Iranian Journal of Pharmacology and Therapeutics Razi Institute for Drug Research (RIDR) of Iran University of Medical Sciences and Health Services (IUMS) ISSN: 1735-2657 Vol. 4, Num. 2, 2005, pp. 76-79

Iranian Journal of Pharmacology & Therapeutics, Vol. 4, No. 2, 2005, pp. 76-79

Effect of Carbontetrachloride-Induced Hepatopathy on the Disposition Kinetics of Closantel in Goats

CHUBASANGLA JAMIR, SHIBEN CHANDRA DEBNATH, SHYAMAL KUMAR DAS, ANIMESH KUMAR CHAKRABORTY and TAPAN KUMAR MONDAL

Department of Pharmacology and Toxicology, Faculty of Veterinary and Animal Science, West Bengal University of Animal and Fishery Sciences, Mohanpur, Nadia, West Bengal, India.

Address correspondence to: Tapan Kumar Mondal, Reader and Head, Department of Pharmacology and Toxicology, Fac-ulty of Veterinary & Animal Science, West Bengal University of Animal and Fishery Sciences, Kolkata, West Bengal, India.E-mail: drtkm@rediffmail.com

Received June 23, 2005; Revised July 29, 2005; Accepted August 30, 2005

Code Number: pt05017

ABSTRACT

Clinically healthy adult black Bengal female goats were used to investigate the pharmacokinetics of closantel following intravenous administration (10 mg/kg) both in healthy and in liver damaged goats. In-duction of liver damage was done by subcutaneous injection of carbontetrachloride at 0.75 mL/kg on three occasion at 48 h interval. After administration of drug, blood samples were collected in heparinized test tubes at pre-determined time. The drug concentration in plasma was measured by double beam UV– Vis spectrophotometer at 222 nm. In vitro plasma protein binding of closantel was also carried out. C_max (118.82 ± 4.85 μg/mL) and C_min (4.69 ± 0.72 μg/mL) of closantel were recorded at 0.08 and 48 h respec-tively after i.v. administration to healthy goats, while the above values were 144.67 ± 2.50 and 3.93 ± 0.05 μg/mL at 0.08 and 48 h respectively in liver damaged goats. The t_½β, Cl_B and Vd_area values were 12.92 ± 1.30 h, 0.008 ± 0.0005 L/kg/h and 0.15 ± 0.01 L/kg respectively in healthy goats, while these values were 12.84 ± 0.38 h, 0.008 ± 0.001 L/kg/h and 0.16 ± 0.005 L/kg respectively in liver damaged goats. Binding capacity and association rate constant of closantel were not altered significantly in liver damaged animal compared to healthy animal. Liver damaged condition did not alter significantly the pharmacokinetic parameters of closantel.

Keywords: Closantel, Disposition kinetics, Liver damage, Goats

Helminthosis is one of the important diseases of domestic animal in India, although the magnitude of incidence may vary in different geographical location. Worms cause various disorders viz., blood loss, nutri-tional deficiencies, urticaria, allergic manifestation, in-testinal obstruction, loss of production, interference of immune system, damage to internal organs, release of toxic substances and secondary bacterial infections. Hence, control of both external and internal parasites of has immense economical and clinical importance.

Closantel N-[5-chloro-4(4-chlorophenyl) cyanomet-hyl]-2-methylphenyl]-2-hydroxy-3, 5-diiodobenzamide] is a typical member of the halogenated salicylanilide group of compound. It is a truly ectendoparasiticidal as it kills both ecto- and endo-parasites. It has been found to be effective against trematodes [ 1 ] and ecto-parasites [ 2 ].

The liver is the main metabolic organ in the body. The efficacy as well as disposition kinetics of anthelmintics used in liver damage condition may be interfered with due to altered metabolism. Besides flukes and round worms, some other parasites may damage the liver and cause cirrhosis during their larval migratory phase. Wherever the larva lodges, chronic inflammatory changes arise with resulting fibrosis. The effects of liver disease on pharmacokinetics of drugs are unpredictable, but clearly, the elimination of many po-tent drugs is impaired in patients with chronic liver dis-ease. There are two distinct approaches to study the bio-transformation of drugs by the diseased liver. One is measuring the activity of drug metabolizing enzymes in liver biopsies, and the other is determining the disposi-tion kinetics of drugs in patient with liver disease [ 3 ].

