Synthesis and in vitro evaluation of fluorine-18 benzimidazole sulfones as CB2 PET-radioligands.

Cannabinoid type 2 receptor (CB2) is up-regulated on activated microglial cells and can potentially be used as a biomarker for PET-imaging of neuroinflammation. In this study the synthesis and pharmacological evaluation of novel fluorinated pyridyl and ethyl sulfone analogues of 2-(tert-butyl)-5-((2-fluoropyridin-4-yl)sulfonyl)-1-(2-methylpentyl)-1H-benzo[d]imidazole (rac-1a) are described. In general, the ligands showed low nanomolar potency (CB2 EC50 < 10 nM) and excellent selectivity over the CB1 subtype (>10 000×). Selected ligands 1d, 1e, 1g and 3l showing high CB2 binding affinity (Ki < 10 nM) were radiolabelled with fluorine-18 from chloropyridyl and alkyl tosylate precursors with good to high isolated radioactive yields (25-44%, non-decay corrected, at the end of synthesis). CB2-specific binding of the radioligand candidates [18F]-1d and [18F]-3l was assessed on rat spleen cryosections using in vitro autoradiography. The results warrant further in vivo evaluation of the tracer candidates as prospective CB2 PET-imaging agents.

Protective cardiorenal effects of spironolactone in a rodent model of polycystic kidney disease.

Studies were performed to examine the contribution of aldosterone to the pathogenesis of cardiovascular and renal disease in a rodent model of genetic kidney disease. Spironolactone (20 mg/kg per day) was administered in water to mixed sex Lewis Polycystic Kidney (LPK) rats (n = 20) and control Lewis rats (n = 27) from 4 to 12 weeks of age. At 12 weeks of age, hypertension was reduced in female LPK rats; systolic blood pressure declined from 226.4 ± 26.8 mmHg in untreated rats and to 179.2 ± 3.2 mmHg in treated rats (P = 0.018). No similar effect on male or control rats was found. Water consumption and urine volume were significantly greater in LPK animals than in Lewis rats, and treatment reduced both variables by ~30% in LPK animals (P < 0.05). Proteinuria and the urinary protein-to-creatinine ratio were normalized in treated LPK relative to Lewis controls, and plasma creatinine levels were significantly reduced by treatment in LPK rats. Spironolactone did not alter kidney morphology in LPK rats (fibrosis or cyst size). Aortic vascular responses to noradrenaline and acetylcholine were sensitized and impaired in the LPK (P < 0.01). Aldosterone antagonism did not alter these responses or indicators of aortic structural remodelling. There was no treatment effect on left ventricular hypertrophy or elevated cardiac messenger RNA for ß-myosin-heavy chain and brain natriuretic peptide in the LPK rats. However, perivascular fibrosis and messenger RNA for α-cardiac actin were normalized by spironolactone in LPK animals relative to Lewis controls. In conclusion, we have shown an important blood pressure independent effect whereby inhibition of aldosterone via spironolactone was able to retard both renal and cardiac disease progression in a rodent model of polycystic kidney disease.

IL-5 promotes induction of antigen-specific CD4+CD25+ T regulatory cells that suppress autoimmunity.

Immune responses to foreign and self-Ags can be controlled by regulatory T cells (Tregs) expressing CD4 and IL-2Rα chain (CD25). Defects in Tregs lead to autoimmunity, whereas induction of Ag-specific CD4+CD25+ Tregs restores tolerance. Ag-specific CD4+CD25+ FOXP3+Tregs activated by the T helper type 2 (Th2) cytokine, IL-4, and specific alloantigen promote allograft tolerance. These Tregs expressed the specific IL-5Rα and in the presence of IL-5 proliferate to specific but not third-party Ag. These findings suggest that recombinant IL-5 (rIL-5) therapy may promote Ag-specific Tregs to mediate tolerance. This study showed normal CD4+CD25+ Tregs cultured with IL-4 and an autoantigen expressed Il-5rα. Treatment of experimental autoimmune neuritis with rIL-5 markedly reduced clinical paralysis, weight loss, demyelination, and infiltration of CD4+ (Th1 and Th17) CD8+ T cells and macrophages in nerves. Clinical improvement was associated with expansion of CD4+CD25+FOXP3+ Tregs that expressed Il-5rα and proliferated only to specific autoantigen that was enhanced by rIL-5. Depletion of CD25+ Tregs or blocking of IL-4 abolished the benefits of rIL-5. Thus, rIL-5 promoted Ag-specific Tregs, activated by autoantigen and IL-4, to control autoimmunity. These findings may explain how Th2 responses, especially to parasitic infestation, induce immune tolerance. rIL-5 therapy may be able to induce Ag-specific tolerance in autoimmunity.

