Difelikefalin, a peripherally restricted KOR (kappa opioid receptor) agonist, produces diuresis through a central KOR pathway.

Difelikefalin is a peripherally restricted kappa opioid receptor (KOR) agonist that was recently approved by the FDA to treat pruritis in dialysis patients. Here, we investigated the cardiovascular and renal responses to difelikefalin, and using the KOR antagonist norbinaltorphimine (norBNI), examined whether any difelikefalin-induced changes in the renal excretion of water and/or electrolytes were mediated through a central or peripheral KOR pathway. The effects of norBNI pretreatment on nalfurafine, a KOR agonist that crosses the blood-brain barrier, were also examined. We hypothesized that difelikefalin would alter urine output differently than nalfurafine, given that KOR agonists produce diuresis via activating central KORs to inhibit vasopressin release. Following catheterization, conscious Sprague-Dawley rats were infused i.v. with isotonic saline and pretreated with norBNI centrally via an intracerebroventricular (ICV) cannula or peripherally via an intravenous catheter. After stabilization, difelikefalin or nalfurafine was administered i.v. and urine output, heart rate and mean arterial pressure (MAP) were recorded for 90 min. Difelikefalin produced a significant increase in urine output, and significant decrease in urinary sodium and potassium excretion, urine osmolality, and MAP. ICV norBNI pretreatment markedly attenuated the increase in urine output caused by difelikefalin and nalfurafine but did not inhibit the electrolyte effects. However, IV norBNI pretreatment prevented all responses to difelikefalin and nalfurafine. Together, these findings demonstrate that difelikefalin and nalfurafine utilize central KOR pathways to elicit diuresis and a decrease in MAP but enhance renal tubular electrolyte reabsorption through a peripheral KOR pathway, providing important insight into two clinically useful KOR agonists.

Cardiovascular and renal effects of novel nonpeptide nociceptin opioid peptide receptor agonists.

BACKGROUND AND PURPOSE: Partial agonists of the nociceptin opioid peptide (NOP) receptor have potential therapeutic use as antihypertensive and water diuretics (aquaretics). To date, peptide NOP receptor ligands have failed to progress in clinical trials due to poor pharmacokinetics and adverse effects. Nonpeptide, small-molecule NOP receptor ligands may be more suitable as therapeutic agents. This study investigated the cardiovascular and renal responses produced by the novel nonpeptide NOP agonists AT-403, AT-090, AT-127, and AT-039. EXPERIMENTAL APPROACH: Changes in mean arterial pressure (MAP), heart rate (HR), renal excretory function and occurrence of sedation and hyperphagia were determined before and after i.v. bolus injection or infusion of the NOP agonists in conscious Sprague-Dawley rats. Additional studies involving (i) measurement of renal sympathetic nerve activity (RSNA) and (ii) renal denervation were conducted to investigate the role of the renal nerves in the cardiorenal responses to AT-039. KEY RESULTS: Bolus i.v. injection of AT-403, AT-090, AT-127 and AT-039 produced significant decreases in MAP and HR and a sodium-sparing diuresis. AT-403, AT-090, and AT-127, but not AT-039, induced sedation and hyperphagia at all doses tested. Infusion i.v. of AT-039 produced hypotension and aquaresis without adverse central nervous system effects or change in HR, responses that were also observed in renal denervated rats. CONCLUSIONS AND IMPLICATIONS: Nonpeptide NOP agonists decrease blood pressure and produce aquaresis in conscious rodents. Due to lack of sedation and hyperphagia, AT-039 represents a novel NOP agonist that may be useful for treatment of hypertension and/or volume overload/hyponatraemic states.

Nalfurafine, a G-Protein-Biased KOR (Kappa Opioid Receptor) Agonist, Enhances the Diuretic Response and Limits Electrolyte Losses to Standard-of-Care Diuretics.

