J. Vet. Med. A 37, 253-258 (1990) 8 1990 Paul Parey Scientific Publishers, Berlin and Hamburg ISSN 093 1- 184X
Institute of Veterinary Physiology, University of Zurich' and Pharmaceutical Research Department F. Hoffmann-La Roche Ltd, BaseP
Plasma Disposition and Tolerance of Aditoprim in Horses after Single Intravenous Injection R.-L.
VON
FELLENBERG', J.-C. JORDAN^, B. LUDWIGZand W. F. R E H M ~
Address of authors: Prof. Dr. R.-L. VON FELLENBERG, Institute for Veterinary Physiology, University of Zurich, WinterthurerstraBe 260, CH-8057 Ziirich/Switzerland J.-C. JORDAN,Dr. B. LUDWIGand PD Dr. W. F. REHM,Pharmaceutical Research Department, F. Hoffmann-La Roche Ltd, GrenzacherstraBe 124, CH-4002 Basel/Switzerland
With 2 figures and 2 tables (Received for publication October 10, 1989)
Summary Aditoprim, a broad spectrum antimicrobial agent acting as a reversible dihydrofolate reductase inhibitor, was intravenously injected into four 12 to 24-year old horses at a dosage of 5 mg/kg b. w. Blood samples were collected over a 48-hour period after drug injection, and the separated plasma samples were assayed for aditoprim by high performance liquid chromatography. The body temperature, heart rate, respiration rate, and behaviour were recorded during the experiment. The bilirubin and urea concentrations were also determined in several plasma samples, and liver function tests were carried out. The concentrations of aditoprim in the plasma of horses were higher than the MIC of this drug against recently isolated pathogens for 6- 13h (Pasteurella haernolytica A 1) to 48 h (E. coli). The main pharmacokinetic characteristics of aditoprim in horses were: a large volume of distribution, reaching a mean value of 7.8 I/kg; a mean plasma clearance of 5.0 I h i n ; a plasma elimination half-life of 12 h. The clinical observations, blood chemistry, and liver function tests all demonstrated that the drug was well tolerated by the horses, although it was injected intravenously as a 25 YO solution. It was concluded that the 25 % aditoprim injection solution could be used in horses without adverse effects at 5 mg/kg. Furthermore, aditoprim should demonstrate good antibacterial effects in horses when intravenously injected once a day.
Introduction Aditoprim, 2,4-diamino-5-(4-[dimethylamino]-3,5-dimethoxybenzyl) pyrimidine (Fig. 1) is a reversible inhibitor of dihydrofolate reductase. The pharmacokinetics of aditoprim have already been studied in cattle (5),pigs (LUDWIG,1983;personal communication), sheep (4),and goats (6),and it was found that this antimicrobial agent exhibited in these species long plasma elimination half-lives (6.7,9, 11.5- 16.0, and 8.0 h, respectively) and a large volume of distribution ( 35 I/kg). The aim of the present study was to determine the plasma disposition characteristics of aditoprim and the tolerance of a 25 % injection solution following a single intravenous injection. U. S. Copyright Clearance Center Code Statement: 0931 - 184X/90/3704-0253$02.50/0
254
VON FELLENBERG, JORDAN, LUDWIG and REHM
OCH Fig. 1. Chemical structure of trimethoprim (R
=
OCH,) and aditoprim (R = N[CH,],)
Material and Methods Experimental animals and trial dosing Four horses, two males and two females, 12 to 24 years old, were available for the study. Their weight ranged from 500 to 620 kg. The animals were located in stalls at the veterinary hospital in Zurich (Switzerland). They received a complete mixed feed, which is routinely given to horses in the hospital, and water ad libitum. Chemicals, test substance, dosing An aqueous 25 70(w/w) aditoprim injection solution prepared by F. Hoffmann-La Roche Ltd, Basel, Switzerland, was used for the study. Products and solvents for the biological fluid analysis were purchased from E. Merck, Darmstadt, Federal Republic of Germany, o r Fluka AG, Buchs, Switzerland. Aditoprim was injected into the V. jugularis sinistra of the animals at a dose of 5 