CHIRALITY 3~165-169 (1991)
Pharmacokinetics of Ibuprofen Enantiomers in Dogs WINFRIED S. BECK, GERD GEISSLINGER, HEIDRUN ENGLER, AND KAY BRUNE Department of Pharmacology and Toxicology (W.S.B., G.G.,K.B.), University of Erlangen-Nuernberg, D-8520 Erlangen, Federal Republic of Germany, and Department of Pharmacology (H.E.),Heumann-Pharma, 0 - 8 5 0 0 Nuernberg, Federal Republic of Germany
ABSTRACT
Inversion of inactive (R)-ibuprofen to active @)-ibuprofen has been suggested to occur presystemically only. In order to investigate the site of inversion in dogs we administered both enantiomers either intravenously or intraduodenally (10 mg/kg) to adult, male beagle dogs (n = 3) in a crossover design. Plasma, urine, and bile were collected for up to 6 h and analyzed stereospecifically by HPLC, according to a previously published method. Pharmacokinetic parameters were calculated using a linear computer program. Absorption after intraduodenal administration occurred rapidly, resulting in maximum plasma concentrations 0.2 h after giving the enantiomer. Approximately 70% of the (R)-enantiomer (according to AUC) was inverted to the S-enantiomer independent of route of administration. No R-ibuprofen could be detected in plasma after @)-ibuprofen administration. Mean residence time was found to be 2 to 3 times longer for (S)than for (R)-ibuprofen. Total systemic clearance from plasma was twice as high for (R)- than for (@ibuprofen. There were no differences between plasma clearances after intravenous and intraduodenal administration. Between 8 and 17% of dose was recovered in bile [especially as free and conjugated (S)-ibuprofen] and 3- 12% in urine [as (S)-ibuprofen, hydroxy- and carboxyibuprofen, free and conjugated forms]. Small amounts of (R)-ibuprofen were detected in bile after intraduodenal administration of (R)-ibuprofen only (1.8%of dose). In short, the unidirectional inversion of R-ibuprofen appears to occur systemically rather than presystemically in dogs.
KEY WORDS: ibuprofen, enantiomer, stereoselectivity, chiral inversion, pharmacokinetics, dog INTRODUCTION
The chiral inversion of 2-arylpropionic acid (2-APA) derivatives has received considerable attention. Hutt and Caldwell reviewed the occurrence and possible mechanisms of the chiral inversion and its implications for pharmacology an d toxicology.',' The process, whereby (R)-ibuprofen undergoes metabolic chiral inversion in man, was studied by Baillie et al.3 Recently, Williams summarized different opinions concerning the site of in vivo inversion of ~ - A P A s On . ~ the one hand, it has been suggested that inversion occurs predominantly in the gut and that the slower the absorpThis contention tion occurs, the greater is is supported by Simmonds et al., who observed the inversion of (Rbbenoxaprofen to the (S)-antipode in rat gut preparations and concluded that no inversion occurred in the liver.7 Other research groups, however, found inversion in liver homogenate, perfused liver, or kidney Ibuprofen, (R,S)-2-(4-isobutylpheny1)propionic acid (IBU), the oldest 2-APA in clinical use, is also the most intensively investigated sub0 1991 Wiley-Liss, Inc.
stance of this group. Stereoselective pharmacokinetic differences of the IBU enantiomers were described in several studies demonstrating that IBU was always unidirectionally inverted from the (R)- to the (S)enantiomer.'',13 Our study was aimed a t (1)assessing the stereoselective pharmacokinetic data of both enantiomers following single intravenous and intraduodenal doses of the individual IBU enantiomers in beagle dogs to show whether inversion occurs predominantly presystemically or systemically and (2) to investigate whether the proposed enterohepatic circulation of IBU in dog is stereo~e1ective.l~ MATERIALS AND METHODS Chemicals (8-and (R)-IBU were donated from Pharma Trans Received for publication August 15, 1990; accepted February 4,1991. Address reprint requests to Dr. Winfried S. Beck, Institut fur Pharmakologie und Toxikologie der Universitat Erlangen-Nurnberg, D8520 Erlangen, F.R.G.
166
BECK ET AL.
