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Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands
Archives of Oral Biology (2004) 49, 895—901
Evaluation of acetaminophen P-glycoproteinmediated salivary secretion by rat submandibular glands Paula Schaiquevicha,*, Niselman Vivianab, Tumilasci Omarc, Rubio Modestoa a
´gicas, ININFA-CONICET, Facultad de Farmacia y Bioqu½´mica, Jun½´n Instituto de Investigaciones Farmacolo 956, 5 piso (1113), Ciudad de Buenos Aires, Argentina b ´ticas, Facultad de Farmacia y Bioqu½´mica, Universidad de Buenos Aires, Departamento de Matema Argentina c ´ndulas salivares, Facultad de Medicina, Universidad de Departamento de Fisiolog½´a, Laboratorio de gla Buenos Aires, Argentina Accepted 3 May 2004
KEYWORDS Acetaminophen; P-glycoprotein; Submandibular gland; S/P ratio
Summary The constant ratio between saliva and plasma acetaminophen concentrations (S/P) during the elimination phase is assumed to result from the equilibrium established among the free-drug concentrations in the arterial blood, venous blood and saliva. Salivary secretion of acetaminophen is assumed to result from a passive diffusion of the drug to saliva from the blood that supplies the salivary glands. However, the constant S/P ratio during acetaminophen disposition and the finding that P-glycoprotein (P-gp), a protein recognized to pump substrates out of the cell, is expressed in duct cells of the submandibular glands questions the mechanisms involved in acetaminophen salivary secretion. Thus, we intended to evaluate the existence of a P-glycoprotein-mediated transport of acetaminophen in rat submandibular glands. Acetaminophen (30 mg/kg, i.v.) pharmacokinetics was assessed in controls and in rats pre-treated with erythromycin (100 mg/kg) as a P-glycoprotein inhibitor. Acetaminophen pharmacokinetic parameters were calculated from saliva and plasma levels considering a non-compartmental analysis. Mean plasma and salivary profiles of control and pre-treated animals were almost superimposable. No difference could be found in S/P ratios in control and erythromycin pre-treated animals (P > 0.05). Moreover, no statistical difference could be found in the kinetic parameters calculated from saliva or plasma drug level (P > 0.05). These observations indicate that acetaminophen salivary secretion in rat submandibular glands is not related to P-glycoprotein-mediated transport under the experimental conditions of the present work. ß 2004 Elsevier Ltd. All rights reserved.
Introduction *
Corresponding author. Tel.: þ54 11 4961 6784; fax: þ54 11 4953 7689. E-mail address: [email protected] (P. Schaiquevich).
After oral administration of acetaminophen, the saliva to plasma concentration ratio (S/P) during the absorption phase is greater than during the
0003–9969/$ — see front matter ß 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.05.003
896
disposition of the drug. The accepted explanation is based on an anatomical—physiological model which assumes that while the drug is being absorbed, the free-drug concentrations in the arterial blood and saliva are in equilibrium and show higher values than those in the venous blood.1,2 When absorption is finished and considering that acetaminophen rapidly distributes in the body, the free-drug concentration is equal in the arterial and venous blood, leading to a constant S/P ratio. In particular, for acetaminophen the constant S/P ratio is equal to unity as previously reported.1—9 This observation and the significant correlation found for the area under the concentration-time profile and half-life elimination time between values derived from venous plasma and saliva levels, has led to the use of saliva as a biological fluid for determining acetaminophen pharmacokinetics in bioavailability and bioequivalence studies.2,5,10—14 Usually, when considering a lipid-soluble acid such as acetaminophen, the S/P ratio could be predicted on the basis of the degree of ionization of the drug in both biological fluids.2,6,9 While the passage of most drugs into saliva occurs by passive diffusion, it has been suggested that an active transport process may operate for some drugs. Then, the S/P acetaminophen ratio during the elimination phase could present two components related to passive and active transport. Recently, the expression of some members of the ATP-binding cassette transporters in major and minor salivary glands of healthy subjects has been demonstrated.15,16 The authors recognized the presence of MDR1 product gene, P-glycoprotein (P-gp), multidrug resistance-associated proteins types 1 and 2 (MRP1 and MRP2, respectively) and lung resistance-related protein (LRP). Particularly, P-glycoprotein has been localised in different types of salivary duct cells. These proteins are classified as members of the ATP-binding cassette superfamily of transport proteins and are related to human cancer resistance to chemotherapeutic agents by pumping substrates out of the malignant tumor cells.17—19 P-glycoprotein is the best-characterized member of the group. The protein recognizes a broad group of amphipathic, lipophilic and cationic compounds, pumping them out of the cell.17,18 The expression of P-glycoprotein in normal tissues of humans and rodents associated with drug absorption, metabolism and excretion led to the hypothesis that the transporter modulates the pharmacokinetic and pharmacological activity of the substrates.20—24 Moreover, the fact that P-glycoprotein has been localised in human salivary glands tumors as well as in normal tissue suggests that the protein is physiologically involved in the trans-
