DPyPE - CAS 201036-16-0

Poly(ethylene glycol), or PEG, conjugation to proteins and peptides is a growing technology used to enhance efficacy of therapeutics. This investigation assesses pharmacodynamic and pharmacokinetic characteristics of PEG-conjugated [D-Pen2,D-Pen5]-enkephalin (DPDPE), a met-enkephalin analog, in rodent (in vivo, in situ) and bovine (in vitro) systems. PEG-DPDPE showed increased analgesia (i.v.) compared with nonconjugated form (p < 0.01), despite a 172-fold lower binding affinity for the delta-opioid receptor. [125I]PEG-DPDPE had a 36-fold greater hydrophilicity (p < 0.01) and 12% increase in the unbound plasma protein fraction (p < 0.01), compared with [(125)I]DPDPE. [125I]PEG-DPDPE had a 2.5-fold increase in elimination half-life (p < 0.01), 2.7-fold decrease in volume of distribution (p < 0.01), and a 7-fold decrease in plasma clearance rate (p < 0.01) to [125I]DPDPE. Time course distribution showed significant concentration differences (p < 0.01) in plasma, whole blood, liver, gallbladder, gastrointestinal (GI) content, GI tract, kidneys, spleen, urine, and brain (brain, p < 0.05), between the conjugated and nonconjugated forms. Increased brain uptake of [(125)I]PEG-DPDPE corresponded to analgesia data. [125I]PEG-DPDPE in brain was shown to be 58.9% intact, with 41.1% existing as [125I]DPDPE (metabolite), whereas [125I]DPDPE was 25.7% intact in the brain (at 30 min). In vitro P-glycoprotein affinity was shown for [125I]DPDPE (p < 0.01) but not shown for [125I]PEG-DPDPE. In vitro saturable uptake, with 100 microM DPDPE, was shown for [125I]PEG-DPDPE (p < 0.05). In this study, PEG-conjugated DPDPE seems to act as a prodrug, enhancing peripheral pharmacokinetics, while undergoing hydrolysis in the brain and allowing nonconjugated DPDPE to act at the receptor.

When agonists to alpha(2)adrenergic receptor (AR) and delta opioid receptor (DOR) are co-administered, they act synergistically to inhibit nociceptive elicited behavior. Some previous studies of synergism have used the DOR-selective agonist [D-Pen(2),D-Pen(5)]-enkehphalin (DPDPE), however, DPDPE has been shown to be less potent in mu opioid receptor-knockout (MOR-KO) mice. It is possible, therefore, that MOR contributes to the synergism of DPDPE with the alpha(2)AR agonists. We compared the interactions of spinally administered DPDPE with an alpha(2)AR-adrenergic agonist in MOR-KO and MOR-wildtype (WT) mice. In these mice, morphine is ineffective and the potency of spinally administered DOR agonists, deltorphin II (DELT II) and DPDPE decreased 16- and 250-fold, respectively. Antagonism studies using the MOR-selective antagonist, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Phe-Thr-NH(2) (CTOP) and the DOR-selective antagonist, naltrindole HCl (naltrindole) demonstrated that while DOR mediates DPDPE-induced antinociception in MOR-KO, both MOR and DOR participate in DPDPE antinociception in WT mice, suggesting that DPDPE is less selective for DOR than previously observed in binding studies when given in vivo. The potency of the alpha(2)AR agonist UK14,304 was equivalent in WT and MOR-KO, demonstrating that the loss of opioid-mediated antinociception in the MOR-KO was not due to generalized impairment of antinociceptive processing. Interestingly, isobolographic analysis showed that, despite substantial loss of DPDPE potency in MOR-KO, DPDPE-UK14,304 synergism is fully retained. Collectively, these experiments demonstrate that although MOR participates in DELT II- and DPDPE-mediated spinal antinociception, DOR independently participates in synergistic antinociception with alpha(2)AR. Resolution of the roles of the opioid receptor subtypes in opioid agonist-induced effects may require comparison of the effects of multiple selective agonists in knockout animals.

The conjugation of fluorescent labels to opioid peptides is an extremely challenging task, which needs to be overcome to create new classes of probes for biological assays. Three opioid peptide analogs of biphalin and [D-Pen2,5]-Enkephalin (DPDPE) containing a fluorescein-maleimide motif were synthesized. The biphalin analog 17 binds to opioid receptors with Kiμ = 530 ± 90 nM and Kiδ = 69.8 ± 16.4 nM. We then tested the ability of the compounds to stimulate G-protein-coupling, 17 activated μ-receptor expressing cells (EC50 = 16.7 ± 6.7 nM, EMax = 76 ± 4%) as well as δ-receptor expressing cells (EC50 = 42 ± 10 nM, EMax = 34 ± 8%). However, 17 was not able to fluorescently label receptor in live or fixed cells. Our data suggest that the biphalin scaffold could be employed to develop fluorescent ligands with the appropriate fluorescent motif, and suggest a means for further probe development.