Influence of hydrogen peroxide in drinking water on diazepam pharmacokinetics in chicks

Aim: Stressful conditions affect drug pharmacokinetics and pharmacodynamics. This study examines the effect of hydrogen peroxide (H O ) in drinking water on the pharmacokinetics of diazepam in a chick model of oxidative stress. 2 2 Materials and Methods: Day old chicks were either provided with plane tap water (control group) or H O in tap water as 2 2 0.5% v/v drinking solution for two weeks in order to produce oxidative stress. On treatment days 7–14, the chicks were treated with a sedative dose of diazepam at 10 mg/kg, intramuscularly. Blood samples were obtained from chicks (5/each sampling time) at times of between 0.17 to 4 h. The concentrations of diazepam in the plasma were determined by an HPLC method with UV-detector. Pharmacokinetic parameters of diazepam were calculated from the mean drug concentrations in the plasma by a non-compartmental analysis using a Windows-based computer program. Results: Injection of diazepam resulted in the appearance of the drug in the plasma of control and H O -treated chicks at mean 2 2 concentrations ranging between 0.11 to 0.444 and 0.131 to 0.535 μg/ml, respectively when measured between 0.17 to 4 h after administration. Diazepam concentrations of the H O -treated chicks were significantly higher than those of the control group 2 2 at the sampling times 0.5, 0.75, 1 and 4 h. The highest concentration of diazepam in the plasma of both the control and H O 2 2 treated chicks occurred one h after the injection. The elimination half-life, mean residence time, maximum plasma concentration, area under the moment curve and area under plasma concentration-time curve in the H O -treated chicks were 2 2 higher than those of the control group by 35, 28, 23, 91 and 49%, respectively. Correspondingly, the steady state volume of distribution, elimination rate constant and total body clearance in the H O -treated chicks decreased from those of the 2 2 respective control values by 15, 24 and 33%. Conclusion: The data suggest that oral exposure of chicks to H O influences the pharmacokinetics of diazepam by 2 2 decreasing its elimination from the body.


Introduction
potentiates the anticholinesterase poisoning induced by the organophosphate insecticides dichlorvos and Many physically-induced stressful conditions diazinon in chicks [12].More recently we found that adversely affect pharmacokinetic and pharmaco-OS induced by H O sensitizes young chicks to the 2 2 dynamic aspects of drugs [1][2][3][4].Reactive oxygen sedative actions of diazepam and xylazine [19].As species are reported to be associated with various stressful conditions might affect drug metabolism and neuronal dysfunctions [5,6].Oxidative stress (OS) kinetics [1][2][3], the purpose of the present study was to produced by ferric-nitrilotriacetate was found to alter examine the effects of H O in drinking water on the 2 2 the pharmacokinetics of clomipramine in rats [7].The pharmacokinetics of diazepam in a chick model of OS. brain is highly vulnerable to OS which modifies the systemic response to central nervous system (CNS)

Materials and Methods
active agents [6,[8][9][10].Hydrogen peroxide (H O ) is 2 2 Animals: Day old broiler chicks of both sexes were used to induce experimental OS in laboratory animals 0 raised in a room with a temperature of 32-35 C, [11-14] and in vitro for experimental models of cellular OS [6,15,16].
constant lighting and wood shavings as floor litter.The H O -induced OS modifies the response of chicks had free access to drinking water and feed 2 2 throughout the experiment.The Scientific Committee rabbits and rats to general anesthetics [17,18].It also

