We hypothesize that the development of peripherally-restricted, mixed-activity opioid receptor agonists will
produce robust analgesia while exhibiting reduced side effects, including a lack of respiratory depression or
reinforcing properties. In preliminary studies, the in vitro screening of novel bis-imidazolidines and fused
heterocyclic lead compounds BIM-22 and FDC-14 identified mixed-opioid receptor agonist activity. After
peripheral (i.p.) administration to mice, both compounds demonstrated antinociception equivalent to morphine
in the 55oC warm-water tail withdrawal (tail-flick) assay, but FDC-14 was 25 times more potent than morphine
in the acetic acid writhing test, with an antinociceptive potency ratio of 72.7, suggestive of peripherally-
restricted activity. Confirming this, LC-MS/MS studies detected BIM-22 and FDC-14 in harvested mouse blood,
but not brain, after i.v. administration. This proposal addresses two research areas of particular interest for this
FOA: i) The development of new and innovative molecular probes for receptors and new strategies for
innovative peptidomimetic design, and ii) The identification of structurally diverse, orally active, metabolically
stable peripherally-restricted opioid agonists. We propose six interacting aims: In Aim 1, we propose the
computationally guided synthesis of heterocyclic peptidomimetics: bis-imidazolidin-2-imines, piperazines, bis-
piperazine, bis-imidazolone and fused heterocyclic libraries. In Aim 2, we will screen all compounds in
competition radioligand binding assays to determine affinity for μ- (MOR), - (DOR), and κ- (KOR) opioid
receptors, and in functional assays to identify dual δ/κ or dual δ/µ agonists. In Aim 3, ten selected compounds
will be evaluated in vitro for pharmacokinetic properties and screened in vivo for antinociception using the
mouse tail-flick assay. Lead compounds identified from this aim will guide 2nd generation SAR studies to
enhance peripherally-selective activity. In Aim 4: we will perform full in vivo antinociceptive characterization of
lead agonist compounds. The most stable, active 4 agonists not crossing the BBB in Aims 2+3 will be
evaluated after i.p. administration with mouse tail-flick and acetic-acid writhing assays for efficacy, duration of
action, and opioid receptor selectivity. In Aim 5: we will characterize the bioavailability in mice of the selected 4
agonists following oral administration and confirm their inability to penetrate the blood brain barrier. In Aim 6:
The two most potent bioavailable novel agonists identified in Aims 1-5 will be examined for liabilities,
specifically antinociceptive tolerance, respiratory and hyperlocomotor effects in the CLAMS physiological and
behavioral assessment system, sedation and disruption of coordinated locomotor activity in the rotorod assay,
and assessment for rewarding or aversive effects in the conditioned place preference (CPP) assay. Effects on
GI transit will also be evaluated to assess effects on constipation. In summary, we expect to generate novel
peripherally-restricted, mixed-activity peptidomimetic opioid receptor agonists as both probes and analgesics
with reduced side effects, thereby significantly impacting analgesic development.