In view of the above, the present research was un-dertaken to determine disposition kinetic behaviour of closantel in healthy and carbontetrachloride-induced liver damaged animals following intravenous admini-stration.

MATERIALS AND METHODS

Chemicals

Closantel, a halogenated salicylanilide derivative (Technical grade, purity > 90%) was obtained as a gift from Sarabhai Zydus Animal Health Pvt. Ltd., Vado-dara, Gujarat, India. All other chemicals used in this experiment were obtained from E. Merck (India) and Sigma Chemical Co (U.S.A.).

Animals

Twelve clinically healthy adult black Bengal nul-liparous female goats weighing between 7 – 10 kg of 1 – 1½ year old were used. They were divided into group I and group II, each containing six animals. The animals were kept in individual custom made stainless steel cage (size - 48″ × 48″ × 36″) in temperature (22 ± 2°C) con-trolled animal room having provision of artificial light. The animals were acclimatized with the laboratory con-dition for 7 days. They were fed with balanced feed and water was supplied ad libitum. The animals were de-wormed with albendazole at 7.5 mg/kg orally 30 days prior to the onset of study. The lower part of the neck of each animal was shaved and the jugular vein was ex-posed. The animals were fasted overnight prior to the start of the experiment.

All the procedures adopted in the study were ap-proved by the ‘Animal Ethical Committee’ of West Bengal University of Animal and Fishery Sciences, In-dia.

Animal Treatment

A single dose of closantel dissolved in 2.5 mL of glycerinformal was administered to each animal of group I through the left jugular vein at 10 mg/kg. Blood samples were collected from the right jugular vein in heparinized test tubes before and after 0.08, 0.16, 0.33, 0.5, 0.66, 1, 2, 4, 6, 8, 12, 24, 36 and 48 h of drug ad-ministration.

Blood samples were (3.5 mL) collected at the above mentioned time period except at 0.08, 0.16, 1 and 2 h where 6 mL of blood were collected for protein binding study of closantel.

Plasma was then separated by centrifugation at 3000 rpm for 30 min. One mL of plasma was utilized for analysis of closantel and 2 mL of plasma for estimation of plasma protein binding of closantel.

Liver of each goat of group II was damaged by sub-cutaneous administration of carbontetrachloride mixed with equal volume of liquid paraffin following over-night fasting. Carbontetrachloride at 0.75 mL/kg was administered on three occasions at 48 h interval. The intensity of liver damage was assessed by performing serum bromosulphophthalein clearance test, icterus in-dex, and serum aspartate and alanine aminotransferase activity.

Bromosulphophthalein (BSP) clearance test and ic-terus index were done by the method of Oser [ 4]. Alanine aminotransferase (ALT) and aspartate ami-notransferase (AST) activities were measured as per method described by Yatazidis [ 5]. Closantel was administered intravenously at 10mg/kg to each of liver damaged goat of group II after 48 h of last dosing of carbontetrachloride. Blood samples were collected and plasma was separated as previously.

Analytical Method

To a centrifuge tube containing 1 mL of plasma, 10 mL of acetonitrile (acidified with concentrate sulfuric acid) was added, shaken vigorously for 2 min and cen-trifuged at 5000 rpm for 20 min. The supernatant was collected and the absorbance was read in double beam UV–Vis spectrophotometer at 222 nm wavelengthagainst blank prepared with plasma collected at ‘0’ h (before administration of drug). A standard curve of closantel in control plasma at different concentration was prepared. Concentration of closantel present in each blood samples was then calculated from standard curve prepared earlier and expressed as μg/mL. The co-efficient of variance of the accuracy and precision of this method was less than 9.50 % of the mean. The sen-sitivity of the method was 0.5 μg/mL. The limit of de-tection of closantel was 1 μg/mL. To avoid photodegra-dation of closantel, the analytical process was done in dark room.

