Absorption kinetics and bioavailability of cephalexin in the dog after oral and intramuscular administration
Carli, S., Anfossi, P., Villa, R., Castellani, G., Mengozzi, G., Montesissa, C. Absorption kinetics and bioavailability of cephalexin in the dog after oral and intramuscular administration. J. vet. Pharmacol. Therap. 22, 308-313.
The pharmacokinetics of cephalexin, a first generation cephalosporin, were investigated in dogs using two formulations marketed for humans, but also often employed by practitioners for pet therapy. Cephalexin was administered to five dogs intravenously and intramuscularly as a sodium salt and by the oral route as a monohydrate. The dosage was always 20 mg/kg of active ingredient. A microbiological assay with Sarcina lutea as the test organism was adopted to measure cephalexin concentrations in serum.
The mean residence time (MRT) median values after intravenous (i.v.), intramuscular (i.m.) and oral administration (p.o.) were 86 min, 200 min, and 279 min, respectively.
After i.m. and oral dosing the peak serum concentrations (24.2 ± 1.8 µg/mL and 20.3 ± 1.7 µg/mL, respectively) were attained at 90 min in all dogs and bioavailabilities were 63 ± 10% and 57 ± 5%, respectively.
The time course of the cephalexin serum concentrations after oral administration was best described by a model incorporating saturable absorption kinetics of the Michaelis-Menten type; thus in the gastrointestinal tract of dogs a carrier mediated transport for cephalexin similar to that reported in humans, may exist. The predicted average serum concentrations of cephalexin after repeated i.m and oral administration indicated that, in order to maintain the therapeutic concentrations, the 20 mg/kg b.w. dosage should be administered every 6-8 h.
Cephalexin is a first generation cephalosporin with bactericidal action and a wide spectrum of activity including Gram-positive and Gram-negative bacteria. It combines resistance to the action of penicillinases produced by Staphylococcus aureus isolates (Muggleton et al., 1968) with activity against the majority of ampicillin-resistant Escherichia coli strains (Foord, 1969).
In vitro, more than 95% of β-haemolytic Streptococci, Pasteurella multocida and Staphylococcus intermedius strains isolated from dogs are inhibited by cephalexin concentrations of ::= 2 µg/mL and more than 50-60% of Bordetella bronchiseptica, Proteus mirabilis, Escherichia coli and Klebsiella pneumoniae strains by 2-4 µg/mL (Silley & Brewster, 1988; Noble & Kent, 1992; Aucoin, 1993; Lloyd et al., 1996; Campbell & Rosin, 1998).
Cephalexin has proved to be effective in the treatment of a wide range of current bacterial infections, and in the dog it is used particularly for pyodermatitis, folliculitis and forunculosis (Griffith & Black, 1970; Pfeffer et al. 1977; Kietzmann et al., 1990; Frank & Kunkle, 1993; Carlotti & Leroy, 1995).
Although some cephalexin preparations are available for veterinary use in Italy, none is confined to the treatment of dogs and cats. Thus the formulations intended for human therapy are usually employed by practitioners for the treatment of pets at dosage schedules extrapolated from those for humans. The purposes of this study were to determine the intramuscular and oral bioavailability and to define the optimal therapeutic dosage regimen of cephalexin in dogs treated with formulations marketed in Italy for human beings.
Data from the literature showed that the intestinal absorption of cephalosporins in humans and rats was a saturable process (Reigner et al., 1990; Liu et al., 1997; Ruiz- Balanguer et al. 1997) and that a carrier-mediated uptake of cephalexin occurred in human intestinal cells (Dantzig & Bergin, 1988, 1990). Therefore a model incorporating the Michaelis-Menten type absorption characteristics was used to fit concentration vs. time data obtained after cephalexin oral administration and compared to the classical model with first order drug input.
Materials And Methods
Five healthy Beagle dogs (two males and three females), 10-14 kg and aged 2-5 years, were used. The animals were purchased from an authorised breeding establishment, Greenhill (Montichiari, Brescia, Italy) and individually caged in temperature-controlled rooms (20-22ºC and 50-60% humidity) with 12 h light cycles.
