Multiple actions of systemic artemin in experimental neuropathy.

The clinical management of neuropathic pain is particularly challenging. Current therapies for neuropathic pain modulate nerve impulse propagation or synaptic transmission; these therapies are of limited benefit and have undesirable side effects. Injuries to peripheral nerves result in a host of pathophysiological changes associated with the sustained expression of abnormal pain. Here we show that systemic, intermittent administration of artemin produces dose- and time-related reversal of nerve injury-induced pain behavior, together with partial to complete normalization of multiple morphological and neurochemical features of the injury state. These effects of artemin were sustained for at least 28 days. Higher doses of artemin than those completely reversing experimental neuropathic pain did not elicit sensory or motor abnormalities. Our results indicate that the behavioral symptoms of neuropathic pain states can be treated successfully, and that partial to complete reversal of associated morphological and neurochemical changes is achievable with artemin.

Neuropeptide FF receptors have opposing modulatory effects on nociception.

The role of neuropeptide FF (NPFF) and its analogs in pain modulation is ambiguous. Although NPFF was first characterized as an antiopioid peptide, both antinociceptive and pronociceptive effects have been reported, depending on the route of administration. Currently, two NPFF receptors, termed FF1 and FF2, have been identified and cloned, but their roles in pain modulation remain elusive because of the lack of availability of selective compounds suitable for systemic administration in in vivo models. Ligand-binding studies confirm ubiquitous expression of both subtypes in brain, whereas only FF2 receptors are expressed spinally. This disparity in localization has served as the foundation of the hypothesis that FF1 receptors mediate the pronociceptive actions of NPFF. We have identified novel small molecule NPFF receptor agonists and antagonists with varying degrees of FF2/FF1 functional selectivity. Using these pharmacological tools in vivo has allowed us to define the roles of NPFF receptor subtypes as pertains to the modulation of nociception. We demonstrate that selective FF2 agonism does not modulate acute pain but instead ameliorates inflammatory and neuropathic pains. Treatment with a nonselective FF1/FF2 agonist potentiates allodynia in neuropathic rats and increases sensitivity to noxious thermal and to non-noxious mechanical stimuli in normal rats in an FF1 antagonist-reversible manner. Treatment with FF1 antagonists reversed established mechanical allodynia, indicating the possibility of increased NPFF tone through FF1 receptors. In conclusion, we provide evidence for the opposing roles of NPFF receptors and highlight selective FF2 agonism and/or selective FF1 antagonism as potential targets warranting further investigation.

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.

Constitutive activity at the cannabinoid CB1 receptor is required for behavioral response to noxious chemical stimulation of TRPV1: antinociceptive actions of CB1 inverse agonists.

The potential modulation of TRPV1 nociceptive activity by the CB(1) receptor was investigated here using CB(1) wild-type (WT) and knock-out (KO) mice as well as selective CB(1) inverse agonists. No significant differences were detected in baseline thermal thresholds of ICR, CB(1)WT or CB(1)KO mice. Intraplantar capsaicin produced dose- and time-related paw flinch responses in ICR and CB(1)WT mice and induced plasma extravasation yet minimal responses were seen in CB(1)KO animals with no apparent differences in TRPV1 channel expression. Capsaicin-evoked CGRP release from spinal cord tissue and capsaicin-evoked action potentials on isolated skin-nerve preparation were significantly decreased in CB(1)KO mice. Pretreatment with intraplantar galanin and bradykinin, compounds known to sensitize TRPV1 receptors, restored capsaicin-induced flinching in CB(1)KO mice. The possibility that constitutive activity at the CB(1) receptor is required to maintain the TRPV1 receptor in a "sensitized" state was tested using CB(1) inverse agonists. The CB(1) inverse agonists elicited concentration-related inhibition of capsaicin-induced calcium influx in F-11 cells and produced dose-related inhibition of capsaicin-induced flinching in ICR mice. These data suggest that constitutive activity at the CB(1) receptor maintains the TRPV1 channel in a sensitized state responsive to noxious chemical stimuli. Treatment with CB(1) inverse agonists may promote desensitization of the channel resulting in antinociceptive actions against chemical stimulus modalities. These studies propose possible therapeutic exploitation of a novel mechanism providing pain relief by CB(1) inverse agonists.

Control of chronic pain by the ubiquitin proteasome system in the spinal cord.

