The Journal of Pharmacology and Experimental Therapeutics

Hind paw-directed assays are commonly used to study the analgesic effects of opioids in mice. However, opioid-induced hyper-locomotion can obscure results of such assays. We aimed to overcome this potential confound by using gait analysis to observe hind paw usage during walking in mice. We measured changes in paw print area following induction of post-surgical pain (using the paw incision model) and treatment with oxycodone. Paw incision surgery reduced the paw print area of the injured hind paw as the mice avoided placing the incised section of the paw on the floor. Surprisingly, oxycodone caused a tiptoe-like gait in mice, resulting in a reduced paw print area in both hind paws. Further investigation of this opioid-induced phenotype revealed that analgesic doses of oxycodone or morphine dose-dependently reduced hind paw print area in uninjured mice. The gait changes were not dependent on opioid-induced increases in locomotor activity; speed and paw print area had no correlation in opioid-treated mice, and other analgesic compounds that alter locomotor activity did not affect paw print area. Unfortunately, the opioid-induced "tiptoe" gait phenotype prevented gait analysis from being a viable metric for demonstrating opioid analgesia in injured mice. However, this work reveals an important, previously uncharacterized effect of treatment with analgesic doses of opioids on paw placement. Our characterization of how opioids affect gait has important implications for the use of mice to study opioid pharmacology and suggests that scientists should use caution when using hind paw-directed nociceptive assays to test opioid analgesia in mice.

Fatal opioid overdoses in the United States have nearly tripled during the past decade, with greater than 92% involving a synthetic opioid like fentanyl. Fentanyl potently activates the -opioid receptor to induce both analgesia and respiratory depression. The danger of illicit fentanyl has recently been exacerbated by adulteration with xylazine, an 2-adrenergic receptor agonist typically used as a veterinary anesthetic. In 2023, over a 1,000% increase in xylazine-positive overdoses was reported in some regions of the U.S. Xylazine has been shown to potentiate the lethality of fentanyl in mice, yet a mechanistic underpinning for this effect has not been defined. Herein, we evaluate fentanyl, xylazine, and their combination in whole-body plethysmography (to measure respiration) and pulse oximetry (to measure blood oxygen saturation and heart rate) in male and female CD-1 mice. We show that xylazine decreases breathing rate more than fentanyl by increasing the expiration time. In contrast, fentanyl primarily reduces breathing by inhibiting inspiration, and xylazine exacerbates these effects. Fentanyl but not xylazine decreased blood oxygen saturation, and when combined, xylazine did not change the maximum level of fentanyl-induced hypoxia. Xylazine also reduced heart rate more than fentanyl. Finally, loss in blood oxygen saturation correlated with the frequency of fentanyl-induced apneas, but not breathing rate. Together, these findings provide insight into how the addition of xylazine to illicit fentanyl may increase the risk of overdose. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=114 SRC="FIGDIR/small/608310v1_ufig1.gif" ALT="Figure 1"> View larger version (23K): org.highwire.dtl.DTLVardef@1a3aabeorg.highwire.dtl.DTLVardef@1eac11org.highwire.dtl.DTLVardef@1b2ceb9org.highwire.dtl.DTLVardef@220f06_HPS_FORMAT_FIGEXP M_FIG C_FIG

Pentilludin is a novel, potent (690 nM) irreversible inhibitor of actions of the receptor type protein tyrosine phosphatase D (PTPRD). Pentilludin displays no in vitro activities in Ames or micronucleus tests, at hERG channels or at targets for currently-licensed drugs. Rats treated with pentilludin doses up to 100 mg/kg/day for two weeks have not been found to display behavioral, hematologic or serum chemistry abnormalities. Treatment with 20 mg/kg sc pentilludin prior to every other M-W-F self-administration session substantially reduces self-administration of amphetamine and more modestly reduces self-administration of remifentanil. Pentilludin provides a novel means for reducing self-administration of psychostimulant and, modestly, opiate drugs in ways that could enhance abstinence in humans.

