Natural products found in Mitragyna speciosa, commonly known as kratom, represent diverse scaffolds (indole, indolenine, and spiro pseudoindoxyl) with opioid activity, providing opportunities to better understand opioid pharmacology. Herein, we report the pharmacology and SAR studies both in vitro and in vivo of mitragynine pseudoindoxyl (3), an oxidative rearrangement product of the corynanthe alkaloid mitragynine. 3 and its corresponding corynantheidine analogs show promise as potent analgesics with a mechanism of action that includes mu opioid receptor agonism/delta opioid receptor antagonism. In vitro, 3 and its analogs were potent agonists in [35S]GTPγS assays at the mu opioid receptor but failed to recruit β-arrestin-2, which is associated with opioid side effects. Additionally, 3 developed analgesic tolerance more slowly than morphine, showed limited physical dependence, respiratory depression, constipation, and displayed no reward or aversion in CPP/CPA assays, suggesting that analogs might represent a promising new generation of novel pain relievers.
Summary
Escalated aggression can have devastating societal consequences, yet underlying neurobiological mechanisms are poorly understood. Here we show significantly increased inter-male mouse aggression when neurotransmission is constitutively blocked from either of two subsets of serotonergic, Pet1+ neurons – one identified by dopamine receptor D1(Drd1a)::cre driven activity perinatally, the other by Drd2::cre from pre-adolescence onward. Blocking neurotransmission from other Pet1+ neuron subsets of similar size and/or overlapping anatomical domains had no effect on aggression compared to controls, suggesting subtype-specific serotonergic neuron influences on aggression. Using established and novel intersectional genetic tools, we further characterized these subtypes across multiple parameters, showing both overlapping and distinct features in axonal projection targets, gene expression, electrophysiological properties, and effects on non-aggressive behaviors. Notably, Drd2::cre-marked 5-HT neurons exhibited D2-dependent inhibitory responses to dopamine in slices, suggesting direct and specific interplay between inhibitory dopaminergic signaling and a serotonergic subpopulation. Thus, we identify specific serotonergic modules that shape aggression.
Buprenorphine has long been classified as a mu analgesic, although its high affinity for other opioid receptor classes and the orphanin FQ/nociceptin ORL1 receptor may contribute to its other actions. The current studies confirmed a mu mechanism for buprenorphine analgesia, implicating several subsets of mu receptor splice variants. Buprenorphine analgesia depended upon the expression of both exon 1-associated traditional full length 7 transmembrane (7TM) and exon 11-associated truncated 6 transmembrane (6TM) MOR-1 variants. In genetic models, disruption of delta, kappa1 or ORL1 receptors had no impact on buprenorphine analgesia, while loss of the traditional 7TM MOR-1 variants in an exon 1 knockout (KO) mouse markedly lowered buprenorphine analgesia. Loss of the truncated 6TM variants in an exon 11 KO mouse totally eliminated buprenorphine analgesia. In distinction to analgesia, the inhibition of gastrointestinal transit and stimulation of locomotor activity were independent of truncated 6TM variants. Restoring expression of a 6TM variant with a lentivirus rescued buprenorphine analgesia in an exon 11 KO mouse that still expressed the 7TM variants. Despite a potent and robust stimulation of 35S-GTPγS binding in MOR-1 expressing CHO cells, buprenorphine failed to recruit β-arrestin-2 binding at doses as high as 10 μM. Buprenorphine was an antagonist in DOR-1 expressing cells and an inverse agonist in KOR-1 cells. Buprenorphine analgesia is complex and requires multiple mu receptor splice variant classes, but other actions may involve alternative receptors.
IBNtxA (39-iodobenzoyl-6b-naltrexamide) is a potent analgesic in mice lacking many traditional opioid side effects. In mice, it displays no respiratory depression, does not produce physical dependence with chronic administration, and shows no crosstolerance to morphine. It has limited effects on gastrointestinal transit and shows no reward behavior. Biochemical studies indicate its actions are mediated through a set of m-opioid receptor clone MOR-1 splice variants associated with exon 11 that lack exon 1 and contain only six transmembrane domains. Like the mouse and human, rats express exon 11-associated splice variants that also contain only six transmembrane domains, raising the question of whether IBNtxA would have a similar pharmacologic profile in rats. When given systemically, IBNtxA is a potent analgesic in rats, with an ED 50 value of 0.89 mg/kg s.c., approximately 4-fold more potent than morphine. It shows no analgesic cross-tolerance in morphine-pelleted rats. IBNtxA displays no respiratory depression as measured by blood oxygen saturation. In contrast, oximetry shows that an equianalgesic dose of morphine lowers blood oxygen saturation values by 30%. IBNtxA binding is present in a number of brain regions, with the thalamus standing out with very high levels and the cerebellum with low levels. As in mice, IBNtxA is a potent analgesic in rats with a favorable pharmacologic profile and reduced side effects.
While levorphanol shares many of the same properties as the classic opioid morphine, it displays subtle differences that may prove helpful in its clinical use. Its G-protein signaling bias is consistent with its diminished respiratory depression, while its incomplete cross tolerance with morphine suggests it may prove valuable clinically with opioid rotation.
At younger ages, women have a lower risk for hypertension than men, but this sexual dimorphism declines with the onset of menopause. These differences are paralleled in rodents following “slow-pressor” angiotensin II (AngII) administration: young male and aged female mice, but not young females, develop hypertension. There is also an established sexual dimorphism both in the cardiovascular response to the neurohypophyseal hormone arginine vasopressin (AVP) and in the expression of oxidative stress. We examined the relationship between AngII-mediated hypertension and the cellular distribution of the superoxide generating NADPH oxidase (NOX) in AVP-expressing hypothalamic paraventricular nucleus (PVN) neurons in “menopausal” female mice. Dual labeling immunoelectron microscopy was used to determine if the subcellular distribution of the organizer/adapter NOX p47phox subunit is altered in PVN dendrites following AngII administered (14 days) during the “postmenopausal” stage of accelerated ovarian failure (AOF) in young female mice treated with 4-vinylcyclohexene diepoxide. Slow-pressor AngII elevated blood pressure in AOF females and induced a significant a significant increase in near plasmalemmal p47phox and a decrease in cytoplasmic p47phox in PVN AVP dendrites. These changes are opposite to those observed in AngII-induced hypertensive male mice (Coleman et al., J. Neuroscience 33: 4308-16, 2013), and may be ascribed in part to baseline differences between young females and males in the near plasmalemmal p47phox on AVP dendrites seen in the present study. These findings highlight fundamental differences in the neural substrates of oxidative stress in the PVN associated with AngII hypertension in postmenopausal females compared with males.
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