The dogs were fed standard laboratory dogfood and were given tap water ad libitum. The care and handling of the animals was in accordance with the provisions of the European Economic Community (EEC) Council Directive 86-609, recognised and adopted by the Italian Government (D.L. 27/01/1992 nº116).
Drug formulations and treatments
Intravenous (i.v.) and intramuscular (i.m.) administrations were carried out using CEPOVEN® (Glaxo, Verona, Italy) which is marketed as vials containing 1 g of dried cephalexin sodium salt together with vials containing 4 mL water for injection. A solution of the active substance (100 mg/mL) in injection water was freshly prepared before each administration. Each dog received cephalexin (20 mg active ingredient per kg bw.) intravenously into the right cephalic vein and intramuscularly in the deep muscles of the thigh.
Oral administration was performed using KEFORAL® (Eli Lilly, Sesto Fiorentino, Firenze, Italy) which is marketed as capsules containing 500 mg of cephalexin monohydrate. The active substance was administered at the dose of 20 mg/kg b.w. by adjusting the amount in each capsule to the body weight of the dogs.
Dogs were fasted for 24 h prior to treatment by i.v., i.m. and oral route according to a crossover design with a washout period of 20 days between each phase.
A venous catheter (Surflo 18C, Terumo Europe N.V., Leuven, Belgium) was placed in the cephalic vein of each dog before starting the trial. Blood samples were collected before each treatment and at 5, 15, 30, 45, 60, 90 min and 2, 3, 4, 6 and 8 h after i.v. injection; the same sampling scheme with an additional sample at 12 h was adopted after i.m. administration.
Following the oral administration blood samples were collected at 0.5, 1, 1.5, 2, 3, 4, 6, 8 and 12 h. After each sampling the catheter was flushed with saline containing 1% heparin.
Serum was obtained by centrifugation at 1500 x g and stored at-20ºC pending assay.
Serum concentrations of cephalexin were determined by the agar plate diffusion method, using 7 mL Antibiotic Medium 1 (Difco Laboratories, Detroit, MI, USA) poured into Petri dishes (90 mm) and Sarcina lutea ATCC 9341 as test organism (Grove & Randall, 1955).
To prepare cephalexin stock solution (1000 µg/mL) 21.2 mg of cephalexin sodium salt (941 µg/mg active ingredient) were dissolved in 20 mL phosphate buffer pH 6.6. The stock solution was diluted with antibiotic-free serum to obtain five calibration concentrations of 2, 1, 0.5, 0.25 and 0. 12 µg/mL.
The paper disks (Schleicher & Schull Dassel, 9 mm) were charged with 50 µL of each standard solution or serum sample. Serum samples with cephalexin concentrations greater than the upper limit of quantification (2 µg/mL) were diluted in control serum to obtain a final concentration within the calibration range. Plates were preincubated for 2 h at room temperature (22ºC) and then incubated overnight at 37ºC.
The method was linear within the range of the calibration curve (r2 = 0.988). Intra-day precision and accuracy were determined for concentrations over the range of the standard curve. The intra-day precision was calculated as the standard deviation (RSD) from six determinations and ranged from 3.8 to 5.4%. The accuracy of the method, calculated as mean error (ME = (observed concentration-added concentration)/added concentration x 100), ranged from -4.1 to 5.3%. The limit of quantitation (LOQ) was 0.12 µg/mL with an RSD of 4.6% and an ME of -3.2%.
Serum vs. time data for each dog after i.v., i.m. and oral administration were fitted to compartmental models by a nonlinear fitting programme (Easy Fit, Istituto Mario Negri, Milano) using a weighted least squares regression analysis. After injection the time course of serum concentrations was given as a sum of exponential terms as:
where Ct is the drug concentration in serum at time t, Ci is the intercept and Ài is the exponential term. Each half-life (t½) was calculated as ln2/Ài and Co (the serum concentration at time 0) as the sum of intercepts. Following i.m. and oral administration the serum vs. time data for each dog were fitted to the general equation:
where ka is the rate constant for the absorption phase and kel is the rate constant for the elimination phase.