Nalfurafine is a G-protein-biased KOR (kappa opioid receptor) agonist that produces analgesia and lacks central nervous system adverse effects. Here, we examined the cardiovascular and renal responses to intravenous and oral nalfurafine alone and in combination with furosemide, hydrochlorothiazide, or amiloride. We hypothesized that nalfurafine, given its distinct mechanism of vasopressin inhibition, would increase urine output to these diuretics and limit electrolyte loss. Following catheterization, conscious Sprague-Dawley rats received an isotonic saline infusion and were then administered an intravenous bolus of nalfurafine, a diuretic, or a combination. Mean arterial pressure, heart rate, and urine output were recorded for 90 minutes. In another study, rats were placed in metabolic cages and administered drug in an oral volume load. Hourly urine samples were then collected for 5 hours. Intravenous and oral nalfurafine produced a marked diuresis, antinatriuresis, antikaliuresis, and a decrease in mean arterial pressure. Compared with diuretic treatment alone, intravenous coadministration with nalfurafine significantly increased urine output to furosemide and hydrochlorothiazide and decreased sodium and potassium excretion. Notably, mean arterial pressure was reduced with nalfurafine/diuretic combination therapy compared to diuretics alone. Similarly, oral coadministration of nalfurafine significantly increased urine output to hydrochlorothiazide and decreased sodium and potassium excretion, whereas combination with furosemide only limited the amount of sodium excreted. Further, both intravenous and oral coadministration of nalfurafine enhanced the diuresis to amiloride and decreased sodium excretion. Together, these findings demonstrate that nalfurafine enhances the diuresis to standard-of-care diuretics without causing an excessive loss of electrolytes, offering a new approach to treat several cardiovascular conditions.

Interactive effects of (±)-trans-U50488 and its stereoisomers with cannabinoids.

The adverse effects of mu opioid agonists have spurred a renewed interest in using kappa opioid receptor (KOR) agonists as analgesics. KOR agonists also have potential for development as diuretics for the treatment of edema and hypertension. Here, we evaluated the discriminative stimulus, antinociceptive, and diuretic effects of the kappa agonist (±)-trans-U-50488 and its stereoisomers (-)-(1S,2S)-U-50488 or (+)-(1R,2R)-U-50488) alone and in combination with the cannabinoid agonist (-)-CP 55,940. To establish (±)-U-50488 as a discriminative stimulus, rats (n = 12) were trained to discriminate intraperitoneal (i.p.) administration of 5.6 mg/kg of (±)-trans-U-50488 from saline under a fixed-ratio 20 (FR-20) schedule of food reinforcement. Then, antinociception was assessed using two procedures: warm water tail withdrawal and von Frey paw withdrawal. Diuretic effects were assessed in separate rats (n = 6/group). Doses of (±)-U-50488 and (-)-U-50488 that served as discriminative stimuli produced significant increases in urine output, but at lower doses than those that produced antinociception. In contrast, (+)-U-50488 alone had no discriminative stimulus or diuretic effects at the doses tested, but did produce antinociception in the von Frey assay. When three cannabinoids and morphine were tested in the (±)-U-50488 discrimination procedure to determine the similarity of these drugs' discriminative stimulus effects to those for (±)-U-50488, the rank order similarity was (-)-CP 55,940 > (-)-trans-THC > (+)-WIN 55,212-2 ≥ morphine. (-)-CP 55,940 alone (0.056 mg/kg) partially substituted for the discriminative stimulus effects of (±)-U-50488 and produced significant diuretic and antinociceptive effects. (-)-CP 55,940 in combination with (±)-U-50488 also produced a two-fold leftward shift in the discriminative stimulus curve for (±)-U-50488, and near-additive antinociception with (±)-U-50488 and (+)-U-50488. Further, the diuretic effect of (-)-CP 55,940 was enhanced by a dose of (+)-U50488, which itself did not alter urine output. These data together indicate that a combination of cannabinoid and kappa opioid agonists can enhance diuresis, but may have limited potential for serving as opioid-sparing pharmacotherapeutics for treatment of pain.

Mouse model systems of autism spectrum disorder: Replicability and informatics signature.