mg a d i t o p r i d k g b. w. during 30-40 seconds. The injected volume ranged from 10 to 12.4 ml/horse. Blood sampling Blood samples were collected from the left jugular vein into glass tubes (Vacutainef, Becton Dickinson-France SA) containing heparine as anticoagulant immediately before drug administration and at various times over a 48-hour period following drug injection. The blood was centrifuged within 30 minutes after collection, at about 1,500 g at 5 "C for 5 minutes. The plasma was removed and stored at - 20 "C pending analysis. Determination of aditoprim in plasma The plasma samples were assayed for aditoprim by high performance liquid chromatography (l), using isocratic reversed-phase technique with a 250 x 4 mm 6- 10 pm Bondapak CIScolumn (Waters Assoc., Milford MA, U. S. A,). Because concentrated biological samples were injected, slight modifications of the H P L C method were necessary: the analytical column had to be protected from highly retained endogenous products by means of a 20 x 4 mm 5 pm MOS-HYPERSIL C8 pre-column (Bischoff Ltd., Leonberg, Fed. Rep. Germany). A short time after the sample injection (approximately 1 minute), the retained plasma products were washed from the pre-column by backflushing with the mobile phase using a column-switching device. The mobile phase was a mixture of 0.3 mol/l diammonium hydrogen phosphate water solution (700 ml, p H 8) and acetonitrile (300 ml) containing 1.5 ml n-hexylmethylamine (12.5 mmol/l). The UV monitor was set at 290 nm (Spectroflow 773, Kratos, Switzerland). The precision of the analytical method was monitored during routine analysis by analyzing plasma samples spiked with a known quantity of aditoprim (quality control samples) together with the biological samples. These test solutions were prepared by a different person from the analyst who carried out the determinations in order to have optimum control of the analytical method. With this H P L C technique, the limit for quantitative determination of aditoprim in plasma was 0.025 pg/ml with precision greater than 20 %. The precision and accuracy of the assay were found equal to k 1 - 4 X and 2.5-11 %, respectively, within a 0.06-2.5pglml concentration range.
*
Blood chemistry and liver function tests The blood chemistry of each horse was examined before aditoprim injection and 48 hours after the injection. The activity of two enzymes, known to be indicative of hepatic parenchymal cell damage
255
Plasma Disposition and Tolerance of Aditoprim in Horses
(aspartate aminotransferase - ASAT - and glutamate dehydrogenase - GLDH -), was determined in several plasma samples during the same period together with the concentrations of bilirubin and urea.
Pharmacokinetic evaluation Equation 1 was fitted to the plasma-concentration time data obtained following a single intravenous injection of aditoprim to horses by use of an extended least squares nonlinear regression programme ELSFIT (7-9):
C, = A e-"'
+ B e-b!
Equ. 1
corresponding to a first order 2-compartment open model (2). C, is the plasma concentration at time t following the drug injection; a/2.303 and 8/2.303 are the slopes, measured in a semi-logarithmic plot, of the first and second phase of the plasma-concentration time curve; A and B are coefficients related to the microconstants k12, k2, and klo, to a and 8, and to the dose D (2). It is assumed that drug elimination takes place exclusively in the central compartment, which is representative of the blood and well perfused organs e. g., liver or kidney.
0
6
12
18
24
30
S6
42
48
Time p . i . ( h ) Fig. 2. Semilogarithmic plot of plasma-concentration time curve of aditoprim in horses after intravenous injection of a 5 mg/kg dose. Values are given as mean f SD of four animals. *: times at which plasma concentrations in one of the four horses were under the limit of quantitation of the assay. The equation was: C, = 2.6812 e-1.9568' + 0.4736 e-0~05s33t.