Sanaq AG, Basel, Switzerland, and had a n optical pu- column (Cyclobond@I, Astec, ICT, Frankfurt/Main, rity of >99%. The two main IBU metabolites, hy- F.R.G.), 250 mm x 4.5 mm i.d. with 5 pm spherical droxyibuprofen (2-[4-(2-hydroxy-2-methylpropyl)phen-packing. The limit of quantification was about 0.1 yllpropionic acid = metabolite A) and carboxyibu- pg/ml. The concentrations of IBU metabolites were deprofen (2-[4-(2-carboxypropyl)phenyllpropionicacid = termined employing a reversed-phase HPLC method. l7 metabolite B), were a gift from The Boots Company For determination of the total amount of metabolites Ltd., Nottingham, U.K. The distilled water was filtered (free and conjugated forms), a n alkaline hydrolysis of (Milli-QB, diameter 45 pm, Millipore, Eschborn, the bile and urine samples was performed by addition F.R.G.). All other chemicals and organic solvents were of 1 M sodium hydroxide solution and incubation at room temperature for 30 min. Afterward extraction of HPLC or reagent grade. was performed with hexanelether (8:2 vlv). Separation Animals was achieved with a prepacked column (Bischoff, LeThree adult, male beagle dogs (Tierhandelsgesell- onberg, F.R.G.), 25 cm x 4.5 mm i.d., Nucleosil 5-pm schaft, Rodenbach, F.R.G.; average body weights over RP 8. the period of the study 13.1, 15.7, 17.1 kg), with free Pharmacokinetic Calculation access to food and water, were used in the study. All All pharmacokinetic parameters were calculated usdogs had implanted permanent duodenal bile fistulas, which allowed repetitive cannulation of the common ing a two-compartment model (program TOPFIT, bile duct and collection of bile. The surgical procedure Thomae, BiberacWRiss, F.R.G).18Standard pharmacowas carried out as described previously by Thomas15 kinetic symbols as described by Aronson et al.” were and Preisig et a1.16 In addition, cholecystectomy was used. Statistical analysis was performed using Student’s t test. performed. Design of study
RESULTS
The dogs were deprived of food for 24 h prior to experiment with free access to water. Bile duct cannulation was performed under light neuroleptic analgesia 1 h before the experiment (0.3 mg fentanyl base and 15 mg fluanisone im per dog; Hypnorma from Janssen, Neuss, F.R.G.). The animals were maintained in a sling restraint during the experiments. In a four-way crossover design the dogs were dosed with 10 mg/kg body weight of (S)- and (R)-IBU, both given intravenously (iv) and intraduodenally (id) (as bolus over 4 min), respectively. The duration of the wash-out period was at least 1 week. The IBU enantiomers were dissolved in 0.03 M phosphate buffer, pH 7.5 (10 mg/ml) which was membrane filtered (diameter 0.22 pm, Millipore, Eschborn, F.R.G.). The administered volume was 1 m l k g for iv a s well as id treatment. Venous blood samples (2.5 ml) were collected from a separate indwelling cannula (foreleg) into tubes containing potassium edetate as a n anticoagulant prior to each administration and at t = 0.083,0.167,0.333,0.5,0.75,1,1.5, 2, 2.5,3, 3.5, 4,4.5, 5, 5.5, 6 h after dosing. Aliquots of plasma samples were immediately frozen and stored at -25°C until analysis. Bile and urine (via catheters) were collected a t 30-min intervals both before and after dosing for up to 6 h. The volume and pH were measured and aliquots were stored at - 25°C until analysis.
