`A R T I C L E
`
`P-glycoprotein is not involved in the
`differential oral potency of naloxone and
`naltrexone
`
`doi: 10.1111/j.1472-8206.2009.00724.x
`
`Mouna Kanaan, Youssef Daali, Pierre Dayer, Jules Desmeules*
`Clinical Pharmacology and Toxicology and Multidisciplinary Pain Center, Department of Anesthesiology, Pharmacology
`and Intensive care, Geneva University Hospitals, Faculty of Medicine, University of Geneva, CH-1211 Geneva 14,
`Switzerland
`
`A B S T R A C T
`
`The poor oral bioavailability of the opioid receptor antagonist naloxone (NA) when
`compared with naltrexone (NX) may be related to a greater interaction of naloxone
`with the efflux drug transporter P-glycoprotein (P-gp). We studied the involvement of
`P-gp in the transepithelial transport of the two opioid receptor antagonists, using a
`validated human in vitro Caco-2 cell monolayer model. The bidirectional transport
`of NA and NX (1, 50 and 100 lM) across the monolayers was investigated in the
`presence and absence of the specific P-gp inhibitor GF120918 (4 lM). NA and NX
`showed equal transport rates between the apical-to-basolateral (A–B) and the
`basolateral-to-apical (B–A) directions and neither the influx nor the efflux transport
`was affected by the P-gp inhibitor (P > 0.05). In conclusion, NA and NX are not P-gp
`substrates. The differential oral bioavailability of the two opioid antagonists is P-gp
`independent.
`
`Keywords
`absorption,
`Caco-2 cells,
`naloxone,
`naltrexone,
`oral bioavailability,
`P-glycoprotein
`
`Received 14 November 2008;
`revised 6 March 2009;
`accepted 13 March 2009
`
`*Correspondence and reprints:
`jules.desmeules@hcuge.ch
`
`I N T R O D U C T I O N
`
`Naloxone (NA), a synthetic N-allyl derivative of oxy-
`morphone, and its analogue naltrexone (NX) are both
`opioid antagonists at the mu, kappa and sigma opioid
`receptor sites [1] (Figure 1). Naloxone is the treatment of
`choice for opioid overdose, regardless of the type of
`opioid, as it permits a dose dependant reversal of opioid
`adverse events in the central nervous system. It is mostly
`administered by intravenous
`injection for complete
`bioavailability and rapid onset of action. Naloxone is
`mainly metabolized in the liver by glucuroconjugation
`into naloxone-3-glucuronide [2] and eliminated by the
`renal route [3]. Naltrexone is approximately twice as
`potent as naloxone [4] and is mostly used orally for the
`treatment of alcohol dependence. Naltrexone is metab-
`olized by dihydrodiol dehydrogenase to the main active
`metabolite 6-beta-naltrexol, via extensive hepatic and
`extra-hepatic routes [5]. Although a weaker opioid
`antagonist than naltrexone, 6-beta-naltrexol possibly
`contributes to the long duration of action observed with
`the parent compound, particularly because plasma
`
`concentrations of the metabolite are higher than those
`of the parent drug. Naltrexone and 6-beta-naltrexol are
`mainly excreted by the renal route [6].
`Naloxone has a low oral bioavailability when com-
`pared to naltrexone [7–10]. It is, however, unclear
`whether the very limited systemic availability of oral
`naloxone is due to a more extensive first pass meta-
`bolism, when compared with naltrexone, or
`to a
`poor gastrointestinal absorption. P-glycoprotein (P-gp)
`(ABCB1/MDR1) is expressed in the major organs asso-
`ciated with drug absorption, distribution and elimina-
`tion, therefore playing a major role in pharmacokinetics
`of several drugs.
