activation of Helicobacter pylori catalase and
nutrophils by Helicobacter pylori. Digest. construction of a catalase-deficient mutant
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10. 10- Zhang, O.; Dawodu, J.B.; Etolhi, G.; 19. Tomb, J.; White, O.; Kerlavage, A.;
Husain, A.; Gemmell, G.G. and Russell, Clayton, R.; Sutton, G.; Fleischmann, R.;
R.I.: Relationship between the mucosal Ketchum, K.; Klenk, H.; Gill, S.;
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Brown, E.; Doig, P.; Smith, D.; Noonan, Venter, J.: The complete genome
B.; Guild, B.; DeJonge, B.; Carmel, G.; sequence of the gastric pathogen
Tummino, P.; Caruso, A.; Uria- Helicobacter pylori. Nature. 1997 ;
Nickelsen, M.; Mills, D.; Ives, C.; 388:539–547.
Gibson, R.; Merberg, D.; Mills, S.; Jiang, 20. Ramarao, N.; Gray – Owen, S.; and
Q.; Taylor, D.; Vovis, G. and Trust, T.: Meyer, T.: Helicobacter pylori induce but
Genomic-sequence comparison of two survive the extracellular release of oxygen
unrelated isolates of the human gastric radicals from professional pheasants using
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12. Pesci, E. and Picket, C.: Genetic 21. Drak, I.; Mapstone, N.; Schorah, C.;
organization and enzymatic activity of a White, K.; Chalmers, D.; Dixon, M. and
superoxide dismutase from the Axon, A.: Reactive oxygen species
microaerophilic human pathogen activity and lipid peroxidation in
Helicobacter pylori. Gene. 1994; Helicobacter pylori associated gastritis:
143:111–116. relation to gastric mucosal ascorbic acid
13. Smoot, D.T.; Elliott, T.B.; Verspaget, concentration and effect of H.pylori
H.W.; Jones, D.; Allen, C.R.; Vernon, eradication. Gut. 1998; 42: 768-771.
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Groupman, J.D. and Ashktorab, H.: Reyes, V.; Patel, J.; Dirden, B.; Boldogh,
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reactive oxygen – induced gastric pylori Infection Induces Oxidative Stress
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oxidative stress and defective in host Gerhard, M.; Goebel, U.; Lehn, N.;
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Enzymatic antioxidant in chronic gastritis patients
31
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
Abstract
A lower extracellular pH is one of the few well-documented physiological differences
between tumour and normal tissues. On the other hand, elevated glutathione (GSH) level has been
detected in many tumours compared with healthy surrounding tissues. The compound II: 3-(9H-purin-
6-yl-thio) carbonothionyl methyl-8-oxo-7-(2-thiophen-2-yl) acetamido-5-thia-1-azabicyclo-4-octo-ene-
carboxylic acid was a cephalothin derivative contain 6-mercaptopurine (6-MP). Compound II react
with general base catalysis in slightly acidic pH or with sulfhydryl nucleophiles to release the
chemotherapeutic drug 6-MP. The generation of compound II was accomplished following multistep
reaction procedures. The structure of compound II and its intermediate was confirmed by their melting
point, infrared spectroscopy, CHN and NMR analysis. The hydrolysis of compound II in aqueous
buffer solution of pH 6 and in the presence of GSH at pH 7.4 was studied. The partition coefficient
(PC) of compound II was also determined. Compound II has acceptable rate of hydrolysis at slightly
acidic medium pH 6 (t1/2 = 56.34 min.) and 80% of compound II had been converted to 6-MP within
30 min in the presence of GSH. And the compound has acceptable stability at pH 7.4(t0.5=639.65
min.) and the rate of hydrolysis was effected by change the buffer concentration. This compound can
selectively deliver 6-MP into the tumour tissues which have acidic pH or elevated GSH level.
Compound II had an improved PC value of 12.23 compared to 1.22 for free drug 6-MP confirming
higher lipophilicity.
Key words: 6-mercaptopurine, cancer, prodrug, targeting
الخالصة
ٰخ٣غززشؿبٛغززغخ ا٣ اال٢ٰ ثز٦ٓزز٪ص٪٠ِٛبسٓزخ اٛغٰخ اِٜغززٛزبد اٟوالٛ احززذ ا٪ز٧ ٰززخٜخٛ) خزبسط اpH( ٮ٤ٰع٩ٰززذس٨ٛخِزبع االط ا٣ ا١ا
ـجٰوٰزخٛغزغخ ا٣زخ ثبال٣ٔبسٟ ٝسا٩ اال٢زٟ ) ُزٮ هزذدGSH( ١٪ٰرزبص٪ٌٜٛ ا٫٪غزٟ حق اسرِبم ُٮ٪ٛ ُٔذ٫خ اخش٨ ع٢ٟ٩ .ـجٰوٰخٛغغخ ا٣اال٩
:II شٗت٠ٛ ا١ ا.حٰـخ٠ٛا
3-(9H-purin-6-yl-thio) carbonothionyl methyl-8-oxo-7-(2-thiophen-2-yl) acetamido-5-thia-1-
azabicyclo-4-octo-ene-carboxylic acid
زخٟوبٛٔبهزذح اٛن اٟ ٚ ٯزِبهII شٗت٠ٛ ا١ ا.)6-MP( ٢سٯ٪ٰث٪شٗجزٟ-6 ـٛشٗت اٟ ٬ٜ٭ ه٪) ٯحزCephalothin( ٢ٰص٪ٛغِٰبٜٛ ْشزٟ ٪٧٩
شٗزت٠ٛ رحؼزٰش اٞ رز.٢سٯ٪ثٰز٪شٗجزٟ-6 زـٛزحشٯزش اٛ اح٪ز٤ٛ ا٢جبحش هٛ) اsulfhydryl( ٚبٯذسٯ٨ِٜن عٟ ٩ؼوَٰ اٛؼٮ اٟحبٛحٰؾ ا٠ُٛٮ ا
اعزـخ ٰٓزبط دسعزبد٪زب ث٨٤ٟ زبٗزذٛا٩ عزـٮ٪ٛا٩ زبئٮ٨٤ٛشٗت ا٠ٜٛ ٯخ٩ٰب٠ٰ٘ٛظٰي اٛ اصجبد اٞٓذ ر٩ اد٪خـٛزوذد اٟ ٚزِبهٛ ثبرجبم ؿشٯٔخ اII
زذاسئ ر٭ االطٛ اٙ٪زٜح٠ٛ ُزٮ اII شٗزت٠ٛ اٚزٜ دساعزخ رحٞر٩ .بطش٤وٜٛ ْٰٓذٛ اٰٜٚزحٛا٩ شاء٠حٛالشوخ رحذ اٛ ـِٰٮٛ اٰٜٚزحٛا٩ بس٨ظ٣اال
٥زز٧ ٢زٟ خٜغزحظز٠ٛززبئظ ا٤ٛػزحذ ا٩ ا.)partition coefficient( II شٗزت٠ٜٛ ٢ززأٯٛ اٚزٟوبٟ دساعزخٖٞ رزٛٗزز٩ )pH 6( ٮ٤ٰع٩ٰذس٨ٛا
) دٰٓٔزخ56.34 َظز٣ زش٠) (ثوpH 6( ٮ٤ٰع٩ٰزذس٨ٛذاسئ ر٭ االط اٛ اٙ٪ٜح٠ٛخ ُٮ اٛ٪ٔجٟ ٜٖٚ عشهخ رحٜز٠ ٯII شٗت٠ٛ ا١ذساعخ اٛا
ٙ٪زٜح٠ٛشٗزت رززبصش ثزٌٰزش رشٰٗزض ا٠ٛ اٚزٜ عزشهخ رح١ ا٢ٰب رجز٠ٗ ، (pH-74) ٮ٤ٰع٩ٰذس٨ٛذاسئ ر٭ االط اٛ اٙ٪ٜح٠ٛخ ُٮ اٛ٪ٔجٟ صجبرٰخ٩
ٮ٤ٰع٩ٰزذس٨ٰٛخ راد االط ا٣غزشؿبٛخالٯزب اٛ ا٬زٛ) ا6-MP( ٢سٯ٪ٰث٪ٰشٗجزٟ-6 ـٛ اٙ ٯغزـٰن اٯظبII شٗت٠ٛ ا١ٓن ا٪ز٠ٛ ا٢ٟ ٖٛزٛ .ذاسئٛا
-6 زـٛ ا٬زٛ اٙ٪شٗزت ٓزذ رحز٠ٛ ا٢زٟ %80 ١ حٰزش ا،II شٗزت٠ٛ اٚزٜ عزشهخ رح٢زٟ ٯضٯزذ١٪ٰرزبص٪ٌٜٛ ا١ٓذ ارؼح اٯؼب ا٩ .) 6( ـٜٛ ٔبسة٠ٛا
١ حٰزش ا،ٞغغزٛ اٚشٗزت داخز٠ِٛبرٯزخ ا٣ ٢ رحغز٬زٜ هٙزب ٯزذ٠ٟ ٢ ٓذ رحغز٢زأٯٛ اٟٚوبٟ ١الحلخ اٟ ٞب ر٠ٗ . دٰٓٔخ30 ٙ خال٢سٯ٪ٰث٪شٗجزٟ
.1.22 ٪٧ ٢سٯ٪ٰ ث٪شٗجزٟ-6 ـٜٛ ٢زأٯٛ اٟٚوبٟ ١ ا٢ٰ ُٮ ح12.23 ٪٧ II شٗت٠ٜٛ ٢زأٯٛ اٟٚوبٟ
Introduction
The use of antineoplastic agent 6-MP antibiotics; their degradation depends on the
accompany by several disadvantages including side chain at C-7 and the substitution on C-3
(9)
sever adverse effects (1), poor absorption, low . The presence of good leaving group at C-3
bioavailability (2), limitation of uses (3), facilitate spontaneous expulsion of the 3-
thiopurine associated leukemia (4,5) and drug substituent by any general nucleophile, -
resistance(6,7,8). Cephalosporins are -lactam lactamase or pH change (10,11), scheme (1).
32
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
O O
SH H H2 H
H2N C C C N CH C NH CH2COOH
H
N COOH CH2
N
S
N H
N N
N
6-Mercaptopurine
N N
O S
H O
CH3 N
N S
H2C C HN
N S O
N N
CH2 O C CH3
O
COOH
AVTP
33
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
O O
6.93 O
S HO
7.40 12 .56
14 12 10 8 6 4 2 0
PPM
Figure ( 1b) : H NMR spectrum of compound II .
34
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
Figure (2a): First order plot for the hydrolysis of compound II in 0.1 M phosphate buffer of pH
6.0 at 37 oC ( =1).
35
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
Time (min)
0
-0.5 0 50 100 150 200 250 300
Log Conc- of 6-MP
-1
-1.5
-2
-2.5
-3
-3.5
-4
-4.5
Figure (2b): First order plot for the hydrolysis of compound II in 0.1 M phosphate buffer of pH
7.4 at 37 oC ( =1).
S
S C SK
SH SK
H H H
N N N
N KOH N CS2 N
N N 15-20 C / 0.5 hr N
N N N
Cpd I
O
S
H2C C HN Heated at 60-70 C
S O for 3 hr at pH 7.0
N
O CH2 O C CH
3
COOH
O
S
H2C C HN
S
S
N
O CH2 S C
S
COOH
H
N
N
N N
Cpd II
36
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
The release of 6-MP from compound II condition used the hydrolysis of compound II
depend on the opening of -lactam ring which followed second order kinetic, since plot of log
in turn depend on the pH of the media and the conc. of 6-MP vs time result in curve line. The
ease with which the substitution at position 3 formation of 6-MP from compound II was
of cephalosporin will leave the molecule (10,11). linear for at least 30 min. (figure 3). 80% of
The tithiocarbonate is better leaving group compound II had been converted to 6-MP
than acetoxy group due to it soft base (23), and within 30 min. Compound II reacted rapidly
this will cause the hydrolysis of compound II with thiolate nuceophile of GSH to yield the 6-
at pH 6 is faster than hydrolysis of cephalothin MP. The thiolate was attack the -lactam ring
at the same pH (Scheme 3). The rate of of cephalosporin resulted in the opening of this
hydrolysis of compound II at pH 6 in the ring with the release of trithiocarbonate
presence of different buffer concentration was derivative from position 3 (24) (scheme 4). The
calculated from figure (4) which represent the partition coefficient has become the most
hydrolysis of compound II at different buffer common physicochemical property (25). The
concentration. The Kobs. Were 0.0133mmin-1. partition coefficient of compound II equal to
0.0154 min-1 and 0.0166 min-1 at buffer 12.23; so this compound had an improved
concentration 0.2, 0.5 and 0.8M respectively. partition coefficient value compared to 1.2 for
The half-lives were 52.13min, 45.00min. and 6-Mp, confirming higher lipophilicity and
41.1 min. for buffer concentration 0.2, 0.5 and improvement of the drug bioavailability.
0.8M respectively. Under experimental
O
S
H2C C HN
S
S
N
CH 2 S C
O
S
COOH
H
N
N
N N
H+
H2O
O
S
H2C C HN
S
N
CH 2
HO
COOH
+
S
S C S
H
N
N
N N
SH
H
N
N
+ CS 2
N N
37
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
O
S
H2C C HN
S
S
N
CH2 S C
O S
COOH
H
N
N
GS
N N
S C S
O
S H
N
H2C C HN N
S +
O N
CH2 N
S N
G COOH
SH
H
N
N
CS 2 +
N N
Figure (3): Plot of the hydrolysis of compound II in the presence of glutathione (0.17 mmole) in
phosphate buffer of pH 7.4 at 37 oC ( =1).
38
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
Time (min)
0
-0.5 0 50 100 150 200 250 300
-1
Log Conc-of 6-MP
-1.5
-2 c
b
-2.5
a
-3
-3.5
-4
-4.5
Figure (4): The effect of buffer concentration on the rate of hydrolysis of compound II at Ph 6 at
37 0C ( =1).