256
VON FELLENBERG, JORDAN, LUDWIG and REHM The terminal plasma elimination half-life tl/2p of aditoprim was then calculated as follows: tl/2li =
Equ.2
0.69YP
The area under the plasma-concentration time curve, AUC, and the systemic clearance, CIS,were calculated according to the equations 3 and 4 (2):
AUC = A/a + B/P CI, = D/AUC
Equ. 3 Equ. 4
The volumes of distribution V,, V,,,,, and V,, were calculated according to equations 5-7 (2, 3) and standardized to the weight of each horse: Equ. 5 V, = D/(A + B) V,,,, = D/(PAUC) Equ. 6 Equ. 7 V,, = VL(ki2 + k21)Ik~i
V, is the volume of the central compartment; V,,,, is a volume term which relates drug concentrations in plasma to the total amount of drug in the body during the terminal exponential phase; V,, is the apparent volume of distribution at steady state.
Results Plasma disposition of aditoprirn Evaluation on plasma concentrations of aditoprim in horses revealed that the plasma disposition of aditoprim was adequately described by the bi-exponential equation 1 . The mean plasma-concentration time profile of aditoprim in horses following a single intravenous dose (5mg/kg) is shown in Fig.2. The mean concentration vs. time curve was calculated from the mean pharmacokinetic parameters obtained in each horse individually because in one horse the plasma concentration of aditoprim was under the limit of quantitation at 48 hours after the drug injection. The median values of the aditoprim concentration in plasma decreased from 2.7pg/ml 8 min p. i., to 0.1 1 pg/ml at 24 h p. i.; they ranged from 0.039 to 0.049 pg/ml plasma 48 h after the injection in horses Nos. 1, 2 and 4, whereas in horse No. 3 they were under the limit for quantitative determination of the analytical method (0.025 pg/ml). The main pharmacokinetic characteristics of aditoprim in horses were (Table 1): large volume of distribution, V,, = 7.0-9.51/kg plasma clearance in the 4.0 to 7.4 I/min range plasma elimination half-life, ranging from 9.1 to 14.5 h. Tolerance Aditoprim was well tolerated by the four horses when administered intravenously as the 25 % injection solution at a dose of 5 mg/kg b. w. The heart race, respiration rate, and Table 1. Pharmacokinetic parameters of aditoprim following a single 5 mg/kg b. w. intravenous injection in horses
Bella Fani Ile Bleue Karat
2.3 1.8 4.1 3.0
0.11 0.13 0.07 0.11
4.5 4.0 7.4 4.2
2.0 2.4 1.o 1.6
7.0 7.0 7.7 9.5
8.1 8.0 9.7 10.5
13.1 12.3 9.1 14.5
Mean
2.8 1 .o 2.7 1.8 4.1
0.1 1 0.03 0.11 0.07 0.13
5.0 1.6 4.3 4.0 7.4
1.8 0.6 1.8 1 .o 2.4
7.8 1.2 7.4 7.0 9.5
9.1 1.2 8.9 8.0 10.5
11.9'> 2.3 12.7 9.1 14.5
k SD Median Minimum Maximum
;*: Harmonic mean. 5 : Plasma concentration measured approximately 8 minutes after the drug injection. +:Plasma concentration measured approximately 24 hours after the drug injection.
Plasma Disposition and Tolerance of Aditoprim in Horses
257
Table 2. I n witro MIC of aditoprim against various strains isolated from diseased animals, and time during which plasma concentrations of aditoprim in horses are higher, or equal to these MIC ~
Strains Pasteurella haemolytica A 1 Staph. aureus
E . coli Salmonella spp.
*
in vitro MIC*
(Pgw
Time interval (hours p. i.)
