Pharmacokinetics of Acemetacin and Its Active Metabolite Indomethacin in Rats During Acute Hepatic Damage and Liver Regeneration
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medigraphic Artemisaen línea AE Chávez-PiñaAnnals of et Hepatology al. Acemetacin 2009; pharmacokinetics 8(2): April-June: and 141-147 liver damage 141 Original Article Annals of Hepatology Pharmacokinetics of acemetacin and its active metabolite indomethacin in rats during acute hepatic damage and liver regeneration Aracely E. Chávez-Piña;1 Liliana Favari;1 Gilberto Castañeda-Hernández1 Abstract and indomethacin was comparable to control values. In conclusion: Indomethacin bioavailability after oral Background and aim: The pharmacokinetics of acem- administration of its precursor, acemetacin, is signifi- etacin, a non-steroidal anti-inflammatory drug which cantly reduced by acute hepatitis produced by CCl4. is biotransformed to indomethacin by hepatic first-pass Pharmacokinetic alterations, as liver damage, are re- effect, was examined during the necrotic and regenera- versible, but do not require complete liver regenera- tion phases resulting from acute hepatitis induced by tion to return to basal conditions. carbon tetrachloride (CCl4). Material and methods: Acute hepatitis was induced by oral CCl4 administra- Key words: Hepatitis, carbon tetrachloride, bioavail- tion to male Wistar rats. On days 0, 1 and 3 after the in- ability, first-pass effect, hepatic metabolism. sult, liver histological analysis was performed, bio- chemical markers of liver damage and regeneration Introduction were measured, and the pharmacokinetics of oral acemetacin and of its active metabolite, indomethacin, Acemetacin, a carboxymethyl ester derivative of in- were determined. Results: One day after CCl4 adminis- domethacin, is currently used in the treatment of inflam- tration, liver necrosis was apparent and there was an mation and pain.1-3 It has been shown that equimolar dos- increase in the circulating levels of indicators of liver es of both agents result in comparable analgesic and anti- damage and regeneration with regard to control condi- inflammatory effects.1,2 Notwithstanding, clinical studies tions. Acemetacin bioavailability was increased, al- have shown that acemetacin produces significantly less though not in a statistically significant manner. On the gastric damage than indomethacin.3,4 Both, experimental other hand, indomethacin bioavailability was signifi- and clinical studies, have shown that acemetacin is me- cantly reduced. By day 3, histological analysis re- tabolized to indomethacin by sterolytic cleavage after vealed liver recovery, although not complete, while oral administration with a relevant participation of he- biochemical indicators of hepatic damage had revert- patic first-pass effect after oral administration.5-9 Notwith- ed either totally or partially. Markers of liver regener- standing, we have provided evidence that acemetacin is ation were still increased. Bioavailability acemetacin not only a prodrug of indomethacin, but an agent en- dowed of gastric-sparing mechanisms, probably involv- ing a reduction in leukocyte adherence.10,11 Liver diseases could change drug disposition, as he- 1 Sección Externa de Farmacología, Centro de Investigación y de patic damage can produce a reduction in the metabolic Estudios Avanzados del Instituto Politécnico Nacional. México, activity of several enzymes and therefore decreases in- D.F., Mexico trinsic drug clearance.12 It has been documented that nu- Address for correspondence: merous therapeutic agents exhibit pharmacokinetic Gilberto Castañeda-Hernández, Ph.D. changes during hepatic injury. Particularly, our group Sección Externa de Farmacología www.medigraphic.comhas reported that experimental liver damage results in al- Centro de Investigación y de Estudios Avanzados del Instituto terations in the bioavailability of several non-steroidal Politécnico Nacional 13 Av. Instituto Politécnico Nacional 2508 anti-inflammatory drugs (NSAIDs), including naproxen, Col. San Pedro Zacatenco ketorolac14 and diclofenac.15 It should be noted, however, 07360 México, D.F. that liver status after an acute damage is continuously E-mail: [email protected] changing, as the necrotic phase is followed by an active Tel: 52 55 5747 3305 16 Fax: 52 55 57 47 38 00 ext. 5415 regeneration phase. Hence, pharmacokinetic alterations after acute hepatic injury are not permanent, but revert.13 Manuscript received and accepted: 17 March and 1 May 2009 Such alterations are particularly relevant for drugs sub- 142 Annals of Hepatology 8(2) 2009: 141-147 mitted to a substantial hepatic first-pass effect after oral function. Finally, animals were sacrificed by an excess of administration.17 anesthesia and the liver was immediately extracted for his- As mentioned above, oral acemetacin is biotrans- tological