- Email: [email protected]
Drug–drug interaction between oxycodone and adjuvant analgesics in blood–brain barrier transport and antinociceptive effect
Drug–Drug Interaction Between Oxycodone and Adjuvant Analgesics in Blood–Brain Barrier Transport and Antinociceptive Effect YUSUKE NAKAZAWA,1 TAKASHI OKURA,1 KEITA SHIMOMURA,1 TETSUYA TERASAKI,2 YOSHIHARU DEGUCHI1 1
Department of Drug Disposition & Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan 2
Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan
Received 5 February 2009; revised 24 March 2009; accepted 8 April 2009 Published online 4 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21807
ABSTRACT: To examine possible blood–brain barrier (BBB) transport interactions between oxycodone and adjuvant analgesics, we firstly screened various candidates in vitro using [3H]pyrilamine, a substrate of the oxycodone transporter, as a probe drug. The uptake of [3H]pyrilamine by conditionally immortalized rat brain capillary endothelial cells (TR-BBB13) was inhibited by antidepressants (amitriptyline, imipramine, clomipramine, amoxapine, and fluvoxamine), antiarrhythmics (mexiletine, lidocaine, and flecainide), and ketamine. On the other hand, antiepileptics (carbamazepine, phenytoin, and clonazepam) and corticosteroids (dexamethasone and prednisolone) did not inhibit [3H]pyrilamine uptake, with the exception of sodium valproate. The uptake of oxycodone was significantly inhibited in a concentrationdependent manner by amitriptyline, fluvoxamine and mexiletine with Ki values of 13, 65, and 44 mM, respectively. These Ki values are 5–300 times greater than the human therapeutic plasma concentrations. Finally, we evaluated in vivo interaction between oxycodone and amitriptyline in mice. Antinociceptive effects of oxycodone were increased by coadministration of amitriptyline. The oxycodone concentrations in plasma and brain were not changed by coadministration of amitriptyline. Overall, the results suggest that several adjuvant analgesics may interact with the BBB transport of oxycodone at relatively high concentrations. However, it is unlikely that there would be any significant interaction at therapeutically or pharmacologically relevant concentrations. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:467–474, 2010
Keywords: adjuvant analgesic; blood–brain barrier; CNS; distribution; drug interactions; oxycodone; pyrilamine; transporters
INTRODUCTION Oxycodone, an opioid agonist, is widely used for the treatment of moderate-to-severe cancer pain.
Correspondence to: Yoshiharu Deguchi (Telephone: þ81426-85-3766; Fax: þ81-426-85-3766; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 467–474 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
Although it has a lower binding affinity than morphine for m-opioid receptor, it has a potent antinociceptive effect with a similar ED50 value to that of morphine.1 This discrepancy between in vivo and in vitro potency can be explained by different blood–brain barrier (BBB) permeability characteristics. The unbound concentration ratio of brain to plasma is 3.0 for oxycodone2 and 0.27 for morphine,3 respectively, suggesting that oxycodone is actively transported into the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
467
468
NAKAZAWA ET AL.
brain across the BBB. We have recently identified an oxycodone transporter at the BBB, and showed that it mediates active transport of oxycodone, as well as pyrilamine.4 Transport of both oxycodone and pyrilamine is sensitive to cationic drugs. Opioid analgesics are often used with several types of adjuvant analgesics, such as antidepressants, antiarrhythmics, antiepileptics, corticosteroids, and NMDA receptor antagonists.5 Adjuvant analgesics are used to improve opioid pain relief, to treat opioid-resistant pain, including neuropathic pain, and to allow reduction of the opioid dose in order to reduce adverse effects. In vivo pharmacological actions of drugs are generally dependent on both pharmacokinetic and pharmacodynamic factors. In addition to the pharmacodynamic interaction between opioid and adjuvant analgesics, pharmacokinetic interaction can modulate drug action. Indeed, inhibition of P-glycoprotein enhances the clinical effect of morphine,6 and increases both the accumulation in the brain and the antinociceptive effect of morphine in rats.7 Among adjuvant analgesics, antidepressants and antiarrhythmics include many cationic drugs that might interact with oxycodone transport across the BBB. However, BBB transport-related interactions of CNS-acting drugs are poorly understood. The purpose of this study is to clarify potential interactions between oxycodone and adjuvant analgesics at the BBB. Initially, we used [3H]pyrilamine as a probe, since oxycodone and pyrilamine share a common transporter at the BBB.4 We investigated the interactions of 15 adjuvant analgesics with [3H]pyrilamine transport using conditionally immortalized rat brain endothelial cells (TR-BBB13) as an in vitro BBB model.8 Based on the results obtained, the interaction of selected adjuvant analgesics with oxycodone was examined using TR-BBB13 cells. Finally, we further examined the influence of amitriptyline on the pharmacokinetics and antinociceptive effect of oxycodone in mice.
MATERIALS AND METHODS
Animals Adult male ddY mice weighing about 30 g were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan); they were housed, 10 per cage, in a laboratory with free access to food and water and were maintained on a 12-h dark/12-h light cycle in a room with controlled temperature (24 28C) and humidity (55 5%). This study was conducted according to guidelines approved by the Experimental Animal Ethical Committee of Teikyo University.
Transport Studies in TR-BBB13 Cells TR-BBB13 cells were seeded on collagen-coated multiwell dishes at a density of 0.1 105 cells/cm2. At 3 days after seeding, the cells were washed twice with 1 mL of PBS and preincubated with incubation buffer (122 mM NaCl, 3 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4) for 20 min at 378C. After preincubation, the buffer (0.25 mL) containing oxycodone (30 mM) or [3H]pyrilamine (74 kBq/mL, 90 nM) was added to initiate uptake in the presence of a test compound. The cells were incubated at 378C for a designated time, and then washed three times with 1 mL of ice-cold incubation buffer to terminate the uptake. For the determination of [3H]pyrilamine radioactivity, the cells were solubilized with 1 N NaOH for 60 min. The radioactivity was measured using a liquid scintillation counter after the addition of scintillation cocktail Hionic Fluor (PerkinElmer Life and Analytical Sciences, Boston, MA). For the determination of oxycodone, the cells were collected and homogenized by sonication, and the homogenate was stored at 208C until determination of oxycodone as described below. Cellular protein content was determined by BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Uptake was expressed as the cell-to-medium ratio (mL/mg protein) obtained by dividing the uptake amount by the concentration of substrate in the incubation buffer.
Chemicals Oxycodone was kindly provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). [3H]Pyrilamine (23–30 Ci/mmol) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other chemicals and reagents were commercial products of reagent grade. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
Antinociception Study The tail-flick latency test in mice was used to quantify antinociception.9 Briefly, the tail was immersed in a water bath at a temperature of 50 18C. The time until the tail was withdrawn was measured as the tail-flick latency. Before DOI 10.1002/jps
DRUG–DRUG INTERACTION BETWEEN OXYCODONE AND ADJUVANT ANALGESICS
drug administration, baseline testing was performed. Then antinociceptive testing was performed at 30, 60, and 90 min after the s.c. administration of saline, amitriptyline (10, 30 mg/kg) or oxycodone (5 mg/kg). Mice were also received the s.c. coadministration of oxycodone (5 mg/kg) and amitriptyline (10, 30 mg/kg) in a mixed solution. A maximum tail-flick latency of 10 s was used to minimize tissue damage to the tail. The tail-flick latency values were converted to percentage of the maximum possible effect (%MPE) as follows: %MPE ¼ (postdrug latency predrug latency)/(maximum latency predrug latency) 100.