0.25 0.5-1.0 0.025 - 1 .O 0.05
6-13 up to 3 up to 48 up to 44
Data from reference 10.
rectal temperature remained in the normal range. The animals continued to eat normally, and normal coloured faeces were formed. Furthermore, the values measured in plasma for the aspartate aminotransferase (ASAT) activity (2.7-6.3 pkat/l), the glutamate dehydrogenase (11.1-27 pmol/l), the urea concentration (4.9- 12.0 mmol/l) were always within the normal range (11). Discussion The mean, and the range, of the in witro minimum inhibitory concentrations (MIC) of aditoprim against Pasteurella haemolytica A 1, Staphylococcus aureus, Escherichiu coli, and Salmonella spp. recently isolated from animal origin are listed in Table 2 (10). The mean duration of plasma aditoprim concentration above, or equal to, these MIC values were determined by graphical interpolation using the plasma concentration-time curves of aditoprim. These values are reported in Table 2. The concentrations of aditoprim measured in the plasma of horses in this experiment were equal to or higher than the in witro MIC of this antimicrobial agent for Salmonella spp. and Escherichia coli for up to 44 h after drug injection. The large volume of distribution of aditoprim which is poorly bound to plasma protein of horses (< 25 %) is probably associated with a high tissue penetration of the drug. Therefore, aditoprim may exhibit an even longer in wiwo antimicrobial activity in horses than can be evaluated from plasma levels. Once a day administration of aditoprim at a dose of 5 mg/kg for therapeutic purposes in horses may be sufficient. Aditoprim was well tolerated by the horses when it was injected as a concentrated 25 70 solution. The injection volume was approximately 10 ml per animal which makes more convenient the injection technique in comparison with drugs which need relatively large injection volumes. Acknowledgements The authors whish to thank PD Dr. P. KELLER-RUPP (F. Hoffmann-La Roche Ltd) for perforrning the blood chemistry analysis and liver function tests. Thanks are due to PD Dr. E. WEIDEKAMM and Dr. D. DELL(F. Hoffmann-La Roche Ltd) for reading and correcting the manuscript.
References 1. ASCALONE, V., J.-C. JORDAN,and B.LUDWIG,1986: Determination of aditoprim, a new dihydrofolate reductase inhibitor, in the plasma of cows and pigs. J. Chromatogr. 383,111 - 118. 2. GIBALDI, M., and D. PERRIER,1975: Pharmacokinetics, 1st edn. pp. 48-69, Marcel Dekker, Inc., New York. 3. GIBALDI,M., and D.PERRIER, 1975: Pharmacokinetics, 1st edn. pp. 175-187, Marcel Dekker, Inc., New York. 4. HAENNI, K., J.-C. JORDAN, and B. LUDWIG,1987: Pharmacokinetics of aditoprim, a dihydrofolate reductase inhibitor, in sheep. J. vet. Pharmacol. Therap. 10, 169-171. 5. JORDAN, J.-C., P. KLAIT, and B. LUDWIG,1986: Pharmacokinetics of aditoprim, a new long-acting dihydrofolate reductase inhibitor, in heifers. J. vet. Med. A 34, 33-41.
258
VON FELLENBERG, JORDAN,LUDWIG and REHM VAN DUIN,~ . K O R S T A N JHE. V , A N GOGH, and A. S. J. P. A.M. VAN MIERT,1988: Some pharmacokinetic data of aditoprim and trimethoprim in healthy and tick-borne fever infected dwarf goats. J. vet. Pharmacol. Therap. 11, 135-144. PECK, C.C., S.L. BEAL, L.B. SHEINER,and A.I. NICHOLS,1984: Extended least squares nonlinear regression: a possible solution to the “choice of the weights” problem in analysis of individual pharmacokinetic data. J. Pharmacokin. Biopharm. 12, 545 -558. SHEINER,L. B., and S. L. BEAL, 1985: Pharmacokinetic parameter estimates from several least squares procedures: superiority of extended least squares. J. Pharmacokin. Biopharm. 13, 185-201. SHEINER,L.B., and S.L. BEAL, 1988: Commentary on “extended least squares (ELS) for pharmacokinetic models”. J. Pharm. Sci. 77, 731 -732. THEN,R. L., and M. KELLER,1988: Properties of aditoprim, a new antibacterial dihydrofolate reductase inhibitor. J. vet. Med. B 35, 114- 120. N. N., 1985: Veterinarmedizinische Laboruntersuchungen fur die Diagnose und Verlaufskontrolle, 3rd edn. Boehringer Mannheim GmbH, Mannheim.