Synthesis and functional evaluation of proteinogenic amino acid-derived synthetic cannabinoid receptor agonists related to MPP-5F-PICA, MMB-5F-PICA, and MDMB-5F-PICA.

Synthetic cannabinoid receptor agonists (SCRAs) comprise the second largest class of new psychoactive substances (NPS), and typically α-amino acid moieties are incorporated as part of their design. Limited investigation has been performed into elucidating structure-activity relationships around commonly used α-amino acid-derived head groups, mainly with valine and tert-leucine-derived compounds previously described. As such, proactive synthesis, characterisation and pharmacological evaluation were performed to explore structure-activity relationships of 15 α-amino acid derivatives, with both the natural isomers and their enantiomers at CB1 and CB2 investigated using a fluorescence-based membrane potential assay. This library was based around the detected SCRAs MPP-5F-PICA, MMB-5F-PICA, and MDMB-5F-PICA, with the latter showing significant receptor activation at CB1 (pEC50 = 8.34 ± 0.05 M; E max = 108 ± 3%) and CB2 (pEC50 = 8.13 ± 0.07 M; E max = 99 ± 2%). Most valine and leucine derivatives were potent and efficacious SCRAs, while smaller derivatives generally showed reduced activity at CB1 and CB2, and larger derivatives also showed reduced activity. SAR trends observed were rationalised via in silico induced fit docking. Overall, while natural enantiomers showed equipotent or greater activity than the unnatural isomers in most cases, this was not universal. As such, a number of these compounds should be monitored as emerging NPS, and various substituents described herein.

Synthesis and Functional Evaluation of Synthetic Cannabinoid Receptor Agonists Related to ADB-HEXINACA.

ADB-HEXINACA has been recently reported as a synthetic cannabinoid receptor agonist (SCRA), one of the largest classes of new psychoactive substances (NPSs). This compound marks the entry of the n-hexyl tail group into the SCRA landscape, which has continued in the market with recent, newly detected SCRAs. As such, a proactive characterization campaign was undertaken, including the synthesis, characterization, and pharmacological evaluation of ADB-HEXINACA and a library of 41 closely related analogues. Two in vitro functional assays were employed to assess activity at CB1 and CB2 cannabinoid receptors, measuring Gßγ-coupled agonism through a fluorescence-based membrane potential assay (MPA) and ß-arrestin 2 (ßarr2) recruitment via a live cell-based nanoluciferase complementation reporter assay. ADB-HEXINACA was a potent and efficacious CB1 agonist (CB1 MPA pEC50 = 7.87 ± 0.12 M; Emax = 124 ± 5%; ßarr2 pEC50 = 8.27 ± 0.14 M; Emax = 793 ± 42.5), as were most compounds assessed. Isolation of the heterocyclic core and alkyl tails allowed for the comprehensive characterization of structure-activity relationships in this compound class, which were rationalized in silico via induced fit docking experiments. Overall, most compounds assessed are possibly emerging NPSs.

Structure-Activity Relationships, Deuteration, and Fluorination of Synthetic Cannabinoid Receptor Agonists Related to AKB48, 5F-AKB-48, and AFUBIATA.

Synthetic cannabinoid receptor agonists (SCRAs) are a growing class of new psychoactive substances (NPS) commonly derived from an N-alkylated indole, indazole, or 7-azaindole scaffold. Diversification of this core (at the 3-position) with amide-linked pendant amino acid groups and modular N-alkylation (of the indole/indazole/7-azaindole core) ensures that novel SCRAs continue to enter the illicit drug market rapidly. In response to the large number of SCRAs that have been detected, pharmacological evaluation of this NPS class has become increasingly common. Adamantane-derived SCRAs have consistently appeared throughout the market since 2011, and as such, a systematic set of these derivatives was synthesized and pharmacologically evaluated. Deuterated and fluorinated adamantane derivatives were prepared to evaluate typical hydrogen bioisosteres, as well as evaluation of the newly detected AFUBIATA.

Comprehensive Characterization of a Systematic Library of Alkyl and Alicyclic Synthetic Cannabinoids Related to CUMYL-PICA, CUMYL-BUTICA, CUMYL-CBMICA, and CUMYL-PINACA.