Brain and Plasma Concentrations of Oxycodone Mice received s.c. administration of oxycodone (5 mg/kg) with or without amitriptyline (10, 30 mg/kg). At 30 min after the administration, blood was collected from the descending aorta under anesthesia with diethyl ether. Then, the brain was removed immediately. The plasma was separated by centrifugation. Plasma and brain samples were stored at 208C until determination of oxycodone.
Determination of Oxycodone Oxycodone was determined by HPLC with electrochemical detection (ECD).4,10 Twenty microliters of codeine solution (250 ng/mL), an internal standard, 100 mL of 4 N NaOH, and 800 mL of butyl chloride were added to 200 mL of cell homogenate. The samples were mixed, and centrifuged for 10 min at 3000 rpm at 48C, then the butyl chloride layer was transferred. The remaining aqueous layer was extracted again with 800 mL of butyl chloride. The combined butyl chloride extract was evaporated to dryness. The residue was reconstituted in 200 mL of mobile phase, and a 40 mL aliquot was injected into the HPLC. In the case of plasma samples, a 50 mL aliquot was mixed with 100 mL of 0.1 M perchloric acid. The mixture was centrifuged for 10 min at 10,000 rpm and the supernatant was collected. The brain was homogenized in five volumes of 0.1 M perchloric acid, and the homogenate was centrifuged for 10 min at 3000 rpm at 48C. Naltrexone solution (250 ng/mL) was added as an internal standard to the supernatants of plasma and brain homogenate, and the solution was neutralized and subjected to solid-phase DOI 10.1002/jps
469
extraction using an Oasis HLB cartridge (Waters, Milford, MA). The Oasis HLB cartridge was prewetted with methanol (1 mL), followed by water (1 mL). A sample was applied to the cartridge, which was washed with 1 mL of 40% methanol, and oxycodone and naltrexone were eluted with methanol. The eluate was dried under a nitrogen stream and the residue was reconstituted in the HPLC mobile phase. The HPLC system consisted of a pump (301E, Eicom, Kyoto, Japan) and an electrochemical detector (ECD-300, Eicom). The HPLC analytical column used was an XTerra1 RP18 (4.6 mm 50 mm, 5 mm particle size, Waters). The HPLC separation of oxycodone, codeine, and naltrexone was carried out at a flow rate of 0.5 mL/min with a mobile phase containing 10% acetonitrile, 20% methanol, and 5 mM phosphate buffer (pH 8.0) at 408C. The retention times of oxycodone, codeine, and naltrexone were 9.1, 6.4, and 23.3 min, respectively. The voltage of the working electrode of the electrochemical detector was set at 800 mV. Standard curves of oxycodone injected onto the column showed good linearity in the range of 0.15–50 pmol (coefficient of determination >0.99). The detection limit for quantification of oxycodone was 3 nM. Runs were accepted if the precision and accuracy of the quality control samples at low (3 nM), moderate (10 nM), and high (100 nM) concentrations each had a coefficient of variation (CV) below 15%.
Data Analysis The 50%-inhibitory concentration (IC50) of test compounds for [3H]pyrilamine and oxycodone uptake by TR-BBB13 cells was calculated by fitting the data to a one-site inhibition model using Prism software (Graphpad, San Diego, CA). The inhibition constant (Ki) values were calculated by using the following equation: Ki ¼
IC50 ð1 þ ½SÞ=Km
where [S] is the concentration of substrate ([3H]pyrilamine or oxycodone) and Km is the Michaelis–Menten constant for [3H]pyrilamine (28 mM) or oxycodone (89 mM).4 Statistical analysis of the data was performed by employing Student’s t-test and by one-way analysis of variance followed by Dunnett’s test for single and multiple comparisons, as appropriate. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
470
NAKAZAWA ET AL.
Differences were considered statistically significant at p < 0.05.
RESULTS Screening for BBB Transport Interactions Using [3H]Pyrilamine Interactions with [3H]pyrilamine transport were investigated for 15 adjuvant analgesics, 2 opioid analgesics, and 2 nonopioid analgesics (Fig. 1). The uptake of [3H]pyrilamine into TR-BBB13 cells was inhibited by antidepressants (amitriptyline, imipramine, clomipramine, amoxapine, and fluvoxamine), antiarrhythmics (mexiletine, lidocaine, and flecainide), and an NMDA receptor antagonist (ketamine) at the concentration of 1 mM, except in the case of amoxapine (0.1 mM). The extents of inhibition were 87–98% by antidepressants, 79–98% by antiarrhythmics, and 89% by ketamine. On the other hand, antiepileptics (carbamazepine, phenytoin, and clonazepam) and corticosteroids (dexamethasone and prednisolone) were not inhibitory, except for sodium valproate. Oxycodone, morphine, and pentazocine significantly inhibited [3H]pyrilamine uptake by 86%, 61%, and 97%, respectively. Buprenorphine did not alter [3H]pyrilamine uptake.
Figure 1. Effect of various adjuvant analgesics on [3H]pyrilamine transport into TR-BBB13 cells. Uptake of [3H]pyrilamine (90 nM) was measured for 10 s at 378C in transport medium (pH 7.4) containing each drug at the concentration of 1 mM, except for amoxapine and buprenorphine (0.1 mM). Each column represents the mean SE of four experiments. Asterisks indicate a significant difference from the control value (
p < 0.01,
p < 0.001). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
Effects of Selected Compounds on the BBB Transport of Oxycodone Six adjuvant analgesics (amitriptyline, imipramine, fluvoxamine, carbamazepine, mexiletine, and ketamine) were selected for investigation of their interaction with oxycodone transport, because these drugs are frequently used as adjuvant analgesics in cancer patients in Japan.11 Oxycodone uptake into TR-BBB13 cells was inhibited by amitriptyline, imipramine, fluvoxamine, mexiletine, and ketamine to the extents of 77%, 46%, 64%, 76%, and 77%, respectively (Fig. 2). Carbamazepine did not inhibit oxycodone uptake, in line with its lack of an inhibitory effect on [3H]pyrilamine uptake. Oxycodone uptake was moderately inhibited by morphine and pentazocine (51% and 65%, respectively), but not by buprenorphine.
Determination of Inhibition Constant (Ki) The concentration dependency of inhibition of [3H]pyrilamine and oxycodone transport by amitriptyline, fluvoxamine, mexiletine, and morphine was examined, because these adjuvant analgesics significantly inhibited the transport of [3H]pyrilamine by 94–98% and that of oxycodone by 76–77%. These analgesics inhibited
Figure 2. Effect of various adjuvant analgesics on oxycodone transport into TR-BBB13 cells. Uptake of oxycodone (30 mM) was measured for 15 s at 378C in transport medium (pH 7.4) containing each drug at the concentration of 1 mM, except for buprenorphine (0.1 mM). Each column represents the mean SE of three experiments. Asterisks indicate a significant difference from the control value (
p < 0.01). DOI 10.1002/jps
DRUG–DRUG INTERACTION BETWEEN OXYCODONE AND ADJUVANT ANALGESICS
[3H]pyrilamine uptake in a concentrationdependent manner with Ki values of 8.51 mM for amitriptyline, 7.37 mM for fluvoxamine, 3.46 mM for mexiletine, and 284 mM for morphine (Fig. 3A). The uptake of oxycodone was also inhibited in a concentration-dependent manner with Ki values of 13 mM for amitriptyline, 65 mM for fluvoxamine, 44 mM for mexiletine, and 470 mM for morphine (Fig. 3B).