6. KNOPPERT,N.W., S.M. NIJMEIJER, C . T . M .
7.
8.
9. 10. 11.
Institute of Veterinary Physiology, University of Zurich' and Pharmaceutical Research Department F. Hoffmann-La Roche Ltd, BaseP
Plasma Disposition and Tolerance of Aditoprim in Horses after Single Intravenous Injection R.-L.
VON
FELLENBERG', J.-C. JORDAN^, B. LUDWIGZand W. F. R E H M ~
Address of authors: Prof. Dr. R.-L. VON FELLENBERG, Institute for Veterinary Physiology, University of Zurich, WinterthurerstraBe 260, CH-8057 Ziirich/Switzerland J.-C. JORDAN,Dr. B. LUDWIGand PD Dr. W. F. REHM,Pharmaceutical Research Department, F. Hoffmann-La Roche Ltd, GrenzacherstraBe 124, CH-4002 Basel/Switzerland
With 2 figures and 2 tables (Received for publication October 10, 1989)
Summary Aditoprim, a broad spectrum antimicrobial agent acting as a reversible dihydrofolate reductase inhibitor, was intravenously injected into four 12 to 24-year old horses at a dosage of 5 mg/kg b. w. Blood samples were collected over a 48-hour period after drug injection, and the separated plasma samples were assayed for aditoprim by high performance liquid chromatography. The body temperature, heart rate, respiration rate, and behaviour were recorded during the experiment. The bilirubin and urea concentrations were also determined in several plasma samples, and liver function tests were carried out. The concentrations of aditoprim in the plasma of horses were higher than the MIC of this drug against recently isolated pathogens for 6- 13h (Pasteurella haernolytica A 1) to 48 h (E. coli). The main pharmacokinetic characteristics of aditoprim in horses were: a large volume of distribution, reaching a mean value of 7.8 I/kg; a mean plasma clearance of 5.0 I h i n ; a plasma elimination half-life of 12 h. The clinical observations, blood chemistry, and liver function tests all demonstrated that the drug was well tolerated by the horses, although it was injected intravenously as a 25 YO solution. It was concluded that the 25 % aditoprim injection solution could be used in horses without adverse effects at 5 mg/kg. Furthermore, aditoprim should demonstrate good antibacterial effects in horses when intravenously injected once a day.
Introduction Aditoprim, 2,4-diamino-5-(4-[dimethylamino]-3,5-dimethoxybenzyl) pyrimidine (Fig. 1) is a reversible inhibitor of dihydrofolate reductase. The pharmacokinetics of aditoprim have already been studied in cattle (5),pigs (LUDWIG,1983;personal communication), sheep (4),and goats (6),and it was found that this antimicrobial agent exhibited in these species long plasma elimination half-lives (6.7,9, 11.5- 16.0, and 8.0 h, respectively) and a large volume of distribution ( 35 I/kg). The aim of the present study was to determine the plasma disposition characteristics of aditoprim and the tolerance of a 25 % injection solution following a single intravenous injection. U. S. Copyright Clearance Center Code Statement: 0931 - 184X/90/3704-0253$02.50/0
254
VON FELLENBERG, JORDAN, LUDWIG and REHM
OCH Fig. 1. Chemical structure of trimethoprim (R
=
OCH,) and aditoprim (R = N[CH,],)
Material and Methods Experimental animals and trial dosing Four horses, two males and two females, 12 to 24 years old, were available for the study. Their weight ranged from 500 to 620 kg. The animals were located in stalls at the veterinary hospital in Zurich (Switzerland). They received a complete mixed feed, which is routinely given to horses in the hospital, and water ad libitum. Chemicals, test substance, dosing An aqueous 25 70(w/w) aditoprim injection solution prepared by F. Hoffmann-La Roche Ltd, Basel, Switzerland, was used for the study. Products and solvents for the biological fluid analysis were purchased from E. Merck, Darmstadt, Federal Republic of Germany, o r Fluka AG, Buchs, Switzerland. Aditoprim was injected into the V. jugularis sinistra of the animals at a dose of 5 mg a d i t o p r i d k g