The plasma protein binding of closantel was deter-mined by equilibrium dialysis technique, as described by Sisodia et al. [ 6 ] and Banerjee et al. [ 7 ], and modi-fied by Mandal et al. [ 8 ]. The protein content of plasma of each sample was also estimated by Biuret method [ 9 ]. The protein binding was then expressed as percentage. The binding capacity, dissociation and association con-stants were also calculated by the method of least re-gression technique described by Pilloud [ 10 ].

Plasma drug concentration versus time for each goat was analyzed using computerized curve fitting pro-gramme ‘PHARMKIT’ (supplied by Department of Pharmacology, JIPMER, Pondicherry, India). The pro-gramme fits the line to a biexponential equation for both healthy and liver damaged groups of animal. Pharma-cokinetic parameters were calculated from the comput-erized curves according to Gibaldi and Perrier [ 11 ].

Statistical Analysis

Students t-test was applied to test the level of sig-nificance in drug concentration, kinetic and different biochemical parameters in different groups of animals.

RESULTS

All the biochemical parameters determined from se-rum sample collected at 2, 4, 5 and 7th day of admini-stration of carbontetrachloride were significantly in-creased from the control values (sample collected before administration of carbontetrachloride) ( Table 1 ). This indicated that carbontetrachloride at the recommended dose level damaged the liver.

Kinetics

The mean plasma concentrations of closantel in healthy and carbontetrachloride-induced liver damaged goats at different time intervals have been incorporated in Fig 1 . The maximum and minimum concentration of closantel in healthy goat were 118.82 ± 4.85 and 4.69 ± 0.72 μg/mL at 0.08 and 48 h while in liver damaged goats, the maximum and minimum concentrations were 144.67 ± 2.50 and 3.93 ± 0.05 μg/mL at the same time, respectively. The maximum concentration of closantel in liver damaged goats was higher than that of healthy goats but the concentrations for different times between both groups of animals were not altered significantly.

Kinetic parameters of closantel in normal and liver damaged goats are presented in Table 2 .

Protein Binding

Results obtained in relation to plasma protein bind-ing of closantel are presented in Table 3 . The binding capacity, association constant and dissociation constant of closantel with plasma protein have been presented in Table 4 .

DISCUSSION

Disposition kinetics of closantel in healthy and hepa-topathic animal after single dose intravenous admini-stration at 10 mg/kg showed a biphasic declination of the concentration in both the groups of animal sugges-tive of “two compartment open model” kinetics of closantel. Swan et al. [ 12 ] also reported two compart-ment model with first order rate constant of closantel in sheep following single dose intravenous administration.

It would be evident from Table 2 that t½β and ClB values of closantel in animals of damaged liver were not altered significantly compared to healthy goats. Swan et al. [ 12 ] observed long elimination half life of 17.0 ± 4 day in adult sheep after i.v. administration. The shorter t_½β (12.92 ± 1.30 and 12.84 ± 0.38 h in normal and liver damaged goats respectively) value in goat might be at-tributed to species variation which has been substanti-ated by Hennessy et al. [ 13 ] during their study on com-parative pharmacokinetics of closantel in sheep and goat following intraruminal or intramuscular administration. The values of AUC and Vd_area were also unaffected in liver damaged goats. Further, these values also sug-gested very limited distribution of closantel in the body. The values of K₁₂ and K₂₁ in both groups of animals also indicated that closantel has lower affinity to accu-mulate in tissue compartment. Halogenated salicylani-lides are extensively plasma bound and poorly distrib-uted to tissues [ 12 ]. In the present experiment, the ex-tensive binding of closantel of plasma albumin (> 97%) has been observed. Results obtained in relation to plasma protein binding of closantel showed that the equilibrium association constant was greater compared to dissociation constant. The binding to plasma albumin has also been characterized by very high affinity and high capacity. Therefore, the extensive plasma binding of closantel is responsible for long elimination half-life which has also been reported in other salicylanilide compound to other species. Michiels et al. [ 14 ] reported that 90% of closantel is excreted unchanged in faeces and urine in sheep and cattle. A reductive monodiiodi-nation reaction appears as the main metabolic pathway for closantel in sheep resulting in the formation of 3, 5 monoiodoclosantel isomers [ 14 ]. Similar results have been confirmed in cattle and goats [ 15 ]. It is expected that the metabolism of drug might be interfered in he-patic damage as liver is the main metabolic organ in the body. Therefore, unaffected pharmacokinetic parame-ters of closantel in liver damaged goat may suggest ex-tra hepatic metabolism of closantel in goats.