In Vivo Effects on Antinociceptive Action and Pharmacokinetics of Oxycodone The in vivo antinociceptive effect of the interaction of oxycodone with amitriptyline was examined, because amitriptyline inhibited oxycodone transport with the lowest Ki value among the tested drugs. Antinociceptive effect was determined by the use of the tail-immersion test after oxycodone administration with or without amitriptyline in mice. The value of %MPE was significantly increased at 30 min after the subcutaneous administration of oxycodone (5 mg/kg) or amitriptyline (30 mg/kg) in mice (Fig. 4). The antinociceptive effect after oxycodone administration (5 mg/kg s.c.) was increased and extended by the coadministration of amitriptyline (10, 30 mg/kg s.c.). The area under the time curve of antinociceptive effect (AUE) was significantly increased by 4.1-, 7.1-, and 10.3-fold after the administration of oxycodone alone, and oxycodone with amitriptyline at doses of 10 and 30 mg/kg, respectively, compared with saline administra-
471
tion (Fig. 5). The AUE after coadministration of oxycodone and amitriptyline at 30 mg/kg was significantly greater than that after administration of oxycodone alone. The oxycodone concentrations in the plasma and brain at 30 min after oxycodone administration (30 mg/kg) were not affected by the coadministration of amitriptyline (10 or 30 mg/kg) (Fig. 6). The brain to plasma concentration ratios were 3.5 0.2, 4.5 0.5, and 4.4 0.4 (mean SE, n ¼ 4) in the rats administered oxycodone alone and oxycodone with amitriptyline at the doses of 10 and 30 mg/kg, respectively, and there was no significant difference among these values.
DISCUSSION Drug–drug interactions are of potential clinical importance, since they may result in adverse effects. Though many drug–drug or drug–food interactions have been suggested on the basis of in vitro studies, it is important to consider whether the interactions might also occur in the in vivo situation. In the present study, potential interactions between oxycodone and adjuvant analgesics were examined using an in vitro BBB cell model, together with in vivo analyses of antinociception and brain distribution. The results of the in vitro experiments indicated that several adjuvant analgesics, including amitriptyline, interact with oxycodone during the BBB transport process at relatively high
Figure 3. Concentration-dependent inhibition of uptake of [3H]pyrilamine (A) and oxycodone (B) into TR-BBB13 cells by amitriptyline, fluvoxamine, mexiletine, and morphine. Uptakes of [3H]pyrilamine (90 mM, 10 s) and oxycodone (30 mM, 15 s) were measured in the presence of various concentrations of amitriptyline (0.1 mM to 1 mM), fluvoxamine (0.1 mM to 1 mM), mexiletine (0.1 mM to 1 mM), and morphine (0.1 mM to 10 mM). Each point represents the mean SE of three to four experiments. DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
472
NAKAZAWA ET AL.
Figure 4. Time course of antinociceptive effects in mice. Mice received subcutaneous administration of saline (*), oxycodone alone (5 mg/kg) (*), amitriptyline alone (10 mg/kg: ~, 30 mg/kg: !), or oxycodone (5 mg/kg) with amitriptyline (10 mg/kg: ~, 30 mg/kg: 5). Each point represents the mean SE of four to five mice. Asterisks indicate a significant difference from the saline value (
p < 0.01).
concentrations. In vivo analysis clearly demonstrated that coadministration of oxycodone with amitriptyline at pharmacologically relevant doses enhanced the antinociceptive effect without causing inhibition of BBB transport of oxycodone. For the initial screening of adjuvant analgesics that interact with BBB transport of oxycodone,
Figure 5. Area under the time curve of antinociceptive effect (AUE) in mice. Mice received subcutaneous administration of saline, oxycodone alone (5 mg/kg), or oxycodone (5 mg/kg) with amitriptyline (10, 30 mg/kg). Each column represents the mean SE of four to five mice. Asterisks indicate a significant difference (
p < 0.01 vs. saline, ##p < 0.01 vs. oxycodone). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
[3H]pyrilamine was used as a probe ligand, because oxycodone and pyrilamine share the same transporter at the BBB.4 The [3H]pyrilamine uptake into TR-BBB13 cells was inhibited by antidepressants (amitriptyline, imipramine, clomipramine, amoxapine, and fluvoxamine), antiarrhythmics (mexiletine, lidocaine, and flecainide), and an NMDA receptor antagonist (ketamine), suggesting that these agents interact with the BBB transport of pyrilamine. The adjuvant analgesics that showed significant inhibition of [3H]pyrilamine transport are cationic drugs with an amine moiety in their structure, except for sodium valproate, an organic anion. Thus, a basic amine structure may play an important role in the interaction with [3H]pyrilamine transport. Sodium valproate significantly inhibited [3H]pyrilamine transport, in contrast with other antiepileptics. Ohashi et al.12 have reported that valproate inhibits OCTN2-mediated organic cation transport, and OCTN2 has an anion-binding site in addition to a cation-binding site. Thus, the inhibitions of [3H]pyrilamine transport by organic cations and by sodium valproate may involve different mechanisms. Among adjuvant analgesics, amitriptyline, imipramine, fluvoxamine, mexiletine, and ketamine are frequently used in patients with cancer pain.11 In the screening using [3H]pyrilamine as a probe ligand, these adjuvant analgesics inhibited [3H]pyrilamine transport by over 89% in TRBBB13 cells. Thus, we examined the BBB transport interactions between oxycodone and these drugs. These five adjuvant analgesics all inhibited oxycodone transport in TR-BBB13 cells. Oxycodone transport was also inhibited by morphine and pentazocine, but not by carbamazepine or buprenorphine. Further, amitriptyline, fluvoxamine, mexiletine, and morphine showed concentration-dependent inhibition of both oxycodone and [3H]pyrilamine transport. We have recently reported that the BBB transport of oxycodone is mediated by a common transporter with pyrilamine.4 Thus, [3H]pyrilamine may be a useful probe for screening of interactions involving oxycodone transporter-mediated BBB transport. Oxycodone transport was inhibited in a concentration-dependent manner with the Ki values of 13 mM for amitriptyline, 65 mM for fluvoxamine, and 44 mM for mexiletine. Ulrich and La¨ uter13 reported that the therapeutic window of serum concentration of amitriptyline was 80–200 mg/L DOI 10.1002/jps
DRUG–DRUG INTERACTION BETWEEN OXYCODONE AND ADJUVANT ANALGESICS
473
Figure 6. Concentrations of oxycodone in the plasma (A) and brain (B) of mice. The concentration was determined at 30 min after subcutaneous administration of oxycodone alone (5 mg/kg) or oxycodone (5 mg/kg) with amitriptyline (10, 30 mg/kg) in mice. Each column represents the mean SE of four mice.