The gold standard pharmacological treatment for opioid use disorder (OUD) consists of maintenance therapy with long-acting opioid agonists such as buprenorphine and methadone. Despite these compounds having demonstrated substantial efficacy, a significant number of patients do not show optimal therapeutic responses. Moreover, the abuse liability of these medications remains a major concern. Cebranopadol, is a new, long-acting pan-opioid agonist that also activates the nociception/orphanin FQ NOP receptor. Here we used rats to explore the therapeutic potential of this agent in OUD. First, in operant intravenous self-administration experiments we compared the potential abuse liability of cebranopadol with the prototypical opioid heroin. Under a fixed ratio 1 (FR1) contingency, rats maintained responding for heroin (1, 7, 20, 60 g/inf) to a larger extent than cebranopadol (0.03, 0.1, 0.3, 1.0, 6.0 g/inf). When the contingency was switched to a progressive ratio (PR) reinforcement schedule, heroin maintained responding at high levels at all except the lowest dose. Conversely, in the cebranopadol groups responding decreased drastically and the break point (BP) did not differ from saline controls. Next, we demonstrated that oral administration of cebranopadol (0, 25, 50 g/kg) significantly attenuated drug self-administration independent of heroin dose (1, 7, 20, 60 g/inf). Cebranopadol also reduced the break point for heroin (20 g/inf). Furthermore, in a heroin self-administration training extinction/reinstatement paradigm, pretreatment with cebranopadol significantly attenuated yohimbine stress-induced reinstatement of drug seeking. Together, these data indicate that cebranopadol has limited abuse liability compared to heroin and is highly efficacious in attenuating opioid self-administration and stress-induced reinstatement, suggesting clinical potential of this compound for OUD treatment.

Opioid analgesics like morphine and fentanyl induce mu-opioid receptor (MOR)-mediated hyperactivity in mice. Here we show that morphine, fentanyl, SR-17018, and oliceridine have submaximal intrinsic efficacy in the mouse striatum using 35S-GTP{gamma}S binding assays. While all of the agonists act as partial agonists for stimulating G protein coupling in striatum, morphine, fentanyl and oliceridine are fully efficacious in stimulating locomotor activity; meanwhile, the noncompetitive biased agonists, SR-17018 and SR-15099 produce submaximal hyperactivity. Moreover, the combination of SR-17018 and morphine attenuates hyperactivity while antinociceptive efficacy is increased. The combination of oliceridine with morphine increases hyperactivity which is maintained over time. These findings provide evidence that noncompetitive agonists at MOR can be used to suppress morphine-induced hyperactivity while enhancing antinociceptive efficacy; moreover, they demonstrate that intrinsic efficacy measured at the receptor level is not directly proportional to drug efficacy in the locomotor activity assay.

BackgroundAnthracyclines such as doxorubicin (Dox) are highly effective anti-tumor agents, but their use is limited by dose-dependent cardiomyopathy and heart failure. Our laboratory previously reported that induction of cytochrome P450 family 1 (Cyp1) enzymes contributes to acute Dox cardiotoxicity in zebrafish and in mice, and that potent Cyp1 inhibitors prevent cardiotoxicity. However, the role of Cyp1 enzymes in chronic Dox cardiomyopathy, as well as the mechanisms underlying cardioprotection associated with Cyp1 inhibition, have not been fully elucidated. MethodsThe Cyp1 pathway was evaluated using a small molecule Cyp1 inhibitor in wild-type (WT) mice, or Cyp1-null mice (Cyp1a1/1a2-/-, Cyp1b1-/-, and Cyp1a1/1a2/1b1-/-). Low-dose Dox was administered by serial intraperitoneal or intravenous injections, respectively. Expression of Cyp1 isoforms was measured by RT-qPCR, and myocardial tissue was isolated from the left ventricle for RNA sequencing. Cardiac function was evaluated by transthoracic echocardiography. ResultsIn WT mice, Dox treatment was associated with a decrease in Cyp1a2 and increase in Cyp1b1 expression in the heart and in the liver. Co-treatment of WT mice with Dox and the novel Cyp1 inhibitor YW-130 protected against cardiac dysfunction compared to Dox treatment alone. Cyp1a1/1a2-/- and Cyp1a1/1a2/1b1-/- mice were protected from Dox cardiomyopathy compared to WT mice. Male, but not female, Cyp1b1-/- mice had increased cardiac dysfunction following Dox treatment compared to WT mice. RNA sequencing of myocardial tissue showed upregulation of Fundc1 and downregulation of Ccl21c in Cyp1a1/1a2-/- mice treated with Dox, implicating changes in mitophagy and chemokine-mediated inflammation as possible mechanisms of Cyp1a-mediated cardioprotection. ConclusionsTaken together, this study highlights the potential therapeutic value of Cyp1a inhibition in mitigating anthracycline cardiomyopathy.