The plasma levels of the individual enantiomers following intravenous or intraduodenal doses of 10 mg/kg (R)-IBU or (S)-IBU to three beagle dogs are given in Figure la-c. The figures clearly demonstrate the unidirectional bioinversion of (R)- to 6)-IBU occurring in t h e dog a s described previously i n o t h e r speties. 1,2,4,8,9,11- 1320 There was no (R)-IBU detectable in plasma after (S)-IBU was given either intravenously or intraduodenally (Fig. lc) a s judged by the plasma samples assayed. Using a n equation suggested by Pang and Kwan,’l approximately 70% of the (R)-enantiomerwas inverted to the (S)-enantiomer independent of administration route (Fig. la-b, Table 1). The calculated pharmacokinetic parameters are listed in Table 1. The absorption after intraduodenal administration occurred very rapidly for both enantiomers yielding maximum plasma concentrations within 0.2 h. At that time absorption of dose appeared to have occurred to completeness. Mean residence time was found to be 2 to 3 times longer for (S)- than for (R)-IBU. Correspondingly, the (S)-enantiomer had a longer terminal elimination half-life. There were no impressive differences between the two enantiomers concerning volume of distribution. Total systemic clearance from plasma was twice as high for (R)- than for (S)-IBU. However, there were no differences in plasma clearances after intravenous or intraduodenal administration. Table 2 shows the fractions of dose recovered in bile and urine as (R)- and (S)-IBU and the two main phase-I metabolites, hydroxy- and carboxyibuprofen (free and conjugated). Between 8 and 17% of dose was recovered in bile predominantly as (S)-IBU and 3-12% in urine. (R)-IBU was detected in bile only after intraduodenal administration of (R)-IBU (1.8% of dose). Only small amounts of the phase I metabolites (free and conju-
Analytical Method
The parent enantiomers and their phase I and I1 metabolites were measured by high-performance liquid chromatography (HPLC). A stereospecific assay for IBU was used to determine simultaneously the concentrations of (R)- and (S)-IBU in plasma, urine and bile, as described previously. l7 The assay involved extraction of IBU with hexane/ether (8:2 vlv). Separation of the enantiomers was achieved using a P-cyclodextrin
INVERSION OF IBUPROFEN IN DOG plasma concentration (vg/ml)
4-
R-IBU
+ S-IBU
1
0
2
4
3
5
6
time (h) plasma concentration (vg/ml) 100 I-~
~-
~
+
R-IBU
+
S-IBU
I
60-l
0
1
2
4
3
5
6
time (h)
4-
2o
S-IBU (after i.v.) S-IBU (after i.d.1
I
OiB
3 4 5 6 time (h) Fig. 1. (a
Pharmacokinetics of Ibuprofen Enantiomers in Dogs WINFRIED S. BECK, GERD GEISSLINGER, HEIDRUN ENGLER, AND KAY BRUNE Department of Pharmacology and Toxicology (W.S.B., G.G.,K.B.), University of Erlangen-Nuernberg, D-8520 Erlangen, Federal Republic of Germany, and Department of Pharmacology (H.E.),Heumann-Pharma, 0 - 8 5 0 0 Nuernberg, Federal Republic of Germany
ABSTRACT
Inversion of inactive (R)-ibuprofen to active @)-ibuprofen has been suggested to occur presystemically only. In order to investigate the site of inversion in dogs we administered both enantiomers either intravenously or intraduodenally (10 mg/kg) to adult, male beagle dogs (n = 3) in a crossover design. Plasma, urine, and bile were collected for up to 6 h and analyzed stereospecifically by HPLC, according to a previously published method. Pharmacokinetic parameters were calculated using a linear computer program. Absorption after intraduodenal administration occurred rapidly, resulting in maximum plasma concentrations 0.2 h after giving the enantiomer. Approximately 70% of the (R)-enantiomer (according to AUC) was inverted to the S-enantiomer independent of route of administration. No R-ibuprofen could be detected in plasma after @)-ibuprofen administration. Mean residence time was found to be 2 to 3 times longer for (S)than for (R)-ibuprofen. Total systemic clearance from plasma was twice as high for (R)- than for (@ibuprofen. There were no differences between plasma clearances after intravenous and intraduodenal administration. Between 8 and 17% of dose was recovered in bile [especially as free and conjugated (S)-ibuprofen] and 3- 12% in urine [as (S)-ibuprofen, hydroxy- and carboxyibuprofen, free and conjugated forms]. Small amounts of (R)-ibuprofen were detected in bile after intraduodenal administration of (R)-ibuprofen only (1.8%of dose). In short, the unidirectional inversion of R-ibuprofen appears to occur systemically rather than presystemically in dogs.