(0.25–0.64 mM). The therapeutic plasma concentrations of fluvoxamine and mexiletine are 0.203–1.26 and 0.2–0.65 mM, respectively. Thus, the Ki values obtained in this study were 5–300 times greater than the human therapeutic plasma concentration ranges for amitriptyline, fluvoxamine, and mexiletine. In addition, the therapeutic concentration ranges of these drugs would be even lower if plasma protein binding is taken into consideration (i.e., the unbound plasma concentrations would be lower). Thus, it seems likely that any interaction between oxycodone and these drugs in the BBB transport process would be clinically irrelevant. To examine the in vivo interaction between oxycodone and amitriptyline, which had the highest activity among the tested drugs for inhibition of oxycodone transport, the antinociceptive effect and brain distribution of oxycodone were measured in mice. The antinociceptive effects, measured with the mouse tail-immersion test, were caused by not only the administration of oxycodone, but also the administration of amitriptyline. It has been suggested that the effect of amitriptyline is mediated by the release of endogenous opioid peptide and activation of the d-opioid receptor in the brain.14 Coadministration of oxycodone and amitryptyline brought about additive antinociceptive effects. Oxycodone concentrations in the plasma and brain were not affected by coadministration of amitriptyline. Indeed, the unbound plasma concentration of amitriptyline at 30 min after s.c. administration of DOI 10.1002/jps
amitriptyline (10 mg/kg) in mice is estimated to be 0.12 mM using free fraction of 0.05, and the concentration is 10 times lower than the Ki value obtained in this study.15 The plasma concentration of nortriptyline, an active metabolite, was 80 times lower than that of amitriptyline.15 It was shown that amitriptyline and its metabolites are substrates of P-glycoprotein,15 which can affect the BBB transport of morphine,7 whereas the pharmacokinetics of oxycodone were not affected by a P-glycoprotein inhibitor.16 Therefore, amitriptyline appears to have little or no effect on BBB transport of oxycodone from the circulating blood into the brain at pharmacologically relevant doses. This finding is consistent with the in vitro finding that amitriptyline inhibits BBB transport of oxycodone at relatively high concentration. These results indicated that the coadministration of amitriptyline enhances the antinociceptive effects of oxycodone with little effect on oxycodone pharmacokinetics, including brain distribution. Thus, this study has clearly demonstrated that interaction between oxycodone and amitriptyline at the oxycodone transporter does occur, but is unlikely to be clinically relevant.
CONCLUSION In conclusion, we have shown that several adjuvant analgesics interact with the BBB transport of oxycodone at relatively high concentrations. However, there is unlikely to be a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
474
NAKAZAWA ET AL.
significant interaction at therapeutically or pharmacologically relevant plasma concentration ranges.
ACKNOWLEDGMENTS This work was supported in part by a Grant-inAid for Scientific Research and a Grant-in-Aid for Young Scientists provided by the Ministry of Education, Culture, Sports, Science and Technology.
REFERENCES 1. Peckham EM, Traynor JR. 2006. Comparison of the antinociceptive response to morphine and morphine-like compounds in male and female Sprague-Dawley rats. J Pharmacol Exp Ther 316: 1195–1201. 2. Bostro¨ m E, Simonsson US, Hammarlund-Udenaes M. 2006. In vivo blood-brain barrier transport of oxycodone in the rat: Indications for active influx and implications for pharmacokinetics/pharmacodynamics. Drug Metab Dispos 34:1624–1631. 3. Tunblad K, Jonsson EN, Hammarlund-Udenaes M. 2003. Morphine blood-brain barrier transport is influenced by probenecid co-administration. Pharm Res 20:618–623. 4. Okura T, Hattori A, Takano Y, Sato T, Hammarlund-Udenaes M, Terasaki T, Deguchi Y. 2008. Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood-brain barrier transport of oxycodone. Drug Metab Dispos 36: 2005–2013. 5. Lussier D, Huskey AG, Portenoy RK. 2004. Adjuvant analgesics in cancer pain management. Oncologist 9:571–591. 6. Kharasch ED, Hoffer C, Whittington D, Sheffels P. 2003. Role of P-glycoprotein in the intestinal absorption and clinical effects of morphine. Clin Pharmacol Ther 74:543–554. 7. Letrent SP, Pollack GM, Brouwer KR, Brouwer KL. 1999. Effects of a potent and specific P-glycoprotein inhibitor on the blood-brain barrier distribution and antinociceptive effect of morphine in the rat. Drug Metab Dispos 270:827–834.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
8. Hosoya K, Takashima T, Tetsuka K, Nagura T, Ohtsuki S, Takanaga H, Ueda M, Yanai N, Obinata M, Terasaki T. 2000. mRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting. J Drug Target 8:357–370. 9. Marinho BG, Miranda LS, Gomes NM, Matheus ME, Leita˜ o SG, Vasconcellos ML, Fernandes PD. 2006. Antinociceptive action of (þ/)-cis-(6-ethyltetrahydropyran-2-yl)formic acid in mice. Eur J Pharmacol 550:47–53. 10. Kokubun H, Ouki M, Matoba M, Kubo H, Hoka S, Yago K. 2005. Determination of oxycodone and hydrocotarnine in cancer patient serum by high-performance liquid chromatography with electrochemical detection. Anal Sci 21:337– 339. 11. Inagaki S, Kato K, Maruyama M, Niimi M, Saito H, Nakano K, Noda Y, Nabeshima T. 2006. Pain assessment for cancer patients based on their pain descriptions (Part 2)—Preliminary study for selecting adequate analgesics and adjuvant analgesics using APQ. Jpn J Pharm Health Care Sci 32:788–804. 12. Ohashi R, Tamai I, Inano A, Katsura M, Sai Y, Nezu J, Tsuji A. 2002. Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein. J Pharmacol Exp Ther 302:1286– 1294. 13. Ulrich S, La¨ uter J. 2002. Comprehensive survey of the relationship between serum concentration and therapeutic effect of amitriptyline in depression. Clin Pharmacokinet 41:853–876. 14. Gray AM, Spencer PS, Sewell RD. 1998. The involvement of the opioidergic system in the antinociceptive mechanism of action of antidepressant compounds. Br J Pharmacol 124:669–674. 15. Uhr M, Grauer MT, Yassouridis A, Ebinger M. 2007. Blood-brain barrier penetration and pharmacokinetics of amitriptyline and its metabolites in pglycoprotein (abcb1ab) knock-out mice and controls. Psychiatr Res 41:179–188. 16. Bostro¨ m E, Simonsson US, Hammarlund-Udenaes M. 2005 Oxycodone pharmacokinetics and pharmacodynamics in the rat in the presence of the P-glycoprotein inhibitor PSC833. J Pharm Sci 94: 1060–1066.