* buffer concentration:
a= 0.2 M , b= 0.5 M , c= 0.8 M
39
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Cephalothin as a carrier of 6-mercaptopurine
14. Blair, S.L.; Heerdt, P.; Sachar, S.; cancer institutes anticancer drug screen.
Abolhda, A.; Hochwald, S.; Cheng, H. and Drug Metab. 2004; 32(3): 321-327.
Burt, M.: Glutathione metabolism in 20. Martin, A.: Physical pharmacy (4th ed.),
patient with non-small cell lung cancer. Leo and Febiger, London, 1993; pp. 241-
Cancer Res. 1997; 57: 152. 242.
15. Yang, W.Z.; Begleiter, A.; Johnston, J.B.; 21. God, T.H. and Wann, R.E.: The synthesis
Israels, L.G. and Mowar, M.R.A.: Role of of organic trithiocarbonates. J. Org.
glutathione and glutathione S-transferase Chem. 1961; 26: 4047-4051.
in chlorambucil resistance. Mol. 22. Takayuki, N.; Jun, O.; Ken-Ichi, K.;
Pharmacol. 1992; 41: 625-630. Kenjj, M.; Hideaki, H.; Hajme, K. and
16. Ali Osman, F.; Brunner, J.M.; Kutluk, Hiroshi, K.: (O-aminomethylethyl phenyl
T.M. and Hess, K.: Prognostic acetamido) cephalosporinic acids with six-
significance of glutathione-S-transferase membered heterocyclics in the C-3 side
Pi expression and subcellular localization chain. Antibiot. J. 1977; 30: 698-703.
in human gliomas. Clin. Cancer Res. 23. Jerry M.: Aliphatic nucleophilic
1997; 3: 2553. substitution. In: Advanced Organic
17. Dillman, R.O.; Johnson, D.E.; Shawler, Chemistry (4th ed.), Mc Graw-Hill,
D.L. and Koziol, J.A.: Cancer Res. 1988; Kogakusha, Ltd, Tokyo, 1992; pp. 327.
48: 6097. 24. Inestrosa, E.P; Suan, R.; Montanez, M.I.;
18. Lash, L.H.; Shirnani, A.; Mai, J.; Rodriguez, R.; Mayorga, C. and Blanca,
Chinnaiyan, P.; Krause, R.J. and Elfarra, M.: Cephalosporine chemical reactivity
A.A.: Renal cellular transport, metabolism and its immunological implication. Curr.
and cytotoxicity of S-(9H-purin-6-yl) Optinallergy, Clin. Immunol. 2005; 5:
glutathione, a prodrug of 6- 323-330.
mercaptopurine. Biochem. Pharmacol. 25. Remers, W.A.: Antineoplastic agents. In:
1997; 54: 1341-1349. Wilson and Gisvold’s textbook of organic
19. Gunnars dottir, S. and Elfarra, A.A.: medical and pharmaceutical chemistry
Cytotoxicity of the novel glutathione- (10th ed.). Delado, J.N. and Remers, W.A.
activated thiopurine prodrugs, cis-AVTP (EDs.), Lippincott, Raven, Philadelphia,
and trans-AVTG, results from the national 1998; pp. 16.
40
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Single dose in oral surgery
Abstract
It is clear that correct application of antibiotic prophylaxis can reduce the incidence of infection
resulting from the bacterial inoculation in a variety of clinical situations; it cannot prevent all
infections any more than it can eliminate all established infections. Optimum antibiotic
prophylaxis depends on: rational selection of the drug(s), adequate concentrations of the drug in
the tissues that are at risk, and attention to timing of administration. Moreover, the risk of
infection in some situations does not outweigh the risks which attend the administration of even the
safest antibiotic drug. The aim of this study was to compare between 2 prophylactic protocols in out
patients undergoing oral surgical procedures. Thirty patients, selected from the attendants of oral
surgery clinic in Al-Karamah Dental Center, were subjected to different oral surgical procedures under
local anesthesia. These patients were given single dose antibiotic prophylaxis in 2 groups; 1st group
were 15 patients given 1 million i.u. of procaine penicillin I.M. 30 minutes before oral surgery, 2 nd
group were 15 patients given 600mg clindamycin orally 1 hours before oral surgery. The maximum
time for all procedures was 2 hours. There was no difference between procaine penicillin (1
million i.u.), and clindamycin (600mg), regimens concerning post operative infection in out patient‘s
oral surgical procedures.
Key words: Antibiotic prophylaxis, outpatient oral surgery
الخالصة
س ااالخزــالؿبد٩غجخ حذ٣ ٜٚ ٯٔــ١ أ٢٘٠ٓبئٰخ ٯ٩ ٯخ إلًشاع٪ٰحٛؼــــبداد ا٠ٛظحٰـح إلهـبء اٛزـجْٰ اٛ ا١اػح أ٪ٛ ا٢ٟ
االخزالؿبدٚٗ ن٤٠ظحٰح ال ٯٛزـجٰـْ أٛ ا١ ئ.هخ٪٤ز٠ٛغشٯشٯــخ اٛحبالد اٛ ا٢ٟ ج٘زٰش٭ ثغجت هــذدٛ ا٪٠٤ٛ ا٢ٟ برغخ٤ٛج٘زٰشٯخ اٛا
٬ٜذ ه٠٭ ٯوز٪ٰحٛؼبد ا٠ٜٛ ٓبئٮ٪ٛ اٟٚزوبٜٛ ٚزٰغخ ااألُؼ٤ٛ ا١ ئ.دح ُوال٪ع٪٠ٛ االخزالؿبد ا٢ٟ ضٜزخٛ ٯغزـٰن ا٦٤٘ٛ٩ ج٘زٰش ٯخٛا
ذ٤ٰٓذ ه٪زٜٛ ٥زجب٣اال٩ ج٘زٰش٭ٛذد ثبالخزالؽ ا٨٠ٛغٰظ ا٤ٛ٭) ُٮ ا٪ٰحٛؼبد ا٠ٛؤــبس (اٜٛ ٘بُٮٛزشٰٗض اٛا٩ ٬وـ٠ٛؤبس اٜٛ ـٔٮ٤٠ٛاالخزٰبس ا
نٟ ٬ حز٦جٰبرٜ ع٦ٛشٟ أ٪٧٩ ؼبد٠ٛج٘زٰشٯخ الر غزذهٮ ئهـبء اٛخبؿش ثوغ االخزالؿبد اٟ زٗشٛغذٯش ثبٛ ا٢ٟ . ٭٪ٰ حٛؼبد ا٠ٛئهـبء ا
زذاخالدٛ ١٪ ٯخؼو٢زٯٛ ا٬شػ٠ٜٛ ٢ٰٰٓبئ٩ ٢ٰٰ هالع٢ٰٛ٪ٗ٪ر٩ ثش٢ٰـخ ث٣ٔبس٠ٛ ا٪٧ ذساعخٛ ا٥ز٧ ًـشع١ئ.ب٣بٟؼبداد أ٠ٛأٗضش ا
١٩س٩ ٯض٢زٯٛ ا٢ٰشاعو٠ٛ اٚ ٓجــ٢ٟ شٯؼبٟ ١٪ اخزٰبس صالصٞ ر.)ػوٮ٪٠ٛزخذٯش اٛ (رحذ ا١ب٤ـت األعٛ غشاحٰخٛوٰبداد اٛعشاحٰخ ُٮ ا
.ػوٮ٪٠ٛزخذٯش اٛهخ رحذ ا٪٤زٟ زذاخالد عشاحٰخٛ ٞــ٨ ئخؼبهٞ حٰش ر١ب٤ـت األعٛ زخظظــٮٛخ اٟ٘شاٛشٗض اٟ غشاحخ ُٮٛ اٞٓغ
هخ٪٠غ٠ٜٛ( ٰبٜب هؼ٧ ئهـبؤٞر٩ ٰخ٠ٛحذح هب٩ ١٪ٰٜٟ ٮ٧٩ ٢ٰٜغ٤ ث٢ٗبٯ٩جشٛ هٔـبس ا٢ٟ ِشدحٟ عشهخٞ٧ ئهـبؤٞ ر، ٬شػ٠ٛإالء ا٧
ص٩ ٯزغبٞــٛ )شٯؼبٟ 15 ٞرؼ٩ ٰخ٣ضبٛهخ ا٪٠غ٠ٜٛ ( ِٞٛ ؿشٯْ ا٢ هٌٜٟٞ 600 ٢ٰبٯغٟذ٤ٜ٘ـٛهٔبس ا٩ )شٯؼبٟ15 ٢ٟ خ٣٪٘٠ٛ ا٬ٛ٩األ
س٩ حذٝبحٰخ هذ٣ ٢ٟ ٢ٰهز٪٠غ٠ٛ ا٢ٰبٕ أ٭ اخزالٍ ث٤٧ ٢٘ ٯٞٛ ٦٣زٰغخ ا٤ٛذ ا٣ٗب.١غشاحٰخ عبهزبٛزذاخالد اٛ إلعشاء اٝالصٛٓـذ ا٪ٛا
.٦اه٪٣٭ ثأ٪٠ِٛغشاحٮ اٛ اٚزذاخٛج٘زٰشٯــخ ثوذ اٛاالخزالؿـبد ا
41
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Single dose in oral surgery
Infection of the incised skin or soft tissues is a common pathogens and their antimicrobial
common but potentially avoidable susceptibility.(12) Procaine penicillin is one of
complication of any surgical procedure. Some the semi-synthetic penicillin obtained by
bacterial contamination of a surgical site is alterations in the prosthetic group differ from
inevitable, either from the patient's own the naturally occurring product (penicillin G )
bacterial flora or from the environment. (7) In in three dimensions: their resistance to acid
procedures that require the insertion of makes oral administration possible, they may
implants or prosthetic devices, the term be resistant to the action of penicillinase
surgical site infection is used to encompass and their spectrum of antimicrobial activity
the surgical wound and the implant. Surgical is usually broadened for many streptococcal
site infection also encompasses infections infections. (14) It is bactericidal, act by
involving the body cavity (e.g. a. subphrenic interfering with bacterial cell wall
abscess ), bones, joints, meninges and other synthesis.(10) Clindamycin is a bacteriostatic
(8)
tissues involved in the operation. act by interfering with protein synthesis of
Prophylactic administration of antibiotics bacteria. It is active against Gram positive
inhibits growth of contaminating bacteria and cocci, including streptococci and penicillin-
their adherence to prosthetic implants, thus resistant staphylococci, and also against
reducing the risk of infection. (9) The goals of many anaerobes, especially B. fragilis( 15).
prophylactic administration of antibiotics to
surgical patients are to: reduce the incidence Subjects and Methods
of surgical site infection, use antibiotics in a After a thorough history taking,
manner that is supported by evidence of clinical, and radiographic examination, thirty
effectiveness, minimize the effect of patients attending Al-karamah Dental Center
antibiotics on the patient‘s normal bacterial were selected to participate in this study.
flora, minimize adverse effects and cause These patients are mostly from the residents
minimal change to the patient‘s host of the neighborhood, which is a relatively a
defenses.(2) It is important to emphasize that low socioeconomic level. None of patients had
surgical antibiotic prophylaxis is an adjunct medical history or active infectious process.
to, not a substitute for, good surgical All patients in this study are not allergic to
technique. Antibiotic prophylaxis should be penicillins. These patients were subjected to
regarded as one component of an effective oral surgical procedures under local anesthesia
policy for the control of hospital-acquired maximally 2 hours the surgical procedures
infection. (10,11) The American college of involved bone and soft tissue and these
surgeons classified wound surgery into 4 includes: removal of impacted lower 3rd molar,
categories: clean, clean-contaminated, Apicectomy for upper central and lateral
contaminated and dirty wound, according to incisors. Patients were classified into two
this classification trans-oral wound is groups according to the antimicrobial agent:
considered Clean contaminated, That is, Class 1. 1st group were 15 patients given single
II, these wounds should receive protection if I.M. doses of 1 million i.u. procaine
(a) the patient has depressed host defenses. penicillin 30 minutes before oral surgery.
(b) A prosthetic device is being inserted. (c) 2. 2nd group were 15 patients given 600mg
The sequel of an infection is serious; and (d) clindamycin orally 1 hour before surgery.
some aspect of the procedure, such as Number of female patients included in our
increased duration or decreased local blood study was 17, while the number of male
supply, makes infection more likely. (8,11) patients was 13. Patients were classified into 3
Prophylactic antimicrobial agents should be groups. Group one (10-19) nine patients, group
administered not more than 30 to 60 minutes two (20-29) thirteen patients and group 3 (30-
before surgery.(8-9) Exceptions to this rule are 39) eight patients. Surgical procedures
cesarean procedures, colonic and urologic included in this study were: removal of
procedures. Therapeutic concentrations of impacted lower RT 3rd molar (11 cases),
antimicrobial agents in tissue should be present removal of impacted lower LT 3rd molar (8
throughout the period that the wound is cases), removal of impacted of upper RT 3 rd
open. The duration of antimicrobial molar (1 case), apicectomy for upper RT
prophylaxis for the majority of procedures is central incisor (5 cases) apicectomy for
controversial; however, experts recommend at upper LT central incisor (4 cases) apicectomy
most one or two postoperative doses. (2,3) The for upper RT lateral incisor (1
antibiotics chosen for prophylaxis can be those case).Meticulous handling of the tissues,
used for active treatment of infection. avoidance of unnecessary surgical trauma and
However, the chosen antibiotics must reflect copious irrigation of the wound before closure
local, disease-specific information about the to remove foreign bodies and debris, leaving
42
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Single dose in oral surgery
12
impaction lower
RT 3rd molar
no potential foci for bacterial infections were
of crucial importance in our measures. Patients 10
impaction lower
were examined 48 hours post-operatively to LT 3rd molar
investigate the presence of any local and 8
general signs of post operative infection these impaction upper
signs are: increased pain or tenderness, post RT 3rd molar
operative swelling at the site of surgery, 6 apicectomy
enlarged, tender regional lymph node and upper RT
fever. The same investigated parameters were centeral incisor
also examined 7th day after surgery, for suture 4 apicectomy
removal. upper LT central
incisor
Results 2 apicectomy
upper RT lateral
Characterization of patients according incisor
to age, gender and type of oral operation is
0
given in figures 1, 2, and 3. No postoperative
infections were recorded in the two groups, Figure( 3) No. of patients according to
and no postoperative complications in the two surgical procedures
groups.