Over 200 synthetic cannabinoid receptor agonists (SCRAs) have been identified as new psychoactive substances. Effective monitoring and characterization of SCRAs are hindered by the rapid pace of structural evolution. Ahead of possible appearance on the illicit drug market, new SCRAs were synthesized to complete a systematic library of cumyl-indole- (e.g., CUMYL-CPrMICA, CUMYL-CPMICA) and cumyl-indazole-carboxamides (e.g., CUMYL-CPrMINACA, CUMYL-CPMINACA), encompassing butyl, pentyl, cyclopropylmethyl, cyclobutylmethyl, cyclopentylmethyl, and cyclohexylmethyl tails. Comprehensive pharmacological characterization was performed with three assay formats, monitoring the recruitment of either wild-type or C-terminally truncated (ßarr2d366) ß-arrestin2 to the activated cannabinoid 1 receptor (CB1) or monitoring Gßγ-mediated membrane hyperpolarization. Altered compound characterization was observed when comparing derived potency (EC50) and efficacy (Emax) values from both assays monitoring the same or a different signaling event, whereas ranges and ranking orders were similar. Structure-activity relationships (SAR) were assessed in threefold, resulting in the identification of the pendant tail as a critical pharmacophore, with the optimal chain length for CB1 activation approximating an n-pentyl (e.g., cyclopentylmethyl or cyclohexylmethyl tail). The activity of the SCRAs encompassing cyclic tails decreased with decreasing number of carbons forming the cyclic moiety, with CUMYL-CPrMICA showing the least CB1 activity in all assay formats. The SARs were rationalized via molecular docking, demonstrating the importance of the optimal steric contribution of the hydrophobic tail. While SAR conclusions remained largely unchanged, the differential compound characterization by both similar and different assay designs emphasizes the importance of detailing specific assay characteristics to allow adequate interpretation of potencies and efficacies.

Off-target pharmacological profiling of synthetic cannabinoid receptor agonists including AMB-FUBINACA, CUMYL-PINACA, PB-22, and XLR-11.

Introduction: Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances that have been associated with multiple instances and types of toxicity. Some SCRAs appear to carry a greater toxicological burden than others, or compared to the prototypical cannabis-derived agonist Δ9-tetrahydrocannabinol (Δ9-THC), despite a common primary mechanism of action via cannabinoid 1 (CB1) receptors. "Off-target" (i.e., non-CB1 receptor) effects could underpin this differential toxicity, although there are limited data around the activity of SCRAs at such targets. Methods: A selection of 7 SCRAs (AMB-FUBINACA, XLR11, PB-22, AKB-48, AB-CHMINICA, CUMYL-PINACA, and 4F-MDMB-BUTINACA), representing several distinct chemotypes and toxicological profiles, underwent a 30 µM single-point screen against 241 G protein-coupled receptor (GPCR) targets in antagonist and agonist mode using a cellular ß-arrestin recruitment assay. Strong screening "hits" at specific GPCRs were followed up in detail using concentration-response assays with AMB-FUBINACA, a SCRA with a particularly notable history of toxicological liability. Results: The single-point screen yielded few hits in agonist mode for any compound aside from CB1 and CB2 receptors, but many hits in antagonist mode, including a range of chemokine receptors, the oxytocin receptor, and histamine receptors. Concentration-response experiments showed that AMB-FUBINACA inhibited most off-targets only at the highest 30 µM concentration, with inhibition of only a small subset of targets, including H1 histamine and α2B adrenergic receptors, at lower concentrations (≥1 µM). AMB-FUBINACA also produced concentration-dependent CB1 receptor signaling disruption at concentrations higher than 1 µM, but did not produce overt cytotoxicity beyond CP55,940 or Δ9-THC in CB1 expressing cells. Discussion: These results suggest that while some "off-targets" could possibly contribute to the SCRA toxidrome, particularly at high concentrations, CB1-mediated cellular dysfunction provides support for hypotheses concerning on-target, rather than off-target, toxicity. Further investigation of non-GPCR off-targets is warranted.

Putative Synthetic Cannabinoids MEPIRAPIM, 5F-BEPIRAPIM (NNL-2), and Their Analogues Are T-Type Calcium Channel (CaV3) Inhibitors.

