Pharmacokinetics of Phenobarbital in the Cat Following Multiple Oral Administration Susan M. Cochrane, Joane M. Parent, William D. Black, Dana G. Allen and John H. Lumsden
tion du produit. La demi-vie d'elimination apres le dernier jour de Phenobarbital was administered traitement etait de 43.3 ± 2.9 h. Le orally to seven healthy cats at a dose grand volume apparent de distribuof 5 mg/kg once a day for 21 days. tion (695.0 ± 43.9 mL/kg) suggere Serum phenobarbital concentrations que le produit se distribue largement were determined using a commercial dans tout le corps. La demi-vie immunoassay technique. A one- d'elimination 'a la fin d'administracompartment model was used to tions successives de phenobarbital describe the final elimination curve. etait significativement plus courte The elimination half-life (t/2 b) after qu'apres une seule administration the final day of treatment was 43.3 + orale chez le chat. L'analyse de la 2.92 h. The large apparent volume of pharmacocinetique du produit apres 1 distribution of 695.0 ± 43.9 mL/kg et 21 jours de traitement montre que la suggests that the drug was widely cinetique d'elimination du phenobardistributed within the body. The t/2 b bital n'a pas significativement change following multiple oral administration apres plusieurs prises orales du was significantly shorter than pre- produit. II semble donc que des viously reported for a single oral dose differences dans la cinetique d'elimiof phenobarbital in the cat. Analysis nation du produit existent entre of pharmacokinetic results after days 1 differentes populations de chats. A and 21 of treatment suggested that the cause de ces differences de population, elimination kinetics of phenobarbital il serait prudent que les niveaux did not change significantly with seriques de phenobarbital soient multiple oral administration. It mesures chez chaque chat apres appears that differences in elimination l'administration du produit. (Traduitpar kinetics can exist between populations Dr Pascal Dubreuil). of cats. These differences emphasize the need for individual monitoring of INTRODUCTION cats receiving phenobarbital.
ABSTRACT
RESUME Du phenobarbital
a
ete administre
per os a sept chats normaux une fois par jour pendant 21 jours a la dose de 5
mg/kg. Les concentrations seriques de phenobarbital ont ete mesurees a l'aide d'une technique commerciale. Un modele a un compartiment decrivait le mieux la courbe d'elimina-
Phenobarbital (PB) is used as an anticonvulsant in several species including the canine (1,2), equine (3), human (4) and feline (2). Pharmacokinetic data for PB are available for dogs (1,5,6), horses (3,7,8) and humans (4,9-12). A single intravenous (IV) and single oral dose study of PB in the cat suggested that this drug is ideally suited for use as an oral anticonvulsant in this species (13).
A dose for multiple oral administration can be calculated from data determined in single IV and oral dose studies, provided that elimination of the drug is not modified with repeated treatment (14,15). An altered rate of elimination can result from changes in the rate of drug metabolism (15). Since PB is a potent inducer of hepatic microsomal enzymes (16), the metabolism of drugs administered in conjunction with PB may potentially be enhanced (17). It is unclear whether or not PB can enhance its own metabolism through enzyme induction. Martin et al (11) demonstrated a shorter elimination half-life (t/2 b) in people who abuse PB, compared with normal volunteers. Other studies in humans (12), dogs (6,18) and cats (19) have failed to show a similar reduction in t½/2 b with repeated PB administration. It has been suggested that the dose and the duration of PB treatment may influence the extent of enzyme induction (18,20). Frey (2) reported a t½/2 b of 34-43 h for PB in the cat following multiple oral administration. This t/2 b was shorter than the value reported by the present authors for a single oral dose of PB in the cat (76.1 ± 6.97 h) (13). While the shorter t/2 b determined for repeated PB administration may be due to changes in enzyme activity, it may also reflect differences in the populations of cats sampled (6). The objective of this study was to determine the effect of multiple oral dose administration on the pharmacokinetics of PB in the cat in order to make recommendations for long-term oral therapy in this species.
Department of Clinical Studies (Cochrane, Parent, Allen), Department of Biomedical Sciences (Black) and Department of Pathology (Lumsden), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. This study was funded by the Ontario Veterinary College Pet Trust. Submitted July 13, 1989.