Phenotyping mouse model systems of human disease has proven to be a difficult task, with frequent poor inter- and intra-laboratory replicability, particularly in behavioral domains such as social and cognitive function. However, establishing robust animal model systems with strong construct validity is of fundamental importance as they are central tools for understanding disease pathophysiology and developing therapeutics. To complete our studies of mouse model systems relevant to autism spectrum disorder (ASD), we present a replication of the main findings from our two published studies of five genetic mouse model systems of ASD. To assess the intra-laboratory robustness of previous results, we chose the two model systems that showed the greatest phenotypic differences, the Shank3/F and Cntnap2, and repeated assessments of general health, activity and social behavior. We additionally explored all five model systems in the same framework, comparing all results obtained in this three-yearlong effort using informatics techniques to assess commonalities and differences. Our results showed high intra-laboratory replicability of results, even for those with effect sizes that were not particularly large, suggesting that discrepancies in the literature may be dependent on subtle but pivotal differences in testing conditions, housing enrichment, or background strains and less so on the variability of the behavioral phenotypes. The overall informatics analysis suggests that in our behavioral assays we can separate the set of tested mouse model system into two main classes that in some aspects lie on opposite ends of the behavioral spectrum, supporting the view that autism is not a unitary concept.

Downregulation of Brain Gα12 Attenuates Angiotensin II-Dependent Hypertension.

BACKGROUND: Angiotensin II (Ang II) activates central Angiotensin II type 1 receptors to increase blood pressure via multiple pathways. However, whether central Gα proteins contribute to Ang II-induced hypertension remains unknown. We hypothesized that Angiotensin II type 1 receptors couple with Gα12 and/or Gαq to produce sympatho-excitation and increase blood pressure and downregulation of these Gα-subunit proteins will attenuate Ang II-dependent hypertension. METHODS AND RESULTS: After chronic infusion of Ang II (s.c. 350 ng/kg/min) or vehicle for 2 weeks, Ang II evoked an increase in Gα12 expression, but not Gαq in the rostral ventrolateral medulla of Sprague-Dawley rats. In other studies, rats that received Ang II or vehicle infusion s.c. were simultaneously infused i.c.v. with a scrambled (SCR) or Gα12 oligodeoxynucleotide (ODN; 50 µg/day). Central Gα12 ODN infusion lowered mean blood pressure in Ang II infused rats compared with SCR ODN infusion (14-day peak; 133 ± 12 vs. 176 ± 11 mm Hg). Compared to the SCR ODN group, Ang II infused rats that received i.c.v. Gα12 ODN showed a greater increase in heart rate to atropine, an attenuated reduction in blood pressure to chlorisondamine, and an improved baroreflex sensitivity. In addition, central Gα12 and Gαq ODN pretreatment blunted the pressor response to an acute i.c.v. injection of Ang II (i.c.v., 200 ng). CONCLUSIONS: These findings suggest that central Gα12 protein signaling pathways play an important role in the development of chronic Ang II-dependent hypertension in rats.

Cognitive Training at a Young Age Attenuates Deficits in the zQ175 Mouse Model of HD.