Synthetic cannabinoid receptor agonists (SCRAs) are a large and growing class of new psychoactive substances (NPSs). Two recently identified compounds, MEPIRAPIM and 5F-BEPIRAPIM (NNL-2), have not been confirmed as agonists of either cannabinoid receptor subtype but share structural similarities with both SCRAs and a class of T-type calcium channel (CaV3) inhibitors under development as new treatments for epilepsy and pain. In this study, MEPIRAPIM and 5F-BEPIRAPIM and 10 systematic analogues were synthesized, analytically characterized, and pharmacologically evaluated using in vitro cannabinoid receptor and CaV3 assays. Several compounds showed micromolar affinities for CB1 and/or CB2, with several functioning as low potency agonists of CB1 and CB2 in a membrane potential assay. 5F-BEPIRAPIM and four other derivatives were identified as potential CaV3 inhibitors through a functional calcium flux assay (>70% inhibition), which was further confirmed using whole-cell patch-clamp electrophysiology. Additionally, MEPIRAPIM and 5F-BEPIRAPIM were evaluated in vivo using a cannabimimetic mouse model. Despite detections of MEPIRAPIM and 5F-BEPIRAPIM in the NPS market, only the highest MEPIRAPIM dose (30 mg/kg) elicited a mild hypothermic response in mice, with no hypothermia observed for 5F-BEPIRAPIM, suggesting minimal central CB1 receptor activity.

Structure-activity relationships of valine, tert-leucine, and phenylalanine amino acid-derived synthetic cannabinoid receptor agonists related to ADB-BUTINACA, APP-BUTINACA, and ADB-P7AICA.

Synthetic cannabinoid receptor agonists (SCRAs) remain one the most prevalent classes of new psychoactive substances (NPS) worldwide, and examples are generally poorly characterised at the time of first detection. We have synthesised a systematic library of amino acid-derived indole-, indazole-, and 7-azaindole-3-carboxamides related to recently detected drugs ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA, and characterised these ligands for in vitro binding and agonist activity at cannabinoid receptor subtypes 1 and 2 (CB1 and CB2), and in vivo cannabimimetic activity. All compounds showed high affinity for CB1 (K i 0.299-538 nM) and most at CB2 (K i = 0.912-2190 nM), and most functioned as high efficacy agonists of CB1 and CB2 in a fluorescence-based membrane potential assay and a ßarr2 recruitment assay (NanoBiT®), with some compounds being partial agonists in the NanoBiT® assay. Key structure-activity relationships (SARs) were identified for CB1/CB2 binding and CB1/CB2 functional activities; (1) for a given core, affinities and potencies for tert-leucinamides (ADB-) > valinamides (AB-) ≫ phenylalaninamides (APP-); (2) for a given amino acid side-chain, affinities and potencies for indazoles > indoles ≫ 7-azaindoles. Radiobiotelemetric evaluation of ADB-BUTINACA, APP-BUTINACA and ADB-P7AICA in mice demonstrated that ADB-BUTINACA and ADB-P7AICA were cannabimimetic at 0.1 mg kg-1 and 10 mg kg-1 doses, respectively, as measured by pronounced decreases in core body temperature. APP-BUTINACA failed to elicit any hypothermic response up to the maximally tested 10 mg kg-1 dose, yielding an in vivo potency ranking of ADB-BUTINACA > ADB-P7AICA > APP-BUTINACA.

Defining Steric Requirements at CB1 and CB2 Cannabinoid Receptors Using Synthetic Cannabinoid Receptor Agonists 5F-AB-PINACA, 5F-ADB-PINACA, PX-1, PX-2, NNL-1, and Their Analogues.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances (NPS). They commonly comprise N-alkylated indole, indazole, or 7-azaindole scaffolds with amide-linked pendant amino acid groups. To explore the contribution of the amino acid side chain to the cannabinoid pharmacology of SCRA NPS, a systematic library of side chain-modified SCRAs was prepared based on the recent detections of amino acid derivatives 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), and 20 (NNL-1). In vitro binding affinities and functional activities at cannabinoid type 1 and 2 receptors (CB1 and CB2, respectively) were determined for all the library members using radioligand competition experiments and a fluorescence-based membrane potential assay. Binding affinities and functional activities varied widely across compounds (Ki = 0.32 to >10 000 nM, EC50 = 0.24-1259 nM), with several clear structure-activity relationships (SARs) emerging. Affinity and potency at CB1 changed as a function of the heterocyclic core (indazole > indole > 7-azaindole) and the pendant amino acid side chain (tert-butyl > iso-propyl > iso-butyl > benzyl > ethyl > methyl > hydrogen). Ensemble docking at CB1 revealed a clear steric basis for observed SAR trends. Interestingly, although 15 (PX-1) and 19 (PX-2) have been detected in recreational drug markets, they failed to induce centrally CB1-mediated effects (e.g., hypothermia) in mice using radiobiotelemetry. Together, these data provide insights regarding structural contributions to the cannabimimetic profiles of 17 (5F-AB-PINACA), 18 (5F-ADB-PINACA), 15 (PX-1), 19 (PX-2), 20 (NNL-1), and other SCRA NPS.