KEY WORDS: ibuprofen, enantiomer, stereoselectivity, chiral inversion, pharmacokinetics, dog INTRODUCTION
The chiral inversion of 2-arylpropionic acid (2-APA) derivatives has received considerable attention. Hutt and Caldwell reviewed the occurrence and possible mechanisms of the chiral inversion and its implications for pharmacology an d toxicology.',' The process, whereby (R)-ibuprofen undergoes metabolic chiral inversion in man, was studied by Baillie et al.3 Recently, Williams summarized different opinions concerning the site of in vivo inversion of ~ - A P A s On . ~ the one hand, it has been suggested that inversion occurs predominantly in the gut and that the slower the absorpThis contention tion occurs, the greater is is supported by Simmonds et al., who observed the inversion of (Rbbenoxaprofen to the (S)-antipode in rat gut preparations and concluded that no inversion occurred in the liver.7 Other research groups, however, found inversion in liver homogenate, perfused liver, or kidney Ibuprofen, (R,S)-2-(4-isobutylpheny1)propionic acid (IBU), the oldest 2-APA in clinical use, is also the most intensively investigated sub0 1991 Wiley-Liss, Inc.
stance of this group. Stereoselective pharmacokinetic differences of the IBU enantiomers were described in several studies demonstrating that IBU was always unidirectionally inverted from the (R)- to the (S)enantiomer.'',13 Our study was aimed a t (1)assessing the stereoselective pharmacokinetic data of both enantiomers following single intravenous and intraduodenal doses of the individual IBU enantiomers in beagle dogs to show whether inversion occurs predominantly presystemically or systemically and (2) to investigate whether the proposed enterohepatic circulation of IBU in dog is stereo~e1ective.l~ MATERIALS AND METHODS Chemicals (8-and (R)-IBU were donated from Pharma Trans Received for publication August 15, 1990; accepted February 4,1991. Address reprint requests to Dr. Winfried S. Beck, Institut fur Pharmakologie und Toxikologie der Universitat Erlangen-Nurnberg, D8520 Erlangen, F.R.G.
166
BECK ET AL.
Sanaq AG, Basel, Switzerland, and had a n optical pu- column (Cyclobond@I, Astec, ICT, Frankfurt/Main, rity of >99%. The two main IBU metabolites, hy- F.R.G.), 250 mm x 4.5 mm i.d. with 5 pm spherical droxyibuprofen (2-[4-(2-hydroxy-2-methylpropyl)phen-packing. The limit of quantification was about 0.1 yllpropionic acid = metabolite A) and carboxyibu- pg/ml. The concentrations of IBU metabolites were deprofen (2-[4-(2-carboxypropyl)phenyllpropionicacid = termined employing a reversed-phase HPLC method. l7 metabolite B), were a gift from The Boots Company For determination of the total amount of metabolites Ltd., Nottingham, U.K. The distilled water was filtered (free and conjugated forms), a n alkaline hydrolysis of (Milli-QB, diameter 45 pm, Millipore, Eschborn, the bile and urine samples was performed by addition F.R.G.). All other chemicals and organic solvents were of 1 M sodium hydroxide solution and incubation at room temperature for 30 min. Afterward extraction of HPLC or reagent grade. was performed with hexanelether (8:2 vlv). Separation Animals was achieved with a prepacked column (Bischoff, LeThree adult, male beagle dogs (Tierhandelsgesell- onberg, F.R.G.), 25 cm x 4.5 mm i.d., Nucleosil 5-pm schaft, Rodenbach, F.R.G.; average body weights over RP 8. the period of the study 13.1, 15.7, 17.1 kg), with free Pharmacokinetic Calculation access to food and water, were used in the study. All All pharmacokinetic parameters were calculated usdogs had implanted permanent duodenal bile fistulas, which allowed repetitive cannulation of the common ing a two-compartment model (program TOPFIT, bile duct and collection of bile. The surgical procedure Thomae, BiberacWRiss, F.R.G).18Standard pharmacowas carried out as described previously by Thomas15 kinetic symbols as described by Aronson et al.” were and Preisig et a1.16 In addition, cholecystectomy was used. Statistical analysis was performed using Student’s t test. performed. Design of study