DOI 10.1002/jps
Department of Drug Disposition & Pharmacokinetics, School of Pharmaceutical Sciences, Teikyo University, 1091-1 Suarashi, Sagamiko, Sagamihara, Kanagawa 229-0195, Japan 2
Department of Molecular Biopharmacy and Genetics, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan
Received 5 February 2009; revised 24 March 2009; accepted 8 April 2009 Published online 4 June 2009 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jps.21807
ABSTRACT: To examine possible blood–brain barrier (BBB) transport interactions between oxycodone and adjuvant analgesics, we firstly screened various candidates in vitro using [3H]pyrilamine, a substrate of the oxycodone transporter, as a probe drug. The uptake of [3H]pyrilamine by conditionally immortalized rat brain capillary endothelial cells (TR-BBB13) was inhibited by antidepressants (amitriptyline, imipramine, clomipramine, amoxapine, and fluvoxamine), antiarrhythmics (mexiletine, lidocaine, and flecainide), and ketamine. On the other hand, antiepileptics (carbamazepine, phenytoin, and clonazepam) and corticosteroids (dexamethasone and prednisolone) did not inhibit [3H]pyrilamine uptake, with the exception of sodium valproate. The uptake of oxycodone was significantly inhibited in a concentrationdependent manner by amitriptyline, fluvoxamine and mexiletine with Ki values of 13, 65, and 44 mM, respectively. These Ki values are 5–300 times greater than the human therapeutic plasma concentrations. Finally, we evaluated in vivo interaction between oxycodone and amitriptyline in mice. Antinociceptive effects of oxycodone were increased by coadministration of amitriptyline. The oxycodone concentrations in plasma and brain were not changed by coadministration of amitriptyline. Overall, the results suggest that several adjuvant analgesics may interact with the BBB transport of oxycodone at relatively high concentrations. However, it is unlikely that there would be any significant interaction at therapeutically or pharmacologically relevant concentrations. ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:467–474, 2010
Keywords: adjuvant analgesic; blood–brain barrier; CNS; distribution; drug interactions; oxycodone; pyrilamine; transporters
INTRODUCTION Oxycodone, an opioid agonist, is widely used for the treatment of moderate-to-severe cancer pain.
Correspondence to: Yoshiharu Deguchi (Telephone: þ81426-85-3766; Fax: þ81-426-85-3766; E-mail: [email protected]) Journal of Pharmaceutical Sciences, Vol. 99, 467–474 (2010) ß 2009 Wiley-Liss, Inc. and the American Pharmacists Association
Although it has a lower binding affinity than morphine for m-opioid receptor, it has a potent antinociceptive effect with a similar ED50 value to that of morphine.1 This discrepancy between in vivo and in vitro potency can be explained by different blood–brain barrier (BBB) permeability characteristics. The unbound concentration ratio of brain to plasma is 3.0 for oxycodone2 and 0.27 for morphine,3 respectively, suggesting that oxycodone is actively transported into the
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
467
468
NAKAZAWA ET AL.
brain across the BBB. We have recently identified an oxycodone transporter at the BBB, and showed that it mediates active transport of oxycodone, as well as pyrilamine.4 Transport of both oxycodone and pyrilamine is sensitive to cationic drugs. Opioid analgesics are often used with several types of adjuvant analgesics, such as antidepressants, antiarrhythmics, antiepileptics, corticosteroids, and NMDA receptor antagonists.5 Adjuvant analgesics are used to improve opioid pain relief, to treat opioid-resistant pain, including neuropathic pain, and to allow reduction of the opioid dose in order to reduce adverse effects. In vivo pharmacological actions of drugs are generally dependent on both pharmacokinetic and pharmacodynamic factors. In addition to the pharmacodynamic interaction between opioid and adjuvant analgesics, pharmacokinetic interaction can modulate drug action. Indeed, inhibition of P-glycoprotein enhances the clinical effect of morphine,6 and increases both the accumulation in the brain and the antinociceptive effect of morphine in rats.7 Among adjuvant analgesics, antidepressants and antiarrhythmics include many cationic drugs that might interact with oxycodone transport across the BBB. However, BBB transport-related interactions of CNS-acting drugs are poorly understood. The purpose of this study is to clarify potential interactions between oxycodone and adjuvant analgesics at the BBB. Initially, we used [3H]pyrilamine as a probe, since oxycodone and pyrilamine share a common transporter at the BBB.4 We investigated the interactions of 15 adjuvant analgesics with [3H]pyrilamine transport using conditionally immortalized rat brain endothelial cells (TR-BBB13) as an in vitro BBB model.8 Based on the results obtained, the interaction of selected adjuvant analgesics with oxycodone was examined using TR-BBB13 cells. Finally, we further examined the influence of amitriptyline on the pharmacokinetics and antinociceptive effect of oxycodone in mice.
MATERIALS AND METHODS
Animals Adult male ddY mice weighing about 30 g were purchased from Japan SLC (Hamamatsu, Shizuoka, Japan); they were housed, 10 per cage, in a laboratory with free access to food and water and were maintained on a 12-h dark/12-h light cycle in a room with controlled temperature (24 28C) and humidity (55 5%). This study was conducted according to guidelines approved by the Experimental Animal Ethical Committee of Teikyo University.
Transport Studies in TR-BBB13 Cells TR-BBB13 cells were seeded on collagen-coated multiwell dishes at a density of 0.1 105 cells/cm2. At 3 days after seeding, the cells were washed twice with 1 mL of PBS and preincubated with incubation buffer (122 mM NaCl, 3 mM KCl, 25 mM NaHCO3, 1.2 mM MgSO4, 1.4 mM CaCl2, 10 mM D-glucose, 10 mM HEPES, pH 7.4) for 20 min at 378C. After preincubation, the buffer (0.25 mL) containing oxycodone (30 mM) or [3H]pyrilamine (74 kBq/mL, 90 nM) was added to initiate uptake in the presence of a test compound. The cells were incubated at 378C for a designated time, and then washed three times with 1 mL of ice-cold incubation buffer to terminate the uptake. For the determination of [3H]pyrilamine radioactivity, the cells were solubilized with 1 N NaOH for 60 min. The radioactivity was measured using a liquid scintillation counter after the addition of scintillation cocktail Hionic Fluor (PerkinElmer Life and Analytical Sciences, Boston, MA). For the determination of oxycodone, the cells were collected and homogenized by sonication, and the homogenate was stored at 208C until determination of oxycodone as described below. Cellular protein content was determined by BCA protein assay kit (Pierce Chemical Co., Rockford, IL). Uptake was expressed as the cell-to-medium ratio (mL/mg protein) obtained by dividing the uptake amount by the concentration of substrate in the incubation buffer.
Chemicals Oxycodone was kindly provided by Takeda Pharmaceutical Co. Ltd. (Osaka, Japan). [3H]Pyrilamine (23–30 Ci/mmol) was purchased from GE Healthcare (Little Chalfont, Buckinghamshire, UK). All other chemicals and reagents were commercial products of reagent grade. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
Antinociception Study The tail-flick latency test in mice was used to quantify antinociception.9 Briefly, the tail was immersed in a water bath at a temperature of 50 18C. The time until the tail was withdrawn was measured as the tail-flick latency. Before DOI 10.1002/jps
DRUG–DRUG INTERACTION BETWEEN OXYCODONE AND ADJUVANT ANALGESICS
drug administration, baseline testing was performed. Then antinociceptive testing was performed at 30, 60, and 90 min after the s.c. administration of saline, amitriptyline (10, 30 mg/kg) or oxycodone (5 mg/kg). Mice were also received the s.c. coadministration of oxycodone (5 mg/kg) and amitriptyline (10, 30 mg/kg) in a mixed solution. A maximum tail-flick latency of 10 s was used to minimize tissue damage to the tail. The tail-flick latency values were converted to percentage of the maximum possible effect (%MPE) as follows: %MPE ¼ (postdrug latency predrug latency)/(maximum latency predrug latency) 100.