`
`M A T E R I A L S A N D M E T H O D S
`
`Materials
`Caco-2 cells (TC7 clone) were kindly provided by Tea
`Fevr, PhD (ISREC, Swiss Institute for Experimental
`Cancer Research, Lausanne, Switzerland). Penicillin–
`streptomycin was purchased from Sigma-Aldrich GmbH
`(Steinheim, Germany) and all other cell culture reagents
`
`ª 2009 The Authors Journal compilation ª 2009 Socie´ te´ Franc¸aise de Pharmacologie et de The´ rapeutique
`Fundamental & Clinical Pharmacology 23 (2009) 543–548
`
`543
`
`Nalox1222
`Nalox-1 Pharmaceuticals, LLC
`Page 1 of 6
`
`
`
`544
`
`M. Kanaan et al.
`
`Naloxone
`
`Naltrexone
`
`Figure 1 Chemical structures of naloxone and naltrexone.
`
`from Gibco BRL (Paisley, Scotland). Naloxone and
`naltrexone were purchased from Sigma-Aldrich GmbH
`and GF120918 was kindly provided by GlaxoSmithKline
`(Middlesex, UK).
`
`Cell culture
`Caco-2 cells (TC7 clone) were used at passages 21–25.
`Cells were cultured in Dulbecco’s modified Eagle’s
`medium (DMEM Glutamax, Gibco BRL) supplemented
`by 20% fetal bovine serum (FBS, Gibco BRL), 1%
`nonessential amino acids (NEA, Gibco BRL), 100 U/mL
`penicillin and 100 lg/mL streptomycin (Sigma-Aldrich),
`at 37 °C in a humidified atmosphere with 5% CO2.
`At 85–95% confluency, Caco-2 cells were treated with
`0.25% trypsin–EDTA (Gibco BRL) and seeded at a
`density of 65 000 cells/cm2 on polycarbonate mem-
`(12-mm diameter, 1.13 cm2,
`branes of Transwells
`0.4 lM pore size, 12-well plates; Costar, Cambridge,
`MA, USA), previously equilibrated for 1 h in the culture
`medium. Medium was changed the day after seeding and
`every other day thereafter [apical volume (A): 0.5 mL,
`basolateral volume (B): 1.5 mL]. Monolayers were used
`for transport studies 20–21 days post seeding to allow
`full maturation of the cells, including P-gp expression
`and appropriate tight junctions.
`
`Measurement of transepithelial electrical
`resistance
`Transepithelial electrical resistance (TEER) was checked
`every 5 days during the 21-day maturation of
`the
`monolayers. Prior to bidirectional
`transport studies,
`medium was removed from both apical and basolateral
`chambers and monolayers were rinsed three times with
`the transport buffer Hank’s balanced salt
`solution
`(HBSS),
`supplemented with 25 mM N-(2-hydroxyl-
`ethyl)piperazine-N¢-2ethane-sulfonic acid (HEPES) (Gib-
`co BRL) and pH adjusted to 7.4 with 0.5 M NaOH. Cells
`were equilibrated in the same buffer for 1 h and the
`integrity of each monolayer was checked by measuring
`
`its TEER with a Millicell-ERS ohmmeter (Millipore Corp.,
`Bedfort, MA, USA). Resistance was also checked imme-
`diately after the experiments.
`
`Transmission electron microscopy and Western
`blotting of P-glycoprotein
`Transmission electron microscopy and Western blotting
`of P-glycoprotein were performed in the context of
`the Caco-2 model validation in our previous study
`(see [11]).
`
`Transport studies
`After measurement of TEERs, HBSS buffer was removed
`from each chamber. Apical to basolateral (A–B) trans-
`port was initiated by replacing basolateral (B) buffer with
`1.5 mL of fresh HBSS supplemented with 25 mM HEPES,
`1% DMSO and pH adjusted to 7.4 with NaOH 0.5 M, and
`replacing apical (A) buffer with 0.5 mL of the drug
`solution in the same buffer (HBSS/HEPES pH 7.4). In
`other wells, B–A transport was initiated by replacing (A)
`buffer with 0.5 mL of fresh HBSS/HEPES pH 7.4 and (B)
`buffer with the drug solution in the same buffer
`(1.5 mL). For the P-gp inhibition studies, GF120918
`(4 lM) was present in both chambers. Samples (100 lL)
`were removed from each receiver chamber at various
`times (30, 60, 90, 120 and 180 min) and replaced with
`buffer to maintain constant volumes. The 3-h transport
`studies were performed at a constant agitation rate
`(50 rpm) using a circular shaker (type SSM1, StuartÒ) in
`an incubator (37 °C, 5% CO2 and humidified atmo-
`sphere). After the transport studies, all aliquots were
`stored at )20 °C until analysis.