analysis. formed to indomethacin by liver first pass effect.5-9 How- ever; there are no available reports on acemetacin phar- Liver function and regeneration assays macokinetics during acute liver injury and subsequent liver regeneration. Thus, the aim of this study was to ex- Liver damage was determined by measuring the activ- amine the pharmacokinetics of acemetacin and of its ac- ities of g-glutamyltranspeptidase (g-GTP) and alanine tive metabolite, indomethacin, during the necrotic and amino-transferase (ALT), as described previously.15 ALT regenerative processes occurring after the induction of is considered an indicator of liver necrosis, whereas g- 19,20 acute hepatitis by CCl4 in the rat, a widely accepted ex- GTP is an indicator of cholestasis. Liver regeneration perimental model of liver damage.15 was estimated by the determination of -fetoprotein by enzyme immune analysis (EMIT) using a commercial kit Material and methods (AxSYM AFP, Abbott, Mexico City), as previously de- scribed.13 Histological analysis of the liver was per- Chemicals formed by hematoxiline- eosin staining as described by Mourelle and coworkers.21 Indomethacin and mineral oil were purchased from Sigma Chemical Company (Saint Louis, MO, USA). CCl4 Determination of acemetacin and indomethacin in and methanol (chromatographic grade) were obtained plasma from J.T. Baker (Xalostoc, Mexico). Acemetacin was a gift from Bayer de México (Mexico City). All other re- Acemetacin and indomethacin concentrations in agents were of analytical grade. High-quality water, em- plasma were determined by reverse-phase high-perfor- ployed to prepare solutions, was obtained using a Milli- mance liquid chromatography with ultraviolet detec- Q Reagent Water System (Continental Waters Systems, tion method developed in our laboratory. Briefly, 100 El Paso, TX, USA). µL of plasma was spiked with 30 µg/mL of carbam- azepine (internal standard) and 1,100 µL of methanol Animals was added. Extraction was carried out by vortex agita- tion during 1 min at maximum speed; then samples were Male Wistar rats (250-300 g) from our own breeding centrifuged. Aliquots of 60 µL of the supernatant were facilities were used in this study. All animals received injected into the chromatographic system. The chro- human care and the study complied with the institution’s matographic system consisted of a Novapak C-18 col- guidelines and the Mexican official regulation regarding umn (150 x 3.9 mm ID, particle size 4 µm, Waters As- technical specifications for production, care and use of soc., Milford, MA, USA) eluted with a mobile phase laboratory animals (NOM-062-ZOO-1999).18 Animals consisting of a mixture of 0.025 M phosphate buffer were housed under controlled conditions at 22 ± 2 ºC (pH 6.0) with methanol, 45:55 v/v at constant flow of temperature, 50-60% relative humidity and 12 h light- 1.0 mL/min at room temperature. The effluent from the dark cycles. Food was withdrawn 12 h before initiation column was monitored spectrophotometrically at 260 of experiments, but animals had free access to drinking nm. Retention times were 2.30, 4.25 and 5.10 min for water. the internal standard, indomethacin and acemetacin re- spectively. Typical chromatograms from a blank rat Study design plasma sample, a blank rat plasma sample spiked with acemetacin, indomethacin and the internal standard car- Rats received either CCl4 (4 g/kg) dissolved in mineral bamazepine, and a plasma sample from a rat treated with oil or vehicle. One or three days after either CCl4 or vehi- acemetacin spiked with the internal standard are shown cle (control) administration, acemetacin (35 mg/kg) was in Figure 1. It can be clearly appreciated that there were administered orally by gavage. The drug was suspended in no peaks due to endogenous compounds which could carboxymethyl cellulose (4 mL/kg).www.medigraphic.com 200-µL blood sam- interfere with the assay. Acemetacin and indomethacin ples were drawn from the caudal artery, as described previ- recovery from plasma samples, as established by com- ously,14 at 0, 0.08, 0.16, 0.25, 0.33, 0.5, 0.75, 1, 2, 4, 8, 10, parison with standard solutions, was 85-95%. Calibra- 24, 27 and 30 h after drug administration. Plasma was ob- tion curves were constructed for acemetacin and in- tained and acemetacin and indomethacin were determined domethacin in the 5–100 µg/mL range, being linear (r2 in plasma samples as described below. Once blood sam- > 0.98) for both compounds. Accuracy and precision of pling was finished, animals were anesthetized with ether the assay, determined by replicate analysis of spiked and submitted to exsanguinations by cardiac puncture. plasma samples of known concentration, was within the This blood was used to determine enzyme markers of liver 100 ± 15% range. AE Chávez-Piña et al. Acemetacin pharmacokinetics and liver damage 143 Results Data