ACKNOWLEDGEMENT

The authors would like to thank M/S Sarabhai Zy-dus, Vadodara, Gujarat, India for their free gift sample of closantel (Technical grade, purity > 90%).

REFERENCES

1. Choudhari SS, Gupta RP, Kumar S, Singh J, Saga AM. Epide-miology and Control of Fasciola Gigantic an Infection Of Cattle And Buffaloes In Eastern Harrying, India. Ind J Anim Sci 1993;63(6):600-5.
2. Khan FA. Comparative Anthelmintic Activity Of Strategic Sus-tained Low Level Administration Of Albendazole In Feed Pel-lets Compared To Single Dose Of Closantel And Tetramisole Against Natural Ovine Parasitic Gastroenteritis. Trop Anim Health Prod 1999;31(4):193-204.
3. Baggot JD. Principles of Drug Disposition in Domestic Animals. In: The Basis of Veterinary Clinical Pharmacology. 1st Ed., Philadelphia: W.B. Saunders Co.; London; 1977. p. 144-87.
4. Oser BL. Hawk’s Physiological Chemistry. 14th Ed, New Delhi : Tata Mcgrow Publishing Ltd; 1965.
5. Yatazidis H. Measurement of Transaminase in Serum. Nature 1960;18:79-80.
6. Sisodia CS, Miller GE, Stowf CM. Protein Binding Of Sul-phonamides and Quinine in Bovine Milk and Plasma. Ind Vet J 1965;42:7-16.
7. Bakeries NC, Miller GE, Stowe CM. Determination of Protein Binding Values of Aminopyrine in Bovine Plasma And Milk. Ind J Exp Biol 1969;7:102-3.
8. Mandal TK, Chakraborty AK, Bhattacharyya A. Disposition Kinetics of Cypermethrin and Fenvalerate in Black Bengal Lac-tating Goats. Paste Sci 1995;45:215-9.
9. Wooton IDP. Estimation of Protein by Biuret Method. In: Mi-croanalysis in Medical Biochemistry. 5th Ed, Edinburgh: Chur-chill Livingstone; 1974. p. 156-8.
10. Pillowed M. Pharmacokinetics, Plasma Protein Binding and Dosage of Oxytetracycline in Cattle and Horses. Res Vet Sci 1973;15:224-30.
11. Gibaldi M, Perrier D. Pharmacokinetics. 2nd Ed, Barcelona: Marcel-Dekker Inc.; 1982.
12. Swan GE, Koeleman HA, Steins HS, Mulders MS. Intravascular Plasma Disposition and Salivary Secretion of Closantel and Ra-foxanide in Sheep. J South Afri Vet Assoc 1999;70(9):75-9.
13. Hennessy DR, Sangster NC, Steel JW, Collins G.H. Compara-tive Pharmacokinetic Disposition of Closantel in Sheep and Goats. J Vet Pharmacol Therap 1993;16(3):254-60.
14. Michiels M, Meuldermans W, Heykans J. The Metabolism and Fate of Closantel (Flukiver) In Sheep and Cattle. Drug Metabo-lism Reviews 1987;18:235-52.
15. Short CR. Consideration of Sheep as a Minor Species: Compari-son of Drug Metabolism and Disposition with Other Domestic Ruminants. Vet Human Toxicol 1994;36:24-40.

Copyright © 2005 by Razi Institute for Drug Research (RIDR)

The following images related to this document are available:

Photo images