P. Schaiquevich et al.
port of a broad spectrum of substrates in the salivary glands.15—18 Even if acetaminophen has not yet been included as a P-gp substrate, the list is daily revised and unrelated new molecules are incorporated. In addition, acetaminophen is highly secreted in saliva. Thus, a component of acetaminophen transport in submandibular glands could be mediated by P-gp pumping function contributing to the observed S/P constant ratio during the disposition phase. The aim of the present work was to study whether a P-glycoprotein-mediated transport of acetaminophen exists in the rat submandibular glands, considering its high salivary secretion previously reported and the existence of the transporter in human salivary glands even if uncertain in roedents. We assessed the kinetics of acetaminophen from saliva and plasma levels of control rats and animals pre-treated with erythromycin as a P-gp inhibitor.24
Materials and methods Animals Adult male Wistar rats of age 2—3 months and weighing 250—350 g were housed in temperaturecontrolled rooms (20 2 8C) with a 12 h light—dark cycle. Rats were fasted for 12 h but allowed free access to water prior to the experiment. Two groups of animals were employed, referred to in the text as control and treated groups. Both groups of animals underwent the same treatment but animals assigned to the treated group also received erythromycin (100 mg/kg, i.p.) 1 h before acetaminophen administration. Erythromycin ethyl succinate suspension was prepared in CMC 0.1%. Urethane (1.2 g/kg, i.p.) was used to induce and maintain anaesthesia in the rats. Following anaesthesia, polyethylene tubes previously heparinized were placed into the femoral artery of one leg and the femoral vein of the opposite. Also, glass cannulas were inserted into the ducts of both right and left submandibular glands of each animal. Acetaminophen was administered via the femoral vein as a 30 mg/kg bolus, dissolved in propylene glycol—saline 50% (v/v). Because anaesthetized animals lack a basal salivary secretion, substance P (10 mg/kg, i.v.) was administered via the femoral vein at each time that salivary samples were collected. The animals were kept under urethane anaesthesia until the end of the experiment. Saliva samples from each gland were collected in different pre-weighed polypropylene tubes. Samples were obtained over 2 min intervals after sub-
Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands
stance P administration at: 0 (blank); 9—11; 19—21; 29—31; 44—46 and 59—61 min after acetaminophen administration. Venous blood samples (200 ml) were collected in heparinized tubes at: 0 (blank); 10; 20; 30; 45; 60 min after acetaminophen intravenous administration. Blood samples were centrifuged for 20 min at 1800 g in order to separate plasma from the rest. Saliva and plasma samples were stored at 40 8C until drug analysis. The present work adhered to the Principles of Laboratory Animal Care (NIH Publication #85-23, 1985) and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council (1996).
HPLC assay of acetaminophen Plasma and saliva acetaminophen levels were quantified by HPLC UV after validating a modified method reported by others.29 Salivary samples were thawed and vortexed for 2 min before precipitation with a mixture of acetonitrile/H3PO4 (5.0:0.135). The sample volume was calculated according to the difference in the weight of the tube before and after saliva collection, assuming a saliva density of 1.00 g/ml. Afterwards, samples were vortexed, centrifuged at 8900 g for 2 min and injected into the chromatographic system. Plasma samples were thawed and vortexed for 2 min. Then, 100 ml of acetonitrile/ZnSO4 10% (w/v) (10:1) was added to the same volume of sample, vortexed for 2 min, centrifuged at 8900 g for 2 min and injected into the chromatographic system. The linear ranges for plasma and saliva assays were [0.1—50.0 mg/ml] and [2.0—30 mg/ml], respectively. Within- and inter-day coefficients of variation for acetaminophen in plasma and saliva were below 5 and 12%, respectively. The recovery percentage of acetaminophen in plasma and saliva was 58 and 88%, respectively. The detection limit was found to be 0.07 mg/ml for both assays. The chromatographic system consisted of a Spectra System P2000 pump, a Thermo Separation sample injector, an UV-100 Spectra Series ultraviolet detector set at 248 nm. Mobile phase consisted of 0.1 M K2HPO4/methanol/acetic acid (90:7:3) and (83:16:1) for saliva and plasma assays, respectively, delivered at 1 ml/min. The analytical column employed was RP-C18, 250 mm 4.6 mm, 5 mm (Symmetry, Waters) maintained at room temperature. In all cases the injected volume was 20 ml.
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Data analysis Mean saliva and plasma profiles were obtained for each group of animals from the individual concentration-time profiles. Individual acetaminophen saliva levels at each time were calculated as the mean value between concentrations calculated from the right and left glands. Individual saliva to plasma concentration ratio (S/P) at each sampling time was measured for the animals of both groups. S/P ratios at each time were compared between groups by MANOVA test with Duncan’s test a posteriori. Significance level was set at 0.05. Mean S/P ratios were calculated for each group. Non-compartmental analysis was used to describe the individual disposition of acetaminophen from saliva or plasma levels. Data were analysed using the non-linear regression program TOPFIT 2.0. The log trapezoidal method and extrapolation to infinity was used to calculate the area under the total concentration-time profile (AUC1) and the area under the concentration-time curve from 0 to 60 min (AUC1 h). Terminal plasma and saliva half-lives for each rat were estimated by linear regression analysis after log transformation of the last data points. Systemic clearance (Cl) and apparent volume of distribution (Vz) were calculated from AUC1, dose, and elimination halflife. The pharmacokinetic parameters were compared between groups by means of a Student t-test and examined for significance at a level of P < 0.05. Also, comparisons were carried out between the parameters calculated from plasma and saliva levels of the control group using the match paired t-test. Finally, mean pharmacokinetic parameters derived from plasma and saliva levels were calculated for both groups of animals.
Results Mean plasma and salivary acetaminophen concentration-time profiles obtained for control rats and animals pre-treated with erythromycin as a P-gp inhibitor are represented in Figs. 1 and 2, respectively. The salivary as well as the plasma profiles are almost superimposable when considering data from control and treated groups. Twenty-three paired samples for control rats were available for saliva-plasma correlation analysis (Fig. 3). Linear regression analysis resulted in a Pearson’s correlation coefficient (r) of 0.838 (P < 0.0001). The relationship between plasma (Cplasma) and saliva (Csaliva) concentrations could
898
P. Schaiquevich et al.
30
Saliva acetaminophen (µg / ml)
Plasma acetaminophen concentration ( µg /ml )
40
35
30
25
20
15
10
25
20
15
10
5
0 0
5
10
15
20
25
30
Plasma acetaminophen (µg /ml)
5
0 0
10
20
30
40
50
60
Figure 3 Saliva vs. plasma concentrations. The line shows the regression line: Csaliva ¼ 1.007Cplasma 1.775.
