Evaluation of the P-glycoprotein (Abcb1) affinity status of a series of morphine analogs: Comparative study with meperidine analogs to identify opioids with minimal P-glycoprotein interactions

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Abstract

One of the major shortcomings of many commonly used opioids is the fact that they are P-gp substrates, which represents a major obstacle towards effective pain management. P-gp can affect opioids’ oral absorption, CNS accumulation, systemic clearance, antinociceptive activity, and tolerance development to their analgesic effects. Moreover, P-gp can be the locus of drug–drug interactions between opioids and other concomitantly administered drugs that are P-gp substrates/inhibitors. The objective of this study was to identify opioids that are non-P-gp substrates to overcome some of the mentioned shortcomings. We evaluated the P-gp affinity status (substrate, non-substrate, or inhibitor) of a series of morphine analogs (10 opioid agonist and 2 opioid antagonists) and compared them to previously reported meperidine analogs. The fold stimulation of the morphine analogs ranged from 1.01 to 1.54 while for the meperidine analogs the fold stimulation ranged from 1.10 to 3.66. From each series (morphine and meperidine analogs) we selected potential candidate opioids that are non-P-gp substrates and conducted in vivo assessments of their antinociceptive effects using P-gp knockout and P-gp competent mice. 6-Desoxymorphine, meperidine and N-phenylbutyl normeperidine did not significantly (p > 0.05) stimulate the basal P-gp ATPase activity, where, the fold stimulations of the basal P-gp ATPase activity were 1.01 ± 0.11, 1.51 ± 0.29 and 1.10 ± 0.23, respectively. Evaluation of the influence of P-gp ablation on their antinociceptive effects indicated that P-gp did not significantly (p > 0.05) affect their antinociceptive effects. Among the evaluated opioids in vivo, 6-desoxymorphine showed high potency and induced no apparent toxicity upon low- and high-dose administration. 6-Desoxymorphine is therefore an ideal lead compound to create a library of opioids that have negligible P-gp affinity for better management of pain.

Introduction

Opioid agonists are potent analgesics that are commonly used for management of moderate to severe pain (Glare and Walsh, 1993). In order for opioids to exert their central action they need to bypass a formidable barrier, the blood brain barrier (BBB), to bind to the opioid receptors (μ, δ, κ) located in the CNS. The BBB is composed of endothelial cells that express a number of efflux transporters such as P-glycoprotein (P-gp, Abcb1), multidrug resistance associated proteins (Mrp, Abcc) and breast cancer related proteins (Bcrp, Abcg2). These efflux transporters act as a defense mechanism that protects the CNS from various xenobiotics such as opioids. Among the efflux transporters, P-gp was the first described and the most extensively studied efflux transporter. P-gp is encoded by mdr1a and mdr1b genes in rodents and MDR1 and MDR3 genes in humans and is well known to play a pivotal role in modulating the PK/PD of many therapeutic agents including opioids (Lin and Yamazaki, 2003, Dagenais et al., 2004).

Extensive studies indicated that P-gp can modulate the permeability, uptake, disposition and antinociceptive activities of opioids. For example, chemical and genetic disruption of P-gp using P-gp inhibitors and P-gp knockout mice suggested that P-gp had a significant impact on the cellular accumulation and the antinociceptive activity of many opioids (e.g., morphine, oxycodone, methadone, fentanyl, loperamide and DPDPE) (Chen and Pollack, 1999, Letrent et al., 1999a, Wandel et al., 2002, Skarke et al., 2003, Dagenais et al., 2004, Hoffmaster et al., 2004, Hassan et al., 2007). Overexpression of P-gp in cultured cells minimized the cellular accumulation of both synthetic and natural opioids (Callaghan and Riordan, 1993). One extreme example that manifests the adverse effects of P-gp on opioids is the active efflux of loperamide from the CNS by P-gp. As a result, loperamide, the potent opioid agonist in vitro, had no centrally mediated antinociceptive effect but rather a gastrointestinal effect (anti-diarrheal effect). These differential effects are believed to be due to the high affinity of loperamide to P-gp which actively extrudes loperamide out of the CNS and negates its antinociceptive activity. Interestingly, when P-gp was inhibited either chemically or genetically, central antinociceptive effects were observed for loperamide (Schinkel et al., 1996, Kalvass et al., 2004).