Discussion
Although some studies found that
18 antibiotic prophylaxis in some oral surgical
16 procedures is controversial (12,16,17).Its generally
14 agreed that when antibiotic prophylaxis is
males
12 decided, the antibiotic must be present in the
females systemic circulation at a high level at the time
10
8 of surgery and is usually given as one dose
(17,18,19)
6 . In spite of the fact that preoperative
antibiotic prophylaxis is an established
4
practice (17, 20), there is no consistent protocol
2
for the method or duration of drug
0 administration in oral surgical procedures, (21)
and this is true for Iraqi dental surgical centers.
Figure (1) No. of patients according to Although it is agreed that procedures entailing
gender entry into the oropharynx or esophagus, need
antibiotic coverage of aerobic cocci is
indicated. Prophylaxis has been shown to
reduce the incidence of severe wound infection
by approximately 50 percent. (22). Our choice
14 for procaine penicillin depends on two factors
1. most of oral infections caused by
12 penicillin sensitive bacteria (23)
group 1 2. The use of penicillin is an established
10
group 2 clinical practice in advanced surgical
8 group 3 centers (22,23), on the other hand some of
the studies select Clindamycin for
6
antimicrobial prophylaxis in oral surgery,
4 clindamycin is occasionally chosen for
orthopedic surgical prophylaxis, where it
2 has an excellent activity against
Staphylococcus spp. and Bacteroides
0
fragilis, but have no activity against
Figure (2) : No. of patients according to age enteric microorganism.(22,24). Also it has
group good reputation for tissue penetration,
with almost the same effectiveness of
penicillin against anaerobes. (13)
The minimum inhibitory concentration (MIC)
of clindamycin is achieved within the first 2-3
dose intervals. Thus, stable drug concentration
is then maintained for greater than 6 hours
43
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Single dose in oral surgery
44
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Single dose in oral surgery
45
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Single dose in oral surgery
46
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
Abstract
Exposure to lead results in significant accumulation in most of vital organs, and free radical
damage has been proposed as a cause of lead-induced tissue damage, where oxidative stress is a likely
molecular mechanism. This study was designed to evaluate therapeutic effects of melatonin in lead-
induced organ toxicity in rats. The therapeutic effects of melatonin on lead induced toxicity in rats
were evaluated using 36 rats, which were allocated into 3 groups and treated as follows: Group I,
includes 12 rats injected subcutaneously with 0.2 ml physiological saline for 30 days, followed by
treatment with a daily dose of 20mg/kg melatonin, administrated I.P for the successive 30 days; groups
II and III, each includes 12 rats , injected with lead acetate 100 mg/kg/day s.c for 30 days, followed by
treatment with intraperotoneal injection of physiological saline (0.2 ml) or melatonin 20mg/kg/day for
the next 30 days. At the end of treatment period, the rats were sacrificed by an overdose (100mg/kg) of
thiopental (twenty-four hour after the last injection). Craniotomy and laparotomy were performed to
obtain the brains, livers and kidneys for the assessment of tissue damage. The changes in total body
weight, weight of major organs (brain, liver and kidney), oxidative stress parameters, hemoglobin
content, liver and renal functions, and histological appearance of the studied organs were evaluated and
compared with that of negative and positive controls. Treatment with melatonin reverses the damage
induced by lead in many organs and tissues through the reduction of MDA levels in RBCs, brain, liver
and kidney; increases GSH levels in all studied organs; in addition to the improvement in the indices of
the functions of the organs studied. These findings demonstrated that melatonin is capable of reversing
damage of rat tissues caused by successive doses of lead acetate, and animals had restored their organ
functions due to treatment with melatonin.
Key words: Melatonin, Lead poisoning, Oxidative stress
الخالصة
حشح ٓذٛس ا٩غزٛ ا٢خ ه٠بع٤ٛ االػشاس ا١ب ا٠ٗ ٯخ٪ٰحٛ االهؼبء اٞولٟ ُٮ٥ ٯزغجت ثزشٗض١ ا٢٘٠شطبص ٯٜٛ زوشعٛ ا١ا
٥ز٧ ٰٞ٠ رظٞر. خٛ٩غإ٠ٰٰٛ٘خ ا٣ٰ٘ب٠ٛ ا١٪ٰ٘ٛ ٰخٛب٠ االٗضش احز٪٧ زأٗغذ٭ٛبد ا٨ االع١ حٰش ا, االهؼبء٥ز٨الػشاس ثٛ غججخ٠ٛٮ ا٧ ١٪٘ر
١ِئشاٛ ا٬ٜ ه٢ٰ٣٪ٰالر٠ٜٛ والعٰخٛزأصٰشاد اٛ ا١ ا.شطبصٛ ثبٞ٠زغٛظبثخ ثب٠ٛ ا١ِئشاٛ ُٮ ا٢ٰ٣٪ٰالر٠ٜٛ والعٰخٰٛخ اِٛوبٛ اٰٰٞٔزٛ ذساعخٛا
٢٠ رزؼ٬ٛ٩هخ اال٪٠غ٠ٛا: ٰخٛزبٛغبد اٛوب٠ٛٔذ اٰٜن رٟغبٟ صالس٬ٛذ ا٠ ٓغ, ُأسا36 ٝب ثبعزخذا٨٠ٰٰٔ رٰٞزٛ شطبصٛ اٞ٠ظبثخ ثزغ٠ٛا
اهـٰذ٢ٰ٣٪ٰالر٠ٛ ا٢ٟ ٌٞٗ/ٌٜٟٞ 20 ب ثغشهخ٧ثوذ٩ , بٟ٪ ٯ٢ٰذح صالص٠ٛ حٮٜ٠ٛ اٙ٪ٜح٠ٛ ا٢ٟ ٟٚ ., 2 ذ ةٜغٛذ رحذ ا٤ٔح. ُأسا12
100 شطبص ثغشهخٛب ثخالد ا٨٤ٔ حٞب ر٠٨٤ٟ ٚ٘ٛ ُأسا12 ذ٠زٮ ػٛا٩ ضخٛضبٛا٩ ٰخ٣ضبٛ ا٢ٰهز٪٠غ٠ٛ ا٩ . بٟ٪ ٯ٢ٰضالصٛ ١٪جشٯزُٛٮ ا
ْ ؿشٯ٢ هٝ٪ ٯ/ ٌٞٗ/ ٌٜٟٞ 20 ٢ٰ٣٪ٰالر٠ٛ ا٩) اٟٚ 0،2 ( حٮٜ٠ٛ اٙ٪ٜح٠ٛ ا٢ٟ ب ارجوذ ثغشمٟ٪ ٯ٢ٰضالصٛ ذٜغٛ رحذ اٝ٪ ٯ/ ٌٞٗ / ٌٜٟٞ
عبهخ24 س٩شٟ ثوذٙزب٤ث٪ صبٯ٢ٟ ٰخٛ عشهخ هبٝبد ثبعزخذا٣ا٪ٰحٛ اٚ ٓزٞغخ رٛوب٠ٛبٯخ ُزشح ا٨٣ ُٮ. ٰخٛزبٛب اٟ٪ ٯ٢ ٰضالصٜٛ ١٪جشٯزٛ ا٢ٔح
ٝ اعغب١صا٩ اٙوذٟ زٌٰٰشاد ُٮٛ ا١ ا. ب٧ِحض رؼشسٛ ٬ٜ٘ٛا٩ االٗجبد٩ ٌخٟ االدٙزششٯح العزحظبٛ اعشاء اٞ ر. غخٛوبٟ اخش٢ٟ
٘جذٛ ا٢ٟ ِٚ٘ػبئ٩ طبد٪خؼبة ُحٛ ا٫٪حزٟ٩ زأٗغذ٭ٛبد ا٨إششاد االعٟ ٩ ) ٬ٜ٘ٛا٩ االٗجبد٩ ٌخٟبد (االد٣ا٪ٰحٛاهؼبء ا٩
غخٛوب٠ٛ ا١ ا. عجخ٪٠ٛا٩ جخٛغبٛخ ا٣ٔبس٠ٰٛن اٟغبٟ ٢ٟ ٚٗ ب ة٨ز٣ٔبسٟ ب ثوذ٨٠ٰٰٔ رٞعخ ر٩ذس٠ٛغٰغٰخ ُٮ االهؼبء ا٤ٛزٌٰشاد اٛا٩ ٬ٜ٘ٛا٩
٫٪غزٟ ( ١٪٧ذٛ اٗغذح اٰٜٚٔ رٙ خال٢ٟ غغخ٣اال٩ االهؼبء٢ٟ و ذٯذٛشطبص ُٮ اٛبرظ ثب٤ٛؼشس اٛ ٯوبٗظ ا١ ا٢٘٠ ٯ٢ٰ٣٪ٰالر٠ٛثب
ف٪حٜٟ ٢ن رحغٟ عخ٩ذس٠ٛ ا١٪ربصبٯ٪ٜ٘ٛ ا٫٪غزٟ صٯبدح٬ٛ اػبُخ ا، ٬ٜ٘ٛا٩ ٘جذٛا٩ بىٟذٛا٩ ش٠حٛ اٝذٛ خالٯب ا٢ٟ ٚٗ بٯذ) ُٮ٨ذٯٛذا٣٪ٛب٠ٛا
غغخ٣خ ُٮ ا٠بع٤ٛ ه٘ظ االػشاس ا٬ٰٜخ هٜٔبثٛ ا٦ٛ ٢ٰ٣٪ٰالر٠ٛ ا١ػح ا٪زبئظ ر٤ٛ ا٥ز٧ ١ا. عخ٩ذس٠ٛكبئَ االهؼبء ا٩ إششادٟ ُٮ
غخٛوب٠ٛزٰغخ ا٣ ب٨ُٰ كبئَ االهؼبء٩ بد ٓذ اعزوبدد٣ا٪ٰحٛ ا١ا٩ ، شطبصٛ خالد ا٢ٟ غشمٛ زوبٓت٠ٛزوشع اٛ ا٢ٟ ١ِئشاٛا
. ٢ٰ٣٪ٰالر٠ٛثب
47
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
48
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
variance (ANOVA). P values less than 0.05 reported when lead acetate was administered
were considered significant for all data with saline (Table 1) . Malondialdehyde
presented in the results. (MDA) levels in the RBCs, brain, liver and
kidney tissues were significantly elevated after
Results exposure of animals to 100mg/kg lead acetate
Administration of 100mg/kg lead acetate (479%, 109%, 178% and 101% respectively,
s.c for one month and treatment with saline for p<0.05) compared with 20 mg/kg melatonin
another month resulted in significant reduction treated animals. Therapeutic treatment with 20
in body weight after two months (25%). mg/kg melatonin resulted in significant
Therapeutic treatment with 20 mg/kg decrease in MDA levels in studied tissues
melatonin I.P for one month after intoxicated (55%, 33%, 54% and 23% respectively,
of rats with lead acetate resulted also in p<0.05) compared with animals challenged
significant reduction in total body weight with 100 mg/kg lead acetate and saline only
(6%), this level seem to be less than that (Table 2).
Table 1. Effects of therapeutic use of 20 mg/kg melatonin on the total body weight and the
weights of brain, liver and kidney in rats previously intoxicated with 100 mg/kg lead acetate.
Malondialdehyde (MDA)
Treatment groups
RBC
Brain Liver Kidney
(nmol/g
(nmol/g tissue) (nmol/g tissue) (nmol/g tissue)
Hb)
Saline +Melatonin (20mg/kg)
5.4 ± 0.12a 48.9 ± 1.62a 52.7 ± 1.31a 24.4 ± 1.21a
(n=12)
Lead acetate (100mg/kg) +
Saline 31.2 ± 2.48b 101.9 ± 4.71b 144.8 ± 5.56b 49.1 ± 2.17b
(n=7)
Lead acetate (100mg/kg) +
Melatonin (20mgkg) 13.9 ± 0.83c 68.2 ± 1.89c 66.5 ± 2.13c 37.8 ± 1.87c
(n=10)
Data are expressed as mean ± SEM; n= number of animals; values with non-identical superscripts (a,
b, c) within the same variable considered significantly different (P<0.05).
49
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
Table 3. Effects of therapeutic use of 20 mg/kg melatonin on the glutathione (GSH) levels in
erythrocytes, brain, liver and kidney in rats previously intoxicated with 100 mg/kg lead acetate.
Glutathione (GSH)
Treatment groups
RBC Brain Liver Kidney
(µmol/g Hb) (µmol/g tissue) (µmol/g tissue) (µmol/g tissue)
Saline +Melatonin
(20mg/kg) 13.9 ± 0.13a 11.8 ± 0.12a 8.9 ± 0.13a 7.8 ± 0.23a
(n=12)
Lead acetate (100mg/kg) +
Saline 3.2 ± 0.19b 4.4 ± 0.18b 3.3 ± 0.12b 4.1 ± 0.12b
(n=7)
Lead acetate (100mg/kg) +
Melatonin (20mgkg) 6.1 ± 0.14c 5.9 ± 0.11c 7.0 ± 0.09c 5.8 ± 0.10c
(n=10)
Data are expressed as mean ± SEM; n= number of animals; values with non-identical superscripts (a,
b, c) within the same variable considered significantly different (P<0.05).