Time (min)
Figure 1 Mean plasmatic profile after acetaminophen administration (30 mg/kg, i.v.) to (&) control and (~) erythromycin (100 mg/kg, i.p.) pre-treated rats. Data are expressed as mean standard mean error (n ¼ 5).
be described by the following equation (mg/ml): Csaliva ¼ 1.007Cplasma 1.775. Mean S/P values for each sampling time are presented in Table 1 for control and treated animals. All mean values calculated for both groups of animals were found to be within the interval 40
Saliva acetaminophen concentration (µg /ml)
35
30
25
[0.7—1.1]. No significant difference was found in the S/P ratios when comparing the two groups (P > 0.05). Acetaminophen mean kinetic parameters calculated from plasma and saliva levels for control and treated animals are shown in Tables 2 and 3, respectively. We did not find statistical significant differences for Vz, Cl or T1/2 calculated from plasma levels when comparing between groups according to the t-test (P > 0.05). Moreover, no differences could be found when comparing between groups the pharmacokinetic parameters derived from salivary levels. Half-life time was approximately 25 min for both groups of rats. Mean areas under the plasma and salivary profiles for the control group calculated from the measured data (AUC1 h) and extrapolated to infinity (AUC1), were slightly higher than those obtained for the treated group (Tables 2 and 3). However, the apparent differences observed were not of statistical significance (P > 0.05).
20
Table 1 Acetaminophen saliva to plasma concentration ratio (S/P) obtained after an intravenous dose (30 mg/kg) administered to control and pre-treated with erythromycin (100 mg/kg, i.p.) rats.
15
10
5
0 0
10
20
30
40
50
60
Time (min)
Figure 2 Mean acetaminophen salivary profile after intravenous dose (30 mg/kg) to (&) control and (~) erythromycin (100 mg/kg, i.p.) pre-treated rats. Data are expressed as mean standard mean error (n ¼ 5).
Time (min)
Control group S/P S.E.M.
10 20 30 45 60
1.10 0.91 0.90 0.71 0.70
0.22 0.12 0.12 0.12 0.14
Treated group S/P S.E.M. 0.95 1.05 0.77 0.85 1.02
0.32 0.16 0.07 0.20 0.19
Data are expressed as mean standard mean error.
Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands
Table 2 Acetaminophen pharmacokinetic parameters calculated from plasma levels after 30 mg/kg i.v. dose administered to control and erythromycintreated animals. Pharmacokinetic parameter
Control group
AUC1 h (mg min/ml) AUC1 (mg min/ml) T1/2 (min) Cl (ml/min/kg) Vz (l/kg)
895.0 1256.2 26.5 24.9 0.94
71.9 148.2 2.8 3.3 0.11
Treated group 774.3 921.9 24.8 31.3 1.08
48.3 50.6 2.3 3.1 0.15
Results are expressed as mean standard mean error.
Table 3 Acetaminophen pharmacokinetic parameters calculated from salivary levels after intravenous administration (30 mg/kg) to control and pretreated animals with erythromycin. Pharmacokinetic parameter
Control group
AUC1 h (mg min/ml) ABC1 (mg min/ml) T1/2 (min) Cl (ml/min kg) Vz (l/kg)
958.8 1089.3 20.6 28.4 0.83
Treated group
94.6 839.0 23.5 101.3 984.3 44.0 2.0 21.5 2.3 2.4 30.7 1.3 0.08 0.96 0.09
Data are expressed as mean standard mean error.
Lastly, no significant differences were found in AUC1h, AUC1, Cl, T1/2 or Vz when comparing the saliva and plasma values obtained from data of the control group (P > 0.05).
Discussion Acetaminophen saliva to plasma concentration ratio (S/P) tends to be higher during the absorption phase. This observation is due to the fact that saliva drug levels reflect free-drug arterial blood concentrations, values that are higher than the corresponding venous blood concentration, until the distribution phase is completely established.1,2 On the contrary, the acetaminophen S/P ratio has been reported to be almost one during the disposition phase with a good correlation between plasma and saliva concentrations.1—9 The constant S/P value has only been hypothesized to result from the passive transfer of the drug into the saliva.1 However, no investigations have been carried out before to evaluate the existence of active transport in the salivary glands of rats or humans that could contribute to or maintain the constant S/P ratio. The presence of a group of transporters related to multidrug resistance of tumor cells has recently
899
been localized in major and minor salivary duct cells of healthy volunteers.15,16 From the identified transporters, the most studied and best-characterized is P-glycoprotein or P-gp.17,18,20 The protein pumps substrates out of the cell and, considering its localization in normal human tissues, it has been proposed that the transporter plays an important role in the pharmacokinetics of its substrates by modulating the absorption, distribution and elimination mechanisms including salivary secretion.16,19,22,23 Even if acetaminophen has not been identified as a P-gp substrate, new different and apparently nonpharmacological or pharmacodynamic-related molecules are daily discovered. Thus, it remains without answer whether the constant value of acetaminophen S/P ratio during the disposition phase is only the result of a passive diffusion process. If P-gp were involved in acetaminophen salivary secretion, the salivary levels of the drug would be expected to be modified by inhibition of the pumping function of the transporter. In addition, P-gp could affect acetaminophen disposition. Acetaminophen pharmacokinetics was assessed from saliva and plasma samples obtained from control rats and animals pre-treated with erythromycin as a