Cannabidiol is widely available and often used for pain management. Individuals with kidney disease or renal allografts have limited analgesia options. We conducted a Phase 1 human study to compare the peripheral immune cell distribution before (pre-cannabidiol) and after exposure to cannabidiol at steady state (post-cannabidiol). This ex vivo study included specimens from 23 participants who received oral cannabidiol (up to 5 mg/kg twice daily) for 11 days. Lymphocytes were isolated and stimulated with anti-CD3/CD28 antibodies, with or without tacrolimus. Pharmacodynamic responses were assessed via CellTiter-Glo(R) proliferation, scRNA-seq, cytokine assays, and flow cytometry. Steady-state plasma concentrations of CBD were quantified via tandem mass spectrometry. We identified an increased proportion of T effector memory (TEM) cells post-cannabidiol (22% increase, P-value of 3.2 x 10-32), which correlated with CBD plasma concentrations (Pearson Corr= 0.77, P-value < 0.01). Post-cannabidiol cytokine assays revealed elevated proinflammatory IL-6 protein levels and anti-inflammatory IL-10 levels (adjusted P-values < 0.0001). Cannabidiol reduced overall T and B lymphocyte proliferation with additive immunosuppressive effects to tacrolimus. In flow cytometry, the proportion of TEM and TEMRA increased post-cannabidiol with tacrolimus (P-values < 0.05). Cannabidiol exhibits mixed immunomodulatory effects with pro- and anti-inflammatory signals. Understanding the clinical safety of cannabidiol use is important given the paucity of pain control options available for immunocompromised transplant populations.

Nociceptive response belongs to a basic animal behavior facilitating adaptability and survival upon external or internal stimuli. Fish, similarly to higher vertebrates, also possess nociceptive machinery. Current protocols involving procedures performed on adult zebrafish including heart cryoinjury do not, however, take into account the adverse effects including pain that may potentially arise from these methodologies. Here, we assess the effect of two analgesics, lidocaine and morphine, followed after heart cryoinjury in zebrafish. Monitoring swimming behavior together with histology and gene expression analysis at the single cell level using scRNA sequencing and RNAscope fluorescent in situ hybridization technology, we show morphine, but not lidocaine, significantly improves animal welfare 6 hours post-cryoinjury, without impairing the heart regeneration process. Altogether, we propose morphine to be considered as the analgesic of choice to reduce post-surgical pain in adult zebrafish. HighlightsO_LICryoinury could be considered as a potential noxious stimulus in adult zebrafish. C_LIO_LIMorphine but not lidocaine treatment effectively alleviates noxious effects post-cryoinjury. C_LIO_LILidocaine treatment delays heart repair and regeneration. C_LIO_LI6 hours Morphine treatment after cryoinjury does not impede heart regeneration. C_LI