`
`Analytical method
`Naloxone and naltrexone analysis for the 50 and 100 lM
`assays was performed by LC-UV at 230 nm. Separation
`was carried out on a Zorbax eclipse XDB-C8 (150 ·
`4.6 mm i.d., particle size: 5 lM) from Agilent, coupled
`with a guard column with the same stationary phase. The
`mobile phase consisted of a mixture of acetonitrile and
`orthophosphoric acid (50 mM) adjusted to pH 4.2 with
`sodium hydroxide 4N and was delivered at 0.9 mL/min. A
`gradient elution was used in which the mobile phase
`composition was changed from 12–88% to 30–70%
`(ACN-Orthophosphoric acid) within 5 min, maintained
`at 30–70% until t = 8 min, and returned to the initial
`composition from t = 8 to t = 9 min. Naloxone and
`naltrexone analysis for 1 and 10 lM assays was achieved
`using an LC–MS system (Esquire 3000 + Ion-Trap) from
`Bruker daltonics (Billerica, MA, USA) equipped with an
`
`ª 2009 The Authors Journal compilation ª 2009 Socie´ te´ Franc¸aise de Pharmacologie et de The´ rapeutique
`Fundamental & Clinical Pharmacology 23 (2009) 543–548
`
`Nalox1222
`Nalox-1 Pharmaceuticals, LLC
`Page 2 of 6
`
`
`
`Naloxone, naltrexone and P-glycoprotein
`
`545
`
`electrospray source working in positive ion mode. The ion
`transitions monitored in multiple reaction-monitoring
`modes were m/z 342 fi 324, and 328 fi 310 for
`naltrexone and naloxone, respectively. Optimized ESI
`source voltages were as
`follows:
`spray needle at
`+4.3 kV, end plate offset at )500 V, capillary exit offset
`at )200 V, skimmer 1 at )107.4 V. Further ion source
`parameters were 70 psi nebulizer gas and 11 L/min of
`drying gas with a temperature of 350 °C. Separation was
`achieved with an XTerraÒ MS C18 column (100 mm ·
`2.1 mm i.d., particle size: 3.5 lM) from Waters (Milford,
`MA, USA) at 0.3 mL/min. Mobile phase consisted of
`ammonium formate 20 mM and acetonitrile (20–80%).
`In all cases, the standard curves were obtained by
`linear regression of measured peak area vs. concentra-
`tion and used to calculate concentrations of the analytes
`in unknown and QC samples. Samples consisted of
`aliquots removed from the receiver chambers (drug
`solution in the aqueous buffer HBSS). No additional
`treatment was needed. No extraction was required and
`the samples were directly injected into the HPLC system
`without need for an internal standard. The performance
`of both methods, in terms of reproducibility, repeatability
`and linearity were assessed before analysis (data not
`shown).
`
`Calculations
`TEER was calculated from the following equation [12]:
`
`TEER ¼ ðTEERmono TEERblankÞ A
`
`where TEERmono is the cell monolayer and polycarbonate
`porous membrane resistance, TEERblank the polycarbon-
`ate porous membrane resistance and A the polycarbonate
`porous membrane surface area (1.13 cm2).
`Apical to basolateral (Papp(A–B)) and basolateral to
`(Papp(B–A)) apparent permeability coefficients
`apical
`were calculated according to Artursson [13] using the
`following equation:
`
`Pappðcm/sÞ ¼ ðdQ=dtÞ=ðA C0 60Þ
`
`where dQ/dt (lg/min) is the permeability rate of the
`drug, calculated from the regression line of the time
`points of sampling, A is the surface area of the monolayer
`(cm2) and C0 the initial drug concentration in the donor
`chamber (lg/L).