P-gp is also believed to be involved in tolerance development to opioids where P-gp expression was induced by 2-fold in brain tissues of morphine tolerant rats (Aquilante et al., 2000). In our laboratory we demonstrated that many opioids are P-gp substrates. For example, we demonstrated that oxycodone is a P-gp substrate and when repeatedly administered, it induced the expression of P-gp in brain, liver, intestine and kidney tissues of rats. This P-gp induction resulted in a P-gp-mediated drug–drug interaction when paclitaxel, the P-gp substrate, was administered to oxycodone treated rats (Hassan et al., 2007). Using microarray analysis, we observed that the expression levels of many efflux transporters were significantly regulated in brain and liver tissues of oxycodone treated rats (unpublished data). In addition, we demonstrated that many novel and known meperidine analogs were P-gp substrates (Mercer et al., 2007). Finally, we evaluated the P-gp affinity status (substrate, non-substrate, or inhibitor) of a representative opioid agonist (methadone), opioid agonist/antagonist (buprenorphine) and opioid antagonist (diprenorphine) using two in vitro (P-gp ATPase activity and monolayer efflux assays) and two in vivo (tissue distribution and antinociceptive monitoring in mdr1a/b (+/+) and mdr1a/b (−/−) mice (Hassan et al., 2009) and there was a good agreement among the four assays. The CNS distribution and the antinociceptive activity of methadone but not buprenorphine or diprenorphine were significantly (p < 0.05) dependant on P-gp. Based on these studies it is clear that one of the major shortcomings of the currently used opioid agonists is the fact that they are P-gp substrates. P-gp affects their (1) oral absorption, (2) CNS accumulation, (3) systemic clearance, (4) antinociceptive effects, and (5) tolerance development to their analgesic effects. In addition, P-gp can be the locus of drug–drug interactions between opioids and other concomitantly administered therapeutic agents that are P-gp substrates. It is therefore of great therapeutic importance to develop opioids that are not P-gp substrates. These new opioids are expected to have better BBB permeability, better antinociceptive activity, delayed development of tolerance and minimal P-gp-mediated drug–drug interactions. In this regard, we previously synthesized and tested the P-gp affinity status of a series of meperidine analogs (n = 11), searching for potent and specific opioids that have minimal P-gp affinity (Mercer et al., 2007). In expansion of our work we evaluated the P-gp ATPase activity of another series of morphine analogs (n = 12) and compared them to the previously synthesized meperidine analogs. From each series (morphine and meperidine analogs) we selected potential candidate opioids that are non-P-gp substrates and conducted in vivo assessments of their antinociceptive effects using P-gp knockout and P-gp competent mice.

Section snippets

Drug-stimulated P-gp ATPase activity

Drug stimulated P-gp ATPase activity was estimated by Pgp-GIo assay system (Promega, Madison, WI). This method relies on the ATP dependence of the light-generating reaction of firefly luciferase. ATP consumption is detected as a decrease in luminescence. In a 96-well plate, recombinant human P-gp (25 μg) was incubated with P-gp-GIo assay buffer™ (20 μl) (control, n = 4), verapamil (200 μM) (n = 4), methadone (100 μM) (n = 4), sodium orthovanadate (100 μM) (n = 4), and morphine analogs listed in Table 1 (200 

Effect of opioids on P-gp ATPase activity

Morphine analogs (Fig. 1A) were examined to determine their effects on the P-gp ATPase activity. Each opioid together with a known excess of ATP was incubated with recombinant human P-gp. ATP consumption due to P-gp stimulation by each opioid was detected as a decrease in luminescence (i.e., the higher the potency of a compound to stimulate the P-gp ATPase activity, the lower the luminescence signal). The positive controls, verapamil and methadone stimulated the P-gp basal activity by 5.41 ± 1.52

Discussion

The objective of this study was to evaluate the P-gp affinity status of a series of morphine and meperidine analogs to identify opioids with minimal P-gp interaction for better and effective management of pain. Since the majority of opioids have been reported to be potential P-gp substrates, P-gp can significantly affect their pharmacokinetics, pharmacodynamics and their therapeutic effects (Letrent et al., 1998, Letrent et al., 1999a, Letrent et al., 1999b, Aquilante et al., 2000, Dagenais et

Acknowledgments

This study was supported in part by a University of Maryland Intramural Research grant (A.C.), a Predoctoral Fellowship from the Egyptian Ministry of Higher Education (H.E.H.), Ralph Shangraw Predoctoral Fellowship from University of Maryland (H.E.H.) and a Ruth L. Kirschstein National Research Service Award from the national Institute on Drug Abuse (N.R.S.A., N.I.D.A.) (C.W.C).

References (41)

  • W.T. Beaver et al.

    Comparisons of the analgesic effects of oral and intramuscular oxymorphone and of intramuscular oxymorphone and morphine in patients with cancer

    J. Clin. Pharmacol.

    (1977)
  • K.W. Bentley et al.

    Novel analgesics and molecular rearrangements in the morphine-thebaine group. II. Alcohols derived from 6,14-endo-etheno- and 6,14-endo-ethanotetrahydrothebaine

    J. Am. Chem. Soc.

    (1967)
  • A. Casy et al.

    Opioid Analgesics: Chemistry and Receptors

    (1986)
  • C. Chen et al.

    Enhanced antinociception of the model opioid peptide [d-penicillamine] enkephalin by P-glycoprotein modulation

    Pharm. Res.

    (1999)
  • F. D’amour et al.

    A method for determining loss of pain sensation

    J. Pharmacol. Exp. Ther.

    (1941)
  • P.A. Glare et al.

    Dose-ranging study of oxycodone for chronic pain in advanced cancer

    J. Clin. Oncol.

    (1993)
  • M.M. Gottesman et al.

    Biochemistry of multidrug resistance mediated by the multidrug transporter

    Annu. Rev. Biochem.

    (1993)
  • Hassan, H.E., Myers, A.L., Coop, A., Eddington, N.D., 2009, April 15. Differential Involvement of P-glycoprotein...
  • K.A. Hoffmaster et al.

    Hepatobiliary disposition of the metabolically stable opioid peptide [D-Pen2, D-Pen5]-enkephalin (DPDPE): pharmacokinetic consequences of the interplay between multiple transport systems

    J. Pharmacol. Exp. Ther.

    (2004)
  • I. Iijima et al.

    Studies in the (+)-morphinan series. 5. Synthesis and biological properties of (+)-naloxone

    J. Med. Chem.

    (1978)
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