Transport Characteristics of Tramadol in the Blood–Brain Barrier

doi:10.1002/jps.24129

Journal of Pharmaceutical Sciences

Volume 103, Issue 10, October 2014, Pages 3335-3341

https://doi.org/10.1002/jps.24129 Get rights and content

Abstract

Tramadol is a centrally acting analgesic whose action is mediated by both agonistic activity at opioid receptors and inhibitory activity on neuronal reuptake of monoamines. The purpose of this study was to characterize the blood–brain barrier (BBB) transport of tramadol by means of microdialysis studies in rat brain and in vitro studies with human immortalized brain capillary endothelial cells (hCMEC/D3). The K_p,uu,brain value of tramadol determined by rat brain microdialysis was greater than unity, indicating that tramadol is actively taken up into the brain across the BBB. Tramadol was transported into hCMEC/D3 cells in a concentration‐dependent manner. The uptake was inhibited by type II cations (pyrilamine, verapamil, etc.), but not by substrates of organic cation transporter OCTs or OCTN2. It was also inhibited by a metabolic inhibitor but was independent of extracellular sodium or membrane potential. The uptake was altered by changes of extracellular pH, and by ammonium chloride‐induced intracellular acidification, suggesting that transport of tramadol is driven by an oppositely directed proton gradient. Thus, our in vitro and in vivo results suggest that tramadol is actively transported, at least in part, from blood to the brain across the BBB by proton‐coupled organic cation antiporter.

Introduction

Tramadol hydrochloride, (1RS,2RS)‐2‐[(dimethylamino)ethyl]‐1‐(3‐methoxyphenyl)‐cyclohexanol hydrochloride, is a widely used centrally acting analgesic. The analgesic effect following parenteral administration of tramadol is due to synergistic interaction between its agonist activity toward opioid receptors and its antinociceptive effect mediated by inhibition of neuronal reuptake of monoamines in the brain.¹ Thus, the analgesic activity should be dependent on the concentration of unbound tramadol in the vicinity of both μ‐opioid receptors and monoamine transporters, such as serotonin transporter (SERT) and norepinephrine transporter (NET), in the brain. Since the concentration of unbound tramadol in the brain would be at least partly determined by the blood–brain barrier (BBB) transport characteristics of tramadol, an understanding of the transport characteristics is important for predicting the onset and duration of analgesic activity of tramadol.

The BBB dynamically regulates the transfer of endogenous nutrients, waste products, and drugs between blood and brain interstitial fluid (ISF),² depending upon the functions of various transporters and receptors localized on the brain capillary endothelial cell membrane.² Opioids, such as morphine and oxycodone,³ and opioid‐like analgesic peptides, such as H‐Tyr‐D‐Arg‐Phe‐beta‐Ala‐OH (TAPA)⁴ and ebiratide,⁵ are transported through the BBB via both identified and unidentified transporters and receptors. Recently, we have shown that oxycodone is actively taken up into rodent brain capillary endothelial cells by proton‐coupled organic cation (H⁺/OC) antiporter.³ Other organic cationic drugs, such as diphenhydramine,6., 7. pyrilamine,3., 7. nicotine,8., 9. and clonidine,¹⁰ are also transported by the H⁺/OC antiporter. Tramadol (pKa 9.41),¹¹ which contains a tertiary amine moiety, is present in cationic form at physiological pH. The tramadol concentration in brain is approximately five times higher than that in plasma¹² and the unbound brain to plasma (K_p,uu,brain) concentration ratio has been indirectly estimated to be greater than unity using the rat brain slice method.¹² Thus, tramadol may be actively transported into the brain by the H⁺/OC antiporter across the BBB.

Functional transporters at the human BBB are important not only for pharmacotherapy to treat cerebral diseases, but also for development of new central nervous system (CNS)‐acting drugs. Human immortalized brain capillary endothelial cells (hCMEC/D3)¹³ retain many of the morphological and functional characteristics of the human BBB in terms of expression of tight‐junction proteins, as well as various ABC and several SLC transporters.2., 14., 15., 16. Our laboratory has shown recently that H⁺/OC antiporter is functionally expressed in hCMEC/D3,⁶ and therefore in vitro uptake studies using hCMEC/D3 cells may be useful to predict tramadol concentration in the human brain, and hence its analgesic effect. However, it is first necessary to establish the transport mechanism of tramadol.

The aim of this study, therefore, was to examine the transport mechanism of tramadol in vivo and in hCMEC/D3 cells. First, we measured the K_p,uu,brain value of tramadol using the rat brain microdialysis technique in order to obtain evidence for active uptake of tramadol through the BBB in vivo. We then investigated the mechanism of tramadol transport using hCMEC/D3 cells in vitro.

Section snippets

Reagents

Tramadol hydrochloride was purchased from Wako Pure Chemical Industries (Osaka, Japan). All other chemicals and reagents were commercial products of reagent grade.