P-gp inhibitor.17,24 Only saliva secreted from the submandibular glands of the rats was collected. Considering that the rat lacks a basal salivary secretion, external substance P was administered to stimulate the salivary flow at each sampling time over 1 h after acetaminophen administration. External SP mediates salivary stimulation by NK1 receptors located on the secretory acini structures. The composition of saliva that results from SP is similar to that obtained after cholinergic salivary stimulation.25—28 After intravenous acetaminophen administration (30 mg/kg), S/P ratio values for control and erythromycin-treated animals were found to be within the interval [0.7—1.1] in agreement with the findings of others.5—7,12 Erythromycin pre-treatment did not modify the S/P ratio, assessed at different times, as no significant difference could be found when comparing the ratios obtained for treated and control animals (P > 0.05). This means that even if the transporter activity of P-gp is partially inhibited, acetaminophen salivary levels are not affected. Then, P-gp is unlikely to be related to acetaminophen salivary secretion. The good correlation between saliva and plasma levels found for the control group is in agreement with findings previously reported by others.2—8 Thus, saliva samples could replace blood samples in assessing the pharmacokinetics of the drug. Acetaminophen parameters (ABC, Cl, T1/2 and Vz) derived from plasma or saliva levels were not mod-
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ified by inhibiting P-gp transporter function as no statistical differences were observed when comparing the parameters between the two groups of animals (P > 0.05). These results suggest that the P-gp inhibitor erythromycin, did not affect acetaminophen kinetics as assessed from saliva or plasma concentrations. If P-gp were related to acetaminophen salivary secretion or to another phase of acetaminophen pharmacokinetics, at least one of the parameters evaluated should have been modified by P-gp inhibition. The results obtained from plasma levels for clearance, apparent volume of distribution and half-life elimination time were similar to those previously reported by others.30,31 Moreover, for the control group, no difference was observed between this set of parameters and that assessed from acetaminophen saliva data. This result confirms once again the possibility of assessing acetaminophen pharmacokinetic parameters from salivary levels rather than plasma concentrations.2,8,13,14 Considering that no differences could be found in S/P ratios when comparing control and erythromycin-treated animals, and the fact that inhibiting Pglycoprotein transport did not modify acetaminophen kinetic parameters, we propose that salivary secretion of acetaminophen is not related to Pglycoprotein pumping activity in the submandibular glands of rats.
Acknowledgements We thank Ms. G. Tumilasci and Ms. C. Garc½´a Bonelli for their technical support. The present study was supported by grant B-090 from the University of Buenos Aires, Argentina.
References 1. Posti J. Saliva-plasma drug concentration ratios during absorption: Theoretical considerations and pharmacokinetic implications. Pharm Acta Helv 1982;57:83—92. 2. Fagiolino P, Va ´zquez M. Bioequivalence study of acetaminophen in saliva. Eur J Drug Metab Pharmacokinet 1994:164—8 [special issue]. 3. Glynn JP, Bastain W. Salivary excretion of acetaminophen in man. J Pharm Pharmacol 1973;25:420—1. 4. Ahmed M, Enever RP. Formulation and evaluation of sustained release acetaminophen tablets. J Clin Hosp Pharm 1981;6:27—38. 5. Adithan C, Thangam J. A comparative study of saliva and serum acetaminophen levels using simple spectrophotometric method. Br J Clin Pharmacol 1982;14:107—9. 6. Kamali F, Fry JR, Bell GD. Salivary secretion of acetaminophen in man. J Pharm Pharmacol 1987;39:150—2.
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7. Smith M, Whitehead E, O’Sullivan G, Reynolds F. A comparison of serum and saliva acetaminophen concentrations. Br J Clin Pharmacol 1991;31:553—5. 8. Hahn TW, Hennenberg SW, Holm-Knudsen RJ, Eriksen K, Rasmussen SN, Rasmussen M. Pharmacokinetics of rectal acetaminophen after repeated dosing in children. Br J Anaesth 2000;85:512—9. 9. Ritschel W, Kearns GL. Excretion and clearance of drugs. Handbook of basic pharmacokinetics: Including clinical applications. 5th ed. Washington, DC: AAPS; 1999. p. 204— 20. 10. Al-Obaidy SS, Wan Po L, McKiernan PJ, Glasgow JFT, Millership J. Assay of paracetamol and its metabolites in urine, plasma and saliva of children with chronic liver disease. J Pharm Biomed Anal 1995;13:1033—9. 11. Sahajwalla CG, Ayres JW. Multiple-dose acetaminophen pharmacokinetics. J Pharm Sci 1991;80:855—60. 12. Vazquez M, Fagiolino P, Nucci G, Parrillo S, Pin ˜eyro A. Postprandial reabsorption of acetaminophen. Eur J Drug Metab Pharmacokinet 1994;19(3):173—293 [1st International Meeting on the Scientific Basis of Modern Pharmacy, Athens, Greece]. 13. Lee HS, Ti TY, Lye WC, Khoo YM, Tan CC. Acetaminophen and its metabolites in saliva and plasma in chronic dialysis patients. Br J Clin Pharmacol 1996;41:41—7. 14. Schaiquevich PS, Niselman AV, Rubio MC. Importance of entero-salivary recirculation in acetaminophen pharmacokinetics. Biopharm Drug Dispos 2002;23:245—9. 15. Uematsu T, Yamaoka M, Matsuura T, Doto R, Hotomi H, Yamada A, et al. P-glycoprotein expression in human major and minor salivary glands. Arch Oral Biol 2001;46:521—7. 