Shou-wu Ma

Dynorphin A activates bradykinin receptors to maintain neuropathic pain

Dynorphin A is an endogenous opioid peptide that produces non-opioid receptor-mediated neural excitation. Here we demonstrate that dynorphin induces calcium influx via voltage-sensitive calcium channels in sensory neurons by activating bradykinin receptors. This action of dynorphin at bradykinin receptors is distinct from the primary signaling pathway activated by bradykinin and underlies the hyperalgesia produced by pharmacological administration of dynorphin by the spinal route in rats and mice. Blockade of spinal B1 or B2 receptor also reverses persistent neuropathic pain but only when there is sustained elevation of endogenous spinal dynorphin, which is required for maintenance of neuropathic pain. These data reveal a mechanism for endogenous dynorphin to promote pain through its agonist action at bradykinin receptors and suggest new avenues for therapeutic intervention.

'Knock-down' of spinal CB1 receptors produces abnormal pain and elevates spinal dynorphin content in mice.

&NA; Recent studies demonstrate the possible existence of tonic modulatory control of nociceptive input mediated by spinal cannabinoid receptors (CB1). Accordingly, it is predicted that a reduction in the spinal CB1 receptors may enhance sensitivity to sensory stimuli and a decrease in spinal antinociceptive potency to cannabinoid agonists. An antisense oligodeoxynucleotide (ODN) specific to the CB1 receptor was used to ‘knock‐down’ CB1 receptors in the lumbar spinal cord and dorsal root ganglia by the local, repeated intrathecal (i.th.) administration of the ODN. This treatment resulted in a decrease in lumbar spinal CB1 receptor expression accompanied by a decrease in the response thresholds to both innocuous tactile and noxious thermal stimuli. The antinociceptive action of the CB1 agonist, WIN 55,212‐2, by i.th. administration was also significantly attenuated after treatment with the antisense ODN. Similar treatment using a mismatch control ODN had no effect on receptor protein or on sensory thresholds. The effects of the antisense ODN treatment on sensory thresholds were fully reversed after discontinuation of the ODN injection. The antisense ODN treated rats also showed a significant increase in lumbar spinal dynorphin A. Acute i.th. injection of MK‐801 or an antidynorphin antiserum blocked the antisense ODN‐induced tactile and thermal hypersensitivity. These data support the possibility of endogenous inhibitory cannabinoid tone to limit spinal afferent input of thermal and tactile stimuli. Lifting of this inhibitory tone through a ‘knock‐down’ of spinal CB1 receptors apparently lowers the thresholds for sensory input, as reflected by the actions of MK‐801 to block tactile and thermal hypersensitivity. The increased spinal dynorphin may act to further promote afferent outflow and abnormal pain because sequestration of spinal dynorphin with antiserum also reverses the manifestations of abnormal pain following knock‐down of CB1 receptors.

Design of novel peptide ligands which have opioid agonist activity and CCK antagonist activity for the treatment of pain

Disease states such as neuropathic pain offer special challenges in drug design due to the system changes which accompany these diseases. In this manuscript we provide an example of a new approach to drug design in which we have modified a potent and selective peptide ligand for the CCK-2 receptor to a peptide which has potent agonist binding affinity and bioactivity at delta and mu opioid receptors, and simultaneous antagonist activity at CCK receptors. De novo design based on the concept of overlapping pharmacophores was a central hypothesis of this design, and led to compounds such as H-Tyr-DPhe-Gly-DTrp-NMeNle-Asp-Phe-NH(2) (i.e., RSA 601) which have the designed properties.

Pharmacological characterization of the cloned kappa opioid receptor as a kappa(1b) subtype

SUBSTANTIAL pharmacological evidence in vitro and in vivo has suggested the existence of subtypes of the kappa opioid receptor. Quantitative radioligand binding techniques resolved the presence of two high affinity binding sites for the k1 ligand [3H]U69,593 in mouse brain membranes, termed k1a and k1b, respectively. Whereas the k1a site has high affinity for fedotozine and oxymorphindole and low affinity for bremazocine and α-neoendorphin, site k1b has high affinity for bremazocine and α-neoendorphin and low affinity for fedotozine and oxymorphindole. CI-977 and 1169,593 bind equally well at both sites. To determine the relationship between these k1 receptor subtypes and the recently cloned mouse k1 receptor (KOR), we examined [3H]U69,593 binding to the KOR in stably transfected cells (KORCHN-8). Competition of [3H]U69,593 binding to the KOR by bremazocine, α-neoendorphin, fedotozine and oxymorphindole resolved a single class of binding sites at which these agents had binding affinities similar to that of the k1b site present in mouse brain. These results suggest that the cloned KOR corresponds to the k1 site in mouse brain defined as k1b.

Designing Peptide Drugs/Ligands for Pathological States. A New Paradigm for Design of Bioactive Peptide Hormones and Neurotransmitters

G-protein coupled receptors and other membrane bound receptors are the target for over 45% of all current drugs. They are important targets because the ligands and receptors modulate or control most biological functions necessary for survival in man, such as fear-flight, feeding behavior, response to stress, response to pain, cardiovascular function, anxiety, learning, sexual behavior, etc. Most of the ligands are polypeptides. The present paradigm for drug discovery and development in this area is to identify the gene/cell/tissue/protein associated with the biological activity and develop a binding assay and functional (2nd messenger) assay. If no endogenous “ligand” is known, assays with “libraries” of “natural products,” peptides and other organic compounds are used. The receptors and the assays chosen utilize receptors, cells, tissues and animals that are “normal.” However, there is growing evidence that the ligands and their receptors/acceptors behave differently in normal and pathological states. Especially in the central nervous system (CNS), many drugs that are effective in normal states become ineffective or less effective in pathological disease states. Evaluation of this problem suggests that a new paradigm is needed.

Design and Synthesis of Bifunctional Peptides: Antagonists at CCKA/CCKB Receptors and Agonists at μ/δ Opioid Receptors

Endogenous CCK has been demonstrated to exhibit anti-opioid effects, particularly in the spinal cord [1], We have hypothesized that the development of novel ligands which possess properties as antagonists at CCK receptors, and as agonists at opioid receptors within the same molecule would present a significant therapeutic advantage in the treatment of pain states refractory to commonly employed opioids such as morphine. Our research has been aimed at the development of ligands that could interact simultaneously and potently with opioid receptors as agonists and with CCK receptors as antagonists.