Synthesis and pharmacological evaluation of newly detected synthetic cannabinoid receptor agonists AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA, MDMB-4F-BUTINACA and their analogs.

Synthetic cannabinoid receptor agonists (SCRAs) continue to make up a significant portion new psychoactive substances (NPS) detected and seized worldwide. Due to their often potent activation of central cannabinoid receptors in vivo, use of SCRAs can result in severe intoxication, in addition to other adverse health effects. Recent detections of AB-4CN-BUTICA, MMB-4CN-BUTINACA, MDMB-4F-BUTICA and MDMB-4F-BUTINACA mark a continuation in the appearance of SCRAs bearing novel tail substituents. The proactive characterization campaign described here has facilitated the detection of several new SCRAs in toxicological case work. Here we detail the synthesis, characterization, and pharmacological evaluation of recently detected SCRAs, as well as a systematic library of 32 compounds bearing head, tail, and core group combinations likely to appear in future. In vitro radioligand binding assays revealed most compounds showed moderate to high affinity at both CB1 (pK i = < 5 to 8.89 ± 0.09 M) and CB2 (pK i = 5.49 ± 0.03 to 9.92 ± 0.09 M) receptors. In vitro functional evaluation using a fluorescence-based membrane potential assay showed that most compounds were sub-micromolar to sub-nanomolar agonists at CB1 (pEC50 = < 5 to 9.48 ± 0.14 M) and CB2 (pEC50 = 5.92 ± 0.16 to 8.64 ± 0.15 M) receptors. An in silico receptor-ligand docking approach was utilized to rationalize binding trends for CB2 with respect to the tail substituent, and indicated that rigidity in this region (i.e., 4-cyanobutyl) was detrimental to affinity.

CD4+CD25+ T cells alloactivated ex vivo by IL-2 or IL-4 become potent alloantigen-specific inhibitors of rejection with different phenotypes, suggesting separate pathways of activation by Th1 and Th2 responses.

CD4(+)CD25(+)Foxp3(+) T cells are regulatory/suppressor cells (Tregs) that include non-antigen (Ag)-specific as well as Ag-specific Tregs. How non-Ag-specific naive CD4(+)CD25(+) Treg develop into specific Tregs is unknown. Here, we generated adaptive Tregs by culture of naive CD4(+)CD25(+)Foxp3(+) T cells with allo-Ag and either interleukin-2 (IL-2) or IL-4. Within days, IL-2 enhanced interferon-gamma receptor (Ifngammar) and Il-5 mRNA and IL-4 induced a reciprocal profile with de novo IL-5Ralpha and increased IFN-gamma mRNA expression. Both IL-2- and IL-4-alloactivated CD4(+)CD25(+) Tregs within 3 to 4 days of culture had enhanced capacity to induce tolerance to specific donor but not to third-party cardiac allografts. These hosts became tolerant as allografts functioned more than 250 days, with a physiologic ratio of less than 10% CD4(+)CD25(+)Foxp3(+) T cells in the CD4(+) population. CD4(+)CD25(+) T cells from tolerant hosts given IL-2-cultured cells had increased Il-5 and Ifngammar mRNA. Those from hosts given IL-4-cultured cells had enhanced IL-5Ralpha mRNA expression and IL-5 enhanced their proliferation to donor but not third-party allo-Ag. Thus, IL-2 and IL-4 activated allo-Ag-specific Tregs with distinct phenotypes that were retained in vivo. These findings suggested that T-helper 1 (Th1) and Th2 responses activate 2 pathways of adaptive Ag-specific Tregs that mediate tolerance. We propose they be known as T-suppressor 1 (Ts1) and Ts2 cells.

Brodifacoum does not modulate human cannabinoid receptor-mediated hyperpolarization of AtT20 cells or inhibition of adenylyl cyclase in HEK 293 cells.