b. w. during 30-40 seconds. The injected volume ranged from 10 to 12.4 ml/horse. Blood sampling Blood samples were collected from the left jugular vein into glass tubes (Vacutainef, Becton Dickinson-France SA) containing heparine as anticoagulant immediately before drug administration and at various times over a 48-hour period following drug injection. The blood was centrifuged within 30 minutes after collection, at about 1,500 g at 5 "C for 5 minutes. The plasma was removed and stored at - 20 "C pending analysis. Determination of aditoprim in plasma The plasma samples were assayed for aditoprim by high performance liquid chromatography (l), using isocratic reversed-phase technique with a 250 x 4 mm 6- 10 pm Bondapak CIScolumn (Waters Assoc., Milford MA, U. S. A,). Because concentrated biological samples were injected, slight modifications of the H P L C method were necessary: the analytical column had to be protected from highly retained endogenous products by means of a 20 x 4 mm 5 pm MOS-HYPERSIL C8 pre-column (Bischoff Ltd., Leonberg, Fed. Rep. Germany). A short time after the sample injection (approximately 1 minute), the retained plasma products were washed from the pre-column by backflushing with the mobile phase using a column-switching device. The mobile phase was a mixture of 0.3 mol/l diammonium hydrogen phosphate water solution (700 ml, p H 8) and acetonitrile (300 ml) containing 1.5 ml n-hexylmethylamine (12.5 mmol/l). The UV monitor was set at 290 nm (Spectroflow 773, Kratos, Switzerland). The precision of the analytical method was monitored during routine analysis by analyzing plasma samples spiked with a known quantity of aditoprim (quality control samples) together with the biological samples. These test solutions were prepared by a different person from the analyst who carried out the determinations in order to have optimum control of the analytical method. With this H P L C technique, the limit for quantitative determination of aditoprim in plasma was 0.025 pg/ml with precision greater than 20 %. The precision and accuracy of the assay were found equal to k 1 - 4 X and 2.5-11 %, respectively, within a 0.06-2.5pglml concentration range.
*
Blood chemistry and liver function tests The blood chemistry of each horse was examined before aditoprim injection and 48 hours after the injection. The activity of two enzymes, known to be indicative of hepatic parenchymal cell damage
255
Plasma Disposition and Tolerance of Aditoprim in Horses
(aspartate aminotransferase - ASAT - and glutamate dehydrogenase - GLDH -), was determined in several plasma samples during the same period together with the concentrations of bilirubin and urea.
Pharmacokinetic evaluation Equation 1 was fitted to the plasma-concentration time data obtained following a single intravenous injection of aditoprim to horses by use of an extended least squares nonlinear regression programme ELSFIT (7-9):
C, = A e-"'
+ B e-b!
Equ. 1
corresponding to a first order 2-compartment open model (2). C, is the plasma concentration at time t following the drug injection; a/2.303 and 8/2.303 are the slopes, measured in a semi-logarithmic plot, of the first and second phase of the plasma-concentration time curve; A and B are coefficients related to the microconstants k12, k2, and klo, to a and 8, and to the dose D (2). It is assumed that drug elimination takes place exclusively in the central compartment, which is representative of the blood and well perfused organs e. g., liver or kidney.
0
6
12
18
24
30
S6
42
48
Time p . i . ( h ) Fig. 2. Semilogarithmic plot of plasma-concentration time curve of aditoprim in horses after intravenous injection of a 5 mg/kg dose. Values are given as mean f SD of four animals. *: times at which plasma concentrations in one of the four horses were under the limit of quantitation of the assay. The equation was: C, = 2.6812 e-1.9568' + 0.4736 e-0~05s33t.