Daily treatment of rats with 100mg/kg lead activities both with respect to lead acetate and
acetate significantly reduces GSH levels in saline treated animal group and between each
RBCs, brain, liver and kidney (77%, 63%, other (table 5). However, therapeutic treatment
64%, and 48% respectively, p<0.05) compared of animals with melatonin, one month after
with 20 mg/kg melatonin treated animals. lead acetate challenge, significantly reduces
Meanwhile therapeutic treatment with 20 serum levels of urea and creatinine in which
mg/kg melatonin, administered one month the reduction were (28% and 25%
after lead acetate results in significant respectively, p<0.05), the reduction in serum
elevation of GSH in the studied tissues (88%, level of their parameters was significantly
34%, 115% and 41% respectively, p<0.05) different when compared with lead acetate and
compared with lead acetate and saline treated saline treated animals and between each
animals (table 3). Administration of 100 others( table 6). Lead acetate, when
mg/kg lead acetate to the rats result in administered subcutaneously, in a consecutive
significant decrease in Hb levels and PCV %( 100 mg/kg doses for one month and saline for
12% and 9% respectively, p<0.05), when another month produces significant elevation
compared with melatonin 20 mg/kg treated in blood lead levels (513%), and lead levels in
group (table 4). Exposure of animals to s.c brain, liver and kidney of these animals were
injections of lead acetate (100 mg/kg) for one also significantly elevated (3810%, 4736%
month and saline for another month produces and 2849% respectively, p<0.05) compared
significant elevation in the serum levels of with 20 mg/kg only melatonin treated animals.
hepatic enzymes activity (AST, ALT, Melatonin reduces lead levels significanty in
ALP)(162%, 232%, and 102% respectively, all studied compartments (blood 28%, brain
p<0.05) compared with 20 mg/kg melatonin 46%, liver 40% and kidney 42%) compared
treated animals. Therapeutic administration of with lead acetate and saline treated animals
melatonin in a dose of 20 mg/kg (39%, 53% (table 7).
and 42%) significantly reduces enzymes
50
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
Table 5. Effects of therapeutic use of 10 or 20 mg/kg melatonin on the liver enzymes (AST, ALT,
and ALP) of rats previously intoxicated with 100 mg/kg lead acetate for one month.
Table 6. Effects of therapeutic use with 10 or 20 mg/kg melatonin on serum urea and creatinine
of rats previously intoxicated with 100 mg/kg lead acetate for one month.
Table 7. Effects of therapeutic use of 10 or 20 mg/kg melatonin on lead levels in blood, brain,
liver and kidney of rats previously intoxicated with 100 mg/kg lead acetate for one month.
Lead level
Treatment groups
Blood Brain Liver Kidney
(μg/dl) (μg/gm) (μg/gm) (μg/gm)
Saline +
Melatonin (20mg/kg) 12.98 ± 0.29 a 0.9 ± 0.05 a 2.18 ± 0.1 a 8.23 ± 0.26 a
(n=12)
Lead acetate (100mg/kg)
+
79.54 ± 3.51 b 35.19 ± 1.33 b 105.43 ± 2.98 b 242.69 ± 2.28 b
Saline
(n=7)
Lead acetate (100mg/kg)
+
57.48 ± 2.15 c 18.92 ± 0.83 c 63.38 ± 1.88 c 141.57 ± 2.1 c
Melatonin (20mgkg)
(n=10)
Data are expressed as mean ± SEM; n= number of animals; values with non-identical superscripts (a,
b, c) within the same variable considered significantly different (P<0.05).
51
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
Sections prepared from livers of rats, Sections prepared from kidneys of rats treated
previously intoxicated with lead acetate 100 with saline and previously intoxicated with
mg/kg, treated with saline for one month, 100 mg/kg lead acetate for one month, showed
showed a wide area of normal appearance with mild degenerative changes and necrosis in the
presence of small area of degeneration and kidney tubules (figure 3). Meanwhile,
necrosis with inflammatory cells infiltration administration of 20 mg/kg melatonin to group
(figure 1). Meanwhile, treatment of rats with of rats previously intoxicated with lead acetate
20 mg/kg melatonin previously intoxicated 100 mg/kg, the kidney sections showed
with 100 mg/kg lead acetate for one month, normal histology but still there is slight
the liver sections showed normal structure dilatation of the renal tubules (figure 4).
appearance with few discrete degenerative
changes (figure 2).
A
B B
A
Figure (3). Section of kidney tissue showing
mild degenerative changes (arrow A) and
Figure (1). Section of liver tissue showing necrosis (arrow B) in the kidney tubules in
a wide area of normal appearance with rats treated with saline previously
presence of small area of degeneration and intoxicated with 100mg/kg lead acetate for
necrosis (arrow A) with inflammatory cells one month. Magnification: 200X
infiltration (arrow B) in rats treated with (hematoxylin and eosin stain).
saline previously intoxicated with 100mg/kg
lead acetate for one month. Magnification:
200X (hematoxylin and eosin stain).
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Melatonin in lead poisoning
54
Iraqi J Pharm Sci, Vol.17 (2) , 2008 Il-6, Il-1, ISH, Breast Cancer
Abstract:
Breast cancer is the second most common cancer in women world. Multiple Cytokines appear to
have a dominant role in human breast cancer formation. Estimation of the in situ expression of IL-6
and IL-1β in breast cancer patients. A sixty patients with breast cancer BC were divided into two
clinical subgroups, (30) with malignant breast cancer MBC and (30) with benign breast tumor as a
control group according to histological examination. In situ hybridization technique used for detection
of IL-6 and IL-1β mRNA sequence in two groups. The results showed that percentages of mRNA
expression of IL-6 and IL-1β were in (≥ 11-50%) for malignant breast cancer. This research also
investigated that (73.3%) of benign breast tumor were expression less than (<10%) for IL-6 and IL-1
β mRNA. The ISH expression of the mean percentages of IL-6 and IL-1 β were higher levels in
malignant breast cancer patients ( 48.13 and 56.07 ,respectively) than benign tumor (2.73 and 1.40
,respectively), with highly significantly differences (P<0.01) of ISH expression for IL-6 and IL-1 β
mRNA among two studied groups., the expression of IL-6 and IL-1 β mRNA are significantly
elevated in the tissue of breast cancer patients compared with benign tumor and was found a significant
correlation between the expression of IL- 6 and IL-1β mRNA in the tissue of breast cancer patients,
thus the results of the present study might be explain the pathological role of these two cytokine in
breast cancer.
Key words: IL-6 mRNA, IL-1β mRNA, Breast cancer, ISH.
الخالصة
سا اعبعزٰب ُزٮ٩بد د٤ٰٗ٪غزبٯزٛ ا٢زٟ وذٯذٛوت اٜر٩ . ٞٛوبٛغبء ُٮ ا٤ٛذ ا٤زشبسا ه٣ٰخ ا٣غشؿبٛشاع اٟٮ اال٣ضذ٭ صبٛ ا١ٯوزجش عشؿب
١ عزشؿب٬شػزٟ ٫زذٛ ثٰززب1 ٩ 6 - ٢ٰٗ٪زٛزش٣ ا٢زٟ ٚ٘زٛ ػزن٪٠ٛزوجٰزش ُزٮ اٛ ا٢زحزش٭ هزٛ ا٪ز٧ جحزشٛ ا٢ٟ ٍذ٨ٛ ا. ضذ٭ٛ ا١ عشؿب٢ٯ٪٘ر
هزخ٪٠غ٠ٛ ا، غزٰغٮ٤ِٛحزض اٛ ا٬زٜزبدا ه٠ اهز٢ٰهز٪ز٠غٟ ٬زٛ اٞ٨٠ٰ رٔغزٞضزذ٭ رزٛ ا١ظبثخ ثغشؿبٟ شٯؼخٟ ) 60( ذساعخٛذ اٜ٠ ش. ضذ٭ٛا
زوجٰزشٰٛزخ ا٤ٔ رٝ اعززخذاٞ ر. ٰذ٠ حٝس٪ظبثخ ثٟ شٯؼخٟ )30( ٰخ٣ضبٛهخ ا٪٠غ٠ٛا٩ ، خجٰشٛضذ٭ اٛ ا١ظبثخ ثغشؿبٟ شٯؼخٟ )30( ٬ٛ٩اال
ززززبٜ٘ٛ غزززٰغٰخ٤ٛٔزززبؿن ا٠ٛ ثٰززززب ُزززٮ ا1 ٩ 6- ٢ٰٗ٪زززٛزش٣ ا٢زززٟ ٚ٘ززٛ ٚشاعززز٠ٛٮ اٟ٪عززز٪شاٯجٛ٭ ا٩٪ززز٤ٛغ اٟحزززبٛ ا٢زحزززش٭ هزززٜٛ ػزززن٪٠ُٛززٮ ا
ضززذ٭ٛ ا١شٯؼززبد عززشؿبٟ هززخ٪٠غٟ ززذ٤ ) ه%50-11 ≥( ززٮ٧ ثٰزززب1٩ 6-٢ٰٗ٪ززٛزش٣الٛ زوجٰززشٛغززجخ ا٣ ١ذساعززخ اٛززشد ا٨ اك. ٢ٰهز٪زز٠غ٠ٛا
. ثٰززب1- ٢ٰٗ٪ٛزش٣ا٩ 6- ٢ٰٗ٪ٛزش٣ ا٢ٟ ٘الٛ ٰذ٠حٛ اٝس٪ٛشٯؼبد اٟ ٢ٟ % 73.3 ـــــــٛ )%10 <( ٮ٧ زوجٰشٛغجخ ا٣ ذ٣ب ٗب٠٤ٰخجٰش ثٛا
٬زٜ )ه56.07 ٩ 48.13 ( خجٰزشٛ اٝس٪زٛهخ ا٪٠غ٠ٛ ثٰزب1٩ 6- ٢ٰٗ٪ٛزش٣ٰخ ألٛٯخ هب٪ئٟ غت٣ ٙوذٟ ػن٪٠ٛزوجٰش ُٮ اٰٛخ ا٤ٔذ ر٤ٰث٩
ٓزبد٩س ُش٪ز٨ ك٬زٛ االخزالُبد ادد ا٥ز٧٩ ٮٛا٪زٛ ا٬ٜ ) ه1.40 ٩ 2.73( غت٤ٛذ اٜ٘زٮ شٛا٩ ٰذ٠حٛ اٝس٪ٛهخ ا٪٠غ٠خ ث٣ٔبسٟ ٮٛا٪زٛا
٢ٰٗ٪زٛزش٣ا٩ 6 - ٢ٰٗ٪زٛزش٣زوجٰزش ألٛ ا٢ٰٯخ ثز٪٤وٟ عجخ٪ٟ د هالٓخ٪ع٩ ذساعخٛذ ا٤ٰٖ ثٛٗز. ) 0.01 < (ٯخ٪٤وٟ ٫٪غزٟ ذ٤ٰخ هٛٯخ هب٪٤وٟ
٢زٟ ٢ٰه٪ز٤ٛ ا٢ززٯ٨ٛ شاػٮٟساال٩ذٛػح ا٪ٰخ ٓذ رٛحبٛذساعخ اٛزبئظ ا٣ ١٘زا ُأ٧٩. ضذ٭ٛ ا١شٯؼبد عشؿب٠ٛ غٰغٰخ٤ٛٔبؿن ا٠ٛ ثٰزب ُٮ ا1-
. ٥س٪رـ٩ ضذ٭ٛ ا١ عشؿب١٪٘بد ُٮ ر٤ٰٗ٪غبٯزٛا
55
Iraqi J Pharm Sci, Vol.17 (2) , 2008 Il-6, Il-1, ISH, Breast Cancer
It is associated with worse survival in patients black signal appears at the specific site of the
with metastatic breast cancer and is correlated hybridized probe (9) . This directly
with the extent of disease (4). In human breast streptavidin-AP conjugate like the biotinylated
cancer, an important role of IL-1β and IL-1RA probe provides a rapid and highly sensitive
mRNA expression was noted in various studies detection method.Evaluation of ISH signal was
(5)
, Interleukin-1β is a highly inflammatory and done with the assistance of a histopathologist
prototypical multifunctional cytokine that .The expression of both IL-6 and IL-1β mRNA
affects nearly all cell types, often in concert was measured by the same scoring system,
with other cytokines or small mediator counting of the number of the positive cells in
molecules. IL-1β elicits important proinfla- the tissue that has given a blue-black
mmatory and immunological responses, such (BCIP/NBT) nuclear staining under the light
as fever, hypotension, increasing circulating microscope. The score was the average from
NO, recruiting neutrophils,and costimulating T 10 distinct high-power fields observed under
cell activation by increasing IL-2R expression ×100 magnification. The percentage of
and inducing IL-2 production (6). The basis of positively stained cell was calculated for each
the various biologic properties of IL-1β case by taking the mean of the percentages of
depends on its regulatory effects on the the positively stained cell in the 10 fields. A
expression of various genes and/or receptors. score of 0 was given when no staining was
IL-1β induces the gene expression of the IL-1 detected, 1 if there was weak to moderate
family, other inflammatory cytokines, colony staining in less than 10% of cells, 2 if
stimulating factors, and mesenchymal growth moderate to strong staining was present in 11
factors (7).This study aimed at estimation of the to 50% of cells, and 3 if strong staining in
in situ expression of IL-6 and IL-1β in more than 50% of cells was detected (10).
malignant breast cancer patients comparing to Statistical Analysis
the benign breast cancer and find out the The suitable statistical methods were
correlation between these two marker in used in order to analyze and assess the results.
malignant and benign patients. Descriptive statistics results presented as
percentages of frequencies ,mean, SD, SEM,
Materials and Methods minimum & maximum levels .Inferential
Sixty Iraqi patients with breast cancer statistics used to accept or reject the statistical
who were admitted to AL -Yarmook and hypotheses, includes: Chi-square (χ2),T-
Baghdad Teaching Hospital. Patients with test.Pearson Correlation (r). P - value < 0.05
breast cancer (BC) were divided into two and P < 0.01 were considered statistically
clinical subgroups according to histological significant. (11).