BACKGROUND: Synthetic cannabinoids are a commonly used class of recreational drugs that can have significant adverse effects. There have been sporadic reports of co-consumption of illicit drugs with rodenticides such as warfarin and brodifacoum (BFC) over the past 20 years but recently, hundreds of people have been reported to have been poisoned with a mixture of synthetic cannabinoids and BFC. We have sought to establish whether BFC directly affects cannabinoid receptors, or their activation by the synthetic cannabinoid CP55940 or the phytocannabinoid Δ9-tetrahydrocannabinol (Δ9-THC). METHODS: The effects of BFC on the hyperpolarization of wild type AtT20 cells, or AtT20 cells stably expressing human CB1- or CB2- receptors, were studied using a fluorescent assay of membrane potential. The effect of BFC on CB1- and CB2-mediated inhibition of forskolin-stimulated adenylyl cyclase (AC) activation was measured using a BRET assay of cAMP levels in HEK 293 cells stably expressing human CB1 or CB2. RESULTS: BFC did not activate CB1 or CB2 receptors, or affect the hyperpolarization of wild type AtT20 cells produced by somatostatin. BFC (1 µM) did not affect the hyperpolarization of AtT20-CB1 or AtT20-CB2 cells produced by CP55940 or Δ9-THC. BFC (1 µM) did not affect the inhibition of forskolin-stimulated AC activity by CP55940 in HEK 293 cells expressing CB1 or CB2. BFC (1 µM) also failed to affect the desensitization of CB1 and CB2 signaling produced by prolonged (30 min) application of CP55940 or Δ9-THC to AtT20 cells. DISCUSSION: BFC is not a cannabinoid receptor agonist, and appeared not to affect cannabinoid receptor activation. Our data suggests there is no pharmacodynamic rationale for mixing BFC with synthetic cannabinoids; however, it does not speak to whether BFC may affect synthetic cannabinoid metabolism or biodistribution. The reasons underlying the mixing of BFC with synthetic cannabinoids are unknown, and it remains to be established whether the "contamination" was deliberate or accidental. However, the consequences for people who ingested the mixture were often serious, and sometimes fatal, but this seems unlikely to be due to BFC action at cannabinoid receptors.

Increased excitatory regulation of the hypothalamic paraventricular nucleus and circulating vasopressin results in the hypertension observed in polycystic kidney disease.

BACKGROUND: Hypertension and baroreflex dysfunction confer poorer outcomes in patients with polycystic kidney disease (PKD). METHOD: We examined whether hypothalamic paraventricular nucleus (PVN) activation or circulating vasopressin contribute to hypertension and baroreflex dysfunction in the Lewis polycystic kidney (LPK) rat. RESULTS: Bilateral PVN inhibition with muscimol reduced SBP further in urethane-anaesthetized adult LPK rats than in control Lewis rats (-43 ±â€Š4 vs. -18 ±â€Š3 mmHg; P < 0.0001, n = 14), but was not associated with a greater reduction in sympathetic nerve activity (SNA) or improvement in HR or SNA baroreflex function. Blockade of ionotropic glutamatergic input to the PVN with kynurenic acid also reduced SBP (P < 0.001), but not SNA, further in both adult and juvenile LPK rats. No differences in AMPA or NMDA receptor mRNA expression were noted. Systemic V1A receptor antagonism using OPC-21268 reduced SBP in adult LPK rats only (P < 0.001) and had no effect on the depressor response to PVN inhibition (P = 0.39). Combined peripheral V1A receptor antagonism and PVN inhibition, however, normalized SBP in adult LPK rats (122 ±â€Š11 vs. 115 ±â€Š6 mmHg; LPK vs. Lewis, P > 0.05, n = 10). CONCLUSION: Our data show that in the LPK rat model of PKD, hypertension is contributed to by increased PVN neuronal activity and, through an independent mechanism, systemic V1A receptor activation. Treatments that reduce PVN neuronal activity and/or inhibit peripheral V1A receptors may provide novel treatment strategies to ameliorate hypertension in individuals with PKD and limit overall disease progression.

CUMYL-4CN-BINACA Is an Efficacious and Potent Pro-Convulsant Synthetic Cannabinoid Receptor Agonist.