Can J Vet Res 1990; 54: 309-312
309
MATERIALS AND METHODS EXPERIMENTAL ANIMALS
Seven healthy mixed breed specific pathogen free cats (six males; one female) ranging from approximately one to two years of age were obtained from the University of Guelph Laboratory Animal Facility. Prior to the study the following tests were performed on each cat: hematological evaluation including hemoglobin, hematocrit, red blood cell count, white blood cell count and differential; serum biochemistry analysis including total protein, albumin, creatinine, urea, glucose, bilirubin, serum alkaline phosphatase (SAP), serum alanine aminotransferase (SALT) serum aspartate aminotransferase (SAST), sodium, potassium and chloride levels (Coulter Dacos, Coulter Electronics of Canada, Burlington, Ontario), and fecal parasite egg count. Throughout the experiment, the cats were housed in individual metal cages in a room isolated from other cats. They were fed commercial dry cat food (Meow Mix®, Ralston Purina, Mississauga, Ontario) and water ad libitum. The cats were fasted for 12 h prior to anesthesia for catheter placement. SURGICAL PROCEDURES
Prior to the oral study an indwelling jugular catheter (16 or 19 gauge, 20.3 cm Intracath, Deseret Co., Sandy, Utah) was placed as previously described to facilitate blood collection (21). To enable catheter placement the cats were placed in a 5 L airtight plexiglass box and anesthesia induced with isoflurane (AErrane®, Anaquest, Pointe Claire, Quebec) and oxygen. The cats were intubated and maintained at a light surgical plane of anesthesia during catheterization. For the two days prior to the start of sample collection, the catheter was flushed once a day with 0. 5 mL heparin solution (100 U/L) The catheter was removed on day 8 of the study and another catheter placed in the opposite jugular vein prior to the final week of the experiment. MULTIPLE ORAL PHENOBARBITAL STUDY
Oral PB administration was initiated two days following catheter
310
placement. Phenobarbital tablets (Phenobarbital acid USP, ParkeDavis, Scarborough, Ontario) were administered at a dose of 5 mg/kg daily at 0830 h for 21 days. The tablets were cut and weighed to ensure an accurate dose. On day 1 of the study, blood samples (0.5 mL) were collected at 0,4,6,8,10 and 12 h after treatment. Prior to sampling, 0.5 mL of blood was drawn and discarded to avoid collecting the residual volume of the catheter. The catheter was flushed with 0.5 mL of heparin solution (100 U/ mL) following the 12 h sample and after subsequent sampling. Sample collection on days 2 through 7 was performed at 4 and 24 h after PB administration. Prior to removal of the catheter on day 8, blood was collected for hematological evaluation and SAP and SALT levels. On day 17, the cats were anesthetized as previously described and a catheter placed in the opposite jugular vein. On days 19 and 20, samples were collected at 4 and 24 h after PB administration. After the final treatment on day 21, samples were drawn at the following times: 4, 6, 8, 12, 24, 32, 48, 56, 72, 80, 96 h. Following the last sample, blood was collected for hematological evaluation and serum biochemistry analysis.
elimination slopes and the overall elimination rate constant (b) were obtained from regression analysis. The elimination half-life was calculated as t¼/2b = 0.693/b, apparent volume of distribution by V'd(B') = Dose/ B', and clearance of drug as C1(B') = V'd(B') x b. Estimated steady state PB levels for the experimental cats were calculated using the following formula published by Wagner (15): CPo = (1.44) t/2 x D x F V'd x T where D represents dose administered, F the fraction of each dose absorbed [F 1 based on previous study (13)] and T the dosage interval. A paired Student's t-test was used to compare hematological, biochemical and pharmacokinetic parameters determined for the first and final treatments. The results of this multiple dose study were compared with the previously reported single dose study using an unpaired Student's t-test (22). Statistical analyses were performed at a confidence level of 95%. All data are presented as mean ± standard error of the mean (SEM).
PHENOBARBITAL ASSAY
The hematological and serum biochemical parameters measured prior to treatment were not significantly different from those determined on day 25, with the exception of glucose. The glucose level prior to start of the study was 7.96 ± 0.333 mmol/L, and at completion of the experiment was 5.92 ± 0.320 mmol/ L. The normal range established at the Ontario Veterinary College for blood glucose in the cat is 3.5-9.0 mmol/ L. The serum drug concentrations measured following oral PB were plotted against time (Fig. 1). Steady state drug concentrations were present when sampling resumed on day 19. The average drug concentration observed at steady state (Cpoo) was approximately 16.4 ,ug/ mL. The maximum (CPOOmax) and minimum (CpoQmin) steady state drug concentrations were approximately 19.3 ,ug/mL and 13.4 pg/mL. The elimination curve for PB following the final treatment on day 21
The blood samples were allowed to clot and the sera were separated by centrifugation (15 min at 1239 g). Phenobarbital analysis was performed by an immunoassay technique (Ames Seralyzer®, Miles Laboratories, Elkhart, Indiana) within 1 h of sampling. This technique was validated for the cat as previously described (13). DATA ANALYSIS
Logarithmic serum PB concentrations were plotted against time (linear). The terminal elimination slopes following the first and final treatments were obtained from this graph and subjected to least squares regression analysis (22). Pharmacokinetic parameters were determined for these elimination phases using a onecompartment model (23). These parameters were calculated using standard formulae described by Baggot (14) and Gibaldi and Perrier (23). The y-intercept (B') of the
RESULTS
administration on days 1 and 21 were 18.8 ,tg/ mL and 18.7 Alg/ mL. These values did not appear to be different from the steady state level of approximately 16.4 ,ug/mL observed in this experiment.