To cite this article:
Mousa YJ, Mohammad FK (2012) Influence of hydrogen peroxide in drinking water on diazepam pharmacokinetics in chicks, Vet World, 5(11): 658-662, doi: 10.5455/vetworld.2012.658-662 of the College of Veterinary Medicine at the University (0.17-4 h) were used to calculate the pharmacokinetic of Mosul has reviewed and approved the protocol of parameters by a non-compartmental analysis [24,25] the study in chicks.All the experiments complied with using a Windows-based computer program [26].institutional regulations addressing animal use, and These were area under plasma concentration-time proper attention and care were given to the chicks used curve (AUC ), area under the moment curve (AUMC ) maximum diazepam concentration (Tmax), mean Ltd., U.K.) in tap water as 0.5% v/v drinking solution residence time (MRT=AUMC/AUC) and total for two weeks in order to produce OS reported earlier clearance (CL=Dose/AUC).Unpaired Student's-t-test [12,19].Each day we supplied the chicks with fresh was used to compare plasma diazepam concentrations drinking water.On treatment days 7-14, the chicks between the control and H O -exposed chicks [27].were treated with a sedative dose of diazepam 2 2 The level of statistical significance was at p < 0.05.(donated by the State Company for Drugs and Medical Appliances-Ninevah, Iraq) given intramuscularly Results (i.m.) at 10 mg/kg [19].We dissolved diazepam in Intramuscular injection of diazepam at the dose warm propylene glycol, and the volume of adminirate of 10 mg/kg, resulted in the appearance of the drug stration was at 5 ml/kg, i.m.Blood samples (1-2 ml) in the plasma of control and H O -treated chicks at were collected from chicks (5/each sampling time) by 2 2 mean concentrations ranging between 0.11 to 0.444 bleeding the jugular vein into heparinized test tubes and 0.131 to 0.535 µg/ml, respectively when measured [20] times of 0.17, 0.33, 0.50, 0.75, 1, 1.50, 2 and 4 h between 0.17 to 4 h after the injection (Table 1).after the diazepam injection.Plasma was obtained by Diazepam concentrations of the H O -treated chicks centrifugation of blood samples at 3000 rpm 2 2 (Centurion, U.K.) for 15 minutes.Plasma samples were significantly higher than those of the control were stored at -18ºC pending diazepam determination group at the sampling times 0.5, 0.75, 1 and 4 h (Table within one week.We extracted diazepam from the 1).The highest concentration of diazepam in the plasma by liquid-liquid extraction method using plasma of both the control and H O -treated chicks 2 2 dietheylether [21].Diazepam concentration in the occurred one h after the injection (Table 1).plasma samples was determined by an HPLC method The pharmacokinetic parameters of diazepam using a C column and UV detector with the HPLC calculated from the means of drug concentrations in 18 pump of Cecil, U.K. [22,23].The mobile phase consisted the plasma at times 0.17 to 4 h in chicks are shown in of acetonitrile: potassium dihydrogen phosphate pH Table 2.The elimination half-life, mean residence 2.1 buffer (70:30); the flow rate was at 1 ml/min and time, maximum plasma concentration, area under the the UV detector wave length was set at 245 nm.moment curve and area under plasma concentration-To reduce the effect of individual variation in time curve in the H O -treated chicks were higher than  under the plasma concentration curve and area under involving the release of free radicals that in turn causes the moment curve increased in the H O -exposed lipid peroxidation and increases intracellular calcium 2 2 chicks.[28,29].The toxic effects of H O , through production 2 2 Although we did not measure tissue diazepam of free radicals, might be related to lipid peroxidation concentration or its urinary excretion, it is also in the CNS and other tissues [16, 28,29].A recent report possible that H O might have decreased diazepam also indicates that H O stimulates G protein alpha 12 excretion rate since the total body clearance and to activate cellular injury [30].The model of H O -2 2 elimination rate constant of the drug were reduced induced OS in the present study appeared to alter the considerably (Table 2).We avoided any stress-induced pharmacokinetics of diazepam in chicks as manifested changes on the gastric emptying and intestinal by higher drug concentration in the plasma compared movement [4] by injecting diazepam systemically to the control group as well by the differences in the (i.m.).Increased sedative response to diazepam in the pharmacokinetics parameters.Oxidative stress H O -treated chicks [19] could be an outcome of these 2 2 induced by ferric-nitrilotriacetate also changed the pharmacokinetics changes.Other clinical changes in pharmacokinetics of clomipramine in rats [7].Other response to diazepam and its own metabolism in forms of stress such as those induced by immobianimals undergoing OS are not yet clear.lization [4], heat and exercise [2], conditioned fear [3], foot shock [31] and isolated physical activities [32] Conclusion were reported to alter drug pharmacokinetics.
In conclusion, the data of the present study Stressful conditions may alter drug disposition and suggest that oral exposure of chicks to H O influences kinetics through changes of plasma protein or tissue 2 2 the pharmacokinetics of diazepam by decreasing its binding, alteration of blood flow rates and cardiovaselimination from the body.cular functions, which might be accompanied with serious clinical outcome [1,2,32].author read and approved the final manuscript.University of Mosul, Iraq.
Radic.Res., 45: 888-905.11.Ahmed, L.I. (2010).Neurobehavioral and Acknowledgements biochemical studies of hydrogen peroxide-induced oxidative stress in chicks.M.Sc.Thesis.University of This report represents a portion of a dissertation Dohuk, Dohuk, Iraq.submitted by the first author to the University of 12. Al-Baggou, B.K., Naser, A.S. and Mohammad, F.K. Mosul, Iraq as partial fulfillment of the requirements (2011).Hydrogen peroxide potentiates organoof PhD degree in Veterinary Pharmacology.The study phosphate toxicosis in chicks.HVM Bioflux, 3: 142was supported by the College of Veterinary Medicine, 149.

Induction of OS and diazepam treatment: Day
ss maximum diazepam concentration (Cmax), time to (control group) or H O (Thomas Baker Chemical2 2 Values are mean ± SE of 5 chicks/each sampling time.Hydrogen peroxide was added to drinking water (0.5%, v/v) of the chicks at one day old and continued for 14 days.*Significantly different from the respective control value, p < 0.05.

Table - 1
[5,8,poral di[35]am concentrations (µg/ml) in the plasma of hydrogen peroxide exposed chicks after a single intramuscular administration at a dose of 10 mg/kg body weight.statevolume of distribution, elimination rate constant and frees oxygen radicals that cause cellular and DNA and total body clearance in the H O -treated chicks damage[5,8,[33][34][35].It is therefore possible that 2 2 altered tissue distribution and elimination of the drug decreased from those of the respective control values might have resulted from the changes in the pharmaby 15, 24 and 33% (Table2).cokinetics of diazepam.In this context the volume of 13. Celik, S. and Ozkaya, A. (2002).Effects of intraperitoneally administered lipoic acid, vitamin E, Competing interestsand linalool on the level of total lipid and fatty acids in guinea pig brain with oxidative stress induced by