`Karlsson et al. [14] suggested that there is involve-
`ment of a drug efflux transporter in Caco-2 cells if the
`efflux ratio (TR = Papp(B–A)/Papp(A–B)) is >2 and if a
`
`decreased secretory transport rate (Papp(B–A)) is ob-
`served in the presence of an inhibitor of the transporter
`in question. For a compound with an efflux ratio of 1.5–
`2.0, a positive effect of
`the inhibitor confirms the
`implication of the efflux transporter [15].
`
`Statistics
`The unpaired bilateral Student’s t test was used for
`statistical comparison of the transport rates in each
`direction and the transport rates in the presence or
`absence of the P-gp inhibitor for a particular direction
`(Xlstat version 5.0; Addinsoft, New York, NY, USA).
`A P value of <0.05 was considered significant.
`
`R E S U L T S
`
`Differentiation and integrity of Caco-2 cell
`monolayers
`Transmission electron microscopy (TEM)
`Histological examination showed a continuous differen-
`tiated cell monolayer presenting microvilli on the apical
`cell surface, interdigitations, numerous desmosomes, and
`tight junctions (see [11]).
`
`Transepithelial electrical resistance
`Caco-2 cell monolayers with TEER values between 300
`and 400 W.cm2 were used in the study. Measurements
`conducted after the experiments displayed similar values
`and confirmed the integrity of the monolayers during all
`of the experiments. No tendency towards an effect on
`TEER was observed under the various experimental
`conditions (pH, substrates and inhibitors).
`
`P-glycoprotein expression and activity
`Immunoblotting and transepithelial transport of P-gp probe
`The Western blot analysis revealed a C219 antibody-
`reactive band of 170 kDa corresponding to P-gp expres-
`sion (see [11]). The P-gp probe rhodamine 123 (5 lM)
`[16] showed a positive interaction with P-glycoprotein in
`our Caco-2 cell monolayers, with a secretory transport
`markedly inhibited by GF120918 (4 lM) ((Papp(B–A):
`
`7.1 ± 0.5.10)6 vs. 1.3 ± 1.4.10
`)6, P < 0.05).
`
`Transepithelial transport of naloxone and
`naltrexone
`The bidirectional transport of naloxone and naltrexone
`(1, 50 and 100 lM) was investigated at pH 7.4/7.4 in
`the presence and absence of 4 lM GF120918. The lowest
`naloxone and naltrexone concentrations tested (1 lM)
`correspond,
`respectively,
`to six times
`the plasma
`
`ª 2009 The Authors Journal compilation ª 2009 Socie´ te´ Franc¸aise de Pharmacologie et de The´ rapeutique
`Fundamental & Clinical Pharmacology 23 (2009) 543–548
`
`Nalox1222
`Nalox-1 Pharmaceuticals, LLC
`Page 3 of 6
`
`
`
`546
`
`M. Kanaan et al.
`
`Control
`
`+ GF120918
`
`(I)
`
`0.12
`
`0.1
`
`0.08
`
`0.06
`
`0.04
`
`0.02
`
`0
`
`Transported quantity (µg/cm2/3 h)
`
`(II)
`
`0.08
`
`0.07
`
`0.06
`
`0.05
`
`0.04
`
`0.03
`
`0.02
`
`0.01
`
`0
`
`Transported quantity (µg/cm2/3 h)
`
`Control
`
`+ GF120918
`
`Figure 2 Transepithelial transport of naloxone (I) and naltrexone
`(II) across Caco-2 cell monolayers in the presence and absence of
`GF120918.
`Naloxone (I) and naltrexone (II) (1 lM) were added to the apical
`side (open columns) or the basolateral side (solid columns) of the
`monolayers in the presence (+GF120918) or absence (control) of
`4 lM of GF120918 at pH 7.4/7.4. Data are mean ± SD of three
`experiments. P < 0.05.
`
`antagonists are well absorbed compounds [19]. Our
`transepithelial bidirectional study indicates that passive
`diffusion seems to be the major mechanism of naloxone
`and naltrexone transmembrane transit.