Cmax was the highest recorded concentration and tmax was the time when Cmax was achieved.
Non compartmental analysis based on statistical moments was also performed (Riegelman & Collier, 1980). The area under serum concentration-time curve (AUC) and the area under the moment curve (AUMC) were calculated by the method of trapezoids and extrapolation to infinity was made as follows:
where tlast is the last time with measurable concentration (Clast) and Àn is the rate constant of the elimination phase. The system moment mean residence time (MRT), the body clearance (Cl), the volume of distribution by area (Vdarea) and the volume of distribution at steady state (Vdss) were determined from the equations:
After the i.m. and oral administration the mean absorption time (MAT) value was calculated as the difference between the MRT obtained after each administration and the MRT after the i.v. administration.
Following the oral administration, the cephalexin concentration time course was fitted to a model incorporating Michaelis- Menten type absorption (Reigner et al., 1990). In this model the rate of absorption is measured as the rate of change of the drug quantity in the gut (Aa):
and the rate of change of drug quantity in the body (A) is:
where Ca and C are the drug concentrations in gut and in serum, respectively, Vmax is the maximum rate of absorption, Km is the value of Ca at which the absorption rate is one-half the maximum, and Cl is the body clearance.
To express the model in terms of concentrations the two equations were divided by the apparent volumes of distribution of the drug in the body (V) and letting Ca' = Va.Ca/V, K' m = Va.Km/V and V'max = Vmax/V, where Va is the gut volume.
As no analytical solutions are available, the set of differential equations was solved using the Runge-Kutta fourth order numerical integration method, with adaptive stepsize to estimate the values of C as function of time (Wolfram, 1991).
The average serum concentrations of cephalexin at steady state after repeated i.m. and oral administrations were estimated according to the following equation:
where is the dosing interval (Gibaldi & Perrier, 1982).
To choose the best representation of the time course plots the weighted (1/observed y) residual sums of squares (WS), r2 and AIC (Akaike information criterion-Yamaoka et al., 1978) were calculated. Serum concentrations of cephalexin, intercepts and exponen- tial terms were given as mean values (± SD).
The other pharmacokinetic parameters, which were not normally distrib- uted, were reported as medians and range. Half-lives were expressed as harmonic mean ± pseudo standard deviations (PSD) determined by the jack-knife technique (Lam et al., 1985).
The serum concentration-time course of cephalexin in the five dogs following i.v., i.m. and oral administration are presented in Fig. 1. Table 1 shows the pharmacokinetic parameters calculated after i.v., i.m. and oral dosing by compartmental and non-compartmental analysis. After i.v. injection the serum concentration-time data were best described by the two-compartment open model and those after i.m. administration by the one
compartment model with first order absorption. After the oral treatment the curve of cephalexin serum concentrations was not adequately described by the compartmental model assuming first order absorption (M1) as the r2 value was > 0.93 in only one out of the five dogs.
The fitting of the oral data to the Michaelis- Menten absorption model (MM) improved r2 (> 0.93 for four animals). Figure 2 compares the curves generated for a representative dog (animal 2) using the M1 and the MM models.
A number of animals had concentrations substantially greater than the LOQ at the last sampling point: range values were 0.27-1.14 µg/mL, 0.30-1.24 µg/mL and 1.06-2.02 µg/mL after i.v., i.m. and oral administration, respectively. However, after i.v. and i.m. injection all the AUCtlast-oo calculated were lower than 5% of the total AUC, and after oral administration only two out of the five AUCtlast-oo were greater (6 and 7% of the total AUC), showing the adequacy of blood sample timing.