Chronic pain is maintained in part by long-lasting neuroplastic changes in synapses and several proteins critical for synaptic plasticity are degraded by the ubiquitin-proteasome system (UPS). Here, we show that proteasome inhibitors administered intrathecally or subcutaneously prevented the development and reversed nerve injury-induced pain behavior. They also blocked pathological pain induced by sustained administration of morphine or spinal injection of dynorphin A, an endogenous mediator of chronic pain. Proteasome inhibitors blocked mechanical allodynia and thermal hyperalgesia in all three pain models although they did not modify responses to mechanical stimuli, but partially inhibited responses to thermal stimuli in control rats. In the spinal cord, these compounds abolished the enhanced capsaicin-evoked calcitonin gene-related peptide (CGRP) release and dynorphin A upregulation, both elicited by nerve injury. Model experiments demonstrated that the inhibitors may act directly on dynorphin-producing cells, blocking dynorphin secretion. Thus, the effects of proteasome inhibitors on chronic pain were apparently mediated through several cellular mechanisms indispensable for chronic pain, including those of dynorphin A release and postsynaptic actions, and of CGRP secretion. Levels of several UPS proteins were reduced in animals with neuropathic pain, suggesting that UPS downregulation, like effects of proteasome inhibitors, counteracts the development of chronic pain. The inhibitors did not produce marked or disabling motor disturbances at doses that were used to modify chronic pain. These results suggest that the UPS is a critical intracellular regulator of pathological pain, and that UPS-mediated protein degradation is required for maintenance of chronic pain and nociceptive, but not non-nociceptive responses in normal animals.

Identification and characterization of novel small-molecule protease-activated receptor 2 agonists.

We report the first small-molecule protease-activated receptor (PAR) 2 agonists, AC-55541 [N-[[1-(3-bromo-phenyl)-eth-(E)-ylidene-hydrazinocarbonyl]-(4-oxo-3,4-dihydro-phthalazin-1-yl)-methyl]-benzamide] and AC-264613 [2-oxo-4-phenylpyrrolidine-3-carboxylic acid [1-(3-bromo-phenyl)-(E/Z)-ethylidene]-hydrazide], each representing a distinct chemical series. AC-55541 and AC-264613 each activated PAR2 signaling in cellular proliferation assays, phosphatidylinositol hydrolysis assays, and Ca(2+) mobilization assays, with potencies ranging from 200 to 1000 nM for AC-55541 and 30 to 100 nM for AC-264613. In comparison, the PAR2-activating peptide 2-furoyl-LIGRLO-NH(2) had similar potency, whereas SLIGRL-NH(2) was 30 to 300 times less potent. Neither AC-55541 nor AC-264613 had activity at any of the other PAR receptor subtypes, nor did they have any significant affinity for over 30 other molecular targets involved in nociception. Visualization of EYFP-tagged PAR2 receptors showed that each compound stimulated internalization of PAR2 receptors. AC-55541 and AC-264613 were well absorbed when administered intraperitoneally to rats, each reaching micromolar peak plasma concentrations. AC-55541 and AC-264613 were each stable to metabolism by liver microsomes and maintained sustained exposure in rats, with elimination half-lives of 6.1 and 2.5 h, respectively. Intrapaw administration of AC-55541 or AC-264613 elicited robust and persistent thermal hyperalgesia and edema. Coadministration of either a tachykinin 1 (neurokinin 1) receptor antagonist or a transient receptor potential vanilloid (TRPV) 1 antagonist completely blocked these effects. Systemic administration of either AC-55541 or AC-264613 produced a similar degree of hyperalgesia as was observed when the compounds were administered locally. These compounds represent novel small-molecule PAR2 agonists that will be useful in probing the physiological functions of PAR2 receptors.

Pharmacological characterization of AC-262536, a novel selective androgen receptor modulator.

Because of the limitations and liabilities of current testosterone therapies, non-steroidal tissue-selective androgen receptor modulators may provide a clinically meaningful advance in therapy. Using a functional cell-based assay AC-262536 was identified as a potent and selective AR ligand, with partial agonist activity relative to the natural androgen testosterone. A 2-week chronic study in castrated male rats indicated that AC-262536 significantly improves anabolic parameters in these animals, especially in stimulating the growth of the levator ani and in suppressing elevated LH levels. In sharp contrast to testosterone, AC-262536 has weak androgenic effects, as measured by prostate and seminal vesicle weights. Thus, AC-262536 represents a novel class of selective androgen receptor modulators (SARMs) with beneficial anabolic effects.

Differential modulation of inflammatory pain by a selective estrogen receptor beta agonist.