256
VON FELLENBERG, JORDAN, LUDWIG and REHM The terminal plasma elimination half-life tl/2p of aditoprim was then calculated as follows: tl/2li =
Equ.2
0.69YP
The area under the plasma-concentration time curve, AUC, and the systemic clearance, CIS,were calculated according to the equations 3 and 4 (2):
AUC = A/a + B/P CI, = D/AUC
Equ. 3 Equ. 4
The volumes of distribution V,, V,,,,, and V,, were calculated according to equations 5-7 (2, 3) and standardized to the weight of each horse: Equ. 5 V, = D/(A + B) V,,,, = D/(PAUC) Equ. 6 Equ. 7 V,, = VL(ki2 + k21)Ik~i
V, is the volume of the central compartment; V,,,, is a volume term which relates drug concentrations in plasma to the total amount of drug in the body during the terminal exponential phase; V,, is the apparent volume of distribution at steady state.
Results Plasma disposition of aditoprirn Evaluation on plasma concentrations of aditoprim in horses revealed that the plasma disposition of aditoprim was adequately described by the bi-exponential equation 1 . The mean plasma-concentration time profile of aditoprim in horses following a single intravenous dose (5mg/kg) is shown in Fig.2. The mean concentration vs. time curve was calculated from the mean pharmacokinetic parameters obtained in each horse individually because in one horse the plasma concentration of aditoprim was under the limit of quantitation at 48 hours after the drug injection. The median values of the aditoprim concentration in plasma decreased from 2.7pg/ml 8 min p. i., to 0.1 1 pg/ml at 24 h p. i.; they ranged from 0.039 to 0.049 pg/ml plasma 48 h after the injection in horses Nos. 1, 2 and 4, whereas in horse No. 3 they were under the limit for quantitative determination of the analytical method (0.025 pg/ml). The main pharmacokinetic characteristics of aditoprim in horses were (Table 1): large volume of distribution, V,, = 7.0-9.51/kg plasma clearance in the 4.0 to 7.4 I/min range plasma elimination half-life, ranging from 9.1 to 14.5 h. Tolerance Aditoprim was well tolerated by the four horses when administered intravenously as the 25 % injection solution at a dose of 5 mg/kg b. w. The heart race, respiration rate, and Table 1. Pharmacokinetic parameters of aditoprim following a single 5 mg/kg b. w. intravenous injection in horses
Bella Fani Ile Bleue Karat
2.3 1.8 4.1 3.0
0.11 0.13 0.07 0.11
4.5 4.0 7.4 4.2
2.0 2.4 1.o 1.6
7.0 7.0 7.7 9.5
8.1 8.0 9.7 10.5
13.1 12.3 9.1 14.5
Mean
2.8 1 .o 2.7 1.8 4.1
0.1 1 0.03 0.11 0.07 0.13
5.0 1.6 4.3 4.0 7.4
1.8 0.6 1.8 1 .o 2.4
7.8 1.2 7.4 7.0 9.5
9.1 1.2 8.9 8.0 10.5
11.9'> 2.3 12.7 9.1 14.5
k SD Median Minimum Maximum
;*: Harmonic mean. 5 : Plasma concentration measured approximately 8 minutes after the drug injection. +:Plasma concentration measured approximately 24 hours after the drug injection.
Plasma Disposition and Tolerance of Aditoprim in Horses
257
Table 2. I n witro MIC of aditoprim against various strains isolated from diseased animals, and time during which plasma concentrations of aditoprim in horses are higher, or equal to these MIC ~
Strains Pasteurella haemolytica A 1 Staph. aureus
E . coli Salmonella spp.
*
in vitro MIC*
(Pgw
Time interval (hours p. i.)
0.25 0.5-1.0 0.025 - 1 .O 0.05
6-13 up to 3 up to 48 up to 44
Data from reference 10.