E C
.20 Cz 0)
DISCUSSION
C
0
During this 21 day oral PB study, no signs of drug toxicity such as ataxia or 0 sedation were observed in the cats. C The hematological and serum biochemistry analyses did not suggest any aadverse effects such as anemia, hypoproteinemia or hepatotoxicity that might have influenced the pharmacokinetic parameters. AlI I I .19I 19 20 2I 225 though there was a significant 24 21 22 23 decrease in the glucose level after Time (doys) Fig. Serum phenobarbital concentrations (i means SEM; means) following oral administration completion of the experiment, it was of 5 mg/kg once a day for 21 days in seven cats. thought not to affect the results. A one-compartment model was corresponded to a one-compartment when B' after treatment on day 21 (20.0 used to describe the elimination slope model (r = 0.992) The pharmacoki- ± 0.977 ,ug/mL) was reduced to 7.34 ± of the drug concentration-time curve netic parameters determined for the 0.397 ,ig/ mL to correct for the amount following the 21 day study. Since elimination curves after day 1 and day of PB already accumulated in the blood sampling on day 21 started at 4 h after 21 of treatment are compared in Table due to repeated drug administration, it PB administration, earlier absorption and distribution compartments were was not significantly different. I. With the exception of B', there were no significant differences between the Steady state serum PB concentra- not detected. However, based on the parameters compared. However, tions (Cpoo) estimated after drug results of a single oral dose study performed by the present authors (13), where sampling began at 5 min after TABLE I. Comparison of pharmacokinetic parameters following first and final dose (5 mg/kg) of drug administration, elimination of phenobarbital in the cat PB after oral administration is described by the oneadequately Final dose First dose compartment model in the cat. The Mean Mean Range ±SEM Parameter +SEM Range half-life of this final elimination phase was at the upper limit of the range of * 20.0 17.9-22.1 B' 6.52 5.72-7.18 ±0.977 ±0.198 34-43 h reported by Frey (2) for PB (,ug/ mL) following multiple oral administration 5.4-8.5 7.34 Adjusted B' ±0.397 in the cat. These values are, however, shorter than previously determined by 0.016 0.012-0.020 0.015 0.012-0.017 b +0.001 ±0.001 (h-') Cochrane et al (13) for a single oral dose of PB in the cat (t/2 b = 76.1 ± 43.3 35.0-56.3 40.8-57.8 47.6 t'/2b ±2.92 ±2.89 (h) 6.96 h). The differences in elimination half-lives between the present multiple 625.0-925.9 696.4-869.6 695.0 773.6 V'd(B') ±43.9 ±23.5 (mL/ kg) oral dose study and the single oral dose study may reflect either enzyme 7.88-14.6 11.3 9.23-12.5 11.4 C1(B') ±0.782 +0.439 (mL/ kg/ h) induction and enhanced PB metabolism, or a preexisting difference in PB 3.5-4.5 4.07 Wt (kg) 4.39 4.0-5.0 ±0.130 +0.172 metabolism between the two groups of = Significant difference between first and final dose cats. * = y-intercept beta curve B' To evaluate the possibility that Adjusted B' = y-intercept corrected for drug accumulation by subtraction of drug concentration multiple PB administration resulted in before treatment on day 21 (12.66,Mg/ mL) from actual B' intercept (20.0 ± 0.977 Mg/ more rapid drug elimination through mL) enzyme induction, the elimination b = overall elimination rate constant = half-life elimination kinetics determined after day 1 of t1/2b = apparent volume of distribution V'd(B') treatment were compared to the same = total body clearance from plasma CI(B') parameters calculated after day 21. Wt = weight of animal 0
0
I
1
±
*
311
