Inhibition of osteoclastogenesis by a phosphodiesterase 4 inhibitor XT-611 through synergistic action with endogenous prostaglandin E2

We examined the effect of a phosphodiesterase 4 (PDE4) inhibitor, 3,4-dipropyl-4,5,7,8-tetrahydro-3H-imidazo[1,2-i]-purin-5-one (XT-611) on osteoclast formation in three different mouse bone-marrow cell (BMC) culture systems. We confirmed that selective inhibitors of PDE4, including XT-611, among several PDE inhibitors decreased osteoclast formation in the BMC culture system. XT-611 also inhibited osteoclast formation in co-culture of mouse bone-marrow stromal cell line ST2 and adherent cell-depleted (ACD)-BMCs. However, it did not inhibit osteoclastogenesis in culture of ACD-BMCs alone in the presence of macrophage-colony stimulating factor (M-CSF) and soluble receptor activator of NF-kappaB ligand (sRANKL). XT-611 significantly increased prostaglandin E(2) (PGE(2)) production from ST2 cells and, in combination with PGE(2), synergistically increased cAMP concentration in osteoclast progenitors. In the ST2 co-culture system, XT-611 did not influence the expression of RANKL, osteoprotegerin and RANK mRNAs. By combined treatment with XT-611 and PGE(2) of ACD-BMCs, osteoclast multinucleation was clearly inhibited with decrease in the expression of calcitonin receptor mRNA, while the expression of RANK and c-fms (an M-CSF receptor) mRNAs was unchanged. These results indicate that the PDE4 inhibitor inhibits osteoclastogenesis by acting on osteoclast progenitors synergistically with PGE(2) secreted from stromal cells, but not by influencing the cell-to-cell interaction between stromal cells and osteoclast progenitors.     

Metabolism of 26,26,26,27,27,27-F6-1alpha,25-dihydroxyvitamin D3 by CYP24: species-based difference between humans and rats

The compound 26,26,26,27,27,27-F(6)-1alpha,25(OH)(2)D(3) is a hexafluorinated analog of the active form of Vitamin D(3). The enhanced biological activity of F(6)-1alpha,25(OH)(2)D(3) is considered to be related to a decreased metabolic inactivation of the compound in target tissues such as the kidneys, small intestine, and bones. Our previous study demonstrated that CYP24 is responsible for the metabolism of F(6)-1alpha,25(OH)(2)D(3) in the target tissues. In this study, we compared the human and rat CYP24-dependent metabolism of F(6)-1alpha,25(OH)(2)D(3) by using the Escherichia coli expression system. In the recombinant E. coli cells expressing human CYP24, bovine adrenodoxin and NADPH-adrenodoxin reductase, F(6)-1alpha,25(OH)(2)D(3) was successively converted to F(6)-1alpha,23S,25(OH)(3)D(3), F(6)-23-oxo-1alpha,25(OH)(2)D(3), and the putative ether compound with the same molecular mass as F(6)-1alpha,25(OH)(2)D(3). The putative ether was not observed in the recombinant E. coli cells expressing rat CYP24. These results indicate species-based difference between human and rat CYP24 in the metabolism of F(6)-1alpha,25(OH)(2)D(3). In addition, the metabolite with a cleavage at the C(24)z.sbnd;C(25) bond of F(6)-1alpha,25(OH)(2)D(3) was detected as a minor metabolite in both human and rat CYP24. Although F(6)-1alpha,23S,25(OH)(3)D(3) and F(6)-23-oxo-1alpha,25(OH)(2)D(3) had a high affinity for Vitamin D receptor, the side-chain cleaved metabolite and the putative ether showed extremely low affinity for Vitamin D receptor. These findings indicate that human CYP24 has a dual pathway for metabolic inactivation of F(6)-1alpha,25(OH)(2)D(3) while rat CYP24 has only one pathway. Judging from the fact that metabolism of F(6)-1alpha,25(OH)(2)D(3) in rat CYP24-harboring E. coli cells is quite similar to that in the target tissues of rat, the metabolism seen in human CYP24-harboring E. coli cells appear to exhibit the same metabolism as in human target tissues. Thus, this recombinant system harboring human CYP24 appears quite useful for predicting the metabolism and efficacy of Vitamin D analogs in human target tissues before clinical trials.     