`Experimental animal data suggest that naloxone may
`interact with P-glycoprotein. Naloxone has been shown
`to stimulate ATPase activity in the plasma membranes of
`the multidrug-resistant Chinese hamster ovary (CHO)
`cell line CR1R12, as well as in purified reconstituted P-gp
`liposomes, although only at very high concentrations
`
`concentration measured 2 min after an injection of
`0.4 mg naloxone (0.01 lg/mL) [17] and 45 times the
`peak plasma concentration (Cmax = 13.7 ng/mL) mea-
`sured after multiple oral doses of 50mg naltrexone [18].
`Naloxone and naltrexone showed no statistical differ-
`ence between apical-to-basolateral
`(Papp(A–B)) and
`basolateral-to-apical (Papp(B–A)) transport. The efflux
`ratio (TR = Papp(B–A)/Papp(A–B)) of the two molecules
`was between 1.1 and 1.3, indicating that naloxone and
`naltrexone are not actively transported by an efflux
`transporter. This was confirmed by the use of the P-gp
`inhibitor, GF120918, which failed to alter the observed
`ratio (Table I, Figure 2).
`
`D I S C U S S I O N
`
`The results of our study clearly show that neither
`naloxone nor naltrexone are P-gp substrates. Indeed, no
`significant efflux ratio was observed for the two mole-
`cules across the P-gp-overexpressing Caco-2 cell mono-
`layers, as their respective fluxes in the absorptive and
`secretory directions were equivalent at all concentrations
`tested. Moreover, a potent P-gp inhibitor, GF120918,
`had no effect on bidirectional flux, either by the
`inhibition of efflux transport or by the enhancement of
`absorptive flux (P > 0.05). The absorptive apparent
`permeability coefficients of naloxone and naltrexone
`)6) show otherwise that the two opioid
`(Papps > 10 · 10
`
`Table I Apparent permeability coefficients Papp (cm/s) of naloxone
`and naltrexone in the presence and absence of GF120918 at pH
`7.4/7.4.
`
`Drug
`
`concentration
`
`± P-gp inhibitor
`
`Papp (A–B)
`)6)
`(cm/s) (10
`pH 7.4/7.4
`
`Papp (B–A)
`
`(cm/s) (10)6)
`pH 7.4/7.4
`
`Naloxone
`1 lM
`+ 4 lM GF120918
`50 lM
`+ 4 lM GF120918
`100 lM
`+ 4 lM GF120918
`Naltrexone
`1 lM
`+ 4 lM GF120918
`50 lM
`+ 4 lM GF120918
`100 lM
`+ 4 lM GF120918
`
`25.4 ± 0.8
`
`22.2 ± 2.6
`
`28.0 ± 0.5
`
`28.7 ± 1.3
`
`28.4 ± 0.3
`
`26.6 ± 1.4
`
`15.1 ± 0.6
`
`13.7 ± 0.8
`
`14.6 ± 0.2
`
`14.7 ± 0.3
`
`13.9 ± 0.4
`
`19.8 ± 5.2
`
`29.2 ± 1.6
`
`30.7 ± 2.0
`
`34.1 ± 0.5
`
`33.8 ± 2.9
`
`31.4 ± 2.1
`
`33.0 ± 1.8
`
`19.7 ± 0.5
`
`19.9 ± 0.9
`
`20.0 ± 1.1
`
`20.1 ± 1.0
`
`17.5 ± 0.6
`
`17.7 ± 0.2
`
`Efflux ratio (TR)
`
`1.1 (no net efflux)
`
`1.3
`
`1.2 (no net efflux)
`
`1.1
`
`1.1 (no net efflux)
`
`1.2
`
`1.3 (no net efflux)
`
`1.4
`
`1.3 (no net efflux)
`
`1.3
`
`1.2 (no net efflux)
`
`0.8
`
`Values are the mean ± SD of three experiments. P < 0.05.