The fact that the pharmacokinetic parameters were not significantly different suggests that for the dose used and the duration of this study, multiple drug administration did not enhance the elimination of PB. In support of this observation, repeated PB administration did not appear to alter estimated steady state concentrations. The observed steady state concentration was similar to the steady state concentrations calculated after both days 1 and 21 of treatment. Steady state serum concentrations are dependent on both input and elimination processes (14). Assuming that input remains constant, enhanced drug elimination would have resulted in a lower steady state PB concentration (15). No differences were, however, demonstrated. This further supports the lack of enzyme induction following repeat oral administration of PB. From these comparisons, it appears that the differences observed between the cats used in this multiple oral dose study and the pound source cats in the previous single dose study (13) may have been due to genetic and/ or environmental differences (16,24). It is of interest that Frey (6) reported a significant difference in half-lives of Beagles (32.0 ± 4.8 h) compared to mongrel dogs (70.0 ± 16 h) after administration of a single IV dose of PB. Frey (25) suggested that mongrel animals may more closely represent clinical cases than do laboratoryraised animals. Therapeutic serum PB concentrations of 15-35 ,ug/mL have been suggested for control of seizures in the cat based on data extrapolated from humans (26,27) and dogs (18,28). In order to achieve these concentrations in the cat, a maintenance dose of approximately 4.2 mg/ kg administered once a day was recommended (13). This dose was calculated from the longer half-life determined in the single oral study. If the PB half-life in an individual cat was shorter, such as recorded in this multiple dose study, the steady state concentration attained after a daily dose of 4.2 mg/kg would be lower and possibly subtherapeutic. Therefore, due to the potential for large differences in PB elimination rates between groups of cats, serum PB steady state concentrations should be measured and the dose adjusted if necessary. 312
In summary, the elimination kinetics of PB did not appear to change with repeated drug administration. It was demonstrated that differences in the elimination kinetics of PB can exist between populations of cats. These differences emphasize the need for monitoring of drug concentrations in individual cats receiving PB.
ACKNOWLEDGMENTS The authors wish to thank Jean Claxton for her excellent technical assistance and guidance during the kinetic analysis and for the use of her computer programs.
REFERENCES 1. AL-TAHAN F, FREY NH. Absorption kinetics and bioavailability of phenobarbital after oral administration to dogs. J Vet Pharmacol Ther 1985; 8: 205-207. 2. FREY HH. Use of anticonvulsants in small animals. Vet Rec 1986; 118: 484-486. 3. SPEHAR AW, HILL MR, MAYHEW IG, HENDELES L. Preliminary studies on the pharmacokinetics of phenobarbital in the neonatal foal. Equine Vet J 1984; 16: 368371. 4. WILENSKY AJ, FRIEL PN, LEVY RH, COMFORT CP, KALUZNY SP. Kinetics of phenobarbital in normal subjects and epileptic patients. Eur J Clin Pharmacol 1982; 23: 87-92. 5. PEDERSOLI W, WIKE JS, RAVIS WR. Pharmacokinetics of single doses of phenobarbital given intravenously and orally to dogs. Am J Vet Res 1987; 48: 679683. 6. FREY HH, G6GEL W, LOSCHER W. Pharmacokinetics of primidone and its active metabolites in the dog. Arch Int Pharmacodyn Ther 1979; 242: 14-30. 7. RAVIS WR, DURAN SH, PEDERSOLI WM, SCHUMACHER J. Pharmacokinetic study of phenobarbital in mature horses after oral dosing. J Vet Pharmacol Ther 1987; 10: 283-289. 8. DURAN SH, RAVIS WR, PEDERSOLI WM, SCHUMACHER J. Pharmacokinetics of phenobarbital in the horse. Am J Vet Res 1987; 48: 807-810. 9. VISWANATHAN CT, BOOKER HE, WELLING PG. Bioavailability of oral and intramuscular phenobarbital. J Clin Pharmacol 1978; 8: 100-105. 10. GARRETTSON LK, DAYTON PG. Disappearance of phenobarbital and diphenylhydantoin from serum of children. Clin Pharmacol Ther 1970; 11: 674-679. 11. MARTIN PR, KAPUR BM, WHITESIDE EA, SELLERS EM. Intravenous phenobarbital therapy in barbiturate and other hypnosedative withdrawal reactions: A kinetic approach. Clin Pharmacol Ther 1979; 26: 256-264.
