Peter J. Greasley

The orphan receptor GPR55 is a novel cannabinoid receptor

The endocannabinoid system functions through two well characterized receptor systems, the CB1 and CB2 receptors. Work by a number of groups in recent years has provided evidence that the system is more complicated and additional receptor types should exist to explain ligand activity in a number of physiological processes.

The putative cannabinoid receptor GPR55 affects osteoclast function in vitro and bone mass in vivo

GPR55 is a G protein-coupled receptor recently shown to be activated by certain cannabinoids and by lysophosphatidylinositol (LPI). However, the physiological role of GPR55 remains unknown. Given the recent finding that the cannabinoid receptors CB1 and CB2 affect bone metabolism, we examined the role of GPR55 in bone biology. GPR55 was expressed in human and mouse osteoclasts and osteoblasts; expression was higher in human osteoclasts than in macrophage progenitors. Although the GPR55 agonists O-1602 and LPI inhibited mouse osteoclast formation in vitro, these ligands stimulated mouse and human osteoclast polarization and resorption in vitro and caused activation of Rho and ERK1/2. These stimulatory effects on osteoclast function were attenuated in osteoclasts generated from GPR55−/− macrophages and by the GPR55 antagonist cannabidiol (CBD). Furthermore, treatment of mice with this non-psychoactive constituent of cannabis significantly reduced bone resorption in vivo. Consistent with the ability of GPR55 to suppress osteoclast formation but stimulate osteoclast function, histomorphometric and microcomputed tomographic analysis of the long bones from male GPR55−/− mice revealed increased numbers of morphologically inactive osteoclasts but a significant increase in the volume and thickness of trabecular bone and the presence of unresorbed cartilage. These data reveal a role of GPR55 in bone physiology by regulating osteoclast number and function. In addition, this study also brings to light an effect of both the endogenous ligand, LPI, on osteoclasts and of the cannabis constituent, CBD, on osteoclasts and bone turnover in vivo.

Identification and characterisation of a novel splice variant of the human CB1 receptor

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNAs are expressed at low levels in a variety of tissues.

Big pharma screening collections: more of the same or unique libraries? The AstraZeneca–Bayer Pharma AG case

In this study, the screening collections of two major pharmaceutical companies (AstraZeneca and Bayer Pharma AG) have been compared using a 2D molecular fingerprint by a nearest neighborhood approach. Results revealed a low overlap between both collections in terms of compound identity and similarity. This emphasizes the value of screening multiple compound collections to expand the chemical space that can be accessed by high-throughput screening (HTS).

N-Arachidonoyl-l-Serine is Neuroprotective after Traumatic Brain Injury by Reducing Apoptosis

N-arachidonoyl-L-serine (AraS) is a brain component structurally related to the endocannabinoid family. We investigated the neuroprotective effects of AraS following closed head injury induced by weight drop onto the exposed fronto-parietal skull and the mechanisms involved. A single injection of AraS following injury led to a significant improvement in functional outcome, and to reduced edema and lesion volume compared with vehicle. Specific antagonists to CB2 receptors, transient receptor potential vanilloid 1 (TRPV1) or large conductance calcium-activated potassium (BK) channels reversed these effects. Specific binding assays did not indicate binding of AraS to the GPR55 cannabinoid receptor. N-arachidonoyl-l-serine blocked the attenuation in phosphorylated extracellular-signal-regulated kinase 1/2 (ERK) levels and led to an increase in pAkt in both the ipsilateral and contralateral cortices. Increased levels of the prosurvival factor Bcl-xL were evident 24 hours after injury in AraS-treated mice, followed by a 30% reduction in caspase-3 activity, measured 3 days after injury. Treatment with a CB2 antagonist, but not with a CB1 antagonist, reversed this effect. Our results suggest that administration of AraS leads to neuroprotection via ERK and Akt phosphorylation and induction of their downstream antiapoptotic pathways. These protective effects are related mostly to indirect signaling via the CB2R and TRPV1 channels but not through CB1 or GPR55 receptors.

Differential Effects of Dapagliflozin on Cardiovascular Risk Factors at Varying Degrees of Renal Function

BACKGROUND AND OBJECTIVE Sodium glucose cotransporter 2 inhibition with dapagliflozin decreases hemoglobin A1c (HbA1c), body weight, BP, and albuminuria (urinary albumin-to-creatinine ratio). Dapagliflozin also modestly increases hematocrit, likely related to osmotic diuresis/natriuresis. Prior studies suggest that the HbA1c-lowering effects of dapagliflozin attenuate at lower eGFR. However, effects on other cardiovascular risk factors at different eGFR levels are incompletely understood. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This pooled analysis of 11 phase 3 clinical trials assessed changes in HbA1c, body weight, BP, hematocrit, and urinary albumin-to-creatinine ratio with placebo (n=2178) or dapagliflozin 10 mg (n=2226) over 24 weeks in patients with type 2 diabetes according to baseline eGFR (eGFR≥45 to <60 ml/min per 1.73 m2, eGFR≥60 to <90 ml/min per 1.73 m2, and eGFR≥90 ml/min per 1.73 m2). RESULTS Compared with placebo, reductions in HbA1c with dapagliflozin were 0.6%, 0.5%, and 0.3%, respectively, for each consecutive lower eGFR subgroup (P value interaction <0.001). Effects of dapagliflozin on hematocrit, body weight, and BP were similar regardless of baseline eGFR, suggesting that effects potentially related to volume and natriuresis are eGFR independent. Moreover, among individuals with baseline urinary albumin-to-creatinine ratio ≥30 mg/g, placebo-adjusted reductions in urinary albumin-to-creatinine ratio were larger in the lowest eGFR subgroup (P value interaction <0.001). Adverse events occurred more frequently in the lowest eGFR subgroup; this was true for both dapagliflozin- and placebo-treated patients. CONCLUSIONS The HbA1c-lowering effects of dapagliflozin decrease as renal function declines. However, dapagliflozin consistently decreases body weight, BP, and urinary albumin-to-creatinine ratio regardless of eGFR. These effects in conjunction with the finding of similar effects on hematocrit, a proxy for volume contraction, suggest that the effects of dapagliflozin are partly mediated via nonglucosuric-dependent mechanisms.