examination: (30) with malignant breast cancer Results
and (30) with benign breast tumor as a control The expression of IL-6 and IL-1β were
group. Fresh samples were obtained during detected by ISH technique. Tables 1 and 2
routine examination of surgically removed show the percentage of frequency scoring for
tissue, each specimen was fixed in 10% IL- 6 and IL-1β mRNA expression among
formalin then processed paraffin wax study groups, respectively. Chi-square test was
embedded section and cut into 5µm thickness, conducted to examine the association between
put on Fisherbrand positively charged slides IL-6 and IL-1β mRNA expression in the tissue
for our research. In situ hybridization: For in in the two groups of investigated women ,it
situ hybridization technique (ISH), DNA Probe was found that highly significant association
Hybridization/Detection System in situ kit (p<0.01) between them among the four
(Maxim Biotech, Inc., USA) was used.The scoring levels. The results showed that
probes were biotin-labeled DNA probes for percentages of mRNA expression of IL-6 and
human IL-6 (360 bp), and human IL-1β (556 IL-1β were in ( ≥ 11-50%) for malignant breast
bp), (Maxim Biotech, Inc., USA). In situ cancer. This research also investigated that
hybridization (ISH) is a technique used the (73.3%) of benign breast tumor were
high specificity of complementary nucleic expression less than (<10%) for IL-6 and
acid binding to detect specific DNA or RNA IL-1 β mRNA. On the other hand, the mean
sequence in the cell (8) For detection of this percentages of these two cytokines was
markers, the biotinylated DNA probe hybridize significantly higher(p<0.001) in malignant
the target sequence (IL-6 and IL-1β mRNA breast cancer compared with benign tumor as
sequence) then a streptavidin-AP (streptavidin demonstrated in (Table 3) . The expression of
- alkaline phosphatase) conjugate is applied IL-6 and IL-1β was heterogeneous blue-black
followed by addition of the substrate promo- nuclear staining in the tissue, as shown in
chloro-indolyl-phosphate /nitro- blutetrazolium Figure (1). In addition, this study demonstrated
(BCIP/NBT)which yields an intense blue- highly significant positive correlation (P<0.01)
56
Iraqi J Pharm Sci, Vol.17 (2) , 2008 Il-6, Il-1, ISH, Breast Cancer
between IL-6 and IL-1β in two studied groups, as shown in (Table 4) and Figure (2).
57
Iraqi J Pharm Sci, Vol.17 (2) , 2008 Il-6, Il-1, ISH, Breast Cancer
0% N 0 8 8
% 0 26.7 13.3
< 10 % N 0 22 22
% 0 73.3 36.7 0.00 Highly Sig.
(P<0.01)
11-50 % N 11 0 11
% 36.7 0 18.3
>50% N 19 0 19
% 63.3 0 31.7
Total N 30 30 60
% 100 100 100
* = breast cancer ** = breast tumor
Table (3): Mean of ISH%IL-6 & IL-1β levels among studied groups
(Malignant breast cancer & Benign breast tumor patients)
Studied groups Malignant Benign (T-test)
BC* BT**
Interleukins N=30 N=30 P-value Sig.
ISH%IL-6
Mean 48.13 21.03 2.73
SD 3.84 2.49 0.00 Highly Sig.
SEM 15 ─ 85 0.45 (P<0.01)
Mini.─ Maxi. 0─8
ISH%IL-1β
Mean 56.07 1.40
SD 17.87 1.13 0.00 Highly Sig.
SEM 3.26 0.21 (P<0.01)
Mini.─ Maxi. 20 ─ 86 0─3
* = breast cancer ** = breast tumor
58
Iraqi J Pharm Sci, Vol.17 (2) , 2008 Il-6, Il-1, ISH, Breast Cancer
Table (4): Correlation between ISH%IL-6 level & ISH%IL-1β level among
total breast cancer patients, Benign BC patients & Malignant BT patients.
Pearson Total Malignant Benign
Correlation B C* BT**
ISH%IL- ISH%IL- ISH%IL- ISH%IL- ISH%IL- ISH%IL-
6 level 1β level 6 level 1β level 6 level 1β level
r 0.893 0.576 0.516
P-value 0.00 0.001 0.004
Sig. Highly Sig. (P<0.01) Highly Sig. (P<0.01) Highly Sig. (P<0.01)
* = breast cancer ** = breast tumor
tissues (5; 14; 15; 16) and in serum (17; 18) .Several
studies suggest that the IL-1 system is vital in
100
the local control of tumor growth, important in
regulating ‗‗protumorigenic‘‘ activities within
80
the tumor microenvironment, and contributes
to angiogenesis, tumor proliferation, and tumor
invasion (19; 20; 21). Furthermore, IL-1ß and IL-6
60
cause tumor regression and increase median
survival time in a variety of cancer patients. In
40
contrast, elevated circulating concentrations of
growth factors such as IGF-I are a surrogate
20
cases risk for cancers of the breast (22; 23; 24). It is
noteworthy that IL-1 β is a prototypical
Control
0 proinflammatory cytokine that exerts a
breast cancer
plethora of biological activities, including
-20 tumor regression (25). The tumor-suppressing
Total Population
-20 0 20 40 60 80 100
property of IL-1 β has been attributed mostly
ISH%IL-6 level to its ability to prime antitumor immunity (26),
but the mechanism for its direct cytostatic
Figure (2): Correlation between ISH%IL-6 actions in suppressing cell cycle progression is
level and ISH%IL-1β level among total largely unknown. The antiproliferative action
breast cancer patients, Benign BT of IL-1 β on human breast cancer cells is
patients& Malignant BC patients. exhibited not by killing the cells but rather by
preventing the ability of the late G1
progression factor, insulin-like growth factor
Discussion (IGF)-I, to promote progression from late G1
Cytokines in general are thought to be
into the S phase of the cell cycle (27) .This
involved in numerous physiologic and
cross-talk between proinflammatory cytokine
pathologic conditions. Among cytokines, IL-1β
and growth factor receptors is similar in
and IL-6 probably seem to play the most
principle to that between the B cell receptor
important role in breast carcinogenesis (12; 13)
and the 2-adrenergic receptor for the
.In the present study, IL-6 and IL-1β mRNA
neurotransmitter norepinephrine (28) and that
expression was examined by in situ
between the IGF-I receptor and integrin-
hybridization technique in tissue of malignant
associated protein for thrombospondin-1 (29)
breast cancer compared with benign tumor .
.Moreover, the role of the IL-1 system in
The IL-6 and IL-1β were expressed in a higher
human breast cancer is conflicting. IL-1 has
percentage in breast cancer tissue compared to
been shown to inhibit growth of breast cancer
benign tumor and we found the positive
cells and to promote cellular differentiation in
expression of IL-6 mRNA and IL-1β mRNA
vitro, but it is equally known to stimulate the
among malignant breast cancer were 46.7%
expression of several proteolytic enzymes in
and 63.3% were more than fifty percent
human cancer (30;31) .The consecutive
(>50%),respectively . This results suggests
degradation of extracellular matrix is a key
that IL-6 and IL-1β are over expressed in
element of local invasion and metastasis (32, 33)
breast carcinoma compared to benign tumor
.In addition, they are many confounding
and might play a pathological role in
studies about the role of IL-6 and IL-1β in
malignant breast cancer. Evidence supporting
tumor cell growth, but its exact role remains
this suggestion includes the fact that in human
varied and unclear (19; 34). It appears that the
breast cancer, the elevated expression of IL-6
effect of IL-6 on tumor cell growth may
and IL-1β were observed in breast carcinoma
depend on the tumor cell type, IL-6 plays a
59
Iraqi J Pharm Sci, Vol.17 (2) , 2008 Il-6, Il-1, ISH, Breast Cancer
new role in cancer biology; it promotes with metastatic breast cancer. Int J cancer.
multidrug resistance (34) and it has been shown (2003)103 (5):642-6.
to be involved in intercellular signaling 5. Pantschenko, A.G., Pushkar, I., Anderson,
between mesenchyme and breast cancer K.H., et al. The interleukin-1family of
epithelium.. These display an oncogenic role cytokines and receptors in human breast
for IL-6; however, lacking is an understanding cancer: implications for tumor
of the mechanisms governing IL-6 production progression.Int JOncol;(2003) 23: 269-84.
in tumors and the biological role of this 6. Saijo, Y.; Tanaka, M.; Miki , M.; Usui,
cytokine in tumorigenesis (19, 35). The human K.; Suzuki, T.; Maemondo, M.; Hong,
IL-6 shows antiadhesive effects, and X.; Tazawa,R.; Kikuchi, T.; Matsushima,
modulates the estrogen receptor and K.and Nukiwa, T. Proinflammatory
progesterone receptor content of these cells (35) Cytokine IL-1_ Promotes Tumor Growth
.The elevated expression of IL-6 has been of Lewis Lung Carcinoma by Induction of
detected in multiple epithelial tumors (36) .An Angiogenic Factors: InVivo Analysis of
interesting finding , in the current study that in Tumor-Stromal Interaction1 The Journal
situ expression of IL-6 was significantly of Immunology, .(2002) 169: 469–475.
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IL-1β (r= 0.576 ; p< 0.01) in malignant breast Takada, H. & Daikuhara. Y. Enhancement
cancer . This results indicating that IL-6 and of human hepatocyte growth factor
IL-1 β are strongly interact with each other and production by interleukin-1β and -1RA
act synergistically, subsequently increasing and tumor necrosis factor-_ by fibroblasts
their effect. This finding in agreement with in culture. J. Biol.Chem. (1993) 268:8140.
that of Robison and colleagues who reported 8. Maritette, P.C.; Roeland. H.D.; Rob, P.M.,
that a significant correlation between IL-6 G.; Van Erica, B.; Clavida, M.H.;
and IL-1 immunoreactivity (13) , thus both ILs, Roelof, A.P.; Jim, E.L. and Anton, K.R
i.e., IL-1β and IL-6 have been shown to be Sensitive mRNA detection fluorescence in
strongly interact and to act additively in breast situ hybridization using horseradish
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10. Nakopoulou, L.; Lazaris, A.C.; Kavantzas,
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62
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity
Abstract
Extract from cell culture of medicinal plant like Nigella sativa have been assessed for its
cytotoxic properties. Thymol is likely responsible for the theraputic effects of Nigella sativa leaf callus
extract. In this short study the inhibitory effect of Nigella sativa leaf callus extract (Thymol) has been
studied on Human Lorgnx Epidrmoid Carcinoma (Hep-2) cell line during different exposure period of
time (24, 48 and 72 hrs.) using different concentration of the extract (1000, 500, 400, 300, 200 and 100
µg/ml). The optical density of the Hep-2 cells has been readed on 492 nm wave length. Thymol –
induced cytotoxicity was (500 µg/ml) which inhibit cell growing compared to the control and this ratio
increased at the 48 hrs of exopsure and stopped at 72 hrs.
Key wards: Nigella sativa, callus extract, cell line, ELISA assay.
الخالصة
٪زز٧ ٯوزجززشٙ٪٠ضززبٯٛ ا.خالٯززبٜٛ ٰخ٠غززٛ ا٦وشُززخ خظبئظزز٠ٛ داء٪غززٛحجززخ اٛـجززٮ اٛجززبد ا٤ٜٛ ضسهززٮٛغززٰظ ا٤ٛ ا٢ززٟ ضٜغزززخٟ ٚزز٠ هٞرزز
ٔظززٰشح دساعززخ رززأصٰشٛذساعززخ اٛ ا٥ززز٧ ُززٮٞ رزز.ظٛ٘ززبٛساّ ا٩ض أٜغزززخٟ ٢ززٟ داء٪غززٛحجززخ اٛجززبد ا٤ٛ والعٰززخٛزززأصٰشاد اٛ ا٢ هززٙ٩غززإ٠ٛا
٩ 48 ,24( ِززخٜخزٟ ٓززبد روززشٯغ٩ أ٢٠ ػززHep-2 ٰخ٣غززشؿبٛخالٯززب اٛ ا٪زز٠٣ ٬ززٜ) هٙ٪٠ضززبٯٛداء (ا٪غززٛحجززخ اٛظ اٛساّ ٗززب٩ض أٜغ ززخٟ
٬زٜخالٯزب هٜٛ O.Dٙ ٰٓزبط اٞ رز.)µg/ml 100٩ 200 ,300 ,400 ,500 ,1000( ضٜغزخ٠ٛ ا٢ٟ ِخٜخزٟ رشاٰٗضٝ عبهخ) ثبعزخذا72
زخ٣ٔبس٠ٛخالٯزب ثبٛ ا٪٠٣ ز٭ صجؾٛا٩ 500µg/ml زشٰٗضٛذ ا٤ٰخ ه٣غشؿبٛخالٯب اٜٛ بٟ عب١٪٘ ٯٙ٪٠ضبٯٛ ا١عذ ا٩ .nm 492 عٮ٪٠ٛ اٙ٪ـٛا
.زوشٯغٛ ا٢ٟ شٛضبٛ اٝ٪ٰٛذ ا٤َٓ ه٪ر٩ زوشٯغٛ ا٢ٟ عبهخ48 ذح٠ٛغجخ ُٮ ا٤ٛ ا٥ز٧ صادد٩ غٰـشحٛخ اٜٟوبٟ نٟ
Introduction
The main inspiration of black seed comes
from the famous saying (Hadith) of our
Prophet Mohammed; (God peace be upon diseases (bacterial and fungal) (5). So, to
him), that ―Habbat Al-soda is remedy for all increase the production of this compound
disease except death.‖ (1).It is an annual (thymol) all the year round without depending
herbaceous plant believed to be endogenous to on the mother plant, plant tissue culture
the Mediteranean region but has been techniques formed callus and then increased
cultivated in other parts of the world including the production of thymol. The anticancer
India and Pakistan (2). Black seed oil contains activity of N. sativa was first revealed by (6)
about 0.5-1.5 % volatile oil including nigellone who observed enhancement of natural killer
and thymoquinone used as anti-histanimic, (NK) cell activity ranging from 200-300% in
antioxidant, antiinfective and bronchodilating advanced cancer patients receiving
effects(3).Thymol, is one of the active multimodality immunotherapy programme in
compounds in N. sativa extract, plays which N. sativa was one of these components.
important role in the inhibition of cancer cells, Thymoquinone and dithymoquinone, active
and can attach with the mutagenic substance, principles of N. sativa, had cytotoxic effect
because thymol is one of the antioxidant against parental and multi-drug resistant
phenolic compounds (4).Plant tissue culture human tumour cell lines which were over 10-
techniques inters in several applications like fold more resistant to doxorubicin and
plant micropropagation, genetic study, plant etoposide (7) . Radiation protection activity of
improvement, study of plant cell physiology, N. sativa in mice against induction of
the production of secondary metabolites in chromosomal aberrations by gamma ray was
addition to production of viruse free plant also reported(8)
63
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity
The using of plant tissue culture techniques vitro assay, the frozen cell line was withdrawn
make the easy of pharmaceutical compounds and maintained in RPMI-1640 containing 10%
production instead of depending on the mother bovine calf serum. When the in vitro cells
plant and become possible to produce these culture forms a monolayer. These cells were
compounds at high amount and at high rate of treated with trypsin/ versine mixture in order to
pure may be over than these isolated from the pursue subculture process.The percentage of
complete mother plant, and its production may inhibition was calculated according to the
be quickly and independent on the season, also following equation: (14)
limit the surface area that is used in the
medicinal plant culturing (9).The objective of Inhibition % = [(OC – OT) /OC] x 100
the present study was to assess the cytotoxic
properties of this extract from cell culure of N.