12. VISWANATHAN CT, BOOKER HE, WELLING PG. Pharmacokinetics of phenobarbital following single and repeated doses. J Clin Pharmacol 1979; 19: 282-289. 13. COCHRANE SM, BLACK WD, PARENT JM, ALLEN DG, LUMSDEN JH. Pharmacokinetics of phenobarbital in the cat following intravenous and oral administration. Can J Vet Res 1990; 54: 132-138. 14. BAGGOT JD. Principles of Drug Disposition in Domestic Animals: The Basis of Veterinary Clinical Pharmacology. Philadelphia: W.B. Saunders, 1977. 15. WAGNER JG, NORTHAM JI, ALWAY CD, CARPENTER OS. Blood levels of drug at the equilibrium state after multiple dosing. Nature (Lond) 1965; 207: 13011302. 16. CONNEY AH. Pharmacological implications of microsomal enzyme induction. Pharmacol Rev 1967; 19: 317-366. 17. BENET LZ, SHEINER LB. Pharmacokinetics: The dynamics of drug absorption, distribution, and elimination. In: Gilman AG, Goodman LS, Rall TW, Murad F, eds. The Pharmacological Basis of Therapeutics. New York: MacMillan Publishing Co., 1985: 3-34. 18. RAVIS WR, NACHREINER RF, PEDERSOLI WM, HOUGHTON NS. Pharmacokinetics of phenobarbital in dogs after multiple oral administration. Am J Vet Res 1984; 45: 1283-1286. 19. TRUHAUT R, FERRANDO R, GRAILLOT C, GAK J-C, FOURLON C, MORAILLON R. Induction du cytochrome p450 par le phenobarbital chez le chat. C R Acad Sci (Paris) 1978; 286: 371-373. 20. PRICE DE, MEHTA A, PARK BK, HAY A, FEELY MP. The effect of low-dose phenobarbital on three indices of hepatic microsomal enzyme induction. Br J Clin Pharmacol 1986; 22: 744-747. 21. COCHRANE SM, PARENT JM, ALLEN DG, BLACK WD, VALLIANT AE, LUMSDEN JH. A method for chronic intravenous catheterization in the cat. Can Vet J 1989; 30: 432-433. 22. STEEL RG, TORRIE JH. Principles and Procedures of Statistics. New York: McGraw-Hill, 1960. 23. GIBALDI M, PERRIER D. Pharmacokinetics. New York: Marcel Dekker, 1975. 24. CONNEY AH, BURNS JJ. Metabolic interactions among environmental chemicals and drugs. Science 1972; 178: 576-586. 25. FREY HH, KAMPMANN E, NIELSEN CK. Study on combined treatment with phenobarbital and diphenylhydantoin. Acta Pharmacol Toxicol 1968; 26: 284-292. 26. BUCHTHAL F, LENNOX-BUCHTHAL MA. Phenobarbital. Relation of serum concentration to control of seizures. In: Woodbury DM, Penry JK, Schmidt RP, eds. Antiepileptic Drugs. New York: Raven Press, 1972: 335-343. 27. LOSCHER W. A comparative study of the protein binding of anticonvulsant drugs in serum of dog and man. J Pharmacol Exp Ther 1979; 208: 429-435. 28. FARNBACH G. Serum concentrations and efficacy of phenytoin, phenobarbital, and primidone in canine epilepsy. J Am Vet Med Assoc 1984; 184: 1117- 1120.
tion du produit. La demi-vie d'elimination apres le dernier jour de Phenobarbital was administered traitement etait de 43.3 ± 2.9 h. Le orally to seven healthy cats at a dose grand volume apparent de distribuof 5 mg/kg once a day for 21 days. tion (695.0 ± 43.9 mL/kg) suggere Serum phenobarbital concentrations que le produit se distribue largement were determined using a commercial dans tout le corps. La demi-vie immunoassay technique. A one- d'elimination 'a la fin d'administracompartment model was used to tions successives de phenobarbital describe the final elimination curve. etait significativement plus courte The elimination half-life (t/2 b) after qu'apres une seule administration the final day of treatment was 43.3 + orale chez le chat. L'analyse de la 2.92 h. The large apparent volume of pharmacocinetique du produit apres 1 distribution of 695.0 ± 43.9 mL/kg et 21 jours de traitement montre que la suggests that the drug was widely cinetique d'elimination du phenobardistributed within the body. The t/2 b bital n'a pas significativement change following multiple oral administration apres plusieurs prises orales du was significantly shorter than pre- produit. II semble donc que des viously reported for a single oral dose differences dans la cinetique d'elimiof phenobarbital in the cat. Analysis nation du produit existent entre of pharmacokinetic results after days 1 differentes populations de chats. A and 21 of treatment suggested that the cause de ces differences de population, elimination kinetics of phenobarbital il serait prudent que les niveaux did not change significantly with seriques de phenobarbital soient multiple oral administration. It mesures chez chaque chat apres appears that differences in elimination l'administration du produit. (Traduitpar kinetics can exist between populations Dr Pascal Dubreuil). of cats. These differences emphasize the need for individual monitoring of INTRODUCTION cats receiving phenobarbital.