Distinction of microsomal prostaglandin E synthase-1 (mPGES-1) inhibition from cyclooxygenase-2 inhibition in cells using a novel, selective mPGES-1 inhibitor

Inflammation-induced microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme that synthesizes prostaglandin E₂ (PGE₂) downstream of cyclooxygenase-2 (COX-2). The efficacy of nonsteroidal anti-inflammatory drugs and COX-2 inhibitors in the treatment of the signs and symptoms of osteoarthritis, rheumatoid arthritis and inflammatory pain, largely attributed to the inhibition of PGE₂ synthesis, provides a rationale for exploring mPGES-1 inhibition as a potential novel therapy for these diseases. Toward this aim, we identified PF-9184 as a novel mPGES-1 inhibitor. PF-9184 potently inhibited recombinant human (rh) mPGES-1 (IC₅₀ = 16.5 ± 3.8 nM), and had no effect against rhCOX-1 and rhCOX-2 (>6500-fold selectivity). In inflammation and clinically relevant biological systems, mPGES-1 expression, like COX-2 expression was induced in cell context- and time-dependent manner, consistent with the kinetics of PGE₂ synthesis. In rationally designed cell systems ideal for determining direct effects of the inhibitors on mPGES-1 function, but not its expression, PF-9184 inhibited PGE₂ synthesis (IC₅₀ in the range of 0.5-5 μM in serum-free cell and human whole blood cultures, respectively) while sparing the synthesis of 6-keto-PGF_1α (PGF_1α) and PGF_2α. In contrast, as expected, the selective COX-2 inhibitor, SC-236, inhibited PGE₂, PGF_1α and PGF_2α synthesis. This profile of mPGES-1 inhibition, distinct from COX-2 inhibition in cells, validates mPGES-1 as an attractive target for therapeutic intervention.     

Inhibition of osteoclast differentiation and bone resorption by a novel lysophosphatidylcholine derivative, SCOH

Osteoclasts are multinucleated cells formed by multiple steps of cell differentiation from progenitor cells of hematopoietic origin. Intervention in osteoclast differentiation is considered as an effective therapeutic approach to the treatment for bone diseases involving osteoclasts. In this study, we found that the organic compound (S)-1-lyso-2-stearoylamino-2-deoxy-sn-glycero-3-phosphatidylcholine (SCOH) inhibited osteoclast differentiation. The inhibitory effect of SCOH was observed in mouse bone marrow cell cultures supported either by coculturing with osteoblasts or by adding macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor kappaB ligand (RANKL). M-CSF and RANKL activate the ERK, Akt, and NF-kappaB signal transduction pathways, and SCOH suppressed this activation. SCOH also inhibited the bone resorptive activity of differentiated osteoclasts. It attenuated bone resorption, actin ring formation, and survival of mature osteoclasts. Reduced activation of Akt and NF-kappaB and decreased induction of XIAP were observed in mature osteoclasts treated with SCOH. Thus, this novel phosphatidylcholine derivative may be useful for treating bone-resorption diseases.     

Discovery of a novel COX-2 inhibitor as an orally potent anti-pyretic and anti-inflammatory drug: design, synthesis, and structure-activity relationship