`
`ª 2009 The Authors Journal compilation ª 2009 Socie´ te´ Franc¸aise de Pharmacologie et de The´ rapeutique
`Fundamental & Clinical Pharmacology 23 (2009) 543–548
`
`Nalox1222
`Nalox-1 Pharmaceuticals, LLC
`Page 4 of 6
`
`
`
`Naloxone, naltrexone and P-glycoprotein
`
`547
`
`(>100 lM) [20]. Our assays at lower concentrations
`(1 and 50 lM) do not confirm these results. Naloxone also
`weakly inhibited vinblastine binding to the plasma
`membranes of multidrug-resistant CHO (B30) cells at
`high concentrations (100 lM) [21]. In another experi-
`mental study in P-gp expressing CHO cells [22], naloxone
`caused a decrease in P-glycoprotein phosphorylation.
`Naloxone seems indeed to interact with animal P-gp, but
`with very low affinity, mainly resulting in weak inhibi-
`tion of the efflux transporter. No data concerning the
`interaction of naltrexone with P-glycoprotein or any
`other ‘‘ATP-binding cassette’’ (ABC) or ‘‘solute-linked
`carriers’’ (SLC) transporter are available in the literature.
`Naloxone and naltrexone are two structurally related
`opioid antagonists, mainly metabolized in the liver into
`naloxone-3-glucuronide and 6-beta-naltrexol, respec-
`tively [2,5]. Naloxone and naltrexone show similar
`potencies when administered parenterally. However,
`after oral administration, naltrexone is thought to be
`significantly more potent and long-lasting than naloxone
`in antagonizing the central effects of opioids [23]. Indeed,
`after oral administration, naloxone bioavailability is less
`than 2% [7,8,24,25], whereas naltrexone is between 5
`and 40% [9,10]. The huge difference observed in
`bioavailability after oral administration renders oral
`naloxone useless in opioid and alcohol addiction treat-
`ment. It has, however,
`lead to the development and
`commercialization of combinations of naloxone and
`opioid agonists, such as pentazocine and buprenorphine,
`to avoid the illicit parenteral use of the opioid agonist
`[26,27], as well as oxycodone to prevent peripherally
`mediated constipation without
`interfering with the
`central analgesic effect [28].
`Naloxone and naltrexone show very similar liposolu-
`bility (LogPs: 1.5 and 1.4,
`respectively), however,
`naltrexone has a higher pKa than naloxone (8.4 vs.
`7.9) [29,30]. Hence, at gastrointestinal pH conditions
`(pH 5–8)
`[31] as well as at physiological pH,
`the
`percentage of the ionized form of naltrexone is greater
`than that of naloxone. Consequently,
`the physico-
`chemical properties of the two opioid antagonists do
`not favour a greater gastrointestinal permeability of
`naltrexone as compared to naloxone. Additionally, our
`experimental study showed an almost twofold greater
`naloxone permeability across Caco-2 cells compared to
`)6 vs.
`naltrexone (ex, at 1 lM; Papp(A–B): 25.4 ± 0.8.10
`)6, P < 0.05).
`15.1 ± 0.6.10
`Evidence largely implicates hepatic first-pass metabo-
`lism in the poor oral bioavailability of naloxone, based
`on studies investigating the systemic biodisposition of
`
`the drug after oral and intravenous administration
`[7,8,24,25,32]. However, other mechanisms such as
`P-glycoprotein or other efflux drug transporters may be
`involved in limiting its gastrointestinal absorption and
`thus oral bioavailability. Our results show that the poor
`oral bioavailability of naloxone is not linked to a limited
`gastrointestinal absorption of the drug.
`In conclusion, our study excludes the involvement of
`P-glycoprotein in the poor oral bioavailability of nalox-
`one as compared to naltrexone. Naloxone gastrointesti-
`nal absorption does not seem to be the major cause of its
`poor oral bioavailability. In fact, rapid and extensive
`metabolism of the opioid antagonist appears as the more
`plausible factor to be considered.
`
`C O N F L I C T S O F I N T E R E S T
`
`The authors state no conflict of interest.
`
`A C K N O W L E D G E M E N T
`
`This work was supported by the Department of Anes-
`thesiology, Pharmacology and Intensive care, Geneva
`University Hospitals, CH-1211 Geneva 14, Switzerland.