Brain and Plasma Concentrations of Oxycodone Mice received s.c. administration of oxycodone (5 mg/kg) with or without amitriptyline (10, 30 mg/kg). At 30 min after the administration, blood was collected from the descending aorta under anesthesia with diethyl ether. Then, the brain was removed immediately. The plasma was separated by centrifugation. Plasma and brain samples were stored at 208C until determination of oxycodone.
Determination of Oxycodone Oxycodone was determined by HPLC with electrochemical detection (ECD).4,10 Twenty microliters of codeine solution (250 ng/mL), an internal standard, 100 mL of 4 N NaOH, and 800 mL of butyl chloride were added to 200 mL of cell homogenate. The samples were mixed, and centrifuged for 10 min at 3000 rpm at 48C, then the butyl chloride layer was transferred. The remaining aqueous layer was extracted again with 800 mL of butyl chloride. The combined butyl chloride extract was evaporated to dryness. The residue was reconstituted in 200 mL of mobile phase, and a 40 mL aliquot was injected into the HPLC. In the case of plasma samples, a 50 mL aliquot was mixed with 100 mL of 0.1 M perchloric acid. The mixture was centrifuged for 10 min at 10,000 rpm and the supernatant was collected. The brain was homogenized in five volumes of 0.1 M perchloric acid, and the homogenate was centrifuged for 10 min at 3000 rpm at 48C. Naltrexone solution (250 ng/mL) was added as an internal standard to the supernatants of plasma and brain homogenate, and the solution was neutralized and subjected to solid-phase DOI 10.1002/jps
469
extraction using an Oasis HLB cartridge (Waters, Milford, MA). The Oasis HLB cartridge was prewetted with methanol (1 mL), followed by water (1 mL). A sample was applied to the cartridge, which was washed with 1 mL of 40% methanol, and oxycodone and naltrexone were eluted with methanol. The eluate was dried under a nitrogen stream and the residue was reconstituted in the HPLC mobile phase. The HPLC system consisted of a pump (301E, Eicom, Kyoto, Japan) and an electrochemical detector (ECD-300, Eicom). The HPLC analytical column used was an XTerra1 RP18 (4.6 mm 50 mm, 5 mm particle size, Waters). The HPLC separation of oxycodone, codeine, and naltrexone was carried out at a flow rate of 0.5 mL/min with a mobile phase containing 10% acetonitrile, 20% methanol, and 5 mM phosphate buffer (pH 8.0) at 408C. The retention times of oxycodone, codeine, and naltrexone were 9.1, 6.4, and 23.3 min, respectively. The voltage of the working electrode of the electrochemical detector was set at 800 mV. Standard curves of oxycodone injected onto the column showed good linearity in the range of 0.15–50 pmol (coefficient of determination >0.99). The detection limit for quantification of oxycodone was 3 nM. Runs were accepted if the precision and accuracy of the quality control samples at low (3 nM), moderate (10 nM), and high (100 nM) concentrations each had a coefficient of variation (CV) below 15%.
Data Analysis The 50%-inhibitory concentration (IC50) of test compounds for [3H]pyrilamine and oxycodone uptake by TR-BBB13 cells was calculated by fitting the data to a one-site inhibition model using Prism software (Graphpad, San Diego, CA). The inhibition constant (Ki) values were calculated by using the following equation: Ki ¼
IC50 ð1 þ ½SÞ=Km
where [S] is the concentration of substrate ([3H]pyrilamine or oxycodone) and Km is the Michaelis–Menten constant for [3H]pyrilamine (28 mM) or oxycodone (89 mM).4 Statistical analysis of the data was performed by employing Student’s t-test and by one-way analysis of variance followed by Dunnett’s test for single and multiple comparisons, as appropriate. JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
470
NAKAZAWA ET AL.
Differences were considered statistically significant at p < 0.05.
RESULTS Screening for BBB Transport Interactions Using [3H]Pyrilamine Interactions with [3H]pyrilamine transport were investigated for 15 adjuvant analgesics, 2 opioid analgesics, and 2 nonopioid analgesics (Fig. 1). The uptake of [3H]pyrilamine into TR-BBB13 cells was inhibited by antidepressants (amitriptyline, imipramine, clomipramine, amoxapine, and fluvoxamine), antiarrhythmics (mexiletine, lidocaine, and flecainide), and an NMDA receptor antagonist (ketamine) at the concentration of 1 mM, except in the case of amoxapine (0.1 mM). The extents of inhibition were 87–98% by antidepressants, 79–98% by antiarrhythmics, and 89% by ketamine. On the other hand, antiepileptics (carbamazepine, phenytoin, and clonazepam) and corticosteroids (dexamethasone and prednisolone) were not inhibitory, except for sodium valproate. Oxycodone, morphine, and pentazocine significantly inhibited [3H]pyrilamine uptake by 86%, 61%, and 97%, respectively. Buprenorphine did not alter [3H]pyrilamine uptake.
Figure 1. Effect of various adjuvant analgesics on [3H]pyrilamine transport into TR-BBB13 cells. Uptake of [3H]pyrilamine (90 nM) was measured for 10 s at 378C in transport medium (pH 7.4) containing each drug at the concentration of 1 mM, except for amoxapine and buprenorphine (0.1 mM). Each column represents the mean SE of four experiments. Asterisks indicate a significant difference from the control value (
p < 0.01,
p < 0.001). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
Effects of Selected Compounds on the BBB Transport of Oxycodone Six adjuvant analgesics (amitriptyline, imipramine, fluvoxamine, carbamazepine, mexiletine, and ketamine) were selected for investigation of their interaction with oxycodone transport, because these drugs are frequently used as adjuvant analgesics in cancer patients in Japan.11 Oxycodone uptake into TR-BBB13 cells was inhibited by amitriptyline, imipramine, fluvoxamine, mexiletine, and ketamine to the extents of 77%, 46%, 64%, 76%, and 77%, respectively (Fig. 2). Carbamazepine did not inhibit oxycodone uptake, in line with its lack of an inhibitory effect on [3H]pyrilamine uptake. Oxycodone uptake was moderately inhibited by morphine and pentazocine (51% and 65%, respectively), but not by buprenorphine.
Determination of Inhibition Constant (Ki) The concentration dependency of inhibition of [3H]pyrilamine and oxycodone transport by amitriptyline, fluvoxamine, mexiletine, and morphine was examined, because these adjuvant analgesics significantly inhibited the transport of [3H]pyrilamine by 94–98% and that of oxycodone by 76–77%. These analgesics inhibited
Figure 2. Effect of various adjuvant analgesics on oxycodone transport into TR-BBB13 cells. Uptake of oxycodone (30 mM) was measured for 15 s at 378C in transport medium (pH 7.4) containing each drug at the concentration of 1 mM, except for buprenorphine (0.1 mM). Each column represents the mean SE of three experiments. Asterisks indicate a significant difference from the control value (
p < 0.01). DOI 10.1002/jps
DRUG–DRUG INTERACTION BETWEEN OXYCODONE AND ADJUVANT ANALGESICS
[3H]pyrilamine uptake in a concentrationdependent manner with Ki values of 8.51 mM for amitriptyline, 7.37 mM for fluvoxamine, 3.46 mM for mexiletine, and 284 mM for morphine (Fig. 3A). The uptake of oxycodone was also inhibited in a concentration-dependent manner with Ki values of 13 mM for amitriptyline, 65 mM for fluvoxamine, 44 mM for mexiletine, and 470 mM for morphine (Fig. 3B).