Synthetic cannabinoid receptor agonists (SCRAs) are the largest class of new psychoactive substances (NPS). New examples are detected constantly, and some are associated with a series of adverse effects, including seizures. CUMYL-4CN-BINACA (1-(4-cyanobutyl)-N-(2-phenylpropan-2-yl)indazole-3-carboxamide) is structurally related to potent, cumylamine-derived SCRAs such as 5F-CUMYL-PINACA, but is unusual due to a terminal aliphatic nitrile group not frequently encountered in SCRAs or pharmaceuticals. We report here that CUMYL-4CN-BINACA is a potent CB1 receptor agonist (K i = 2.6 nM; EC50 = 0.58 nM) that produces pro-convulsant effects in mice at a lower dose than reported for any SCRA to date (0.3 mg/kg, i.p). Hypothermic and pro-convulsant effects in mice could be reduced or blocked, respectively, by pretreatment with CB1 receptor antagonist SR141716, pointing to at least partial involvement of CB1 receptors in vivo. Pretreatment with CB2 receptor antagonist AM-630 had no effect on pro-convulsant activity. The pro-convulsant properties and potency of CUMYL-4CN-BINACA may underpin the toxicity associated with this compound in humans.

The chemistry and pharmacology of putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS) 5F-PY-PICA, 5F-PY-PINACA, and their analogs.

5F-PY-PICA and 5F-PY-PINACA are pyrrolidinyl 1-(5-fluoropentyl)ind (az)ole-3-carboxamides identified in 2015 as putative synthetic cannabinoid receptor agonist (SCRA) new psychoactive substances (NPS). 5F-PY-PICA, 5F-PY-PINACA, and analogs featuring variation of the 1-alkyl substituent or contraction, expansion, or scission of the pyrrolidine ring were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-quadrupole time-of-flight-mass spectrometry (LC-QTOF-MS). In competitive binding experiments against HEK293 cells expressing human cannabinoid receptor type 1 (hCB1 ) or type 2 (hCB2 ), all analogs showed minimal affinity for CB1 (pKi  < 5), although several demonstrated moderate CB2 binding (pKi 5.45-6.99). In fluorescence-based membrane potential assays using AtT20-hCB1 or -hCB2 cells, none of the compounds (at 10 µM) produced an effect >50% of the classical cannabinoid agonist CP55,940 (at 1 µM) at hCB1 , although several showed slightly higher relative efficacy at hCB2 . Expansion of the pyrrolidine ring of 5F-PY-PICA to an azepane (8) conferred the greatest hCB2 affinity (pKi 6.99) and activity (pEC50 7.54, Emax 72%) within the series. Unlike other SCRA NPS evaluated in vivo using radio biotelemetry, 5F-PY-PICA and 5F-PY-PINACA did not produce cannabimimetic effects (hypothermia, bradycardia) in mice at doses up to 10 mg/kg.

Synthesis and pharmacology of new psychoactive substance 5F-CUMYL-P7AICA, a scaffold- hopping analog of synthetic cannabinoid receptor agonists 5F-CUMYL-PICA and 5F-CUMYL-PINACA.

Synthetic cannabinoid receptor agonists (SCRAs) are a dynamic class of new psychoactive substances (NPS), with novel chemotypes emerging each year. Following the putative detection of 5F-CUMYL-P7AICA in Australia in 2016, the scaffold-hopping SCRAs 5F-CUMYL-PICA, 5F-CUMYL-PINACA, and 5F-CUMYL-P7AICA were synthesized and characterized by nuclear magnetic resonance (NMR) spectroscopy, gas chromatography-mass spectrometry (GC-MS), and liquid chromatography-quadrupole time-of-flight-MS (LC-QTOF-MS). Since little is known of the pharmacology of 7-azaindole SCRAs like 5F-CUMYL-P7AICA, the binding affinities and functional activities of all compounds at cannabinoid type 1 and type 2 receptors (CB1 and CB2 , respectively) were assessed using tritiated radioligand competition experiments and fluorescence-based plate reader membrane potential assays. Despite CB1 binding affinities differing by over two orders of magnitude (Ki  = 2.95-174 nM), all compounds were potent and efficacious CB1 agonists (EC50  = 0.43-4.7 nM), with consistent rank order for binding and functional activity (5F-CUMYL-PINACA >5F-CUMYL-PICA >5F-CUMYL-P7AICA). Additionally, 5F-CUMYL-P7AICA was found to exert potent cannabimimetic effects in mice, inducing hypothermia (6°C, 3 mg/kg) through a CB1 -dependent mechanism.

Studies on naïve CD4+CD25+T cells inhibition of naïve CD4+CD25- T cells in mixed lymphocyte cultures.