The predicted steady state serum concentrations of cephalexin during a multiple dosage regimen were estimated as follows: 14.3 ± 0.5 µg/mL and 10.7 ± 0.4 µg/mL after i.m. administration at time intervals of 6 and 8 h, respectively; 13.0 ± 1.3 µg/mL and 9.8 ± 1.0 µg/mL after oral administration every 6 and 8 h, respectively.
The time course of cephalexin in serum of dogs treated by the i.v. route was best described by a two-compartment model; both distribution and elimination were fast, as already reported in the literature for other animal species (Carli et al., 1983; Soback et al., 1987; Garg et al., 1990, 1996). In humans, the urinary recovery of cephalexin accounts for about 80% of the dose and the drug is cleared from the kidneys mainly by glomerular filtration combined with active tubular secretion (Barbhaiya, 1996). In the present study the mean value of Cl (2.5 mL/min/kg) is lower than the average glo- merular filtration rate (GFP) reported for dogs (4.5 ± 2.0 mL/ min/kg-Baggot, 1977; 2.8 ± 0.96 mL/min/kg-Finco, 1997), suggesting the involvement of tubular reabsorption. Granero et al. (1994) reported that in the rat cephalexin acted as a competitive inhibitor of cefadroxil tubular reabsorption.
The concentration-time data obtained after the oral administration of cephalexin to dogs fitted the MM absorption model. The Michaelis-Menten equation commonly describes capacity limited processes such as the carrier-mediated transport, and underlies the absorption mechanism of many β-lactam antibiotics including cephalexin. However, cephalexin concentrations were only measured in serum of dogs and absorption rate reflects more than just the movements of the drug across gastrointestinal membranes. Carrier-mediated transport in the gastrointestinal tract of the dog is the hypothesis suggested by the results of the present study and has been reported for humans and rats (Dantzig & Bergin, 1988, 1990; Reigner et al., 1990; Liu et al., 1997; Ruiz-Balanguer et al., 1997). After i.m. administration the bioavailability of cephalexin was 63 ± 10%; similar values have been reported for the cephalexin sodium salt (73.9 ± 6.3%-Carli et al., 1983), monohydrate
(67.5 ± 2.9%-Garg et al., 1990; 81.9 ± 4.2%-Garg et al., 1992) and lysine salt (89.6 ± 1.0%-Carli et al., 1983) in other species. The oral bioavailability of cephalexin in dogs (57 ± 5%) calculated in the present experiment was greater than that calculated in calves (about 35%) by Soback et al. (1987), but low compared to the urinary recovery (74.1-88.7%) of oral doses in humans reported by Barbhaiya (1996).
The serum Cmax found by Campbell & Rosin (1998) in fasting and non fasting dogs, treated orally with 30 mg/kg of cephalexin every 12 h, were higher than in the present study and ranged from 23.8 to 46.2 µg/mL and from 21.9 to 84.6 µg/mL. Moreover, after the i.m. (oily suspension) and oral (tablets) administration of cephalexin sodium salt (10 mg/kg) to dogs the mean serum concentrations were 31.9 ± 1.08 µg/mL and 18.6 ± 1.70 µg/mL, respectively, with tmax at 1.8 ± 0.11 and 0.9 ± 0.11 h (Silley et al., 1988).
In veterinary practice, several regimens are reported for the treatment of recurrent pyoderma with cephalexin: 15-20 mg/kg administered every 12 h, as well as 20-22 mg/kg every 8 h for at least 3 weeks are recommended (Guague' re & Picard, 1990; Frank & Kunkle, 1993; Carlotti & Leroy, 1995; Rosser, 1997).
The persistence of antibiotic concentrations in serum and tissues above the minimum inhibitory concentrations is a pharmacodynamic variable related to the clinical efficacy.
The predicted average serum concentrations of cephalexin in dogs after repeated i.m. and oral administration, suggested that the 20 mg/kg b.w. dosage should be administered every 6-8 h in order to attain serum concentrations greater than 5 µg/mL. Longer time intervals between dosing cause a drop in serum concentrations below the MIC values (::= 2 µg/mL) reported for cephalexin-sensitive bacteria (Silley & Brewster, 1988; Noble & Kent, 1992; Aucoin, 1993; Lloyd et al., 1996; Campbell & Rosin, 1998).