16. Uematsu T, Yamaoka M, Doto R, Tanaka H, Matsuura T, Furusawa K. Expression of ATP-binding cassette transporter in human salivary ducts. Arch Oral Biol 2003;48:87—90. 17. Matheny CJ, Lamb MW, Brower KL, Pollack GM. Pharmacokinetic and pharmacodynamic implications of P-glycoprotein modulation. Pharmacotherapy 2001;21:778—96. 18. Fojo AT, Ueda K, Slamon DJ, Gottesman MM, Pastan I. Expression of multidrug resistance gene in human tumors and tissues. Proc Natl Acad Sci USA 1987;84:265—9. 19. Sikic BI, Fisher GA, Lum BL, Halsey J, Beketic-Reskovic L, Chen G. Modulation and prevention of multidrug resistance by inhibitors of P-glycoprotein. Cancer Chemother Phamacol 1997;40(Suppl):S13—9. 20. Ambudkar SV, Dey S, Hrycyna CA, Ramachandra M, Pastan I, Gottesman MM. Biochemical, cellular and pharmacological aspects of the multidrug transporter. Annu Rev Pharmacol Toxicol 1999;39:361—98. 21. Thiebaut F, Tsuruo T, Hamada H, Gottesman MM, Pastan I, Willingham MC. Cellular localization of the multidrugresistance gene product P-glycoprotein in normal human tissues. Proc Natl Acad Sci USA 1987;84:7735—8. 22. Lown KS, Mayo RR, Leichtman AB, Hsiao H-L, Turgeon DK, Schmiedlin-Ren P, et al. Role of intestinal P-glycoprotein (mdr1) in interpatient variation in the oral bioavailability of cyclosporine. Clin Pharmacol Ther 1997;62:248—60. 23. Schinkel AH, Wagenaar E, Van Deemter L, Mol CA, Borst P. Absence of the mdr1a P-glycoprotein in mice affects tissue distribution and pharmacokinetics of dexamethasone, digoxin, and cyclosporin. J Clin Invest 1995;96:1698—705. 24. Schwarz UI, Gramatte ´ T, Krappweis J, Oertel R, Kirch W. Pglycoprotein inhibitor erythromycin increases oral bioavailability of talinol in humans. Int J Clin Pharmacol Ther 2000;38:161—7. 25. Holzer P, Holzer-Petsche U. Tachykinins in the gut. Part II. Roles in neural excitation, secretion and inflammation. Pharmacol Ther 1997;73:219—63.
Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands
26. Martinez JR, Martinez AM. Stimulatory and inhibitory effects of substance P on rat submandibular secretion. J Dent Res 1981;60:1031—8. 27. Mansson B, Ekstro ¨m J. On the non-adrenergic, non-cholinergic contribution to the parasympathetic nerve-evoked secretion of parotid saliva in the rat. Acta Physiol Scand 1991;141:197—205. 28. Bobyock E, Barbieri EJ, Chernick WS. Effects of substance P and substance P antagonists on rat salivary secretion. J Dent Res 1986;65:1427—31.
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29. Goicoechea AG, Vila-Jato JL. Acetaminophen presystemic biotransformation versus bioavailability in therapeutical. Eur J Drug Metab Pharmacokinet 1998;23:333—8. 30. Hjelle JJ, Klaassen CD. Glucuronidation and biliary excretion of acetaminophen in rats. J Pharmacol Exp Ther 1984;228:407—13. 31. Galinsky RE, Levy G. Dose- and time-dependent elimination of acetaminophen in rats: Pharmacokinetic implications of cosubstrate depletion. J Pharmacol Exp Ther 1981;219: 14—20.
Evaluation of acetaminophen P-glycoproteinmediated salivary secretion by rat submandibular glands Paula Schaiquevicha,*, Niselman Vivianab, Tumilasci Omarc, Rubio Modestoa a
´gicas, ININFA-CONICET, Facultad de Farmacia y Bioqu½´mica, Jun½´n Instituto de Investigaciones Farmacolo 956, 5 piso (1113), Ciudad de Buenos Aires, Argentina b ´ticas, Facultad de Farmacia y Bioqu½´mica, Universidad de Buenos Aires, Departamento de Matema Argentina c ´ndulas salivares, Facultad de Medicina, Universidad de Departamento de Fisiolog½´a, Laboratorio de gla Buenos Aires, Argentina Accepted 3 May 2004
KEYWORDS Acetaminophen; P-glycoprotein; Submandibular gland; S/P ratio
Summary The constant ratio between saliva and plasma acetaminophen concentrations (S/P) during the elimination phase is assumed to result from the equilibrium established among the free-drug concentrations in the arterial blood, venous blood and saliva. Salivary secretion of acetaminophen is assumed to result from a passive diffusion of the drug to saliva from the blood that supplies the salivary glands. However, the constant S/P ratio during acetaminophen disposition and the finding that P-glycoprotein (P-gp), a protein recognized to pump substrates out of the cell, is expressed in duct cells of the submandibular glands questions the mechanisms involved in acetaminophen salivary secretion. Thus, we intended to evaluate the existence of a P-glycoprotein-mediated transport of acetaminophen in rat submandibular glands. Acetaminophen (30 mg/kg, i.v.) pharmacokinetics was assessed in controls and in rats pre-treated with erythromycin (100 mg/kg) as a P-glycoprotein inhibitor. Acetaminophen pharmacokinetic parameters were calculated from saliva and plasma levels considering a non-compartmental analysis. Mean plasma and salivary profiles of control and pre-treated animals were almost superimposable. No difference could be found in S/P ratios in control and erythromycin pre-treated animals (P > 0.05). Moreover, no statistical difference could be found in the kinetic parameters calculated from saliva or plasma drug level (P > 0.05). These observations indicate that acetaminophen salivary secretion in rat submandibular glands is not related to P-glycoprotein-mediated transport under the experimental conditions of the present work. ß 2004 Elsevier Ltd. All rights reserved.
Introduction *
Corresponding author. Tel.: þ54 11 4961 6784; fax: þ54 11 4953 7689. E-mail address: [email protected] (P. Schaiquevich).