Xylazine has emerged in recent years as an adulterant in an increasing number of opioid-positive overdose deaths in the United States. Although its exact role in opioid-induced overdose deaths is largely unknown, xylazine is known to depress vital functions and cause hypotension, bradycardia, hypothermia, and respiratory depression. In this study, we examined the brain-specific hypothermic and hypoxic effects of xylazine and its mixtures with fentanyl and heroin in freely moving rats. In the temperature experiment, we found that intravenous xylazine at low, human-relevant doses (0.33, 1.0, 3.0 mg/kg) dose-dependently decreases locomotor activity and induces modest but prolonged brain and body hypothermia. In the electrochemical experiment, we found that xylazine at the same doses dose-dependently decreases nucleus accumbens oxygenation. In contrast to relatively weak and prolonged decreases induced by xylazine, intravenous fentanyl (20 g/kg) and heroin (600 g/kg) induce stronger biphasic brain oxygen responses, with the initial rapid and strong decrease, resulting from respiratory depression, followed by a slower, more prolonged increase reflecting a post-hypoxic compensatory phase, with fentanyl acting much quicker than heroin. The xylazine-fentanyl mixture eliminated the hyperoxic phase of oxygen response and prolonged brain hypoxia, suggesting xylazine-induced attenuation of the brains compensatory mechanisms to counteract brain hypoxia. The xylazine-heroin mixture strongly potentiated the initial oxygen decrease, and the pattern lacked the hyperoxic portion of the biphasic oxygen response, suggesting more robust and prolonged brain hypoxia. These findings suggest that xylazine exacerbates the life-threatening effects of opioids, proposing worsened brain hypoxia as the mechanism contributing to xylazine-positive opioid-overdose deaths.

Fentanyl leads to tens of thousands of overdose deaths every year despite widespread availability of naloxone. Like other opioids, fentanyl causes respiratory depression. Unlike morphine, high dose fentanyl rapidly produces airway obstruction, muscle rigidity, and cardiovascular failure. Using a rat model of opioid overdose, we compared the physiological effects of fentanyl and morphine and studied the efficacy of a novel rescue strategy. In contrast to morphine, we report that fentanyl more frequently causes respiratory failure secondary to vocal cord closure and leads to more severe cardiovascular disruption, including the blockade of baroreflex-like rebound in blood pressure. We also show that administration of intramuscular naloxone immediately after intravenous infusion of fentanyl did not improve survival. However, combining intramuscular naloxone with the alpha-2 adrenergic agonist clonidine rescued vocal cord function and stabilized cardiovascular and respiratory physiology from fentanyl-induced effects. Our findings demonstrate that fentanyl is associated with a unique and more severe toxidrome compared to morphine. Also, supplementing naloxone with drugs targeting the adrenergic system improves survival primarily by reopening the upper airway, implicating airway obstruction as a significant component of fentanyl-induced respiratory depression. Therefore, reversal of vocal cord closure appears to be the necessary precursor to the restoration of not only respiration, but also vascular autoregulation, a significant determinant of survival from fentanyl overdose.

The endocannabinoid system is widely expressed throughout the body and is comprised of receptors, ligands, and enzymes that maintain metabolic, immune, and reproductive homeostasis. Increasing interest in the endocannabinoid system has arisen due to these physiologic roles, policy changes leading to more widespread recreational use, and the therapeutic potential of Cannabis and phytocannabinoids. Rodents have been the primary preclinical model of focus due to their relative low cost, short gestational period, genetic manipulation strategies, and gold-standard behavioral tests. However, the potential for lack of clinical translation to non-human primates and humans is high as cross-species comparisons of the endocannabinoid system has not been evaluated. To bridge this gap in knowledge, we evaluate the relative gene expression of 14 canonical and extended endocannabinoid receptors in seven peripheral organs of C57/BL6 mice, Sprague-Dawley rats, and non-human primate rhesus macaques. Notably, we identify species- and organ-specific heterogeneity in endocannabinoid receptor distribution where there is surprisingly limited overlap among the preclinical models. Importantly, we determined there were only five receptors (CB2, GPR18, GPR55, TRPV2, and FAAH) that had identical expression patterns in mice, rats, and rhesus macaques. Our findings demonstrate a critical, yet previously unappreciated, contributor to challenges of rigor and reproducibility in the cannabinoid field, which has profound implications in hampering progress in understanding the complexity of the endocannabinoid system and development of cannabinoid-based therapies.