rectal temperature remained in the normal range. The animals continued to eat normally, and normal coloured faeces were formed. Furthermore, the values measured in plasma for the aspartate aminotransferase (ASAT) activity (2.7-6.3 pkat/l), the glutamate dehydrogenase (11.1-27 pmol/l), the urea concentration (4.9- 12.0 mmol/l) were always within the normal range (11). Discussion The mean, and the range, of the in witro minimum inhibitory concentrations (MIC) of aditoprim against Pasteurella haemolytica A 1, Staphylococcus aureus, Escherichiu coli, and Salmonella spp. recently isolated from animal origin are listed in Table 2 (10). The mean duration of plasma aditoprim concentration above, or equal to, these MIC values were determined by graphical interpolation using the plasma concentration-time curves of aditoprim. These values are reported in Table 2. The concentrations of aditoprim measured in the plasma of horses in this experiment were equal to or higher than the in witro MIC of this antimicrobial agent for Salmonella spp. and Escherichia coli for up to 44 h after drug injection. The large volume of distribution of aditoprim which is poorly bound to plasma protein of horses (< 25 %) is probably associated with a high tissue penetration of the drug. Therefore, aditoprim may exhibit an even longer in wiwo antimicrobial activity in horses than can be evaluated from plasma levels. Once a day administration of aditoprim at a dose of 5 mg/kg for therapeutic purposes in horses may be sufficient. Aditoprim was well tolerated by the horses when it was injected as a concentrated 25 70 solution. The injection volume was approximately 10 ml per animal which makes more convenient the injection technique in comparison with drugs which need relatively large injection volumes. Acknowledgements The authors whish to thank PD Dr. P. KELLER-RUPP (F. Hoffmann-La Roche Ltd) for perforrning the blood chemistry analysis and liver function tests. Thanks are due to PD Dr. E. WEIDEKAMM and Dr. D. DELL(F. Hoffmann-La Roche Ltd) for reading and correcting the manuscript.
References 1. ASCALONE, V., J.-C. JORDAN,and B.LUDWIG,1986: Determination of aditoprim, a new dihydrofolate reductase inhibitor, in the plasma of cows and pigs. J. Chromatogr. 383,111 - 118. 2. GIBALDI, M., and D. PERRIER,1975: Pharmacokinetics, 1st edn. pp. 48-69, Marcel Dekker, Inc., New York. 3. GIBALDI,M., and D.PERRIER, 1975: Pharmacokinetics, 1st edn. pp. 175-187, Marcel Dekker, Inc., New York. 4. HAENNI, K., J.-C. JORDAN, and B. LUDWIG,1987: Pharmacokinetics of aditoprim, a dihydrofolate reductase inhibitor, in sheep. J. vet. Pharmacol. Therap. 10, 169-171. 5. JORDAN, J.-C., P. KLAIT, and B. LUDWIG,1986: Pharmacokinetics of aditoprim, a new long-acting dihydrofolate reductase inhibitor, in heifers. J. vet. Med. A 34, 33-41.
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VON FELLENBERG, JORDAN,LUDWIG and REHM VAN DUIN,~ . K O R S T A N JHE. V , A N GOGH, and A. S. J. P. A.M. VAN MIERT,1988: Some pharmacokinetic data of aditoprim and trimethoprim in healthy and tick-borne fever infected dwarf goats. J. vet. Pharmacol. Therap. 11, 135-144. PECK, C.C., S.L. BEAL, L.B. SHEINER,and A.I. NICHOLS,1984: Extended least squares nonlinear regression: a possible solution to the “choice of the weights” problem in analysis of individual pharmacokinetic data. J. Pharmacokin. Biopharm. 12, 545 -558. SHEINER,L. B., and S. L. BEAL, 1985: Pharmacokinetic parameter estimates from several least squares procedures: superiority of extended least squares. J. Pharmacokin. Biopharm. 13, 185-201. SHEINER,L.B., and S.L. BEAL, 1988: Commentary on “extended least squares (ELS) for pharmacokinetic models”. J. Pharm. Sci. 77, 731 -732. THEN,R. L., and M. KELLER,1988: Properties of aditoprim, a new antibacterial dihydrofolate reductase inhibitor. J. vet. Med. B 35, 114- 120. N. N., 1985: Veterinarmedizinische Laboruntersuchungen fur die Diagnose und Verlaufskontrolle, 3rd edn. Boehringer Mannheim GmbH, Mannheim.
6. KNOPPERT,N.W., S.M. NIJMEIJER, C . T . M .
7.
8.
9. 10. 11.