After oral administration of acetaminophen, the saliva to plasma concentration ratio (S/P) during the absorption phase is greater than during the
0003–9969/$ — see front matter ß 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.archoralbio.2004.05.003
896
disposition of the drug. The accepted explanation is based on an anatomical—physiological model which assumes that while the drug is being absorbed, the free-drug concentrations in the arterial blood and saliva are in equilibrium and show higher values than those in the venous blood.1,2 When absorption is finished and considering that acetaminophen rapidly distributes in the body, the free-drug concentration is equal in the arterial and venous blood, leading to a constant S/P ratio. In particular, for acetaminophen the constant S/P ratio is equal to unity as previously reported.1—9 This observation and the significant correlation found for the area under the concentration-time profile and half-life elimination time between values derived from venous plasma and saliva levels, has led to the use of saliva as a biological fluid for determining acetaminophen pharmacokinetics in bioavailability and bioequivalence studies.2,5,10—14 Usually, when considering a lipid-soluble acid such as acetaminophen, the S/P ratio could be predicted on the basis of the degree of ionization of the drug in both biological fluids.2,6,9 While the passage of most drugs into saliva occurs by passive diffusion, it has been suggested that an active transport process may operate for some drugs. Then, the S/P acetaminophen ratio during the elimination phase could present two components related to passive and active transport. Recently, the expression of some members of the ATP-binding cassette transporters in major and minor salivary glands of healthy subjects has been demonstrated.15,16 The authors recognized the presence of MDR1 product gene, P-glycoprotein (P-gp), multidrug resistance-associated proteins types 1 and 2 (MRP1 and MRP2, respectively) and lung resistance-related protein (LRP). Particularly, P-glycoprotein has been localised in different types of salivary duct cells. These proteins are classified as members of the ATP-binding cassette superfamily of transport proteins and are related to human cancer resistance to chemotherapeutic agents by pumping substrates out of the malignant tumor cells.17—19 P-glycoprotein is the best-characterized member of the group. The protein recognizes a broad group of amphipathic, lipophilic and cationic compounds, pumping them out of the cell.17,18 The expression of P-glycoprotein in normal tissues of humans and rodents associated with drug absorption, metabolism and excretion led to the hypothesis that the transporter modulates the pharmacokinetic and pharmacological activity of the substrates.20—24 Moreover, the fact that P-glycoprotein has been localised in human salivary glands tumors as well as in normal tissue suggests that the protein is physiologically involved in the trans-
P. Schaiquevich et al.
port of a broad spectrum of substrates in the salivary glands.15—18 Even if acetaminophen has not yet been included as a P-gp substrate, the list is daily revised and unrelated new molecules are incorporated. In addition, acetaminophen is highly secreted in saliva. Thus, a component of acetaminophen transport in submandibular glands could be mediated by P-gp pumping function contributing to the observed S/P constant ratio during the disposition phase. The aim of the present work was to study whether a P-glycoprotein-mediated transport of acetaminophen exists in the rat submandibular glands, considering its high salivary secretion previously reported and the existence of the transporter in human salivary glands even if uncertain in roedents. We assessed the kinetics of acetaminophen from saliva and plasma levels of control rats and animals pre-treated with erythromycin as a P-gp inhibitor.24
Materials and methods Animals Adult male Wistar rats of age 2—3 months and weighing 250—350 g were housed in temperaturecontrolled rooms (20 2 8C) with a 12 h light—dark cycle. Rats were fasted for 12 h but allowed free access to water prior to the experiment. Two groups of animals were employed, referred to in the text as control and treated groups. Both groups of animals underwent the same treatment but animals assigned to the treated group also received erythromycin (100 mg/kg, i.p.) 1 h before acetaminophen administration. Erythromycin ethyl succinate suspension was prepared in CMC 0.1%. Urethane (1.2 g/kg, i.p.) was used to induce and maintain anaesthesia in the rats. Following anaesthesia, polyethylene tubes previously heparinized were placed into the femoral artery of one leg and the femoral vein of the opposite. Also, glass cannulas were inserted into the ducts of both right and left submandibular glands of each animal. Acetaminophen was administered via the femoral vein as a 30 mg/kg bolus, dissolved in propylene glycol—saline 50% (v/v). Because anaesthetized animals lack a basal salivary secretion, substance P (10 mg/kg, i.v.) was administered via the femoral vein at each time that salivary samples were collected. The animals were kept under urethane anaesthesia until the end of the experiment. Saliva samples from each gland were collected in different pre-weighed polypropylene tubes. Samples were obtained over 2 min intervals after sub-
Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands
stance P administration at: 0 (blank); 9—11; 19—21; 29—31; 44—46 and 59—61 min after acetaminophen administration. Venous blood samples (200 ml) were collected in heparinized tubes at: 0 (blank); 10; 20; 30; 45; 60 min after acetaminophen intravenous administration. Blood samples were centrifuged for 20 min at 1800 g in order to separate plasma from the rest. Saliva and plasma samples were stored at 40 8C until drug analysis. The present work adhered to the Principles of Laboratory Animal Care (NIH Publication #85-23, 1985) and the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Science, National Research Council (1996).