Where :
sativa leaf callus using against Hep-2 cell line
OC: optical density of control wells
using ELISA (enzyme linked immunosorbant
OT : optical density of test wells
assay) assay.
From the above calculation, a graph was
plotted for the precentage of growth inhibition
Material and Methods: against each extract concentration. Activity
Collection of plant material: against Hep-2 cell line was determined by the
inhibition assay using an ELISA assay. In
Seeds of Nigella sativa gotten from Dr. short, cell cultures in the microtitration plate
Aws Al-Ani (Directorate of Agriculture were exposed to a range of plant extract
Research and Food Technology/ Ministry of concentrations during the log phase of growth
Science and Technology/ Baghdad/ Iraq) to be and the effect determined after recovery time.
used. The following protocol as descriped in (15) was
Callus culture condition: performed the extracts of Nigella sativa leaf
callus :
The establishment and maintenance of a) After trypsinization, cell suspension seed
callus were carried out using the procedures in a micro titration plates at 50000
described before(10). cells/ml RPMI-1640 growth medium with
Extraction prepration: serum 5% was used for seeding.
b) Plates then incubated for 24 hours.
For preliminary screening, the seeds were c) By using maintenance medium, two-fold
cultured and callus induced and material from serial dilution were prepared starting from
callus culture were lyophilzed and extracted by 1000 μg/ml ending with 100 μg/ml.
a method described elsewhere (11). In short, d) After incubation for 24 hrs, cells exposed
one g of callus was mixed with 30 ml of NaOH to different extract dilutions. Only 200
solution 5% and then diethyl ether was then μl of each concentration added for each
added in a ratio of (2:1) (v /v) and mixed well well (6-replicates for each tested
as described elsewhere (12). The extract was concentration). 200 μl of maintenance
then filtered and concentrated in vacum at 45º medium added to each well of control
C and then kept in the dark at 4º C until tested. group.The times of exposure were (24, 48
Cytotoxicity test using ELISA assay: and 72 hrs). The plates sealed with self
adhesive film then returned to the
For this test, the extract were weighed incubator at 37º C.ْ
(0.05 g) and dissolved in phosphate buffer e) After the end of the exposure period, the
saline (PBS) and dimethylsulphoxide (DMSO) medium and the cells decanted off and
to prepare extract solution at 1000 µg/ml. The replaced by 200 μl of 0.01% crystal violet
following dilutions of extract were then dye. After 20 min. the stain was washed
prepared: 500, 400, 300, 200 and 100 gently with tap water for three times. The
µg/ml.Hep-2 cell line, obtained from Iraqi plate was left until become dry.
Center For Cancer and Medical Genetics The optical density of each well was read
Researches at the passage level 326 were used by using a micro-ELISA reader at 492
in this study. The origin and description of this nm transmitting wave length (15 , 16).
cell line was mentioned by (13). It was a human Statisitical analyses:
laryngeal carcinoma excised from 57 years old A one-way analysis of variance was
man, then transplanted in immune suppressed performed to test whether group variance was
rat by cortisone. After growth of the tumour in sifgnificant or not, the comparison between
the rat, it was then excised and transplanted as groups were used analyses of variance Least
an in vitro tissue culture. It was kept at -169˚C Significant Differences Test (L.S.D.)(16).
(in liquid nitrogen). In preparation to any in
64
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity
65
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity
inhibition increased when reacrhed 48 hr of 8. Shubber, E.K., Ibrahim, S.A.M. and Al-
exopsure and stopped at the 72 hr of exposure. Azawi, A.F.N. Reduction of gamma ray
The using of these concentrations (1000, 500, induced chromosomal abnormalities in
400, 300, 200 and 100 µg/ml) in this study mouse bone marrow cells by Nigella
affect on the growth of Hep-2 cell line with sativa. 2nd Arabian Conference of
slight differences between them , the OD of Biotechnology, and Genetic Science.
Hep-2 cell line ratio was the lowest on the Al-Mennia, Egypt (2000). Oct.20-23.
concentration 500 µg/ml compared with other 9. Robins, R. J.; payne, J. and Rhodes, M. J.
concentrations. So, the extract make an Cell suspension culture of Cinchona
inhibition on the growth of Hep-2 cell line ladgerinna; I Growth and quinoline
compared the control depending on the alkaloid production. Plant Media (1985).
concentration that is used and the length of 3:163-246.
incubation period. The result of this study 10. Sokmen, A.; Jones, B.M. and Erturk, M.
suggest that thymol inhibited proliferation of Antimicrobial activity of extracts from the
tumor cell line by a mechanism that involves cell cultures of some medicinal plants.
cytotoxicity, in fact, it is known that Phytother. Res. (1999). 13, 355-357.
thymoquinone (a quinone from Nigella sativa) 11. Pacheco, P. ; Sierra, J.;
inhibited the proliferation of COS 31 (canine Schmedahirschmann, G.; Potter, C.W.;
osteosarcoma) at concentration 100µM by Jones, B.M.; Moshref, M. Antiviral
inducing apoptosis and cell cycle arrest at G1. activity of Chilean medicinal plant-
Non-cancerous cells are relatively resistance to extracts. Phytotherapy Research (1993).
thymoquinone (19) . Nigella sativa and other 7, 415-418.
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cell line, the effect were determined on 24 hr .)1989( .خ٠٘حٛ ثٰذ ا.وخ ثٌذادٟ عب.ـجٰخٛا
of incubation. The greatest inhibitory effects 13. Moree, AE.; Sabachewsky, L. and Toolan,
were observed on Nigella sativa plant extract HW. Culture characteristic of four
even at low concentration (20) . permanent lines of cancer cells. Cancer
Res. (1955). 1: 598- 605.
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vitro cytotoxic antiviral and
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Younis and KW, AL- Morani. Principle of
review of the pharmaco- therapeutic
statistics. (1986) Al-Mosil University.
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66
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Nigella sativa callus cytotoxicity
67
Iraqi J Pharm Sci Vol.17 (2) , 2008 Permeation of chlopheniramine maleate from gels
Abstract
Chlopheniramine maleate ( CPM ) , is one of the H- receptor antagonist , widely used in
allergic diseases ,like skin rash and pruritis .CPM 3%w/w was successfully loaded in 2%w/w
sodium alginate (SA) as a gel base , and to be considered as a selected formula .It was found
that the diffusion of CPM through the skin of albino rat was increased as the
concentration of CPM increased from 2 %w/w sodium alginate , More over , the addition of
Triethanolamine 5 % w/w, to sodium alginate 2 % w/w , loaded by CPM 3 % w/w , enhanced the
amount of CPM diffuse through the skin of albino rat . Mean while the addition of PEG 1000
2% w/w , and urea 5 % w/w, separately to sodium alginate 2 % w/w , loaded by CPM 3 % w/w ,
hindered significantly P<0.05 the amount of the drug diffused through the skin of the rat .The
selected formula of sodium alginate 2% w/w as a base loaded by CPM 3% w/w was physically
acceptable , with shelf life approximately 3.3 years .
Key wards: chlopheniramine maleate , gel , skin permeation
الخالصة
ٔذٛ. خبسعٰخٛح٘خ اٛا٩ ذ٭ٜغٛـِح اٛاعن ُٮ ا٩ ٚ٘خ ثشٜ٠غزو٠ٛا٩ 1 ٦٧ غزٔجالد٠ٛ ضجـبد٠ٛ احذ ا٪٧ ٰٰذٛبٟ ٢ٰٰٟشا٤ُس٪ٜ٘ٛا
, ٝال٧ ٗٔبهذحٝ٪دٯ٪ظٛبد ا٤ٰغٛ ا٢ٟ )١ص٩\١ص٩( %2 ) ُٮ١ص٩\ ١ص٩(%3 ٰٰذٛبٟ ٢ٰٰٟشا٤ُس٪ٜ٘ٛالد ثزشٰٗت ا٩حب٠ٛغحذ ا٣
ؤبس ُٮٛغشر االثشػ ٯضداد ثضٯبدح رشٰٗض اٛذ اٜ عٙ خال٢ٟ ٰٰذٛبٟ ٢ٰٰٟشا٤ُس٪ٜ٘ٛ اٜٚ رخ١عذ ا٩ ٔذٛ . خزبسحٟ اهزجشد ٗزشٰٗجخ٩
ٓبهذح٢ٟ )١ص٩\١ص٩( %2٬ٛ) ا١ص٩\١ص٩( %5 ٢ٰٟال٪٣زشاٯٰضبٛ اػبُخ ا١ٖ ُبٛ ر٬ٜح ه٩هال, ٝ٪دٯ٪ظٛبد ا٤ٰغٛ ا٢ٟ )١ص٩ \١ص٩(%2
غشرٛذ اٜ عٙبُزح خال٤ٛؤبس اٰٛخ ا٠ٗ صٯبدح٬ٛ ا٫ اد, ٰٰذٛبٟ ٢ٰٰٟشا٤ُس٪ٜ٘ٛ ا٢ٟ )١ص٩\١ص٩(%3 خ ةٜ٠حٟ ٝ٪دٯ٪ظٛبد ا٤ٰغٛا
ٚ٘سٯب ثش٪ٰٛ ا٢ٟ )١ص٩\١ص٩(%5 ٩ )1000(ٙ٪٘ ٗالٯ٢ٰٜ أص٬ٛ٪بدح ثٟ ٢ٟ )١ص٩\١ص٩(%2 أػبُخ١ ُأ, طوٰذ اخش٬ٜه٩ .االثشػ
ٚ٘ االهبٓخ ثش٬ٛ ا٫ اد,ٰٰذٛبٟ ٢ٰٰٟشا٤ُس٪ٜ٘ٛ ا٢ٟ )١ص٩ \١ص٩( %3 خ ةٜ٠حٟ, ٝ٪دٯ٪ظٛبد ا٤ٰغٛ ا٢ٟ )١ص٩\١ص٩( %2 ٬ٛ اِٚظ٤ٟ
بد٤ٰغٛ ا٢ٟ )١ص٩\١ص٩( %2 ٬ٜ٭ ه٪ذ رحز٣خزبسح ٗب٠ٛزشٰٗجخ اٛ ا١ أ. الثشػٛ غشرٛذ اٜ عٙبُزح خال٤ٛؤبس اٰٛخ ا٠ٗ P<0.05 ٞ٨ٟ
ؤبسٜٛ ِبرٯخ٣ ُزشح٩ ٯخ عٰذح٩ طِبد ُٰضٯب٬ٛ اػبُخ ا, ٰٰذٛبٟ ٢ٰٰٟشا٤ُس٪ٜ٘ٛ ا٢ٟ )١ص٩\١ص٩( %3 خ ةٜ٠حٟ ٝال٧ ٗٔبهذحٝ٪دٯ٪ظٛا
. خ٤ ع3,3د٩ثحذ
Introduction
Gels are semi solids consisting of dispersions disintegrant and gelling agent in
made up of either small inorganic particles or pharmaceutical preparations (3) . It has several
large organic molecules enclosing and unique properties that have been enabled it to
interpenetrated by a liquid (1) .The delivery of be used as a gel matrix for delivery of many
the drug into and through the skin is drug (4) . Chlorpheniramine maleate as a
recognized an effective means of therapy for potent H1-receptor antagonist can be indicate
local dermatological and systemic disease ,In for many types of allergy such as rhinitis and
recent years, the development of transdermal pruritis , it can prevent but does not reverse
permeation has been attracting an attention histamine mediated response (5) . This study
due to several advantages , Such as better aimed to both suggest new alternative dosage
control of blood levels , reducing systemic form for enhancing topical penetration of
toxicity and avoid firs pass metabolism (2) . CPM , and to evaluate the potential and
Sodium alginate , a naturally occurring poly transdermal absorption .
saccharide has been widely used as a
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Iraqi J Pharm Sci Vol.17 (2) , 2008 Permeation of chlopheniramine maleate from gels
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Iraqi J Pharm Sci Vol.17 (2) , 2008 Permeation of chlopheniramine maleate from gels
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Iraqi J Pharm Sci Vol.17 (2) , 2008 Permeation of chlopheniramine maleate from gels
20 20
NaCMC4%w/w
15 sod.Alginate 2%w/w
15 Na Alginate
4%w/w sod. Alginate 4%w/w
10 10
5 5
0 0
0 1 2 3 4 5 6 0 1 2 3 4 5 6
Tim e (hour) Time (hour)
Figure (1) . The effect of different bases on Figure (2). The effect of polymer
the release of CPM 1%w/w at pH 7.4 and concentration on the release of CPM 1%
37˚C w/w through rat skin at pH 7.4 and
37˚C
Effect of polymer concentrations on the
release of 1%w/w CPM gel :
Table 2. and figures 2 and 3 ,
demonstrate the effect of SA concentrations 20
on the release profiles of the CPM through sod. Alginate1%w/w
Amount of CPM released (mg.)
