Pharmacological characterization of the dermorphin analog [Dmt1]DALDA, a highly potent and selective μ-opioid peptide
Introduction
Opioid analgesics have remained the mainstay in the management of pain and μ-opioids are the most widely used. μ-Opioids comprise a wide range of drugs and structures. These agents are defined by their high affinity and selectivity for μ-opioid sites in traditional receptor binding assays and they share many pharmacological properties, such as tolerance, dependence, respiratory depression and inhibition of gastrointestinal transit. Yet, these drugs also display many differences, both clinically and in animal studies (Pasternak, 2001). Patients often respond better to one drug than another and the presence of incomplete cross-tolerance among these drugs has led to the use of opioid rotation in pain management. Animal studies confirm many differences among μ-opioid analgesics. A number of μ-opioids, including morphine-6β-glucuronide, heroin, 6-acetylmorphine and fentanyl, show incomplete cross-tolerance to morphine and retain their potencies in CXBK mice that are insensitive to morphine (Rossi et al., 1996). The analgesia elicited by various μ-opioids also involve different G-proteins Standifer et al., 1996, Standifer and Pasternak, 1997. The cloning of a μ-opioid receptor, MOR-1, was soon followed by antisense studies revealing dramatic differences between morphine and other μ-opioids Rossi et al., 1994, Rossi et al., 1995a, Rossi et al., 1997. In these experiments, antisense oligodeoxynucleotide probes, targeting various exons within the μ-opioid receptor gene (Oprm), differentially block the analgesic actions of either morphine or morphine-6β-glucuronide. A knockout model in which exon 1 of the MOR-1 receptor was disrupted, further separated the actions of morphine from both morphine-6β-glucuronide and heroin (Schuller et al., 1999). Like other knockout models disrupting the μ-opioid receptor gene Loh et al., 1998, Matthes et al., 1996, Sora et al., 1997, morphine was totally inactive in these mice. Yet, morphine-6β-glucuronide, heroin and 6-acteylmorphine all retained full analgesic activity, although their potency was slightly diminished. Together, these observations illustrate the marked pharmacological differences among μ-opioid analgesics. These differences also raise the possibility of additional new μ-opioid drugs with unusual pharmacological profiles.
The tetrapeptide DALDA (H-Tyr-d-Arg-Phe-Lys-NH2), an analog of the tetrapeptide segment of the highly selective μ-opioid peptide dermorphin, possesses high selectivity for the μ-opioid receptor (Schiller et al., 1989). DALDA is highly polar and metabolically stable, with limited penetration of the blood–brain barrier (Samii et al., 1994) and the placental barrier (Holsey et al., 1999). Its inability to traverse these barriers, along with its metabolic stability, may lead to a potential utility as a peripheral analgesic, particularly in obstetrics.
More recently, in an effort to further increase the potency of DALDA, a number of analogues containing structural modifications of the tyrosine residue were synthesised. One such analog, [Dmt1]DALDA ([2,6-dimethyl-Tyr]-d-Arg-Phe-Lys-NH2), has superior potency and selectivity for the μ-opioid receptor (Schiller et al., 2000). Interestingly, [Dmt1]DALDA produces systemic analgesia with a potency approximately 100-fold greater than morphine (Schiller, personal communication). This was clearly unexpected due to the very polar nature of the compound. The following study was undertaken to further characterize [Dmt1]DALDA pharmacology.
Section snippets
Drugs
All radioligands were purchased from DuPont NEN (Boston, MA), with the exception of [3H]naloxone benzoylhydrazone, which was synthesized as previously described (Price et al., 1989). [Dmt1]DALDA (MW 981) was synthesized in the laboratory of P.W.S. Morphine sulfate, morphine-6-glucoronide, naloxone, β-funaltrexamine, naloxonazine, and 3-methoxynaltrexone were obtained from the Research Technology Branch of the National Institute of Drug Abuse (Rockville, MD). Antisense oligodeoxynucleotides
Radioligand binding
In competition binding assays, [Dmt1]DALDA bound with high selectivity to the μ receptor, affording Ki values in the low nanomolar range (Table 1) with somewhat greater selectivity for μ1. Analysis of binding to κ1 and κ3 receptors revealed a best fit to a two-site curve. The higher affinity [Dmt1]DALDA binding component in the κ3 receptor competition most likely represents competition of residual μ-opioid receptor binding in the assay (Clark et al., 1989). The selectivity ratio of [Dmt1]DALDA
Discussion
[Dmt1]DALDA is a highly potent and selective μ-opioid analgesic with a unique mechanism of action distinct from that of morphine. It was surprising to see the extraordinary analgesic potency of [Dmt1]DALDA both spinally and systemically, being 5000- and 200-fold, respectively, more potent than morphine. The activity of systemic [Dmt1]DALDA could not result solely from peripheral actions for a number of reasons. First, in a model of peripheral analgesia the potency of [Dmt1]DALDA intradermally
Acknowledgements
The authors would like to thank Drs. Michael King, Ricardo Cruciani, and Wendy Su for their additional input with the animal studies. This work was supported, in part, by a research grant (DA07242) and a Senior Scientist Award (DA00220) to GWP from the National Institute on Drug Abuse, and a Center Award (CA08748) from the National Cancer Institute.
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