Non-selective antagonists of muscarinic acetylcholine receptors (mAChRs) that broadly inhibit all five mAChR subtypes provide an efficacious treatment for some movement disorders, including Parkinson disease and dystonia. Despite their efficacy in these and other central nervous system disorders, anti-muscarinic therapy has limited utility due to severe adverse effects that often limit their tolerability by patients. Recent advances in understanding the roles that each mAChR subtype plays in disease pathology suggest that highly selective ligands for individual subtypes may underlie the anti-parkinsonian and anti-dystonic efficacy observed with the use of non-selective anti-muscarinic therapeutics. Our recent work has indicated that the M4 muscarinic acetylcholine receptor has several important roles in opposing aberrant neurotransmitter release, intracellular signaling pathways, and brain circuits associated with movement disorders. This raises the possibility that selective antagonists of M4 may recapitulate the efficacy of non-selective anti-muscarinic therapeutics and may decrease or eliminate the adverse effects associated with these drugs. However, this has not been directly tested due to lack of selective antagonists of M4. Here we utilize genetic mAChR knockout animals in combination with non-selective mAChR antagonists to confirm that the M4 receptor underlies the locomotor-stimulating and anti-parkinsonian efficacy in rodent models. We also report the synthesis, discovery, and characterization of the first-in-class selective M4 antagonists VU6013720, VU6021302, and VU6021625 and confirm that these optimized compounds have anti-parkinsonian and anti-dystonic efficacy in pharmacological and genetic models of movement disorders.

1.The gene SLC6A15 encodes the protein B0AT2, a transporter for neutral amino acids. It is highly expressed in the brain and has been associated with depression but little is otherwise know about its function. In this study, we identified the first inhibitors of this protein to pharmacologically investigate its function. The transporter activity was evaluated using a cellular uptake of the substrate 3H-proline. A miniaturized assay was developed and used for a High Throughput Screening (HTS) of 200,000 compounds. Hits were tested for cell toxicity and selectivity versus related transporters of the SLC6 family. The most promising inhibitors were validated by proline uptake and neurite outgrowth in primary hippocampal neurons. Of the 10 chemical scaffolds identified, a 1,5-benzodiazepine series had the most promising selectivity and structure-activity relationship (SAR) profile. The best compounds showed drug-like properties and inhibited B0AT2 with an IC50 of 250 nM both in SLC6A15-overexpressing HEK293 cells and primary neurons, with no detectable inhibition (> 80{micro}M) of SERT, DAT, GAT1, or NTT4/SLC6A17. These compounds also dose-dependently stimulated neurite outgrowth in primary neurons. The identified compounds are the first inhibitors of the amino acids transporter B0AT2/SLC6A15. Their potency, selectivity and physicochemical properties allow to target the transporter in relevant biological systems and to initiate new studies to understand its role and implication in diseases.

Kappa opioid receptor agonists are clinically used to treat pruritis and have therapeutic potential for the treatment of pain and neuropsychiatric disorders. We have previously shown that triazole 1.1 is a G protein signaling-biased KOR agonist, that can suppress itch without producing signs of sedation in mice. This profile was recapitulated in rats and non-human primates however, triazole 1.1 had limited potency as an antipruritic. Here we describe a more potent, G protein signaling-biased agonist, triazole 187. Triazole 187 is a potent antipruritic agent and does not decrease spontaneous locomotor activity; interestingly, it produces anxiolytic-like behaviors in mice, an effect not observed for triazole 1.1. In addition to curbing sedation, triazole 187 produces only mild diuresis, resulting in 30% of urine output induced by U50,488H at dose that is 188-fold the antipruritic potency dose. Compounds like triazole 187 may present a means to treat anxiety accompanied by persistent chronic itch while avoiding sedation and diuresis accompanied by typical KOR agonists. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=107 SRC="FIGDIR/small/638680v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@17e9e6aorg.highwire.dtl.DTLVardef@1d27741org.highwire.dtl.DTLVardef@9389dcorg.highwire.dtl.DTLVardef@3fd825_HPS_FORMAT_FIGEXP M_FIG Graphic Abstract C_FIG