ABSTRACT
RESUME Du phenobarbital
a
ete administre
per os a sept chats normaux une fois par jour pendant 21 jours a la dose de 5
mg/kg. Les concentrations seriques de phenobarbital ont ete mesurees a l'aide d'une technique commerciale. Un modele a un compartiment decrivait le mieux la courbe d'elimina-
Phenobarbital (PB) is used as an anticonvulsant in several species including the canine (1,2), equine (3), human (4) and feline (2). Pharmacokinetic data for PB are available for dogs (1,5,6), horses (3,7,8) and humans (4,9-12). A single intravenous (IV) and single oral dose study of PB in the cat suggested that this drug is ideally suited for use as an oral anticonvulsant in this species (13).
A dose for multiple oral administration can be calculated from data determined in single IV and oral dose studies, provided that elimination of the drug is not modified with repeated treatment (14,15). An altered rate of elimination can result from changes in the rate of drug metabolism (15). Since PB is a potent inducer of hepatic microsomal enzymes (16), the metabolism of drugs administered in conjunction with PB may potentially be enhanced (17). It is unclear whether or not PB can enhance its own metabolism through enzyme induction. Martin et al (11) demonstrated a shorter elimination half-life (t/2 b) in people who abuse PB, compared with normal volunteers. Other studies in humans (12), dogs (6,18) and cats (19) have failed to show a similar reduction in t½/2 b with repeated PB administration. It has been suggested that the dose and the duration of PB treatment may influence the extent of enzyme induction (18,20). Frey (2) reported a t½/2 b of 34-43 h for PB in the cat following multiple oral administration. This t/2 b was shorter than the value reported by the present authors for a single oral dose of PB in the cat (76.1 ± 6.97 h) (13). While the shorter t/2 b determined for repeated PB administration may be due to changes in enzyme activity, it may also reflect differences in the populations of cats sampled (6). The objective of this study was to determine the effect of multiple oral dose administration on the pharmacokinetics of PB in the cat in order to make recommendations for long-term oral therapy in this species.
Department of Clinical Studies (Cochrane, Parent, Allen), Department of Biomedical Sciences (Black) and Department of Pathology (Lumsden), Ontario Veterinary College, University of Guelph, Guelph, Ontario NIG 2W1. This study was funded by the Ontario Veterinary College Pet Trust. Submitted July 13, 1989.
Can J Vet Res 1990; 54: 309-312
309
MATERIALS AND METHODS EXPERIMENTAL ANIMALS
Seven healthy mixed breed specific pathogen free cats (six males; one female) ranging from approximately one to two years of age were obtained from the University of Guelph Laboratory Animal Facility. Prior to the study the following tests were performed on each cat: hematological evaluation including hemoglobin, hematocrit, red blood cell count, white blood cell count and differential; serum biochemistry analysis including total protein, albumin, creatinine, urea, glucose, bilirubin, serum alkaline phosphatase (SAP), serum alanine aminotransferase (SALT) serum aspartate aminotransferase (SAST), sodium, potassium and chloride levels (Coulter Dacos, Coulter Electronics of Canada, Burlington, Ontario), and fecal parasite egg count. Throughout the experiment, the cats were housed in individual metal cages in a room isolated from other cats. They were fed commercial dry cat food (Meow Mix®, Ralston Purina, Mississauga, Ontario) and water ad libitum. The cats were fasted for 12 h prior to anesthesia for catheter placement. SURGICAL PROCEDURES
Prior to the oral study an indwelling jugular catheter (16 or 19 gauge, 20.3 cm Intracath, Deseret Co., Sandy, Utah) was placed as previously described to facilitate blood collection (21). To enable catheter placement the cats were placed in a 5 L airtight plexiglass box and anesthesia induced with isoflurane (AErrane®, Anaquest, Pointe Claire, Quebec) and oxygen. The cats were intubated and maintained at a light surgical plane of anesthesia during catheterization. For the two days prior to the start of sample collection, the catheter was flushed once a day with 0. 5 mL heparin solution (100 U/L) The catheter was removed on day 8 of the study and another catheter placed in the opposite jugular vein prior to the final week of the experiment. MULTIPLE ORAL PHENOBARBITAL STUDY
Oral PB administration was initiated two days following catheter
310
placement. Phenobarbital tablets (Phenobarbital acid USP, ParkeDavis, Scarborough, Ontario) were administered at a dose of 5 mg/kg daily at 0830 h for 21 days. The tablets were cut and weighed to ensure an accurate dose. On day 1 of the study, blood samples (0.5 mL) were collected at 0,4,6,8,10 and 12 h after treatment. Prior to sampling, 0.5 mL of blood was drawn and discarded to avoid collecting the residual volume of the catheter. The catheter was flushed with 0.5 mL of heparin solution (100 U/ mL) following the 12 h sample and after subsequent sampling. Sample collection on days 2 through 7 was performed at 4 and 24 h after PB administration. Prior to removal of the catheter on day 8, blood was collected for hematological evaluation and SAP and SALT levels. On day 17, the cats were anesthetized as previously described and a catheter placed in the opposite jugular vein. On days 19 and 20, samples were collected at 4 and 24 h after PB administration. After the final treatment on day 21, samples were drawn at the following times: 4, 6, 8, 12, 24, 32, 48, 56, 72, 80, 96 h. Following the last sample, blood was collected for hematological evaluation and serum biochemistry analysis.