RESULTS
The dogs were deprived of food for 24 h prior to experiment with free access to water. Bile duct cannulation was performed under light neuroleptic analgesia 1 h before the experiment (0.3 mg fentanyl base and 15 mg fluanisone im per dog; Hypnorma from Janssen, Neuss, F.R.G.). The animals were maintained in a sling restraint during the experiments. In a four-way crossover design the dogs were dosed with 10 mg/kg body weight of (S)- and (R)-IBU, both given intravenously (iv) and intraduodenally (id) (as bolus over 4 min), respectively. The duration of the wash-out period was at least 1 week. The IBU enantiomers were dissolved in 0.03 M phosphate buffer, pH 7.5 (10 mg/ml) which was membrane filtered (diameter 0.22 pm, Millipore, Eschborn, F.R.G.). The administered volume was 1 m l k g for iv a s well as id treatment. Venous blood samples (2.5 ml) were collected from a separate indwelling cannula (foreleg) into tubes containing potassium edetate as a n anticoagulant prior to each administration and at t = 0.083,0.167,0.333,0.5,0.75,1,1.5, 2, 2.5,3, 3.5, 4,4.5, 5, 5.5, 6 h after dosing. Aliquots of plasma samples were immediately frozen and stored at -25°C until analysis. Bile and urine (via catheters) were collected a t 30-min intervals both before and after dosing for up to 6 h. The volume and pH were measured and aliquots were stored at - 25°C until analysis.
The plasma levels of the individual enantiomers following intravenous or intraduodenal doses of 10 mg/kg (R)-IBU or (S)-IBU to three beagle dogs are given in Figure la-c. The figures clearly demonstrate the unidirectional bioinversion of (R)- to 6)-IBU occurring in t h e dog a s described previously i n o t h e r speties. 1,2,4,8,9,11- 1320 There was no (R)-IBU detectable in plasma after (S)-IBU was given either intravenously or intraduodenally (Fig. lc) a s judged by the plasma samples assayed. Using a n equation suggested by Pang and Kwan,’l approximately 70% of the (R)-enantiomerwas inverted to the (S)-enantiomer independent of administration route (Fig. la-b, Table 1). The calculated pharmacokinetic parameters are listed in Table 1. The absorption after intraduodenal administration occurred very rapidly for both enantiomers yielding maximum plasma concentrations within 0.2 h. At that time absorption of dose appeared to have occurred to completeness. Mean residence time was found to be 2 to 3 times longer for (S)- than for (R)-IBU. Correspondingly, the (S)-enantiomer had a longer terminal elimination half-life. There were no impressive differences between the two enantiomers concerning volume of distribution. Total systemic clearance from plasma was twice as high for (R)- than for (S)-IBU. However, there were no differences in plasma clearances after intravenous or intraduodenal administration. Table 2 shows the fractions of dose recovered in bile and urine as (R)- and (S)-IBU and the two main phase-I metabolites, hydroxy- and carboxyibuprofen (free and conjugated). Between 8 and 17% of dose was recovered in bile predominantly as (S)-IBU and 3-12% in urine. (R)-IBU was detected in bile only after intraduodenal administration of (R)-IBU (1.8% of dose). Only small amounts of the phase I metabolites (free and conju-
Analytical Method
The parent enantiomers and their phase I and I1 metabolites were measured by high-performance liquid chromatography (HPLC). A stereospecific assay for IBU was used to determine simultaneously the concentrations of (R)- and (S)-IBU in plasma, urine and bile, as described previously. l7 The assay involved extraction of IBU with hexane/ether (8:2 vlv). Separation of the enantiomers was achieved using a P-cyclodextrin
INVERSION OF IBUPROFEN IN DOG plasma concentration (vg/ml)
4-
R-IBU
+ S-IBU
1
0
2
4
3
5
6
time (h) plasma concentration (vg/ml) 100 I-~
~-
~
+
R-IBU
+
S-IBU
I
60-l
0
1
2
4
3
5
6
time (h)
4-
2o
S-IBU (after i.v.) S-IBU (after i.d.1
I
OiB
3 4 5 6 time (h) Fig. 1. (a