Cyclooxygenase (COX) has been considered as a significant pharmacological target because of its pivotal roles in the prostaglandin biosynthesis and following cascades that lead to various (patho)physiological effects. Non-steroidal anti-inflammatory drugs (NSAIDs) that suppress COX activities have been used clinically for the treatment of fever, inflammation, and pain; however, nonselective COX inhibitors exhibit serious side-effects such as gastrointestinal damage because of their inhibitory activities against COX-1. Thus, COX-1 is constitutive and expressed ubiquitously and serves a housekeeping role, while COX-2 is inducible or upregulated by inflammatory/injury stimuli such as interleukin-1β, tumor necrosis factor-α, and lipopolysaccharide in macrophage, monocyte, synovial, liver, and lung, and is associated with prostaglandin E₂ and prostacyclin production that evokes or sustains systemic/peripheral inflammatory symptoms. Also, hypersensitivity of aspirin is a significant concern clinically. Hence, design, synthesis, and structure–activity relationship of [2-{[(4-substituted)-pyridin-2-yl]carbonyl}-(6- or 5-substituted)-1H-indol-3-yl]acetic acid analogues were investigated to discover novel acid-type COX-2 inhibitor as an orally potent new-class anti-pyretic and anti-inflammatory drug. As significant findings, compounds 1–3 demonstrated potent COX-2 inhibitory activities with high selectivities for COX-2 over COX-1 in human cells or whole-blood in vitro, and demonstrated orally potent anti-pyretic activity against lipopolysaccharide-induced systemic-inflammatory fever model in F344 rats. Also compound 1 demonstrated orally potent anti-inflammatory activity against edema formation and a suppressive effect against PGE₂ production in carrageenan-induced peripheral-inflammation model on the paw of SD rats. These results suggest that compounds 1–3 are potential agents for the treatment of inflammatory disease and are useful for further pharmacological COX-2 inhibitor investigations.     

Effects of FK228, a novel histone deacetylase inhibitor,on human lymphoma U-937 cells in vitro and in vivo

FK228 [(E)-(1S,4S,10S,21R)-7-[(Z)-ethylidene]-4,21-diisopropyl-2-oxa-12,13-dithia-5,8,20,23-tetraazabicyclo-[8,7,6]-tricos-16-ene-3,6,9,19,22-pentanone; FR901228, depsipeptide] is a novel histone deacetylase inhibitor that shows therapeutic efficacy in Phase I trials of patients with malignant lymphoma. However, its mechanism of action has not been characterized. In this study, we examined the in vitro and in vivo effects of FK228 on human lymphoma U-937 cells. FK228 very strongly inhibited the growth of U-937 cells with an IC(50) value of 5.92 nM. In a scid mouse lymphoma model, mice treated with FK228 once or twice a week survived longer than control mice, with median survival times of 30.5 (0.56 mg/kg) and 33 days (0.32 mg/kg), respectively (vs. 20 days in control mice). Remarkably, 2 out of 12 mice treated with FK228 (0.56 mg/kg once or twice a week) survived past the observation period of 60 days. The apoptotic population of U-937 cells time-dependently increased to 37.7% after 48 hr of treatment with FK228. In addition, FK228 induced G1 and G2/M arrest and the differentiation of U-937 cells to the CD11b(+)/CD14(+) phenotype. Expression of p21(WAF1/Cip1) and gelsolin mRNA increased up to 654- and 152-fold, respectively, after 24hr of treatment with FK228. FK228 caused histone acetylation in p21(WAF1/Cip1) promoter regions, including the Sp1-binding sites. In conclusion, (i) FK228 prolonged the survival time of scid mice in a lymphoma model, and (ii) the beneficial effects of FK228 on human lymphoma may be exerted through the induction of apoptosis, cell cycle arrest, and differentiation via the modulation of gene expression by histone acetylation.     

LAAE-14, a new in vitro inhibitor of intracellular calcium mobilization,modulates acute and chronic inflammation

A new lipidic acid-amido ether derivative (LAAE-14) able to reduce dose-dependently the calcium increases mediated either by calcium ionophore ionomycin, by the endoplasmic reticular Ca(2+)-ATPase inhibitor thapsigargin, or by the chemotactic tripeptide N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLP), in human neutrophils as well as in murine peritoneal macrophages, but not ATP, has been evaluated as a potential anti-inflammatory drug. This compound attenuated leukocyte activation by means of its inhibitory effect on the respiratory burst elicited in both types of cells by 12-O-tetradecanoyl phorbol 13-acetate, by inhibition of the degranulation process induced by cytochalasin B+fMLP or cytochalasin B+platelet activating factor, as well as by reduction of leukotriene B(4) synthesis induced by the calcium ionophore A23187. In addition, in zymosan-stimulated mouse peritoneal macrophages LAAE-14 caused a potent inhibition of nitrite and prostaglandin E(2) production. This compound exerted acute and chronic anti-inflammatory effects by oral route, that may be related with several mechanisms such as attenuation of leukocyte activation, inhibition of inducible nitric oxide synthase, cyclo-oxygenase-2 and cytosolic phospholipase A(2) expression as well as reduction in tumour necrosis factor-alpha production. Its anti-inflammatory profile is clearly correlated with its behavior as inhibitor of intracellular calcium mobilization. The profile and potency of this compound may have relevance for the inhibition of the inflammatory response at different levels and may represent a new approach to the development of new anti-inflammatory drugs.     