Huntington's Disease (HD) is a progressive neurodegenerative disorder that causes motor, cognitive, and psychiatric symptoms. In these experiments, we tested if operant training at an early age affected adult cognitive deficits in the zQ175 KI Het (zQ175) mouse model of HD. In Experiment 1 we trained zQ175 mice in a fixed-ratio/progressive ratio (FR/PR) task to assay learning and motivational deficits. We found pronounced deficits in response rates and task engagement in naïve adult zQ175 mice (32-33 weeks age), while deficits in zQ175 mice trained from 6-7 weeks age were either absent or less severe. When those mice were re-tested as adults, FR/PR performance deficits were absent or otherwise less severe than deficits observed in naïve adult zQ175 relative to wild type (WT) mice. In Experiment 2, we used a Go/No-go operant task to assess the effects of early cognitive testing on response inhibition deficits in zQ175 mice. We found that zQ175 mice that began testing at 7-8 weeks did not exhibit deficits in Go/No-go testing, but when re-tested at 28-29 weeks age exhibited an initial impairment that diminished with training. These transient deficits were nonetheless mild relative to deficits observed among adult zQ175 mice without prior testing experience. In Experiment 3 we trained mice in a two-choice visual discrimination test to evaluate cognitive flexibility. As in prior experiments, we found performance deficits were mild or absent in mice that started training at 6-9 weeks of age, while deficits in naive mice exposed to training at 28-29 weeks were severe. Re-testing mice at 28-29 weeks age, were previously trained starting at 6-9 weeks, revealed that deficits in learning and cognitive flexibility were absent or reduced relative to effects observed in naive adults. In Experiment 4, we tested working memory deficits with a delayed non-match to position (DNMTP) test. Mice with prior experience exhibited mild working memory deficits, with males zQ175 exhibiting no deficits, and females performing significantly worse than WT mice at a single delay interval, whereas naive zQ175 exhibited severe delay-dependent deficits at all intervals exceeding 1 s. In sum, these experiments indicate that CAG-dependent impairments in motivation, motor control, cognitive flexibility, and working memory are sensitive to the environmental enrichment and experience. These findings are of clinical relevance, as HD carrier status can potentially be detected at an early age.

Deficits in a Simple Visual Go/No-go Discrimination Task in Two Mouse Models of Huntington's Disease.

Huntington's disease (HD), a devastating neurodegenerative disorder caused by a CAG repeat expansion on the HTT gene located on chromosome 4, is associated with a characteristic pattern of progressive cognitive dysfunction known to involve early deficits in executive function. A modified Go/No-go successive discrimination task was designed to assess the type of online response control/executive function known to be disrupted in patients with HD. The present studies show that this simple discrimination assay revealed early and robust deficits in two mouse models of HD, the zQ175 KI mouse (deficits from 28 weeks of age) and the R6/2 mouse, carrying ~240 CAG repeats (deficits from 9 weeks of age). These deficits are not due to gross motor dysfunction in the test animals, but instead appear to measure some inability to inhibit responding in the HD mouse models, suggesting this assay may measure deficits in underlying attentional and/or behavioral inhibition processes. Accordingly, this assay may be well suited to evaluation of simple deficits in cognitive function in mouse HD models, providing a potential platform for preclinical screening.

A mixed fixed ratio/progressive ratio procedure reveals an apathy phenotype in the BAC HD and the z_Q175 KI mouse models of Huntington's disease.

Apathy, characterized by generally reduced interest in and likelihood to perform goal-directed actions, is a recognized symptom of Huntington's disease (HD), a devastating neurological disorder caused by a CAG repeat expansion of the Htt gene located on chromosome 4. The present experiments used a modified progressive ratio task that incorporated a fixed-ratio schedule of reinforcement component to assess consummatory behavior, and a progressive-ratio schedule component that required increasing numbers of lever-presses for successive reinforcers (0.01 ml of evaporated milk). The studies revealed an apathetic phenotype in two mouse models of HD, with decreased response rates either overall or only at higher ratio requirements in the progressive-ratio component relative to wild-type controls. Based on the procedure used (within-session fixed- and progressive-ratio components), it is proposed that an observed phenotype can be ascribed either specifically to reduced motivation to work for food reinforcement or more generally to deficits in consummatory behavior. This procedure provides a simple means to assess this type of phenotype in rodents, with issues in consummatory vs. incentive motivation reflected in general alterations in fixed- versus progressive alterations on an escalating-ratio schedules respectively, providing translational measures of the amotivation/apathy construct of the human realm to the homologous construct of incentive motivation in preclinical models of human disease.

HD mouse models reveal clear deficits in learning to perform a simple instrumental response.

Mouse models of Huntington's disease (HD) were trained to acquire one of two simple instrumental responses (a lever press or a nosepoke) to obtain food reinforcement. Animals from several HD strains revealed apparently progressive deficits in this task, being significantly less able than littermate controls to perform the required responses, at ages where motor function is only mildly affected. These data could provide a simple way to measure learning deficits in these mouse models, likely related to the characteristic pattern of neural damage observed in HD mouse models.