elimination slopes and the overall elimination rate constant (b) were obtained from regression analysis. The elimination half-life was calculated as t¼/2b = 0.693/b, apparent volume of distribution by V'd(B') = Dose/ B', and clearance of drug as C1(B') = V'd(B') x b. Estimated steady state PB levels for the experimental cats were calculated using the following formula published by Wagner (15): CPo = (1.44) t/2 x D x F V'd x T where D represents dose administered, F the fraction of each dose absorbed [F 1 based on previous study (13)] and T the dosage interval. A paired Student's t-test was used to compare hematological, biochemical and pharmacokinetic parameters determined for the first and final treatments. The results of this multiple dose study were compared with the previously reported single dose study using an unpaired Student's t-test (22). Statistical analyses were performed at a confidence level of 95%. All data are presented as mean ± standard error of the mean (SEM).
PHENOBARBITAL ASSAY
The hematological and serum biochemical parameters measured prior to treatment were not significantly different from those determined on day 25, with the exception of glucose. The glucose level prior to start of the study was 7.96 ± 0.333 mmol/L, and at completion of the experiment was 5.92 ± 0.320 mmol/ L. The normal range established at the Ontario Veterinary College for blood glucose in the cat is 3.5-9.0 mmol/ L. The serum drug concentrations measured following oral PB were plotted against time (Fig. 1). Steady state drug concentrations were present when sampling resumed on day 19. The average drug concentration observed at steady state (Cpoo) was approximately 16.4 ,ug/ mL. The maximum (CPOOmax) and minimum (CpoQmin) steady state drug concentrations were approximately 19.3 ,ug/mL and 13.4 pg/mL. The elimination curve for PB following the final treatment on day 21
The blood samples were allowed to clot and the sera were separated by centrifugation (15 min at 1239 g). Phenobarbital analysis was performed by an immunoassay technique (Ames Seralyzer®, Miles Laboratories, Elkhart, Indiana) within 1 h of sampling. This technique was validated for the cat as previously described (13). DATA ANALYSIS
Logarithmic serum PB concentrations were plotted against time (linear). The terminal elimination slopes following the first and final treatments were obtained from this graph and subjected to least squares regression analysis (22). Pharmacokinetic parameters were determined for these elimination phases using a onecompartment model (23). These parameters were calculated using standard formulae described by Baggot (14) and Gibaldi and Perrier (23). The y-intercept (B') of the
RESULTS
administration on days 1 and 21 were 18.8 ,tg/ mL and 18.7 Alg/ mL. These values did not appear to be different from the steady state level of approximately 16.4 ,ug/mL observed in this experiment.