4-chlorokynurenine (4-Cl-KYN) is in clinical development for potential CNS indications. We have sought to further understand the distribution and metabolism of 4-Cl-KYN as this information might provide a strategy to enhance the clinical development of this drug. We used excretion studies in rats, in vitro transporter assays and pharmacogenetic analysis of clinical trial data to determine how 4-Cl-KYN and metabolites are distributed. Our data indicated that a novel acetylated metabolite (N-acetyl-4-Cl-KYN) did not affect the uptake of 4-Cl-KYN across the blood-brain barrier via LAT1. 4-Cl-KYN and metabolites were found to be renally excreted in rodents. In addition, we found that N-acetyl-4-Cl-KYN inhibited renal and hepatic transporters involved in excretion. Thus, this metabolite had the potential to limit the excretion of a range of compounds. Our pharmacogenetic analysis found that a SNP in N-acetyltransferase 8 (NAT8, rs13538) was linked to levels of N-acetyl-4-Cl-KYN relative to 4-Cl-KYN found in the plasma and that a SNP in SLC7A5 (rs28582913) was associated with the plasma levels of the active metabolite, 7-Cl-KYNA. Thus, we have a pharmacogenetics-based association for plasma drug level that could aid in the drug development of 4-Cl-KYN and have investigated the interaction of a novel metabolite with drug transporters.

Alcohol induced peripheral neuropathy (AIPN) is a neurodegenerative disease caused by chronic alcohol intake and is associated with peripheral nerve damage and somatosensory symptoms, such as allodynia. Current treatments lack efficacy and do not target underlying pathology emphasizing the need for preclinical models of AIPN to elucidate mechanisms and novel targets. Thus, we performed a detailed characterization of a mouse model of AIPN and candidate mechanistic associations including the role of neuroinflammation and acetaldehyde. Our studies showed chronic alcohol induced mechanical and cold hypersensitivity and deficits in spontaneous behaviors in EtOH concentration-, time- and sex-dependent manners. Female mice drank more alcohol and developed more rapid and severe hypersensitivity but less robust deficits of spontaneous behaviors. The grimace test demonstrated chronic alcohol promoted spontaneous pain independent of sex. Duration of intake impacted alcohol-induced deficits in peripheral nerve electrophysiology amplitude and intra-epidermal nerve fiber density. We characterized an extensive time-course of chronic alcohol-induced neuroinflammation in the DRG and spinal cord and found significant time, sex and tissue effects. Polymorphisms of ALDH2 have been associated with alcohol-induced neuroinflammation, alcohol-related pain, and alcohol-induced peripheral neuropathy. We investigated the role of acetaldehyde, via inhibition of ALDH2, in the development of AIPN and showed that ALDH2 inhibition accelerated and exacerbated development of chronic alcohol-induced hypersensitivity in male and female mice. Overall, our studies in a well-controlled model of AIPN strongly point to neuroinflammation and inflammatory modulators such as acetaldehyde as important mechanistic targets for possible intervention in AIPN.

Previous studies show ATP-sensitive potassium (KATP) channel openers can reduce hypersensitivity associated with chronic pain models in rodents, and reduce morphine tolerance. Many agonists of KATP channels are not soluble in physiologically relevant vehicles, requiring adaptation for clinical use. This study compared the antinociception activity of novel KATP channel targeting prodrugs, CKLP1, CKLP2, and CF3-CKLP. These prodrugs are activated by endogenous alkaline phosphatase enzymes present in the peripheral and central nervous systems. Analgesic capabilities of intrathecally injected prodrugs were tested in rodent models of spinal nerve ligation (SNL) and Complete Freunds Adjuvant (CFA) as models for neuropathic and inflammatory pain, respectively. CKLP1 and CKLP2 significantly increased mechanical paw withdrawal thresholds 1-2 hours after intrathecal administration in the SNL model, but all three prodrugs were able to attenuate hypersensitivity up to 7 days after CFA treatment. The reduction of opioid tolerance and opioid-induced hypersensitivity in mice treated chronically with morphine was significantly reduced in CKLP1 and CKLP2 treated animals. Prodrug cleavage was confirmed in mouse spinal cords using liquid chromatography. These studies may aid in the further development of KATP channel prodrugs for use in treatments of chronic pain, opioid tolerance, and withdrawal.