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Iraqi J Pharm Sci Vol.17 (2) , 2008 Permeation of chlopheniramine maleate from gels
diffusion through the rat skin can't be 3% w/w CPM loaded in 2% w/w SA gels
estimated , because there are two types through rat skin . It was seen that both urea
of partitioning , one of the partition of 5% w/w and PEG 1000 2% w/w significantly
the drug for the skin ( Ds ) , and the other for decrease P < 0.05 the amount of the drug
vehicle ( Dv ) ,or gel base . so these two diffused through rat skin. Since incorporation
magnitudes of the two diffusion coefficient of urea in hydro gel may activate the
Ds and Dv , determines whether the release hydrolysis process of urea to form ammonia
from vehicle or skin is the rate limiting and carbon dioxide , which lead to elevate the
step, and by this approach, the pH of the medium of environment , which in
concentration of incorporated drug in the turn dissociate the CPM into maleate anion
gel base may solve this problem , regardless and chlorpheniramine cation , these ionized
the diffusion or partition coefficient (11 ,14 ). species may hinder the diffusion process of
the drug (15) . Moreover , the alkyl amine
Table 3. Effect of CPM concentration on group , of the drug may complex ethylene
the diffusion rate constant (K) using oxide ( CH2-Ö-CH ) group of PEG1000 that
2%w/w S0dium Alginate (SA) gel . decrease the amount of the drug available for
permeation, this result is in a consistent with
CPM that result obtained , when lidocaine was
CPM Rate formulated as a topical gel (16) . On the other
amount
concentration constant hand , incorporation of 2.5%w/w of TEA
diffused
% (mg.min ˉ½) which counter irritant to the skin as an
mg./5hr. *
enhancer , showed a slight increase of CPM
1%w/w 7.54±0.09 0.582 permeation through the rat skin , this effect
may be attributed to both effects of TEA as a
3%w/w 11.765±0.21 0.8217 basic tertiary amine enhancer once that is
compatible with alkyl amine anti histamine
Each value represents the mean SD
(CPM ) , and second may be referred to the
( n=3 readings in each group ) *
effect of emulsification of TEA with malic
acid to form water soluble salt that easily
20 allow the drug penetration through the rat
skin (17).
Amount of CPM diffused (mg.)
CPM 1%w /w
15 Table (4). Effect of different enhancers on
CPM 3%w /w the diffusion rate constant ( K ) of CPM
3%w/w through the rat skin
10
CPM
Enhancer amount Rate constant
5 type diffused (mg.min ˉ½)
mg./5hr.
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Iraqi J Pharm Sci Vol.17 (2) , 2008 Permeation of chlopheniramine maleate from gels
1.94
20
1.93 50 C
Amount of CPM diffused (mg.)
No addition
1.92
Urea 5% w/w
15 1.91
PEG 1000 2% w/w 60 C
1.9
TEA 5% w/w
0 7 14 21 28
10 خطي Tim e (Days)
40(
)C
Figure (6) . Accelerated degradation of
5 خطي
CPM in a selected formula at different
50( exaggerated temperatures
)C
0 خطي
0
0 1 2 3 4 5 6 60(
Log rate constant (K) day
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Iraqi J Pharm Sci, Vol.17 (2) ,2008 Permeation of chlopheniramine maleate from gels
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Interleukins and abortion
Abstract
Recurrent Spontaneous Abortion (RSA) is the most painful experience for couples expecting
a child. This study aimed to determine the relevance of IL-2 and IL-6 in recurrent spontaneous abortion
(RSA). Serum samples were collected from 90 women attend Al Kadhmiya teaching hospital in
Baghdad. 60 women (first group) had recurrent abortion the women were negative for rubella virus,
herpes simplex virus and toxplasma gondii. And they were negative from bacterial infection eg.
Niesseria gonorrhea and Chlamydia trachomatis. The histopathological tests for fetus abnormalities
were negative in this group, and 30 women (second group) with successful pregnancy (normal
delivery). All samples were analyzed for IL-2 and IL-6 by commercially available Enzyme-Linked
Immunosorbent Assay (ELISA) kits. The data showed highly significant increase in the serum level of
IL-2 in group 1 compared with group 2 (P<0.001). However, IL-6 showed highly significant increase
level in group 2 compared with group 1. In addition, there was no significant correlation between these
two markers in studied groups. The data of this study strengthen the possibility that high level of IL-2
and low level of IL-6 may explain the role of type-1 cytokines in the pathogenicity of recurrent
spontaneous abortion.
Key words: - interleukin 2, interleukin 6 and recurrent spontaneous abortion (RSA)
الخالصة
٢زحش٭ هٜٛ ذساعخٛ ا٥ز٧ أعشٯذ.ٙغبة األؿِب٣ ُٮ ئ٢ٰشاًجٛ ا٢ٰع٩ضٛخ ُٮ حٰبح ا٠ٰٛزغبسة األٛ ا٢ٟ ز٘شس٠ٛزارٮ اٛٯوذ اإلعٔبؽ ا
٬ِغزشٟ ٬ٛ ئ٢ٜغبء ادخ٣ ٢ٟ ٢ٰهز٪٠غٟ ٢ٟ ٚظٟ خ٤ٰ ه١٪وذ رغو٠ز٘شس حٰش ع٠ٛزارٮ اٛ ُٮ ئحذاس اإلعٔبؽ ا6٩ 2 ٢ٰٗ٪ٛزش٣س اال٩د
٢ٟ ٢٧٪ٜاصجذ خ٩ ْالرٮ عجٛا٩ ز٘شس٠ٛزارٮ اٛبع ا٨ اإلع٢ٟ ا٪٣غبء هب٣ ٢ٟ ذ٣خ ٗب٤ٰ ه١٪ عز٬ٛ٩هخ األ٪٠غ٠ٛٮ ا٠ٰٜزوٰٛخ ا٠٘بكٛا
داء٩ Herpes simplex virus ٮٜبع٤زٛحأل اٛط ا٩ُٰش٩ ,Rubella virus )ٰشاء٠حٰٛخ (ا٣ب٠ٛحظجخ األٛط ا٩اإلطبثخ ثِٰش
ٰذٯبٟ٘الٛا٩ , Niesseria gonorrhea ١غٰالٛغججخ ا٠ٛ ثج٘زشٯب ا٢٨ ئطبثزٖٝ هذٛٗز٩ Toxoplasma gondii عبد٪ٔ٠ٛا
ذ٣ٰخ ٗب٣ضبٛهخ ا٪٠غ٠ٛ ا.هخ٪٠غ٠ٛ ا٥ز٧ خ٤بد ُٮ أع٧٪د رش٪ع٩ ٝ هذ٢ٟ غٰغٰخ٤ٛذساعخ اٖٛ أصجزذ اٛٗز٩ Chlamydia trachomatis
٢زحش٭ هٜٛ )ELIZA( ٰضاٛبد ُحظذ ثـشٯٔخ اال٤ٰوٰٛن ا٠ ع.سح ؿجٰوٰخ٪ ثظ٢٨ٜ٠ ح٢ػو٩ غبء٣ ٢ٟ ذ٣خ ٗب٤ٰ ه١٪ صالص٢ٟ
نٟ خ٣ٔبسٟ ٬ٛ٩هخ األ٪٠غ٠ٛ اٙ٪ظٟ ُٮ2 ٢ٰٗ٪ٛزش٣ٯبد اال٪غزٟ ٰخ ُٮٛٯخ هب٪٤وٟ ّ٩زبئظ ُش٤ٛشد ا٨ أك, 6 ٩ 2 ٢ٰٗ٪ٛزش٣اال
هخ٪٠غ٠ٛن اٟ خ٣ٔبسٟ شرِوخٟ ٰخ٣ضبٛهخ ا٪٠غ٠ٛ اٙ٪ظٟ ُٮ6 ٢ٰٗ٪ٛزش٣وذالد االٟ ذ٣ٖ ٗبٛٗز٩ , )p< 0.001( ٰخ٣ضبٛهخ ا٪٠غ٠ٛا
ذ٠ذساعخ دهٛ ا٥ز٧ زبئظ٣ . ذساعخٰٛن ٰٓذ اٟغب٠ٛ ُٮ ا٢ٰ٠اط٪ٛ ا٢زٯ٧ ٢ٰ٭ ث٪٤وٟ زبئظ اسرجبؽ٤ٛش ا٨ رلٞٛ ٖٛ ر٬ٛ ثبإلػبُخ ئ.٬ٛ٩األ
.6 ٢ٰٗ٪ٛزش٣خِبع اال٣ا٩ 2 ٢ٰٗ٪ٛزش٣ز٘شس ُٮ حبالد اسرِبم اال٠ٛٔبئٮ اٜزٛشاػٰخ اإلعٔبؽ اٟ) ُٮ أ1 ٢ٗبٯ٪غبٯزٛس(ا٩ٰخ دٛب٠احز
Introduction
Recurrent spontaneous abortion (RSA) is defects include coagulation disorders,
one of the important complications in autoimmune defects, endocrine disorders and
pregnancy. Half of recurrent miscarriages loss endometrial defects (2). Mammalian pregnancy
is multifactorial, can be explained by genetic, is thought to be a state of immunological
hormonal,anatomical, metabolic abnormalities tolerance. The mechanisms underlying this
infections or autoimmune mechanisms and an phenomenon are still poorly understood,
be divided into embryological driven causes Successful mammalian pregnancy depends
(mainly due to abnormal embryonic upon tolerance of a genetically incompatible
karyotypes) and maternally driven causes fetus by the maternal immune system. When
which affect the endometrium and/or placental tolerance is not achieved pregnancies fail (3) .
development (1). Known causes of maternal
74
Iraqi J Pharm Sci , Vol.17 (2) , 2008 Interleukins and abortion
Immunological rejection of the fetus due to to the success of pregnancy by down regulating
recognition of paternal antigens by the potential Th-1 reactivity (12; 13). Protect the
maternal immune system, resulting in fetus and placenta from being rejected and to
abnormal immune cells and cytokine aid in the maintenance of normal pregnancy. In
production, is postulated to be one cause of humans an important role for the T-helper 2
unexplained pregnancy loss (4). Cytokines have immune response has also been reported
traditionally been divided into families during normal pregnancy (14; 15).
dependent upon the immune cell of origin and Methods : Studied group
the immunological effects that they bring This study included ninety (90) women from
about. CD4+ T-helper cells are the major the Obstetrics and Gynecology Department of
immune cells involved in cytokine production, Al- Kadhmiya teaching hospital in Baghdad.
and these can be divided into functional Patients' ages ranged between (18-36) years
subsets based on their cytokine production, T- with a mean of (27.5 − 30.1) year. The patients
helper 1 (Th1) cells produce interferon gamma were divided into two groups:
(IFNg), IL-2 and tumor necrosis factor beta Group 1: sixty (60) women were admitted to
(TNFb) are the main effectors of cell mediated the hospital for recurrent spontaneous abortion
immune response (5) T-helper 2 (Th2) cells (3-6 numbers of abortions) for evacuation.
produce IL-4, IL-5, IL-6 and IL-10, which are Group 2: thirty (30) women with successful
the main effectors of antibody-mediated pregnancy (normal delivery) as control group.
humoral responses (6). Local mechanisms may Sample collection
play an important role in evading immune From each women included in the study blood
attack because maternal alloreactive samples were collected to obtain the serum.
lymphocytes are not systemically depleted. The Procedure
specialized fetal tissue in contact with maternal
* Enzyme Linked Immunosorbent Assay
uterine tissue might contribute to tolerance by
(ELISA) for the detection of IL-2, IL-6 in
several mechanisms, such as depleting
serum: IL-2, IL-6: ELISA Test Kits provided
tryptophan,(7) by inactivating natural killer cells
by (Mabtech Australia Pty Ltd). Product cod:
through HLA-G expression,(8) or by provoking
(3460-IA-6) IL-6, (3430-IA-6) IL-2.
apoptosis of activated maternal lymphocytes (9).
Estimation of IL-2, IL-6 level in serum or
Incomplete tolerance might therefore result in
plasma by ELISA method. This method has
disturbed pregnancy such as spontaneous
two immunological steps. In the first step, the
abortion and pre-eclampsia. Further, Th1/Th2
cytokine is captured by monoclonal antibody
cytokine balance has been seen as a very
bound to the wells of a micro titer plate. In the
important mechanism determining the survival
second step a monoclonal antibody linked to a
of the fetus in the maternal uterus. Recent
biotinylated monoclonal antibody is add
evidence suggests that maternal tolerance is
together with streptavidine-peroxidase
established at the feto-maternal interface, by
conjugate. The solid phase antibody-antigen
factors deriving from the decidualized
complex and in turn, binds the conjugate. After
endometrium and from the trophoblast itself, is
incubation, the wells are washed and the
maintained throughout gestation in
antigen complex bound to the well detected by
physiological pregnancy (10). Cytokines
addition of a chromogenic substrate. The
released at the feto-maternal interface have
intensity of the color developed is directly
been proposed to play an important role in
related to the specific monoclonal antibodies
regulating embryo survival controlling not
concentration of the sample (16).
only the maternal immune response but also
Statistical analysis
angiogenesis and vascular remodeling (10; 11).