COX-2-mediated PGD2 synthesis regulates phosphatidylcholine biosynthesis in rat renal papillary tissue

Phosphatidylcholine (PC) is the major membrane phospholipid in mammalian cells. Previous works from our laboratory demonstrated a close metabolic relationship between the maintenance of PC biosynthesis and the prostaglandins endogenously synthesized by cyclooxygenase (COX) in rat renal papilla. In the present work, we studied the COX isoform involved in papillary PC biosynthesis regulation. The incorporation of [methyl-3H]choline and [32P]orthophosphate to PC was determined in the absence and presence of SC-560 and NS-398, COX-1 and COX-2 specific inhibitors. PC synthesis was highly sensitive to COX-2 inhibition, while COX-1 inhibition only reduced PC synthesis at high SC-560 concentration. The analysis of choline-containing metabolites showed that COX-2 inhibition affected the formation of CDP-choline intermediary. The evaluation of PC biosynthetic enzymes revealed that microsomal, as well as nuclear, CTP:phosphocholine cytidylyltransferase (CCT), and nuclear-CDP-choline:1,2-diacylglycerol cholinephosphotransferase (CTP) activities were affected by COX-2 inhibition. The addition of exogenous prostaglandin D(2) (PGD(2)) restored nuclear-CCT and -CPT activities but not microsomal CCT. Papillary synthesis of PGD(2) was only detected in nuclear fraction where it was blocked by COX-2 inhibitor NS-398, but not by COX-1 inhibitor. All together, the present results demonstrated that COX-2-mediated PGD(2) synthesis is a PC biosynthesis regulator in rat renal papilla. Considering the importance of the maintenance of PC biosynthesis for the preservation of cell membrane homeostasis to ensure cell viability, and the extensive use of COX-2 inhibitors in therapeutics, the present results could have great pharmacological implications, and can constitute a biochemical explanation for the nephrotoxic effect of non-steroidal anti-inflammatory drugs.     

Inhibition of prostaglandin D2 clearance in rat hepatocytes by the thromboxane receptor antagonists daltroban and ifetroban and the thromboxane synthase inhibitor furegrelate

Prostanoids, i.e. prostaglandins and thromboxane, regulate liver-specific functions both in homeostasis and during defense reactions. For example, prostanoids are released from Kupffer cells, the resident liver macrophages, in response to the inflammatory mediator anaphylatoxin C5a, and mediate an enhanced glucose output from hepatocytes as energy supply. In perfused rat livers, the thromboxane receptor antagonist daltroban enhanced C5a-induced prostanoid overflow and reduced glucose output. It was the aim of this study to elucidate whether daltroban interfered with prostanoid release from Kupffer cells or prostanoid clearance by hepatocytes, and/or whether it directly influenced prostanoid-dependent glucose metabolism in these cells. In perfused rat livers, daltroban enhanced prostaglandin (PG)D(2) overflow not only after infusion of C5a (15-fold), but also after PGD(2) (10-fold). Neither daltroban nor another receptor antagonist, ifetroban, or the thromboxane synthase inhibitor furegrelate enhanced prostanoid release from Kupffer cells. In contrast, all inhibitors reduced clearance, i.e. uptake and degradation, of PGD(2) by hepatocytes: within 5 min uptake of 1 nmol/L PGD(2) was reduced from 43+/-5 fmol (controls) to 22+/-6 fmol (daltroban), 24+/-6 fmol (ifetroban) and 21+/-6 fmol (furegrelate). PGD(2) in the medium was reduced to 39+/-7% in the controls, but remained at 93+/-9%, 93+/-11% and 60+/-3% in the presence of the inhibitors. PGD(2)-dependent glucose output in the perfused liver or activation of glycogen phosphorylase in isolated hepatocytes remained unaffected by daltroban. These data clearly demonstrate that the thromboxane-inhibitors reduced PGD(2) clearance by hepatocytes, presumably by inhibition of prostanoid transport into the cells. In contrast, they did not interfere with PGD(2)-dependent glucose metabolism, suggesting an independent mechanism for the inhibition of glucose output from the liver.     