This work was supported in part by a fund supplied by the University Ministry for Scientific and Technological Research (MURST-60% 1995).
Aucoin, D. (1993) Target. The Antimicrobial Reference Guide to Effective Treatment. pp. 77-78. North American Compendiums, Inc., Port Huron. Baggot, J. (1977) Principles of drug disposition in domestic animals: the basis of veterinary clinical pharmacology. pp. 113-143. W.B. Saunders, Co., Philadelphia.
Barbhaiya, R.H.A. (1996) Pharmacokinetic comparison of cefadroxil and cephalexin after administration of 250, 500 and 1000 mg solution doses. Biopharmaceutics and Drug Disposition, 17, 319-330.
Campbell, B.G. & Rosin, E. (1998) Effect of food on absorption of cefadroxil and cephalexin in dogs. Journal of Veterinary Pharmacology and Therapeutics, 21, 418-420.
Carli, S., Perretta, G., Brusa, T., Invernizzi, A. & Faustini, R. (1983) Comparison of pharmacokinetics of sodium and lysine cephalexin in calves. Journal of Veterinary Pharmacology and Therapeutics, 6, 181-185. Carlotti, D.N. & Leroy, S. (1995) Actualite' s an antibiothe' rapie cutane'e syste' mique chez le chien. Pratique Me'dicale & Chirurgicale de l'animal de Compagnie, 30, 263-271.
Dantzig, A.H. & Bergin, L. (1988) Carrier-mediated uptake of cephalexin in human intestinal cells. Biochemical Biophysical Research Communica- tions, 155, 1082-1087.
Dantzig, A.H. & Bergin, L. (1990) Uptake of the cephalosporin, cephalexin, by a dipeptide transport carrier in the human intestinal cell line, Caco-2. Biochimica Biophysica Acta, 1027, 211-217.
Finco, D.R. (1997) Kidney Function. In Clinical Biochemistry of Domestic Animals. 5th edn. Eds Kaneko, J.J., Harvey, J.W. & Bruss, M.L. pp. 441-484. Academic Press, London.
Foord, R.D. (1969) Proceedings of a Symposium on the Clinical Evaluation of Cephalexin London. June 1969. Glaxo Laboratories Ltd. pp. 4-5.
Frank, L.A. & Kunkle, G.A. (1993) Comparison of the efficacy of cefadroxil and generic and proprietary cephalexin in the treatment of pyoderma in dogs. Journal of American Veterinary Medical Association, 203, 530-533.
Garg, S.K., Chaudhary, R.K. & Srivastava, A.K. (1992) Disposition kinetics and dosage of cephalexin in cow calves following intramus- cular administration. Annales de Recherches Ve'te'rinaires, 23, 399-401. Garg, S.K., Chaudhary, R.K. & Srivastava, A.K. (1996) Pharmacokinetics of cephalexin in calves after intravenous and subcutaneous administration. Acta Veterinaria Hungarica, 44, 195-201.
Garg, S.K., Chaudhary, R.K., Srivastava, A.K. & Garg, B.D. (1990) Pharmacokinetics and dosage regimen of cephalexin in buffalo calves Gibaldi, M. & Perrier, D. (1982) Noncompartmental analysis based on statistical moment theory. In Pharmacoâinetics. 2nd edn. pp. 409-424. Marcel Dekker, Inc., New York.
Granero, L., Gimeno, M.J., Torres-Molina, R., Chesa-Jimenez, J. & Peris, J.E. (1994) Studies on the renal excretion mechanisms of cefadroxil.
Drug Metabolism and Disposition, 22, 447-450.
Griffith, R.S. & Black, H.R. (1970) Cephalexin. Medical Clinics of North America, 54, 1229-1253.
Grove, D. & Randall, W. (1955) Assay methods of antibiotics. A laboratory manual. 1st edn. pp. 14-16. Medical Encyclopaedic Inc., New York.