To understand the contribution of the estrogen receptor beta, the potent and selective agonist ERb-131 was evaluated in animal models of inflammatory pain. In paradigms of acute and persistent inflammatory pain, ERb-131 did not alleviate the nociception induced by either carrageenan or formalin. However, in the chronic complete Freund's adjuvant model, ERb-131 resolved both inflammatory and hyperalgesic components. Thus, ERb-131 is sufficient to alleviate chronic but not acute inflammatory pain states.

Broad modulation of neuropathic pain states by a selective estrogen receptor beta agonist.

The effects of estrogens on pain perception remain controversial. In animal models, both beneficial and detrimental effects of non-selective estrogens have been reported. ERb-131 a non-steroidal estrogen receptor beta ligand was evaluated in several pain animal models involving nerve injury or sensitization. Using functional and binding assays, ERb-131 was characterized as a potent and selective estrogen receptor beta agonist. In vivo, ERb-131 was devoid of estrogen receptor alpha activity as assessed in a rat uterotrophic assay. ERb-131 alleviated tactile hyperalgesia induced by capsaicin, and reversed tactile allodynia caused by spinal nerve ligation and various chemical insults. Moreover, ERb-131 did not influence the pain threshold of normal healthy animals. Thus, estrogen receptor beta agonism is a critical effector in attenuating a broad range of anti-nociceptive states.

Cholecystokinin in the rostral ventromedial medulla mediates opioid-induced hyperalgesia and antinociceptive tolerance.

Opioid-induced hyperalgesia is characterized by hypersensitivity to innocuous or noxious stimuli during sustained opiate administration. Microinjection of lidocaine into the rostral ventromedial medulla (RVM), or dorsolateral funiculus (DLF) lesion, abolishes opioid-induced hyperalgesia, suggesting the importance of descending pain facilitation mechanisms. Here, we investigate the possibility that cholecystokinin (CCK), a pronociceptive peptide, may drive such descending facilitation from the RVM during continuous opioid administration. In opioid-naive rats, CCK in the RVM produced acute tactile and thermal hypersensitivity that was antagonized by the CCK2 receptor antagonist L365,260 or by DLF lesion. CCK in the RVM also acutely displaced the spinal morphine antinociceptive dose-response curve to the right. Continuous systemic morphine elicited sustained tactile and thermal hypersensitivity within 3 d. Such hypersensitivity was reversed in a time-dependent manner by L365,260 in the RVM, and blockade of CCK2 receptors in the RVM also blocked the rightward displacement of the spinal morphine antinociceptive dose-response curve. Microdialysis studies in rats receiving continuous morphine showed an approximately fivefold increase in the basal levels of CCK in the RVM when compared with controls. These data suggest that activation of CCK2 receptors in the RVM promotes mechanical and thermal hypersensitivity and antinociceptive tolerance to morphine. Enhanced, endogenous CCK activity in the RVM during sustained morphine exposure may diminish spinal morphine antinociceptive potency by activating descending pain facilitatory mechanisms to exacerbate spinal nociceptive sensitivity. Prevention of opioid-dose escalation in chronic pain states by CCK receptor antagonism represents a potentially important strategy to limit unintended enhanced clinical pain and analgesic tolerance

Opioid receptor-mediated hyperalgesia and antinociceptive tolerance induced by sustained opiate delivery.

Opiates are commonly used to treat moderate to severe pain and can be used over prolonged periods in states of chronic pain such as those associated with cancer. In addition, to analgesic actions, studies show that opiate administration can paradoxically induce hyperalgesia. At the pre-clinical level, such hyperalgesia is associated with numerous pronociceptive neuroplastic changes within the primary afferent fibers and the spinal cord. In rodents, sustained opiate administration also induces antinociceptive tolerance. The mechanisms by which prolonged opiate exposure induces hyperalgesia and the relationship of this state to antinociceptive tolerance remain unclear. The present study was aimed at determining whether sustained opiate-induced hyperalgesia, associated neuroplasticity and antinociceptive tolerance are the result of specific opiate interaction at opiate receptors. Enantiomers of oxymorphone, a mu opioid receptor agonist, were administered to rats by spinal infusion across 7 days. Sustained spinal administration of (-)-oxymorphone, but not its inactive enantiomer (+)-oxymorphone or vehicle, upregulated spinal dynorphin content, produced thermal and tactile hypersensitivity, and produced antinociceptive tolerance. These results indicate that these pronociceptive actions of sustained opiate administration require specific interaction with opiate receptors and are unlikely to be the result of accumulation of potentially excitatory metabolic products. While the precise mechanisms, which may account for these pronociceptive changes remain to be unraveled, the present data point to plasticity initiated by opiate receptor interaction.