In Vivo Effects on Antinociceptive Action and Pharmacokinetics of Oxycodone The in vivo antinociceptive effect of the interaction of oxycodone with amitriptyline was examined, because amitriptyline inhibited oxycodone transport with the lowest Ki value among the tested drugs. Antinociceptive effect was determined by the use of the tail-immersion test after oxycodone administration with or without amitriptyline in mice. The value of %MPE was significantly increased at 30 min after the subcutaneous administration of oxycodone (5 mg/kg) or amitriptyline (30 mg/kg) in mice (Fig. 4). The antinociceptive effect after oxycodone administration (5 mg/kg s.c.) was increased and extended by the coadministration of amitriptyline (10, 30 mg/kg s.c.). The area under the time curve of antinociceptive effect (AUE) was significantly increased by 4.1-, 7.1-, and 10.3-fold after the administration of oxycodone alone, and oxycodone with amitriptyline at doses of 10 and 30 mg/kg, respectively, compared with saline administra-
471
tion (Fig. 5). The AUE after coadministration of oxycodone and amitriptyline at 30 mg/kg was significantly greater than that after administration of oxycodone alone. The oxycodone concentrations in the plasma and brain at 30 min after oxycodone administration (30 mg/kg) were not affected by the coadministration of amitriptyline (10 or 30 mg/kg) (Fig. 6). The brain to plasma concentration ratios were 3.5 0.2, 4.5 0.5, and 4.4 0.4 (mean SE, n ¼ 4) in the rats administered oxycodone alone and oxycodone with amitriptyline at the doses of 10 and 30 mg/kg, respectively, and there was no significant difference among these values.
DISCUSSION Drug–drug interactions are of potential clinical importance, since they may result in adverse effects. Though many drug–drug or drug–food interactions have been suggested on the basis of in vitro studies, it is important to consider whether the interactions might also occur in the in vivo situation. In the present study, potential interactions between oxycodone and adjuvant analgesics were examined using an in vitro BBB cell model, together with in vivo analyses of antinociception and brain distribution. The results of the in vitro experiments indicated that several adjuvant analgesics, including amitriptyline, interact with oxycodone during the BBB transport process at relatively high
Figure 3. Concentration-dependent inhibition of uptake of [3H]pyrilamine (A) and oxycodone (B) into TR-BBB13 cells by amitriptyline, fluvoxamine, mexiletine, and morphine. Uptakes of [3H]pyrilamine (90 mM, 10 s) and oxycodone (30 mM, 15 s) were measured in the presence of various concentrations of amitriptyline (0.1 mM to 1 mM), fluvoxamine (0.1 mM to 1 mM), mexiletine (0.1 mM to 1 mM), and morphine (0.1 mM to 10 mM). Each point represents the mean SE of three to four experiments. DOI 10.1002/jps
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
472
NAKAZAWA ET AL.
Figure 4. Time course of antinociceptive effects in mice. Mice received subcutaneous administration of saline (*), oxycodone alone (5 mg/kg) (*), amitriptyline alone (10 mg/kg: ~, 30 mg/kg: !), or oxycodone (5 mg/kg) with amitriptyline (10 mg/kg: ~, 30 mg/kg: 5). Each point represents the mean SE of four to five mice. Asterisks indicate a significant difference from the saline value (
p < 0.01).
concentrations. In vivo analysis clearly demonstrated that coadministration of oxycodone with amitriptyline at pharmacologically relevant doses enhanced the antinociceptive effect without causing inhibition of BBB transport of oxycodone. For the initial screening of adjuvant analgesics that interact with BBB transport of oxycodone,
Figure 5. Area under the time curve of antinociceptive effect (AUE) in mice. Mice received subcutaneous administration of saline, oxycodone alone (5 mg/kg), or oxycodone (5 mg/kg) with amitriptyline (10, 30 mg/kg). Each column represents the mean SE of four to five mice. Asterisks indicate a significant difference (
p < 0.01 vs. saline, ##p < 0.01 vs. oxycodone). JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
[3H]pyrilamine was used as a probe ligand, because oxycodone and pyrilamine share the same transporter at the BBB.4 The [3H]pyrilamine uptake into TR-BBB13 cells was inhibited by antidepressants (amitriptyline, imipramine, clomipramine, amoxapine, and fluvoxamine), antiarrhythmics (mexiletine, lidocaine, and flecainide), and an NMDA receptor antagonist (ketamine), suggesting that these agents interact with the BBB transport of pyrilamine. The adjuvant analgesics that showed significant inhibition of [3H]pyrilamine transport are cationic drugs with an amine moiety in their structure, except for sodium valproate, an organic anion. Thus, a basic amine structure may play an important role in the interaction with [3H]pyrilamine transport. Sodium valproate significantly inhibited [3H]pyrilamine transport, in contrast with other antiepileptics. Ohashi et al.12 have reported that valproate inhibits OCTN2-mediated organic cation transport, and OCTN2 has an anion-binding site in addition to a cation-binding site. Thus, the inhibitions of [3H]pyrilamine transport by organic cations and by sodium valproate may involve different mechanisms. Among adjuvant analgesics, amitriptyline, imipramine, fluvoxamine, mexiletine, and ketamine are frequently used in patients with cancer pain.11 In the screening using [3H]pyrilamine as a probe ligand, these adjuvant analgesics inhibited [3H]pyrilamine transport by over 89% in TRBBB13 cells. Thus, we examined the BBB transport interactions between oxycodone and these drugs. These five adjuvant analgesics all inhibited oxycodone transport in TR-BBB13 cells. Oxycodone transport was also inhibited by morphine and pentazocine, but not by carbamazepine or buprenorphine. Further, amitriptyline, fluvoxamine, mexiletine, and morphine showed concentration-dependent inhibition of both oxycodone and [3H]pyrilamine transport. We have recently reported that the BBB transport of oxycodone is mediated by a common transporter with pyrilamine.4 Thus, [3H]pyrilamine may be a useful probe for screening of interactions involving oxycodone transporter-mediated BBB transport. Oxycodone transport was inhibited in a concentration-dependent manner with the Ki values of 13 mM for amitriptyline, 65 mM for fluvoxamine, and 44 mM for mexiletine. Ulrich and La¨ uter13 reported that the therapeutic window of serum concentration of amitriptyline was 80–200 mg/L DOI 10.1002/jps
DRUG–DRUG INTERACTION BETWEEN OXYCODONE AND ADJUVANT ANALGESICS
473
Figure 6. Concentrations of oxycodone in the plasma (A) and brain (B) of mice. The concentration was determined at 30 min after subcutaneous administration of oxycodone alone (5 mg/kg) or oxycodone (5 mg/kg) with amitriptyline (10, 30 mg/kg) in mice. Each column represents the mean SE of four mice.