Guague' re, E. & Picard, G. (1990) Utilisation de la ce' falexine et du lactate d'e' thyle dans le traitement des pyodermites canines. Practique Me'dicale et Chirurgicale de l'Animal de Compagnie, 25, 547-551.
Kietzmann, M., Mischke, R., Albrecht, N. & Nolte, I. (1990) Vertra" glich- keit und Pharmakokinetik von Cefalexin (Cefaseptin® Dragees) beim Hund. Kleintiepraxis, 35, 390-398.
Lam, F.C., Hung, C.T. & Perrier, D.G. (1985) Estimation of variance for harmonic mean half-lives. Journal of Pharmaceutical Science, 74, 229-231.
Liu, X.D., Xie, L., Gao, J.P., Lai, L.S. & Liu, G.Q. (1997) Cefixime absorption kinetics after oral administration to humans. European Journal of Drug Metabolism and Pharmacoâinetics, 22, 185-188.
Lloyd, D.H., Lamport, A.I. & Feeney, C. (1996) Sensitivity to antibiotics amongst cutaneous and mucosal isolates of canine pathogenic staphylococci in the UK, 1980-96. Veterinary Dermatology, 7, 171-175.
Muggleton, P.W., O'Callagan, C.H., Foord, R.D., Kirby, S.M. & Ryan, D.M. (1968) Laboratory appraisal of cephalexin. Antimicrobial Agents and Chemotherapy, 8, 353-360.
Noble, W.C. & Kent, L.E. (1992) Antibiotic resistance in Staphylococcus intermedius isolated from cases of pyoderma in the dog. Veterinary Dermatology, 3, 71-74.
Pfeffer, M., Jackson, A., Ximenes, J. & Menezes, J.P.D. (1977) Comparative human oral clinical pharmacology of cefadroxil, cephalexin, and cephradine. Antimicrobial Agents And Chemotherapy, 11, 331-338.
Reigner, B.J., Couet, W., Guedes, J.-R., Fourtillan, J.-B. & Tozer, T.N. (1990) Saturable rate of cefatrizine absorption after oral administra- tion to humans. Journal of Pharmacoâinetics and Biopharmaceutics, 18, 17-34.
Riegelman, S. & Collier, P. (1980) The application of statistical moment theory to the evaluation of in vivo dissolution time and absorption time. Journal of Pharmacoâinetics and Biopharmaceutics, 8, 509-534.
Rosser, E.J. (1997) German Shepherd dog pyoderma: a prospective study of 12 dogs. Journal of the American Animal Hospital Association, 33, 355-363.
Ruiz-Balanguer, N., Nacher, A., Casabo, V.J. & Merino, M. (1997) Nonlinear intestinal absorption kinetics of cefuroxime axetil in rats. Antimicrobial Agents and Chemotherapy, 41, 445-448.
Silley, P. & Brewster, G. (1988) Kill kinetics of the cephalosporin antibiotics cephalexin and cefuroxime agains bacteria of veterinary importance. Veterinary Record, 123, 343-345.
Silley, P., Rudd, A.P., Symington, W.M. & Tait, A.J. (1988) Pharmaco- kinetics of cephalexin in dogs and cats after oral, subcutaneous and intramuscular administration. Veterinary Record, 122, 15-17.
Soback, S., Ziv, G., Kurtz, B. & Paz, R. (1987) Clinical pharmacokinetics of five cephalosporins in calves. Research in Veterinary Science, 43, 166-172.
Wolfram, S. (1991) Numerical solution of differential equations. In Mathematica® A system for doing mathematics by computer. 2nd edn. pp. 696-703. Addison Wesley Publications Co., London.
Yamaoka, K., Tanigawara, Y., Nakagawa, T. & Uno, T. (1978) Application of Akaike's information criterion (AIC) in the evaluation of linear pharmacokinetic equations. Journal of Pharmacoâinetics and Biopharmaceutics, 6, 165-175.