HPLC assay of acetaminophen Plasma and saliva acetaminophen levels were quantified by HPLC UV after validating a modified method reported by others.29 Salivary samples were thawed and vortexed for 2 min before precipitation with a mixture of acetonitrile/H3PO4 (5.0:0.135). The sample volume was calculated according to the difference in the weight of the tube before and after saliva collection, assuming a saliva density of 1.00 g/ml. Afterwards, samples were vortexed, centrifuged at 8900 g for 2 min and injected into the chromatographic system. Plasma samples were thawed and vortexed for 2 min. Then, 100 ml of acetonitrile/ZnSO4 10% (w/v) (10:1) was added to the same volume of sample, vortexed for 2 min, centrifuged at 8900 g for 2 min and injected into the chromatographic system. The linear ranges for plasma and saliva assays were [0.1—50.0 mg/ml] and [2.0—30 mg/ml], respectively. Within- and inter-day coefficients of variation for acetaminophen in plasma and saliva were below 5 and 12%, respectively. The recovery percentage of acetaminophen in plasma and saliva was 58 and 88%, respectively. The detection limit was found to be 0.07 mg/ml for both assays. The chromatographic system consisted of a Spectra System P2000 pump, a Thermo Separation sample injector, an UV-100 Spectra Series ultraviolet detector set at 248 nm. Mobile phase consisted of 0.1 M K2HPO4/methanol/acetic acid (90:7:3) and (83:16:1) for saliva and plasma assays, respectively, delivered at 1 ml/min. The analytical column employed was RP-C18, 250 mm 4.6 mm, 5 mm (Symmetry, Waters) maintained at room temperature. In all cases the injected volume was 20 ml.
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Data analysis Mean saliva and plasma profiles were obtained for each group of animals from the individual concentration-time profiles. Individual acetaminophen saliva levels at each time were calculated as the mean value between concentrations calculated from the right and left glands. Individual saliva to plasma concentration ratio (S/P) at each sampling time was measured for the animals of both groups. S/P ratios at each time were compared between groups by MANOVA test with Duncan’s test a posteriori. Significance level was set at 0.05. Mean S/P ratios were calculated for each group. Non-compartmental analysis was used to describe the individual disposition of acetaminophen from saliva or plasma levels. Data were analysed using the non-linear regression program TOPFIT 2.0. The log trapezoidal method and extrapolation to infinity was used to calculate the area under the total concentration-time profile (AUC1) and the area under the concentration-time curve from 0 to 60 min (AUC1 h). Terminal plasma and saliva half-lives for each rat were estimated by linear regression analysis after log transformation of the last data points. Systemic clearance (Cl) and apparent volume of distribution (Vz) were calculated from AUC1, dose, and elimination halflife. The pharmacokinetic parameters were compared between groups by means of a Student t-test and examined for significance at a level of P < 0.05. Also, comparisons were carried out between the parameters calculated from plasma and saliva levels of the control group using the match paired t-test. Finally, mean pharmacokinetic parameters derived from plasma and saliva levels were calculated for both groups of animals.
Results Mean plasma and salivary acetaminophen concentration-time profiles obtained for control rats and animals pre-treated with erythromycin as a P-gp inhibitor are represented in Figs. 1 and 2, respectively. The salivary as well as the plasma profiles are almost superimposable when considering data from control and treated groups. Twenty-three paired samples for control rats were available for saliva-plasma correlation analysis (Fig. 3). Linear regression analysis resulted in a Pearson’s correlation coefficient (r) of 0.838 (P < 0.0001). The relationship between plasma (Cplasma) and saliva (Csaliva) concentrations could
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30
Saliva acetaminophen (µg / ml)
Plasma acetaminophen concentration ( µg /ml )
40
35
30
25
20
15
10
25
20
15
10
5
0 0
5
10
15
20
25
30
Plasma acetaminophen (µg /ml)
5
0 0
10
20
30
40
50
60
Figure 3 Saliva vs. plasma concentrations. The line shows the regression line: Csaliva ¼ 1.007Cplasma 1.775.
Time (min)
Figure 1 Mean plasmatic profile after acetaminophen administration (30 mg/kg, i.v.) to (&) control and (~) erythromycin (100 mg/kg, i.p.) pre-treated rats. Data are expressed as mean standard mean error (n ¼ 5).
be described by the following equation (mg/ml): Csaliva ¼ 1.007Cplasma 1.775. Mean S/P values for each sampling time are presented in Table 1 for control and treated animals. All mean values calculated for both groups of animals were found to be within the interval 40
Saliva acetaminophen concentration (µg /ml)
35
30
25
[0.7—1.1]. No significant difference was found in the S/P ratios when comparing the two groups (P > 0.05). Acetaminophen mean kinetic parameters calculated from plasma and saliva levels for control and treated animals are shown in Tables 2 and 3, respectively. We did not find statistical significant differences for Vz, Cl or T1/2 calculated from plasma levels when comparing between groups according to the t-test (P > 0.05). Moreover, no differences could be found when comparing between groups the pharmacokinetic parameters derived from salivary levels. Half-life time was approximately 25 min for both groups of rats. Mean areas under the plasma and salivary profiles for the control group calculated from the measured data (AUC1 h) and extrapolated to infinity (AUC1), were slightly higher than those obtained for the treated group (Tables 2 and 3). However, the apparent differences observed were not of statistical significance (P > 0.05).
20
Table 1 Acetaminophen saliva to plasma concentration ratio (S/P) obtained after an intravenous dose (30 mg/kg) administered to control and pre-treated with erythromycin (100 mg/kg, i.p.) rats.
15
10
5
0 0
10
20
30
40
50
60
Time (min)
Figure 2 Mean acetaminophen salivary profile after intravenous dose (30 mg/kg) to (&) control and (~) erythromycin (100 mg/kg, i.p.) pre-treated rats. Data are expressed as mean standard mean error (n ¼ 5).
Time (min)
Control group S/P S.E.M.
10 20 30 45 60
1.10 0.91 0.90 0.71 0.70
0.22 0.12 0.12 0.12 0.14
Treated group S/P S.E.M. 0.95 1.05 0.77 0.85 1.02
0.32 0.16 0.07 0.20 0.19
Data are expressed as mean standard mean error.