Cell Culture

hCMEC/D3 cells had been immortalized by lentiviral transduction of the catalytic subunit of human telomerase and SV40‐T antigen.¹³ The cells were cultivated at 37°C in EBM‐2 medium (Lonza, Basel, Switzerland) supplemented with 2.5% fetal bovine serum, 0.025% VEGF, 0.025% R3‐IGF, 0.025% hEGF, 0.01% hydrocortisone, 5 μg/mL bFGF, 1%

In Vitro and In Vivo Permeability Rate Constants and In Vivo Microdialysis Study

The in vitro permeability rate constants (PA_vitro) of tramadol and antipyrine, determined in KRP buffer at 37°C, were 0.435 ± 0.0152 and 0.497 ± 0.0159, respectively. The in vivo permeability rate constant (PA_vivo) of antipyrine was 0.0929 ± 0.0080. The R_d value was therefore calculated to be 0.190 ± 0.0178.

The brain ISF concentration (C_isf) of tramadol was extrapolated using the reference method (Eq. 5) from the dialysate concentration (C_d,vivo), PA_vitro, and R_d values. The unbound fraction in

Discussion

In the present study, BBB transport of tramadol was characterized by means of both rat microdialysis and in vitro uptake studies using immortalized human brain capillary endothelial hCMEC/D3 cells. The unbound concentration of tramadol in brain ISF was larger than that in plasma and the K_p,uu,brain value was 2.30 ± 0.37, suggesting active uptake into the brain across the BBB. Tramadol exists as over 98% cationic form at physiological pH,¹¹ and might be actively transported through the BBB by

Conclusions

Our results indicate that tramadol has 2.3‐fold higher unbound concentration in the brain than in plasma in rats, suggesting that it is actively taken up into the brain from circulating blood. In vitro uptake study using hCMEC/D3 cells suggested that H⁺/OC antiporter plays a major role in active transport of tramadol across the human BBB.

Acknowledgments

We thank Dr. Pierre‐Olivier Couraud (Institut Cochin, Paris, France) for supplying hCMEC/D3 cells under license from INSERM. This work was supported in part by a Grant‐in‐Aid for Scientific Research and by the MEXT‐Supported Program for the Strategic Research Foundation at Private Universities provided by The Ministry of Education, Culture, Sports, Science and Technology.

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RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and MetabolismTransport Characteristics of Tramadol in the Blood–Brain Barrier

Abstract

Introduction

Section snippets

Reagents

Cell Culture

In Vitro and In Vivo Permeability Rate Constants and In Vivo Microdialysis Study

Discussion

Conclusions

Acknowledgments

Opioid and nonopioid components independently contribute to the mechanism of action of tramadol, an “atypical” opioid analgesic

J Pharmacol Exp Ther

Contribution of carrier‐mediated transport systems to the blood–brain barrier as a supporting and protecting interface for the brain; importance for CNS drug discovery and development

Pharm Res

Involvement of the pyrilamine transporter, a putative organic cation transporter, in blood–brain barrier transport of oxycodone

Drug Metab Dispos

Blood–brain barrier transport of a novel micro 1‐specific opioid peptide, H‐Tyr‐D‐Arg‐Phe‐beta‐Ala‐OH (TAPA)

J Neurochem

In‐vivo blood–brain barrier transport of a novel adrenocorticotropic hormone analogue, ebiratide, demonstrated by brain microdialysis and capillary depletion methods

J Pharm Pharmacol

Diphenhydramine active uptake at the blood–brain barrier and its interaction with oxycodone in vitro and in vivo

J Pharm Sci

Functional expression of a proton‐coupled organic cation (H+/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model

Fluids Barriers CNS

Coexistence of passive and proton antiporter‐mediated processes in nicotine transport at the mouse blood–brain barrier

AAPS J

Blood‐to‐brain influx transport of nicotine at the rat blood–brain barrier: Involvement of a pyrilamine‐sensitive organic cation transport process

Neurochem Int

Clonidine transport at the mouse blood–brain barrier by a new H+ antiporter that interacts with addictive drugs

J Cereb Blood Flow Metab

Synthesis and characterization of molecularly imprinted polymer for controlled release of tramadol

Cent Eur J Chem

Structure‐brain exposure relationships in rat and human using a novel data set of unbound drug concentrations in brain interstitial and cerebrospinal fluids

J Med Chem

RESEARCH ARTICLE – Pharmacokinetics, Pharmacodynamics and Drug Transport and Metabolism
Transport Characteristics of Tramadol in the Blood–Brain Barrier

Functional expression of a proton‐coupled organic cation (H⁺/OC) antiporter in human brain capillary endothelial cell line hCMEC/D3, a human blood–brain barrier model

Clonidine transport at the mouse blood–brain barrier by a new H⁺ antiporter that interacts with addictive drugs