E C
.20 Cz 0)
DISCUSSION
C
0
During this 21 day oral PB study, no signs of drug toxicity such as ataxia or 0 sedation were observed in the cats. C The hematological and serum biochemistry analyses did not suggest any aadverse effects such as anemia, hypoproteinemia or hepatotoxicity that might have influenced the pharmacokinetic parameters. AlI I I .19I 19 20 2I 225 though there was a significant 24 21 22 23 decrease in the glucose level after Time (doys) Fig. Serum phenobarbital concentrations (i means SEM; means) following oral administration completion of the experiment, it was of 5 mg/kg once a day for 21 days in seven cats. thought not to affect the results. A one-compartment model was corresponded to a one-compartment when B' after treatment on day 21 (20.0 used to describe the elimination slope model (r = 0.992) The pharmacoki- ± 0.977 ,ug/mL) was reduced to 7.34 ± of the drug concentration-time curve netic parameters determined for the 0.397 ,ig/ mL to correct for the amount following the 21 day study. Since elimination curves after day 1 and day of PB already accumulated in the blood sampling on day 21 started at 4 h after 21 of treatment are compared in Table due to repeated drug administration, it PB administration, earlier absorption and distribution compartments were was not significantly different. I. With the exception of B', there were no significant differences between the Steady state serum PB concentra- not detected. However, based on the parameters compared. However, tions (Cpoo) estimated after drug results of a single oral dose study performed by the present authors (13), where sampling began at 5 min after TABLE I. Comparison of pharmacokinetic parameters following first and final dose (5 mg/kg) of drug administration, elimination of phenobarbital in the cat PB after oral administration is described by the oneadequately Final dose First dose compartment model in the cat. The Mean Mean Range ±SEM Parameter +SEM Range half-life of this final elimination phase was at the upper limit of the range of * 20.0 17.9-22.1 B' 6.52 5.72-7.18 ±0.977 ±0.198 34-43 h reported by Frey (2) for PB (,ug/ mL) following multiple oral administration 5.4-8.5 7.34 Adjusted B' ±0.397 in the cat. These values are, however, shorter than previously determined by 0.016 0.012-0.020 0.015 0.012-0.017 b +0.001 ±0.001 (h-') Cochrane et al (13) for a single oral dose of PB in the cat (t/2 b = 76.1 ± 43.3 35.0-56.3 40.8-57.8 47.6 t'/2b ±2.92 ±2.89 (h) 6.96 h). The differences in elimination half-lives between the present multiple 625.0-925.9 696.4-869.6 695.0 773.6 V'd(B') ±43.9 ±23.5 (mL/ kg) oral dose study and the single oral dose study may reflect either enzyme 7.88-14.6 11.3 9.23-12.5 11.4 C1(B') ±0.782 +0.439 (mL/ kg/ h) induction and enhanced PB metabolism, or a preexisting difference in PB 3.5-4.5 4.07 Wt (kg) 4.39 4.0-5.0 ±0.130 +0.172 metabolism between the two groups of = Significant difference between first and final dose cats. * = y-intercept beta curve B' To evaluate the possibility that Adjusted B' = y-intercept corrected for drug accumulation by subtraction of drug concentration multiple PB administration resulted in before treatment on day 21 (12.66,Mg/ mL) from actual B' intercept (20.0 ± 0.977 Mg/ more rapid drug elimination through mL) enzyme induction, the elimination b = overall elimination rate constant = half-life elimination kinetics determined after day 1 of t1/2b = apparent volume of distribution V'd(B') treatment were compared to the same = total body clearance from plasma CI(B') parameters calculated after day 21. Wt = weight of animal 0
0
I
1
±
*
311
The fact that the pharmacokinetic parameters were not significantly different suggests that for the dose used and the duration of this study, multiple drug administration did not enhance the elimination of PB. In support of this observation, repeated PB administration did not appear to alter estimated steady state concentrations. The observed steady state concentration was similar to the steady state concentrations calculated after both days 1 and 21 of treatment. Steady state serum concentrations are dependent on both input and elimination processes (14). Assuming that input remains constant, enhanced drug elimination would have resulted in a lower steady state PB concentration (15). No differences were, however, demonstrated. This further supports the lack of enzyme induction following repeat oral administration of PB. From these comparisons, it appears that the differences observed between the cats used in this multiple oral dose study and the pound source cats in the previous single dose study (13) may have been due to genetic and/ or environmental differences (16,24). It is of interest that Frey (6) reported a significant difference in half-lives of Beagles (32.0 ± 4.8 h) compared to mongrel dogs (70.0 ± 16 h) after administration of a single IV dose of PB. Frey (25) suggested that mongrel animals may more closely represent clinical cases than do laboratoryraised animals. Therapeutic serum PB concentrations of 15-35 ,ug/mL have been suggested for control of seizures in the cat based on data extrapolated from humans (26,27) and dogs (18,28). In order to achieve these concentrations in the cat, a maintenance dose of approximately 4.2 mg/ kg administered once a day was recommended (13). This dose was calculated from the longer half-life determined in the single oral study. If the PB half-life in an individual cat was shorter, such as recorded in this multiple dose study, the steady state concentration attained after a daily dose of 4.2 mg/kg would be lower and possibly subtherapeutic. Therefore, due to the potential for large differences in PB elimination rates between groups of cats, serum PB steady state concentrations should be measured and the dose adjusted if necessary. 312
In summary, the elimination kinetics of PB did not appear to change with repeated drug administration. It was demonstrated that differences in the elimination kinetics of PB can exist between populations of cats. These differences emphasize the need for monitoring of drug concentrations in individual cats receiving PB.
ACKNOWLEDGMENTS The authors wish to thank Jean Claxton for her excellent technical assistance and guidance during the kinetic analysis and for the use of her computer programs.
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