Sustained morphine exposure induces a spinal dynorphin-dependent enhancement of excitatory transmitter release from primary afferent fibers.

Paradoxical opioid-induced pain has been demonstrated repeatedly in humans and animals. The mechanisms of such pain are unknown but may relate to opioid-induced activation of descending pain facilitatory systems and enhanced expression and pronociceptive actions of spinal dynorphin. Here, the possibility that these opioid-induced central changes might mediate increased excitability to the spinal cord was tested. Tactile and thermal hypersensitivity was observed at 7, but not 1, days after subcutaneous morphine pellet implantation; placebo pellets produced no effects. Basal and capsaicin-evoked release of calcitonin gene-related peptide (CGRP) was measured in minced spinal tissues taken from naive rats or rats on post-pellet days 1 and 7. The content and evoked release of CGRP were significantly increased in tissues from morphine-exposed rats at 7, but not 1, days after implantation. Morphine increased spinal dynorphin content on day 7 in rats with sham bilateral lesions of the dorsolateral funiculus (DLF) but not in rats with DLF lesions. Pharmacological application of dynorphin A(2-13), a non-opioid fragment, to tissues from naive rats enhanced the evoked release of CGRP. Enhanced evoked release of CGRP from morphine-pelleted rats was blocked by dynorphin antiserum or by previous lesions of the DLF. Sustained morphine induces plasticity in both primary afferents and spinal cord, including increased CGRP and dynorphin content. Morphine-induced elevation of spinal dynorphin content depends on descending influences and enhances stimulated CGRP release. Enhanced transmitter release may allow increased stimulus-evoked spinal excitation, which is likely to be critical for opioid-induced paradoxical pain. Such pain may manifest behaviorally as antinociceptive tolerance.

Time-dependent descending facilitation from the rostral ventromedial medulla maintains, but does not initiate, neuropathic pain.

Although injury-induced afferent discharge declines significantly over time, experimental neuropathic pain persists unchanged for long periods. These observations suggest that processes that initiate experimental neuropathic pain may differ from those that maintain such pain. Here, the role of descending facilitation arising from developing plasticity in the rostral ventromedial medulla (RVM) in the initiation and maintenance of experimental neuropathic pain was explored. Tactile and thermal hypersensitivity were induced in rats by spinal nerve ligation (SNL). RVM lidocaine blocked SNL-induced tactile and thermal hypersensitivity on post-SNL days 6-12 but not on post-SNL day 3. Lesion of RVM cells expressing mu-opioid receptors with dermorphin-saporin did not prevent the onset of SNL-induced tactile and thermal hypersensitivity, but these signs reversed to baseline levels beginning on post-SNL day 4. Similarly, lesions of the dorsolateral funiculus (DLF) did not prevent the onset of SNL-induced tactile and thermal hypersensitivity, but these signs reversed to baseline levels beginning on post-SNL day 4. Lesions of the DLF also blocked the SNL-induced increase in spinal dynorphin content, which has been suggested to promote neuropathic pain. These data distinguish mechanisms that initiate the neuropathic state as independent of descending supraspinal influences and additional mechanism(s) that require supraspinal facilitation to maintain such pain. In addition, the data indicate that these time-dependent descending influences can underlie some of the SNL-induced plasticity at the spinal level. Such time-dependent descending influences driving associated spinal changes, such as the upregulation of dynorphin, are key elements in the maintenance, but not initiation, of neuropathic states.

Role of NK-1 neurotransmission in opioid-induced hyperalgesia.

Opiates are among the most important drugs for treatment of moderate to severe pain and prolonged opiate administration is often required to treat chronic pain states. We investigated the neurobiological actions of sustained opiate administration revealing paradoxical pronociceptive adaptations associated with NK-1 receptor function. Sustained morphine delivered over 6 days elicited hyperalgesia in rats and mice during the period of opiate delivery. Sustained morphine administration increased substance P (SP) and NK-1 receptor expression in the spinal dorsal horn. Sustained morphine treatment also enhanced capsaicin-evoked SP release in vitro, and increased internalization of NK-1 receptors in response to noxious stimulation. While NK-1 receptor internalization was observed primarily in the superficial laminae of placebo-treated rats, NK-1 receptor internalization was seen in both superficial and deep lamina of the dorsal horn in morphine-treated animals. Morphine-induced hyperalgesia was reversed by spinal administration of an NK-1 receptor antagonist in rats and mice, and was observed in wildtype (NK-1(+/+)), but not NK-1 receptor knockout (NK-1(-/-)), mice. These data support a critical role for the NK-1 receptor in the expression of sustained morphine-induced hyperalgesia. Additionally, these data indicate that sustained opiate administration induces changes reminiscent of those associated with inflammatory pain. These opiate-induced changes might produce unintended deleterious actions in the course of pain treatment in patients. Understanding of sustained morphine-induced neurochemical changes will help identify approaches that limit the deleterious actions of opiates.