(0.25–0.64 mM). The therapeutic plasma concentrations of fluvoxamine and mexiletine are 0.203–1.26 and 0.2–0.65 mM, respectively. Thus, the Ki values obtained in this study were 5–300 times greater than the human therapeutic plasma concentration ranges for amitriptyline, fluvoxamine, and mexiletine. In addition, the therapeutic concentration ranges of these drugs would be even lower if plasma protein binding is taken into consideration (i.e., the unbound plasma concentrations would be lower). Thus, it seems likely that any interaction between oxycodone and these drugs in the BBB transport process would be clinically irrelevant. To examine the in vivo interaction between oxycodone and amitriptyline, which had the highest activity among the tested drugs for inhibition of oxycodone transport, the antinociceptive effect and brain distribution of oxycodone were measured in mice. The antinociceptive effects, measured with the mouse tail-immersion test, were caused by not only the administration of oxycodone, but also the administration of amitriptyline. It has been suggested that the effect of amitriptyline is mediated by the release of endogenous opioid peptide and activation of the d-opioid receptor in the brain.14 Coadministration of oxycodone and amitryptyline brought about additive antinociceptive effects. Oxycodone concentrations in the plasma and brain were not affected by coadministration of amitriptyline. Indeed, the unbound plasma concentration of amitriptyline at 30 min after s.c. administration of DOI 10.1002/jps
amitriptyline (10 mg/kg) in mice is estimated to be 0.12 mM using free fraction of 0.05, and the concentration is 10 times lower than the Ki value obtained in this study.15 The plasma concentration of nortriptyline, an active metabolite, was 80 times lower than that of amitriptyline.15 It was shown that amitriptyline and its metabolites are substrates of P-glycoprotein,15 which can affect the BBB transport of morphine,7 whereas the pharmacokinetics of oxycodone were not affected by a P-glycoprotein inhibitor.16 Therefore, amitriptyline appears to have little or no effect on BBB transport of oxycodone from the circulating blood into the brain at pharmacologically relevant doses. This finding is consistent with the in vitro finding that amitriptyline inhibits BBB transport of oxycodone at relatively high concentration. These results indicated that the coadministration of amitriptyline enhances the antinociceptive effects of oxycodone with little effect on oxycodone pharmacokinetics, including brain distribution. Thus, this study has clearly demonstrated that interaction between oxycodone and amitriptyline at the oxycodone transporter does occur, but is unlikely to be clinically relevant.
CONCLUSION In conclusion, we have shown that several adjuvant analgesics interact with the BBB transport of oxycodone at relatively high concentrations. However, there is unlikely to be a JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
474
NAKAZAWA ET AL.
significant interaction at therapeutically or pharmacologically relevant plasma concentration ranges.
ACKNOWLEDGMENTS This work was supported in part by a Grant-inAid for Scientific Research and a Grant-in-Aid for Young Scientists provided by the Ministry of Education, Culture, Sports, Science and Technology.
REFERENCES 1. Peckham EM, Traynor JR. 2006. Comparison of the antinociceptive response to morphine and morphine-like compounds in male and female Sprague-Dawley rats. J Pharmacol Exp Ther 316: 1195–1201. 2. Bostro¨ m E, Simonsson US, Hammarlund-Udenaes M. 2006. In vivo blood-brain barrier transport of oxycodone in the rat: Indications for active influx and implications for pharmacokinetics/pharmacodynamics. Drug Metab Dispos 34:1624–1631. 3. Tunblad K, Jonsson EN, Hammarlund-Udenaes M. 2003. Morphine blood-brain barrier transport is influenced by probenecid co-administration. Pharm Res 20:618–623. 4. Okura T, Hattori A, Takano Y, Sato T, Hammarlund-Udenaes M, Terasaki T, Deguchi Y. 2008. Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood-brain barrier transport of oxycodone. Drug Metab Dispos 36: 2005–2013. 5. Lussier D, Huskey AG, Portenoy RK. 2004. Adjuvant analgesics in cancer pain management. Oncologist 9:571–591. 6. Kharasch ED, Hoffer C, Whittington D, Sheffels P. 2003. Role of P-glycoprotein in the intestinal absorption and clinical effects of morphine. Clin Pharmacol Ther 74:543–554. 7. Letrent SP, Pollack GM, Brouwer KR, Brouwer KL. 1999. Effects of a potent and specific P-glycoprotein inhibitor on the blood-brain barrier distribution and antinociceptive effect of morphine in the rat. Drug Metab Dispos 270:827–834.
JOURNAL OF PHARMACEUTICAL SCIENCES, VOL. 99, NO. 1, JANUARY 2010
8. Hosoya K, Takashima T, Tetsuka K, Nagura T, Ohtsuki S, Takanaga H, Ueda M, Yanai N, Obinata M, Terasaki T. 2000. mRNA expression and transport characterization of conditionally immortalized rat brain capillary endothelial cell lines; a new in vitro BBB model for drug targeting. J Drug Target 8:357–370. 9. Marinho BG, Miranda LS, Gomes NM, Matheus ME, Leita˜ o SG, Vasconcellos ML, Fernandes PD. 2006. Antinociceptive action of (þ/)-cis-(6-ethyltetrahydropyran-2-yl)formic acid in mice. Eur J Pharmacol 550:47–53. 10. Kokubun H, Ouki M, Matoba M, Kubo H, Hoka S, Yago K. 2005. Determination of oxycodone and hydrocotarnine in cancer patient serum by high-performance liquid chromatography with electrochemical detection. Anal Sci 21:337– 339. 11. Inagaki S, Kato K, Maruyama M, Niimi M, Saito H, Nakano K, Noda Y, Nabeshima T. 2006. Pain assessment for cancer patients based on their pain descriptions (Part 2)—Preliminary study for selecting adequate analgesics and adjuvant analgesics using APQ. Jpn J Pharm Health Care Sci 32:788–804. 12. Ohashi R, Tamai I, Inano A, Katsura M, Sai Y, Nezu J, Tsuji A. 2002. Studies on functional sites of organic cation/carnitine transporter OCTN2 (SLC22A5) using a Ser467Cys mutant protein. J Pharmacol Exp Ther 302:1286– 1294. 13. Ulrich S, La¨ uter J. 2002. Comprehensive survey of the relationship between serum concentration and therapeutic effect of amitriptyline in depression. Clin Pharmacokinet 41:853–876. 14. Gray AM, Spencer PS, Sewell RD. 1998. The involvement of the opioidergic system in the antinociceptive mechanism of action of antidepressant compounds. Br J Pharmacol 124:669–674. 15. Uhr M, Grauer MT, Yassouridis A, Ebinger M. 2007. Blood-brain barrier penetration and pharmacokinetics of amitriptyline and its metabolites in pglycoprotein (abcb1ab) knock-out mice and controls. Psychiatr Res 41:179–188. 16. Bostro¨ m E, Simonsson US, Hammarlund-Udenaes M. 2005 Oxycodone pharmacokinetics and pharmacodynamics in the rat in the presence of the P-glycoprotein inhibitor PSC833. J Pharm Sci 94: 1060–1066.
DOI 10.1002/jps