Nepicastat is a potent dopamine-beta-hydroxylase inhibitor that modulates the sympathetic nervous system by inhibiting the synthesis of norepinephrine. Nepicastat is a potential drug for the treatment of congestive heart failure. We sought to investigate the mechanistic role of Nepicastsat in the heart of Spontaneous Hypertensive Rats (SHR) rats. Here, we investigated if Nepicastat at both acute (7 days) and chronic administration (14 days) decrease blood pressure and echocardiography parameters in SHR rats. SHR 3-4 months male rats were administered either Nepicastsat (30mg/kg, orally), Enalapril (10 mg/kg, orally), or vehicle for 7 days or 14 days. Blood pressure and echocardiography parameters were recorded on day 0, day 3, day 7, and day 14 of drug administration. The animals were sacrificed, and tissues are collected for histology, qRTPCR, and flow cytometry analysis. At both acute and chronic administration, Nepicastat decreased systolic blood pressure and intraventricular septal thickness of SHR rats compared to vehicle groups. The decrease in blood pressure was comparable to Enalapril treated rats. Interestingly, Nepicastat also decreased the infiltrating macrophages and B cells in the hearts of SHR rats. In conclusion, Nepicastsat consistently decreased the systolic blood pressure but increased the macrophages and B cell infiltration in the heart of SHR rats.

The cannabinoid CB2 receptor (CB2R) is a potential therapeutic target for distinct forms of tissue injury and inflammatory diseases. To thoroughly investigate the role of CB2R in pathophysiological conditions and for target validation in vivo, optimal pharmacological tool compounds are essential. Despite the sizable progress in the generation of potent and selective CB2R ligands, pharmacokinetic parameters are often neglected for in vivo studies. Here, we report the generation and characterization of a tetra-substituted pyrazole CB2R full agonist named RNB-61 with high potency (Ki 0.13-1.81 nM, depending on species) and a peripherally restricted action due to P-glycoprotein mediated efflux from the brain. 3H and 14C labelled RNB-61 showed apparent Kd values < 4 nM towards human CB2R in both cell and tissue experiments. The >6000-fold selectivity over CB1 receptors and negligible off-targets in vitro, combined with high oral bioavailability and suitable systemic pharmacokinetic (PK) properties, prompted the assessment of RNB-61 in a mouse ischemia-reperfusion model of acute kidney injury (AKI) and in a rat model of chronic kidney injury/inflammation and fibrosis (CKI) induced by unilateral ureteral obstruction. RNB-61 exerted dose-dependent nephroprotective and/or antifibrotic effects in the AKI/CKI models. Thus, RNB-61 is an optimal CB2R tool compound for preclinical in vivo studies with superior biophysical and PK properties over generally used CB2R ligands.

It is estimated that chronic neuropathic pain conditions exhibit up to 10% prevalence in the general population, with increased incidence in females. However, nonsteroidal inflammatory drugs (NSAIDs) are ineffective, and currently indicated prescription treatments such as opioids, anticonvulsants, and antidepressants provide only limited therapeutic benefit. In the current work, we extended previous studies in male rats utilizing a paradigm of central Toll-like receptor 4 (TLR4)-dependent, NSAID-unresponsive neuropathic-like pain hypersensitivity to male and female C57BL/6N mice, uncovering an unexpected hyperalgesic phenotype in female mice following intrathecal (IT) LPS. In contrast to previous reports in female C57BL/6J mice, female C57BL/6N mice displayed tactile and cold allodynia, grip force deficits, and locomotor hyperactivity in response to IT LPS. Congruent with our previous observations in male rats, systemic inhibition of 12/15-Lipoxygenases (12/15-LOX) in female B6N mice with selective inhibitors - ML355 (targeting 12-LOX-p) and ML351 (targeting 15-LOX-1) - completely reversed allodynia and grip force deficits. We demonstrate here that 12/15-LOX enzymes also are expressed in mouse spinal cord and that 12/15-LOX metabolites produce tactile allodynia when administered spinally (IT) or peripherally (intraplantar in the paw, IPLT) in a hyperalgesic priming model, similar to others observations with the cyclooxygenase (COX) metabolite Prostaglandin E2 (PGE2). Surprisingly, we did not detect hyperalgesic priming following IT administration of LPS, indicating that this phenomenon likely requires peripheral activation of nociceptors. Collectively, these data suggest that 12/15-LOX enzymes contribute to neuropathic-like pain hypersensitivity in rodents, with potential translatability as druggable targets across sexes and species using multiple reflexive and non-reflexive outcome measures.