Opioids and Efflux Transporters. 1. P-Glycoprotein Substrate Activity of N-Substituted Analogs of Meperidine.

Abstract

P-Glycoprotein (P-gp) is an efflux transporter which is up-regulated at the blood brain barrier in both morphine and oxycodone tolerant rats. Numerous studies have shown that many clinically employed opioid analgesics are substrates for P-gp, suggesting that up-regulation of P-gp may contribute to the development of central tolerance to opioids. The studies herein focus on the development of SAR for P-gp substrate activity in the meperidine series of compounds, and show that a meperidine analog of greater potency, N-phenylbutyl-N-normeperidine, has low activity as a P-gp substrate and has the potential to be utilized as a tool to study the contribution of P-gp on the development of central tolerance to opioids.

The development of improved agents for the treatment of chronic pain remains an important goal in public health¹. The vast majority of currently employed agents for the treatment of severe chronic pain are opioid analgesics, which act as agonists at mu opioid receptors in the CNS.² Unfortunately, clinically employed opioid analgesics suffer from the development of tolerance, necessitating escalating doses to maintain the patient in a pain-free state³, thereby leading to escalated side-effects such as constipation^{4, 5}. Numerous mechanisms at the receptor and cellular level have been indicated in the development of tolerance to mu opioid receptor agonists⁶, but recent reports have suggested that efflux transporters at the blood-brain barrier (BBB) may also contribute towards the development of central tolerance^{7, 8}. P-glycoprotein (P-gp) is an efflux transporter which is located in numerous tissues⁹, and its function at the BBB is to actively remove xenobiotics from the CNS⁹. Two commonly employed opioid analgesics, morphine (1) and oxycodone (2) (Figure 1), are known substrates for this transporter and rats tolerant to both morphine⁸ and oxycodone⁷ show up-regulation in P-gp level at the BBB Thus, on chronic administration, the up-regulated P-gp would be expected to result in lower brain concentrations of opioid thereby exacerbating tolerance to the central analgesic effects. P-gp knockout animals are available and offer a useful model to study the effects of P-gp on opioids¹⁰, but an alternative approach in wild-type animals is the development of mu opioid receptor agonists which are not substrates for P-gp. These compounds would allow a full investigation of the contribution of up-regulated P-gp to opioid tolerance as full cross-tolerance between morphine and the opioid with no P-gp substrate activity would not be anticipated to occur, and also potentially be developed into opioid analgesics with lower degrees of tolerance.

Figure 1.

Morphine (1) and oxycodone (2)

The mu opioid analgesic meperidine (3) has been shown to possess low activity as a P-gp substrate¹¹, but only moderate antinociceptive activity in vivo^{12, 13}, thus initial investigations described herein are focused on delineating the structure-activity relationships (SAR) of the N-substituent in this class for low P-gp substrate activity, while increasing mu opioid potency based on known SAR for potency in this series¹³.

A range of previously reported and novel N-substituted analogs of meperidine were prepared from nitrile 4 (obtained from Sigma-Aldrich, Inc), via alkylation with alkyl halides in DMF in the presence of K₂CO₃, followed by hydrolysis of the nitrile to the ethyl esters (5–14) through treatment with H₂SO₄ and EtOH¹⁴ (Scheme 1). The alkyl substituents were chosen based on known active merperidine analogs and also following a series as previously described for other classes of opioids¹⁵, and include arylalky, alkyl, and branched alkyl groups (Table 1).

Scheme 1.

Table 1.

Compounds prepared, salt form, yield, and melting points. Citations reference previously known compounds.

R	Ester	Salt, Yield, mp
CH3	314	Oxalate, 7%, 190–192°C
(CH2)2(C6H5)	516	Oxalate, 33%, 205–20°C
(CH2)3(C6H5)	617	Oxalate, 14%, 225°C
(CH2)4(C6H5)	717	Oxalate, 25%, 170°C
CH2(C6H5)	814	Oxalate, 46%, 204–205°C
CH2CH=CH2	918	Oxalate, 40%, 213–214°C
(CH2)2CH3	1019	Oxalate, 57%, 215–216°C
CH2CH=CHCH3	11	Oxalate, 67%, 173–177°C
(CH2)3CH3	1219	Oxalate, 24%, 190–192°C
CH2C(CH3)=CH2	13	Oxalate, 35%, 180–181°C
CH2CH(CH3)2	14	Oxalate, 55%, 165–167°C

All esters were converted to oxalate salts (Scheme 1). Drug stimulated P-gp ATPase activity was estimated by Pgp-Glo assay system²⁰ (Promega, Madison, WI) and the results were shown in Figure 2. This method relies on the ATP dependence of the light-generating reaction of firefly luciferase where ATP consumption is detected as a decrease in luminescence, the greater the decrease in signal the higher the P-gp activity. Sodium orthovanadate was used as a P-gp ATPase inhibitor, whereas verapamil was used as a positive control. Test compounds (all tested at 200 μM) which are significantly lower than the control (NT) are P-gp substrates, whereas test compounds significantly higher than the NT are P-gp inhibitors. Compounds which are not significantly different from the NT are neither P-gp substrates nor inhibitors.

Figure 2.

Results of compounds and standards in the Pgp-Glo assay system; all compounds assayed at 200 μM. Data are represented as mean ± SEM (n=4). * indicates significant difference from the control at p <0.05 as indicated by t-test.

The P-gp substrate activity of the esters showed differences depending on the nature of the N-substituent. Most analogs were substrates for P-gp like verapermil, with the exception of meperidine itself and N-phenylbutyl-N-normeperidine (7). These results show a distinct SAR for P-gp substrate activity in this series as N-phenylalkyl analogs of shorter length (phenethyl (5), phenylpropyl (6)) were substrates.

Previous studies have shown that 7 has twice the antinociceptive activity as meperidine¹³. Thus 7 has the profile of low P-gp substrate activity and greater potency than meperidine required for use as a tool to study the contribution of P-gp upregulation on the development of opioid tolerance and cross tolerance between opioids with P-gp substrate activity and opioids that are not P-gp substrates. Studies on this compound are currently underway, and will be reported in due course.