`
`R E F E R E N C E S
`
`1 Helm S., Trescot A.M., Colson J., Sehgal N., Silverman S. Opioid
`antagonists, partial agonists, and agonists/antagonists: the role
`of office-based detoxification. Pain Physician (2008) 11
`225–235.
`2 Weinstein S.H., Pfeffer M., Schor J.M., Indindoli L., Mintz M.
`Metabolites of naloxone in human urine. J. Pharm. Sci. (1971)
`60 1567–1568.
`3 Fishman J., Roffwarg H., Hellman L. Disposition of naloxone-
`7,8,3H in normal and narcotic-dependent men. J. Pharmacol.
`Exp. Ther. (1973) 187 575–580.
`4 Crabtree B.L. Review of naltrexone, a long-acting opiate
`antagonist. Clin. Pharm. (1984) 3 273–280.
`5 Porter S.J., Somogyi A.A., White J.M. Kinetics and inhibition of
`the formation of 6beta-naltrexol from naltrexone in human
`liver cytosol. Br. J. Clin. Pharmacol. (2000) 50 465–471.
`6 Bullingham R.E., McQuay H.J., Moore R.A. Clinical pharmaco-
`kinetics of narcotic agonist-antagonist drugs. Clin. Pharmaco-
`kinet. (1983) 8 332–343.
`7 Kreek M.J. Opioid antagonists and partial antagonists, in:
`Schuster C.R., Kuhar M.J. (Eds), Pharmacological aspects of drug
`dependence. Towards an integrated neurobehavioral approach,
`Springer-Verlag, Berlin Heidelberg, New York, 1996, p. 565.
`8 De Schepper H.U., Cremonini F., Park M.I., Camilleri M.
`Opioids and the gut: pharmacology and current clinical
`experience. Neurogastroenterol. Motil. (2004) 16 383–394.
`Review.
`
`ª 2009 The Authors Journal compilation ª 2009 Socie´ te´ Franc¸aise de Pharmacologie et de The´ rapeutique
`Fundamental & Clinical Pharmacology 23 (2009) 543–548
`
`Nalox1222
`Nalox-1 Pharmaceuticals, LLC
`Page 5 of 6
`
`
`
`548
`
`M. Kanaan et al.
`
`9 Meyer M.C., Straughn A.B., Lo M.W., Schary W.L., Whitney
`C.C. Bioequivalence, dose-proportionality, and pharmacokinet-
`ics of naltrexone after oral administration. J. Clin. Psychiatry
`(1984) 45 15–19.
`10 Wall M.E., Brine D.R., Perez-Reyes M. Metabolism and dispo-
`sition of naltrexone in man after oral and intravenous admin-
`istration. Drug Metab. Dispos. (1981) 9 369–375.
`11 Kanaan M., Daali Y., Dayer P., Desmeules J. Lack of interaction
`of the NMDA receptor antagonists dextromethorphan and
`dextrorphan with P-glycoprotein. Curr. Drug Metab. (2008) 9
`144–151.
`12 Amidon G.L., Lennernas H., Shah V.P., Crison J.R. A theoretical
`basis for a biopharmaceutic drug classification: the correlation
`of in vitro drug product dissolution and in vivo bioavailability.
`Pharm. Res. (1995) 12 413–420.
`13 Artursson P. Epithelial transport of drugs in cell culture. I: A
`model for studying the passive diffusion of drugs over intestinal
`absorptive (Caco-2) cells. J. Pharm. Sci. (1990) 79 476–482.
`14 Karlsson J., Kuo S.M., Ziemniak J., Artursson P. Transport of
`celiprolol across human intestinal epithelial (Caco-2) cells:
`mediation of secretion by multiple transporters including
`P-glycoprotein. Br. J. Pharmacol. (1993) 110 1009–1016.
`15 Polli J.W., Wring S.A., Humphreys J.E., Huang L., Morgan J.B.,
`Webster L.O., Serabjit-Singh C.S. Rational use of in vitro P-
`glycoprotein assays in drug discovery. J. Pharmacol. Exp. Ther.
`(2001) 299 620–628.