Evaluation of acetaminophen P-glycoprotein-mediated salivary secretion by rat submandibular glands
Table 2 Acetaminophen pharmacokinetic parameters calculated from plasma levels after 30 mg/kg i.v. dose administered to control and erythromycintreated animals. Pharmacokinetic parameter
Control group
AUC1 h (mg min/ml) AUC1 (mg min/ml) T1/2 (min) Cl (ml/min/kg) Vz (l/kg)
895.0 1256.2 26.5 24.9 0.94
71.9 148.2 2.8 3.3 0.11
Treated group 774.3 921.9 24.8 31.3 1.08
48.3 50.6 2.3 3.1 0.15
Results are expressed as mean standard mean error.
Table 3 Acetaminophen pharmacokinetic parameters calculated from salivary levels after intravenous administration (30 mg/kg) to control and pretreated animals with erythromycin. Pharmacokinetic parameter
Control group
AUC1 h (mg min/ml) ABC1 (mg min/ml) T1/2 (min) Cl (ml/min kg) Vz (l/kg)
958.8 1089.3 20.6 28.4 0.83
Treated group
94.6 839.0 23.5 101.3 984.3 44.0 2.0 21.5 2.3 2.4 30.7 1.3 0.08 0.96 0.09
Data are expressed as mean standard mean error.
Lastly, no significant differences were found in AUC1h, AUC1, Cl, T1/2 or Vz when comparing the saliva and plasma values obtained from data of the control group (P > 0.05).
Discussion Acetaminophen saliva to plasma concentration ratio (S/P) tends to be higher during the absorption phase. This observation is due to the fact that saliva drug levels reflect free-drug arterial blood concentrations, values that are higher than the corresponding venous blood concentration, until the distribution phase is completely established.1,2 On the contrary, the acetaminophen S/P ratio has been reported to be almost one during the disposition phase with a good correlation between plasma and saliva concentrations.1—9 The constant S/P value has only been hypothesized to result from the passive transfer of the drug into the saliva.1 However, no investigations have been carried out before to evaluate the existence of active transport in the salivary glands of rats or humans that could contribute to or maintain the constant S/P ratio. The presence of a group of transporters related to multidrug resistance of tumor cells has recently
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been localized in major and minor salivary duct cells of healthy volunteers.15,16 From the identified transporters, the most studied and best-characterized is P-glycoprotein or P-gp.17,18,20 The protein pumps substrates out of the cell and, considering its localization in normal human tissues, it has been proposed that the transporter plays an important role in the pharmacokinetics of its substrates by modulating the absorption, distribution and elimination mechanisms including salivary secretion.16,19,22,23 Even if acetaminophen has not been identified as a P-gp substrate, new different and apparently nonpharmacological or pharmacodynamic-related molecules are daily discovered. Thus, it remains without answer whether the constant value of acetaminophen S/P ratio during the disposition phase is only the result of a passive diffusion process. If P-gp were involved in acetaminophen salivary secretion, the salivary levels of the drug would be expected to be modified by inhibition of the pumping function of the transporter. In addition, P-gp could affect acetaminophen disposition. Acetaminophen pharmacokinetics was assessed from saliva and plasma samples obtained from control rats and animals pre-treated with erythromycin as a P-gp inhibitor.17,24 Only saliva secreted from the submandibular glands of the rats was collected. Considering that the rat lacks a basal salivary secretion, external substance P was administered to stimulate the salivary flow at each sampling time over 1 h after acetaminophen administration. External SP mediates salivary stimulation by NK1 receptors located on the secretory acini structures. The composition of saliva that results from SP is similar to that obtained after cholinergic salivary stimulation.25—28 After intravenous acetaminophen administration (30 mg/kg), S/P ratio values for control and erythromycin-treated animals were found to be within the interval [0.7—1.1] in agreement with the findings of others.5—7,12 Erythromycin pre-treatment did not modify the S/P ratio, assessed at different times, as no significant difference could be found when comparing the ratios obtained for treated and control animals (P > 0.05). This means that even if the transporter activity of P-gp is partially inhibited, acetaminophen salivary levels are not affected. Then, P-gp is unlikely to be related to acetaminophen salivary secretion. The good correlation between saliva and plasma levels found for the control group is in agreement with findings previously reported by others.2—8 Thus, saliva samples could replace blood samples in assessing the pharmacokinetics of the drug. Acetaminophen parameters (ABC, Cl, T1/2 and Vz) derived from plasma or saliva levels were not mod-
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ified by inhibiting P-gp transporter function as no statistical differences were observed when comparing the parameters between the two groups of animals (P > 0.05). These results suggest that the P-gp inhibitor erythromycin, did not affect acetaminophen kinetics as assessed from saliva or plasma concentrations. If P-gp were related to acetaminophen salivary secretion or to another phase of acetaminophen pharmacokinetics, at least one of the parameters evaluated should have been modified by P-gp inhibition. The results obtained from plasma levels for clearance, apparent volume of distribution and half-life elimination time were similar to those previously reported by others.30,31 Moreover, for the control group, no difference was observed between this set of parameters and that assessed from acetaminophen saliva data. This result confirms once again the possibility of assessing acetaminophen pharmacokinetic parameters from salivary levels rather than plasma concentrations.2,8,13,14 Considering that no differences could be found in S/P ratios when comparing control and erythromycin-treated animals, and the fact that inhibiting Pglycoprotein transport did not modify acetaminophen kinetic parameters, we propose that salivary secretion of acetaminophen is not related to Pglycoprotein pumping activity in the submandibular glands of rats.
Acknowledgements We thank Ms. G. Tumilasci and Ms. C. Garc½´a Bonelli for their technical support. The present study was supported by grant B-090 from the University of Buenos Aires, Argentina.
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