BACKGROUND: Naïve CD4+CD25+T cells suppress immune responses in a non-antigen specific manner. The effects of naïve CD4+CD25+T cells in suppressing alloimmune responses as assayed in the mixed lymphocyte culture (MLC) is poorly understood. METHOD: The alloreactivity of naïve CD4+CD25+, CD4+CD25(-) and unfractionated CD4+T cells from DA rats was compared in MLC with MHC incompatible stimulator cells. The response of Lewis and PVG cells to semi-allogeneic (LewisxPVG)F1 cells and fully allogeneic stimulators were compared. Potential mechanisms of suppression were examined by blocking T cell cytokines, produced by activated CD4+CD25+T cells. RESULTS: Proliferation of CD4+CD25(-)T cells was significantly greater than unfractionated CD4+T cells to both allogeneic and syngeneic stimulator cells. CD4+CD25+T cells had no response to syngeneic stimulators and very low proliferative responses to alloantigen due to the Foxp3(-) cells. Admixing CD4+CD25+T cells with CD4+CD25(-)T cells at a ratio of 1:10 reduced the proliferation to that of unfractionated CD4+ T cells. At a ratio of 1:1 proliferation was nearly totally suppressed, IL-2, IL-4 and IL-5 mRNA induction was reduced but IFN-gamma, IL-10, TGF-beta and inducible nitric oxide (iNOS) mRNA induction was spared. The inhibition by CD4+CD25+ T cells was not due to their consumption of IL-2 nor to anti-CD25mAb that had been used to enrich the cells being releases and blocking the IL-2 receptor on CD4+CD25(-)T cells that had been activated by alloantigen and induced to express CD25. Blocking IFN-gamma, IL-10, TGF-beta, IL-5 or iNOS did not prevent CD4+CD25+T cell's inhibition of CD4+CD25(-)T cell proliferation. Blocking IFN-gamma or iNOS enhanced CD4+CD25(-)T cell proliferation only in the absence of CD4+CD25+T cells. Depletion of CD4+CD25+T cells enhanced responses to syngeneic stimulator cells, but this anti-self suppression did not regulate the response to alloantigen on semi-allogeneic stimulators. CONCLUSIONS: Two independent mechanisms that control proliferation of CD4+CD25(-)T cells in MLC were identified that naive CD4+CD25+T cells mediated by cell to cell contact and not release of cytokines produced in the cultures, and that CD4+CD25(-)T cells producing IFN-gamma to induce iNOS.

Transfer of allograft specific tolerance requires CD4+CD25+T cells but not interleukin-4 or transforming growth factor-beta and cannot induce tolerance to linked antigens.

BACKGROUND: The mechanisms by which CD4+T cells, especially CD4+ CD25+T cells, transfer allograft specific tolerance are poorly defined. The role of cytokines and the effect on antigen-presenting cells is not resolved. METHODS: Anti-CD3 monoclonal antibody (mAb) therapy induced tolerance to PVG heterotopic cardiac transplantation in DA rats. Peripheral CD4+T cells or CD4+ CD25+ and CD4+ CD25-T cell subsets were adoptively transferred to irradiated DA hosts grafted with PVG heart grafts. For specificity studies, tolerant CD4+T cells were transferred to hosts with Lewis or (PVGxLewis)F1 heart grafts. Cytokine mRNA induction and the requirement for interleukin (IL)-4 and transforming growth factor (TGF)-beta in the transfer of tolerance was assessed. RESULTS: CD4+T cells transferred specific tolerance and suppressed naïve CD4+T cells capacity to effect rejection of PVG but not Lewis grafts. (PVGxLewis)F1 grafts had a major rejection episode but recovered. Later these hosts accepted PVG but not Lewis skin grafts. Adoptive hosts restored with tolerant or naïve cells had similar levels of mRNA expression for all Th1 and Th2 cytokines and effector molecules assayed. Transfer of tolerance by CD4+T cells was not blocked by mAb to IL-4 or TGF-beta. CD4+ CD25-T cells from either naïve or tolerant hosts effected rejection. In contrast neither tolerant nor naïve CD4+ CD25+T cells restored rejection. CONCLUSIONS: Specific tolerance transfer required CD4+ containing CD4+ CD25+T cells. An inflammatory response with induction of mRNA for Th1 and Th2 cytokines plus cytotoxic effector molecules occurred, but IL-4 and TGF-beta were not essential. Inhibition of antigen presenting cells was not the sole mechanism as there was no linked tolerance.