The Student test (t-test) analysis program was
Th-1 cytokines are considered to be
used to calculate the values, Mean, and
detrimental to pregnancy, via direct embryo
standard error were all used in the analysis and
toxic activity, or via damage to the placental
the relationship between the indicators was
trophoblast, or possibly by activating cells that
measured qualitatively by using the correlation
are deleterious to the conceptus, whereas Th-2
coefficient
cytokines may directly or indirectly contribute
75
Iraqi J Pharm Sc, Vol.17 (2) , 2008 Interleukins and abortion
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Iraqi J Pharm Sc, Vol.17 (2) , 2008 Interleukins and abortion
trophoblast antigens activate the PBMC of Makhseed and colleagues, demonstrated that
women with a history of unexplained recurrent IL-6 concentrations were lower in women with
spontaneous abortion (RSA) to produce the RSA than in those undergoing normal delivery,
embryotoxic cytokines IFN- and TNF-ß(22,23). considering that IL-6 is a Th2-type cytokine
Interleukin-2, tumor necrosis factor- , and and that normal pregnancy appears to be a
interferon- are deleterious and used to Th2-biased condition (13). Th1 and Th2 cells
stimulate the apoptosis of human primary are mutually inhibitory to each other when Th1
villous trophoblast cells. In addition this study reactivity is high, Th2 reactivity is usually low
reveals in table 2 that the expression of IL-6 and vice versa (30). The current study shows in
proteins in circulation of women with table (3) no significant correlation (p>0.05)
successful pregnancy was significantly between IL-2 and IL-6 in women with
higher (p<0.001) than that of women with recurrent spontaneous abortion (RSA) and
abortion. Further studies showed that there is a successful pregnancy. This un relation might
greater increase in IL-6 secretion during be associated with different role of these two
pregnant compared to not pregnant state that cytokine during pregnancy thus we suggested
may be detected by ELISA of endometrial further study focusing on the role of IL-2 and
biopsy samples (24). human trophoblasts IL-6 in pregnancy and RSA in placental tissue.
express IL-6 receptor and produce IL-6, which In conclusion, IL-2 might play a pathological
induces the production of hCG in an autocrine role in pregnancy in contrast IL-6 might play a
manner, suggesting a role of IL-6 in early role in successful pregnancy.
implantation and its continuation in early
pregnancy(25). IL-6 may play a role in References
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
الخالصة
ِشدا٤ٟ اء٩ دٚٗ ٙب٠ اعزو٬ٜ ه١٩ٰزبصٜ٘صٯ٩شٛا٩ ٢ٰٟس٪ِٰز٠ٛ ا٢ٟ ٰؾٜ خٙب٠ اعزو٢ٟ ائذ٪ِٛوشع اٛ ذ٠٠ذساعخ طٛ ا٥ز٧
٬ٛ ا٢٠زوذد االٰٗبط ٓغٟ جٰغ٠ٛخ اٟزالص٠ظبثخ ثٟ شأحٟ ا١٪اسثو٩ اسثن.زوذد االٰٗبطٟ جٰغ٠ٛخ اٟزالص٠ظبثبد ث٠ٛغبء ا٤ٛذ هالط ا٤ه
ذٛ٩ب٤ٰخ ر٣ضبٛهخ ا٪٠غ٠ٛ ا٩ ش٨ذح صالصخ اش٠ٛ ٰبٟ٪ي ٯٜٟ4 ثغشهخ١٩ٰزبصٜ٘صٯ٩شٛذ اٛ٩ب٤ ر٬ٛ٩هخ اال٪٠غ٠ٛ ا: هبد٪٠غٟ صالس
ٰبٟ٪ي ٯٜٟ4 ثغشهخ١٩ٰزبصٜ٘صٯ٩شٛ ا٢ٟ ٰؾٜذ خٛ٩ب٤ضخ ُٔذ رٛضبٛهخ ا٪٠غ٠ٛب اٟش ا٨ذح صالصخ اش٠ٛ ٰبٟ٪ي ٯٜٟ 1500 ثغشهخ٢ٰٟس٪ِٰز٠ٛا
زٌٰشُٮٛ ا١ا.والطٛ ا٢ٟ ش٨ثوذ صالصخ اش٩ والطٛ اٚ عحجذ ٓجٝذٛبرط ا٠٣ .والطٛذح اٟ ِظ٤ٛ ٰبٟ٪ي ٯٜٟ 1500 ثغشهخ٢ٰٟس٪ِٰز٠ٛا٩
٩ ١٩ٰزبصٜ٘صٯ٩شٰٛؾ اٜكب اٗضشُٮ خ٪حٜٟ ١ ٗب١٩عزٰش٪زغزٛا٩ ٮ٤ٰر٪ٜٛ ا١٪ٟس٪٨ٛ ا, ١٩عٰغزغش٩جشٛ ا,٢ٰٛ٪غ٣ اال, ص٪ٗ٪ٜ٘ٛٯبد ا٪غزٟ
. شٗت٠ٛوالط ثبٛم ثوذ ا٩ششٛ ا٫٪غزٟ ٢ٯب ه٪٤وٟ ٚٔ ٯ١٩عزٰش٪زغزٛ ا٫٪غزٟ ١ب ا٠ٗ.ِشدا٤ٟ اء٩ دٚٗ خ اخزٛب ُٮ حب٠ٟ ٢ٰٟس٪ِٰز٠ٛا
ٰؾٜ خ١زشرٰت اٛ ا٬ٜوب هٟ ب٠٧زبٜٗ٩ ٢ٰٟس٪ِٰز٠ٛا٩ ١٩ٰزبصٜ٘صٯ٩شٛ ُٮ ا62,5 , 36,4, 29,4ٮ٧ جٰغٛزبط ا٣ ا٬ٰٜخ هٜٔبثٛغجخ ا٣ ذ٣ٗب٩
.جٰغٛزبط ا٣غجخ ا٣ ٬ٜبُن ه٣ رأصٰش٦ٛ ٢ٰٟس٪ِٰز٠ٛ ا٩ ١٩ٰزبصٜ٘ صٯ٩شٛا
Introduction
Polycystic ovary syndrome (PCOS) is the
most common abnormality in women during cause androgen excess which are intrinsically
reproductive age, it is a hetrogenous disorder programmed to produce more androgens (6) .
of uncertain etiology (1). It is characterized by Excess androgens are known to interfere with
chronic anovulation and hyperandrogenism (2) the process of follicular maturation , thus
affecting approximately 5-10 % of inhibiting ovulation and producing more
reproductive age women. The association arrested follicles. It has been postulated that in
between hyperinsulinemic insulin resistance PCOS ovaries there is an increased rsistance to
and PCOS is well recognizes and may play an all insulin functions , except for steroidogenic
important pathogenic role in development of effect and the ultimate result is excess
PCOS (3). Obese and lean women with PCOS androgen production even with normal insulin
manifest insulin resistance independent on level(7). Metformin is abiguanide
body weight, although obesity is an additive hypoglycemic agent that is approved for the
factor which aggravates insulin resistance (4). management of type ΙΙ diabetes (8). Its main
There is some data to suggest that insulin mechanism of action is the reduction of hepatic
enhances the effect of LH on preovulatory glucose production ( hnhibition of
ovarian follicle arrest (5). It is possible that gluconeogenesis ). It also increases insulin
hyperinsulinemia due to insulin resistance mediated glucose utilization in peripheral
drives the LH affect on ovarian theca cells to tissue and decreases intestinal absorbtion of
glucose(9).
1 Corresponding author : E-mail : mohammed_taher43@yahoo.com
Received : 19/9/2007
Accepted : 30/12/2008
Several authors (10,11) have demonstrated the
additional benefits of using metformin such as
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
these related to menstrual cycle regulation and 2. -Group ΙΙ included (11) patients (BMI
induction of ovulation, protection from 29.1±5.2Kg/m2), age 24.9± years). The
pregnancy losses ,improvement of patients received metformin 1500mg daily
cardiovascular risk factors ,moreover in three divided doses (500mg after each
metformin markedly increases both spontenous meal ) for three months.
ovulation rate and clomiphene-induced 3. -Group ΙΙΙ included (16) patients (BMI
ovulation rate for obese women with PCOS (12) 34.2±6 Kg/m2) , age 26.5±4 years. The
. Many studies have shown improvements in patients received a combination of the two
ovulatory function , development of normal drugs ( rosiglitazone 4mg/day +
menses , and restoring of fertility (13). In spite metformin 1500mg/day ) for three months.
all of these benefits , many workers reported 4. -Control group included (16) normal
that metformin effect may be to some extent women (BMI 30±4.8 Kg/m2), age 27.1±6
transient and some cellular adaptation may years.
occur during more prolonged therapy (14).
Rosiglitazone was approved in (1999) by food Sample collection:
and drug administration (FDA) as an oral Eight millilitrs (8ml) of venous blood
antidiabetic agent for the management of type samples used in this study were drawn from
ΙΙ dibetes as montherapy and in combination PCOS patients.The first sample was collected
(15)
with oral hypoglycemic agents . before treatment as a baseline level, and after
Rosiglitazone increases insulin sensitivity three months of treatment with insulin
without stimulating insulin secretion, its mode sensitizing agents.Fasting blood samples were
of action is by binding and activation of the used for the measurement of glucose , insulin,
nuclear peroxisome proliferators activator hormones (LH, testosterone and progesterone).
gamma (PPAR-γ) which is found in key target Blood samples were left at room temperature
tissues for insulin action as adipose tissue, for 30 minutes for clotting, centrifugated and
skeletal muscle and liver. Activation of then serum was separated and collected in
(PPAR-γ) regulates the transcription of insulin small aliquots(0.5ml) and stored at (-20 C)
–responsive gens involved in control of until biochemical and hormonal analysis was
glucose and fatty acid metabolism (9,11) . performed. The serum was used for
Therefore this study was designed to show measurement of fasting blood sugar,insulin
whether combination of ( rosiglitazone and ,testosterone, LH and progesterone levels.
metformin ) has advantages over using each
drug alone in the treatment of women with Biochemical and hormonal assay:
PCOS. Fasting insulin levels were determined
using a commercial kit obtained from Randox,
Methods and Materials using Radioimmunoassay (RIA) method (17,18).
This study was conducted at Baghdad Serum testosterone levels were determined
city in AL-Elwia maternity teaching hospital using a commercial kit obtained from
from October-2004 till June-2005.The study Immunotech, based also on (RIA) (19). By
groups included 60 raqi women, (44) case with using of a kit from Immunotech, the
PCOS aged 17-40 years with a mean of age radioimmunoassay of progesterone is
27.3±5.07 years , and 16 normal control compitiotion assay (20). Serum LH determined
subjects aged 18-38 years with a mean of age using kit from Immunotech, by the
27.1±6 years. The patients included in this immunoradiometric assay (IRMA) which is
study were diagnosed with PCOS were non- sandwich type assay (21).Fasting serum glucose
diabetic, non-hypertensive and non- was measured by a commercial kit obtained
pregnant.The patients were under gynecologist from BIOmghreb, using the enzymatic
supervision during period of treatment. The method(22).
diagnosis of PCOS was made by gynecologist Diagnosis of infertility depends on that
depending on ultrasound examination, clinical inability of any couple to conceive a child
features and laboratory tests (hormonal assay). within a 12 months period of unprotected
The patients involved in this study were on coitus ( sexual intercourse) (23).
normal diet.They were divided randomly into 3 Body mass index (BM I) was calculated using
groups: the standard formula :
1. Groiup 1 included 17 patients (BMI BMI=weight (kg)/hight (m2). Obses patients
28.8±3.9 Kg/m2), age 29.7±6.4 years. The were defined as having BMH> 27 Kg/m2(24,25).
patients received rosiglitazone 4mg daily Homeostasis model assessment of insulin
in two divided doses (2mg) in the morning rsistance (HOMA_IR) was calculated using
and (2mg) in the evening after meals for 3 the following formula:
months. HOMA-IR= basal glucose (mmol/L).basal
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
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Iraqi J Pharm Sci , Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
Table (4): Effect of treatment with the combination of metformin(1500 mg /d) and rosiglitazone
(4mg/d) on the levels of insulin , glucose , HOMA-IR,LH and testosterone in group ΙΙ.
Variables Control levels Baseline levels (before treatment) After treatment
(n=16) (n=16) (n=16)(%)
Table (5): Ovulation rate in PCOS patients for treatment with insulin sensitizing agents.
Total 17 11 16 44
n=no. of women
Group 1: women treated with rosiglitazone 4mg/d alone.
Grouop ΙΙ: women treated with metformin 1500mg/d alone.
Group ΙΙΙ : women treated with combination of roziglitazone 4mg/d and metformin 1500mg/d.
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Iraqi J Pharm Sc, Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
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Iraqi J Pharm Sc, Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
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Iraqi J Pharm Sc, Vol.17 (2) , 2008 Rosiglitazone , metformin in polycystic ovary syndrome
A, et al. Effect of metformin treatment on 35. Nicolas A. Cataldo , Fahim Abbasi , Tracy
luteal phase progesterone concentration in L. Metabolic and ovarian effects of
polycystic ovary syndrome. Brazelian J rosiglitazone treatment for 12 weeks in
Medical and /biological Research. insulin resistance women with polycystic
2004:1637-1644. ovary syndrome . Hum Reprod.
34. Aziz R, Ehrmann D, Lergo RS et al . 2006;21(1) : 109-120.
troglitazone improves ovulation and 36. Dereli D, Dereli T, Bayraktar F, et al.
hirsutism in polycystic ovary syndrome Endocrinol and metabolic effects of
multicenter, double blind, placebo- rosiglitazone in non-obese women with
controlled trial. J Clin Endocrinol Metab polycystic ovary syndrome . Endocrinol
2002;86:1626-1632. Journal. 2005;52(3):299-308.
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