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

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.

Reversal of experimental neuropathic pain by T-type calcium channel blockers.

Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca(2+) current is associated with increased neuronal activity and blockade of N- and P-types, but not L-type, calcium channels have been found to block experimental neuropathic pain. While T-type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L(5)/L(6) spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose-dependent blockade of both tactile and thermal hypersensitivities in nerve-injured rats; responses of sham-operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T-type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T-type calcium channel blockers may have significant potential in the treatment of neuropathic pain states.

Discovery of selective nonpeptidergic neuropeptide FF2 receptor agonists.

We report the discovery and initial characterization of a novel class of selective NPFF2 agonists. HTS screening using R-SAT, a whole cell based functional assay, identified a class of aryliminoguanidines as NPFF1 and NPFF2 ligands. Subsequent optimization led to molecules exhibiting selective NPFF2 agonistic activity. Systemic administration showed that selective NPFF2 agonists (1 and 3) are active in various pain models in vivo, whereas administration of a nonselective NPFF1 and NPFF2 agonist (9) increases sensitivity to noxious and non-noxious stimuli.

Synthesis, structure-activity relationships, and characterization of novel nonsteroidal and selective androgen receptor modulators.

Herein we describe the discovery of ACP-105 (1), a novel and potent nonsteroidal selective androgen receptor modulator (SARM) with partial agonist activity relative to the natural androgen testosterone. Compound 1 was developed from a series of compounds found in a HTS screen using the receptor selection and amplification technology (R-SAT). In vivo, 1 improved anabolic parameters in a 2-week chronic study in castrated male rats. In addition to compound 1, a number of potent antiandrogens were discovered from the same series of compounds whereof one compound, 13, had antagonist activity at the AR T877A mutant involved in prostate cancer.

Synthesis and evaluation of dibenzothiazepines: a novel class of selective cannabinoid-1 receptor inverse agonists.

A novel class of CB1 inverse agonists was discovered. To efficiently establish structure-activity relationships (SARs), new synthetic methodologies amenable for parallel synthesis were developed. The compounds were evaluated in a mammalian cell-based functional assay and in radioligand binding assays expressing recombinant human cannabinoid receptors (CB1 and CB2). In general, all of the compounds exhibited high binding selectivity at CB1 vs CB2 and the general SAR revealed a lead compound 11-(4-chlorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12e) which showed excellent in vivo activity in pharmacodynamic models related to CB1 receptor activity. The low solubility that hampered the development of 12e was solved leading to a potential preclinical candidate 11-(3-chloro-4-fluorophenyl)dibenzo[b,f][1,4]thiazepine-8-carboxylic acid butylamide (12h).

Identification of the atypical L-type Ca2+ channel blocker diltiazem and its metabolites as ghrelin receptor agonists.

Using a high-throughput functional screen, the atypical L-type Ca2+ channel blocker diltiazem was discovered to be an agonist at the human ghrelin (GHSR1a) receptor. In cellular proliferation, Ca2+ mobilization, and bioluminescence resonance energy transfer (BRET-2) assays, diltiazem was a partial agonist at GHSR1a receptors, with 50 to 80% relative efficacy compared with the GHSR1a peptide agonist GHRP-6, and high nanomolar to low micromolar potency, depending upon the assay. Seven of the known primary metabolites of diltiazem were synthesized, and three of them (MA, M1, and M2) were more efficacious and/or more potent than diltiazem at GHSR1a receptors, with a rank order of agonist activity of M2 > M1 > MA > diltiazem, whereas M4 and M6 metabolites displayed weak agonist activity, and the M8 and M9 metabolites were inactive. Binding affinities of diltiazem and these metabolites to GHSR1a receptors followed a similar rank order. In vivo tests showed that diltiazem and M2 each stimulated growth hormone release in male Sprague-Dawley neonatal rats, although to a lesser degree than GHRP-6. Thus, diltiazem and chemical analogs of diltiazem represent a new class of GHSR1a receptor agonists. The possible contributions of GHSR1a receptor activation to the clinical actions of diltiazem are discussed in the context of the known beneficial cardiovascular effects of ghrelin.