Succinate receptor GPR91, a Gαi coupled receptor that increases intracellular calcium concentrations through PLCβ

Succinate has been reported as the endogenous ligand for GPR91. In this study, succinate was confirmed to activate GPR91 resulting in both 3′–5′‐cyclic adenosine monophosphate (cAMP) inhibition and inositol phosphate formation in a pertussis toxin (PTX)‐sensitive manner. GPR91 agonist‐mediated effects detected using dynamic mass redistribution (DMR) were inhibited with PTX, edelfosine and U73122 demonstrating the importance of not only the Gαi pathway but also PLCβ. These results show that GPR91 when expressed in HEK293s cells couples exclusively through the Gαi pathway and acts through Gαi not only to inhibit cAMP production but also to increase intracellular Ca2+ in an inositol phosphate dependent mechanism via PLCβ activation.

Novel thioamide derivatives as neutral CB1 receptor antagonists.

A novel class of cannabinoid-1 (CB1) receptor antagonists for the treatment of obesity is presented. The carboxamide linker in a set of 5,6-diaryl-pyrazine-2-amide derivatives was transformed into the corresponding thioamide, by using a one-pot synthesis. The structural series of thioamides not only showed retained CB1 potency (below 10nM), but also showed improved solubility. In addition, the neutral antagonist 2c significantly reduced body weight in cafeteria diet obese mice.

Why do SGLT2 inhibitors reduce heart failure hospitalization? A differential volume regulation hypothesis

The effect of a sodium glucose cotransporter 2 inhibitor (SGLT2i) in reducing heart failure hospitalization in the EMPA‐REG OUTCOMES trial has raised the possibility of using these agents to treat established heart failure. We hypothesize that osmotic diuresis induced by SGLT2 inhibition, a distinctly different diuretic mechanism than that of other diuretic classes, results in greater electrolyte‐free water clearance and, ultimately, in greater fluid clearance from the interstitial fluid (IF) space than from the circulation, potentially resulting in congestion relief with minimal impact on blood volume, arterial filling and organ perfusion. We utilize a mathematical model to illustrate that electrolyte‐free water clearance results in a greater reduction in IF volume compared to blood volume, and that this difference may be mediated by peripheral sequestration of osmotically inactive sodium. By coupling the model with data on plasma and urinary sodium and water in healthy subjects who received either the SGLT2i dapagliflozin or loop diuretic bumetanide, we predict that dapagliflozin produces a 2‐fold greater reduction in IF volume compared to blood volume, while the reduction in IF volume with bumetanide is only 78% of the reduction in blood volume. Heart failure is characterized by excess fluid accumulation, in both the vascular compartment and interstitial space, yet many heart failure patients have arterial underfilling because of low cardiac output, which may be aggravated by conventional diuretic treatment. Thus, we hypothesize that, by reducing IF volume to a greater extent than blood volume, SGLT2 inhibitors might provide better control of congestion without reducing arterial filling and perfusion.

Effect of Tenapanor on Interdialytic Weight Gain in Patients on Hemodialysis

BACKGROUND AND OBJECTIVES Interdialytic weight gain in patients on hemodialysis is associated with adverse cardiovascular outcomes and increased mortality. The degree of interdialytic weight gain is influenced by sodium intake. We evaluated the effects of tenapanor (AZD1722 and RDX5791), a minimally systemically available inhibitor of the sodium/hydrogen exchanger isoform 3, on interdialytic weight gain in patients with CKD stage 5D treated with hemodialysis. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS This phase 2, randomized, double-blind study (NCT01764854; conducted January to September of 2013) enrolled adults on maintenance hemodialysis with interdialytic weight gain ≥3.0% of postdialysis weight and ≥2 kg. Patients were randomly assigned (1:1) to receive tenapanor or placebo. The primary end point was change in mean interdialytic weight gain (percentage of baseline postdialysis weight) from baseline (mean across a 2-week run-in period) to week 4. In a subgroup of inpatients, 24-hour stool sodium and stool weight were assessed for 1 week. RESULTS Sixteen patients received 1 week of inpatient treatment (tenapanor, eight; placebo, eight), and 72 patients received 4 weeks of treatment in an outpatient setting (tenapanor, 37; placebo, 35; completers: tenapanor, 31; placebo, 33). In the outpatient cohort, no significant effect on interdialytic weight gain was detected; least squares mean changes in relative interdialytic weight gain from baseline to week 4 were tenapanor, -0.26% (95% confidence interval, -0.57% to 0.06%) and placebo, -0.23% (95% confidence interval, -0.54% to 0.07%; P=0.46). During week 1 (inpatient cohort only), compared with placebo, tenapanor treatment resulted in higher stool sodium content (mean [±SD]: tenapanor, 36.6 [±21.8] mmol/d; placebo, 2.8 [±2.7] mmol/d; P<0.001) and higher stool weight (tenapanor, 172.5 [±68.1] g/d; placebo, 86.3 [±30.0] g/d; P<0.01). A similar safety profile was observed across treatment groups with the exception of diarrhea, which occurred more frequently with tenapanor treatment. CONCLUSIONS Tenapanor treatment increased stool sodium and weight over placebo in patients undergoing hemodialysis. However, over 4 weeks of treatment, there was no difference in interdialytic weight gain between patients treated with tenapanor and those receiving placebo.