`16 Takano M., Hasegawa R., Fukuda T., Yumoto R., Nagai J.,
`Murakami T. Interaction with P-glycoprotein and transport of
`erythromycin, midazolam and ketoconazole in Caco-2 cells.
`Eur. J. Pharmacol. (1998) 358 289–294.
`17 Naloxone product information. http://www.documed.ch.
`18 Dunbar J.L., Turncliff R.Z., Dong Q., Silverman B.L., Ehrich
`E.W., Lasseter K.C. Single- and multiple-dose pharmacokinetics
`of long-acting injectable naltrexone. Alcohol Clin. Exp. Res.
`(2006) 30 480–490.
`19 Yee S. In vitro permeability across Caco-2 cells (colonic) can
`predict in vivo (small intestinal) absorption in man – fact or
`myth. Pharm. Res. (1997) 14 763–766.
`20 Rebbeor J.F., Senior A.E. Effects of cardiovascular drugs on
`ATPase activity of P-glycoprotein in plasma membranes and in
`purified reconstituted form. Biochim. Biophys. Acta. (1998)
`1369 85–93.
`
`21 Callaghan R., Riordan J.R. Synthetic and natural opiates
`interact with P-glycoprotein in multidrug-resistant cells. J. Biol.
`Chem. (1993) 268 16059–16064.
`22 Callaghan R., Riordan J.R. Collateral sensitivity of multidrug
`resistant cells to narcotic analgesics is due to effects on the
`plasma membrane. Biochim. Biophys. Acta. (1995) 1236
`155–162.
`23 Gonzalez J.P., Brogden R.N. Naltrexone. A review of its
`pharmacodynamic and pharmacokinetic properties and thera-
`peutic efficacy in the management of opioid dependence. Drugs
`(1988) 35 192–213.
`24 Fishman J., Roffwarg H., Hellman L. Disposition of naloxone-
`7,8,3H in normal and narcotic-dependent men. J. Pharmacol.
`Exp. Ther. (1973) 187 575–580.
`25 Foss J.F. A review of the potential role of methylnaltrexone in
`opioid bowel dysfunction. Am. J. Surg. (2001) 182 19S–26S.
`26 Baum C., Hsu J.P., Nelson R.C. The impact of the addition of
`naloxone on the use and abuse of pentazocine. Public Health
`Rep. (1987) 102 426–429.
`27 Simojoki K., Vorma H., Alho H. A retrospective evaluation of
`patients switched from buprenorphine (Subutex) to the bupr-
`enorphine/naloxone combination (Suboxone). Subst. Abuse
`Treat Prev. Policy (2008) 17 16.
`28 Meissner W., Leyendecker P., Mueller-Lissner S., et al. A
`randomised controlled trial with prolonged-release oral oxyco-
`done and naloxone to prevent and reverse opioid-induced
`constipation. Eur. J. Pain (2009) 13 56–64.
`29 Clark’s. Isolation and Identification of Drugs in pharmaceuti-
`cals, body fluids, and post-mortem material, The Parmaceutical
`Press, London, UK, 1986.
`30 Gerebtzoff G., Seelig A. In silico prediction of blood-brain
`barrier permeation using the calculated molecular cross-
`sectional area as main parameter. J. Chem. Inf. Model. (2006)
`46 2638–2650.
`31 Neuhoff S., Ungell A.L., Zamora I., Artursson P. pH-dependent
`bidirectional transport of weakly basic drugs across Caco-2
`monolayers: implications for drug-drug interactions. Pharm.
`Res. (2003) 20 1141–1148.
`32 Weinstein S.H., Pfeffer M., Schor J.M., Franklin L., Mintz M.,
`Tutko E.R. Absorption and distribution of naloxone in rats after
`oral and intravenous administration. J. Pharm. Sci. (1973) 62
`1416–1419.
`
`ª 2009 The Authors Journal compilation ª 2009 Socie´ te´ Franc¸aise de Pharmacologie et de The´ rapeutique
`Fundamental & Clinical Pharmacology 23 (2009) 543–548
`
`Nalox1222
`Nalox-1 Pharmaceuticals, LLC
`Page 6 of 6
`
`