Characterization of the recombinant human prostanoid DP receptor and identification of L-644,698, a novel selective DP agonist

1. A human embryonic kidney cell line [HEK 293(EBNA)] stably expressing the human recombinant prostaglandin D₂ (PGD₂) receptor (hDP) has been characterized with respect to radioligand binding and signal transduction properties by use of prostanoids and prostanoid analogues. Radioligand binding studies included saturation analyses, the effects of nucleotide analogues, the initial rate of ligand-receptor association and equilibrium competition assays. In addition, adenosine 3′:5′-cyclic monophosphate (cyclic AMP) generation in response to ligand challenge was also measured, as this is the predominant hDP signalling pathway.
2. L-644,698 ((4-(3-(3-(3-hydroxyoctyl)-4-oxo-2-thiazolidinyl) propyl) benzoic acid) (racemate)) was identified as a novel ligand having high affinity for hDP with an inhibitor constant (K_i) of 0.9 nM. This K_i value was comparable to the K_i values obtained in this study for ligands that have previously shown high affinity for DP: PGD₂ (0.6 nM), ZK 110841 (0.3 nM), BW245C (0.4 nM), and BW A868C (2.3 nM).
3. L-644,698 was found to be a full agonist with an EC₅₀ value of 0.5 nM in generating cyclic AMP following activation of hDP. L-644,698 is, therefore, comparable to those agonists with known efficacy at the DP receptor (EC₅₀): PGD₂ (0.5 nM), ZK 110841 (0.2 nM) and BW245C (0.3 nM).
4. L-644,698 displayed a high degree of selectivity for hDP when compared to the family of cloned human prostanoid receptors: EP₁ (>25,400 fold), EP₂ (∼300 fold), EP_3-III (∼4100 fold), EP₄ (∼10000 fold), FP (>25,400 fold), IP (>25,400 fold) and TP (>25,400 fold). L-644,698 is, therefore, one of the most selective DP agonists as yet described.
5. PGJ₂ and Δ¹²-PGJ₂, two endogenous metabolites of PGD₂, were also tested in this system and shown to be effective agonists with K_i and EC₅₀ values in the nanomolar range for both compounds. In particular, PGJ₂ was equipotent to known DP specific agonists with a K_i value of 0.9 nM and an EC₅₀ value of 1.2 nM.

     

Effects of KP-496, a novel dual antagonist at the cysteinyl leukotriene receptor 1 and the thromboxane A(2) receptor, on airway obstruction in guinea pigs

BACKGROUND AND PURPOSE: KP-496 is a novel dual antagonist for cysteinyl leukotriene receptor 1 (CysLT(1)) and thromboxane A(2) (TXA(2)) receptor (TP). The aim of this study was to evaluate the pharmacological profile of inhaled KP-496 and its effects on airway obstruction.EXPERIMENTAL APPROACH: Antagonist activities of inhaled KP-496 were investigated using bronchoconstriction induced in guinea pigs by LTD(4) or U46619, a stable TXA(2) mimetic. Guinea pigs sensitized with injections of ovalbumin were used to assess the effects of inhaled KP-496 on bronchoconstriction induced by antigen (i.v.). Another set of guinea pigs were sensitized and challenged with ovalbumin by inhalation and the effects of inhaled KP-496 on immediate and late airway responses and airway hyperresponsiveness were investigated.KEY RESULTS: KP-496 significantly inhibited LTD(4)- and U46619-induced bronchoconstriction in a dose-dependent manner. The inhibitory effects of KP-496 (1%) were comparable to those of montelukast (a CysLT(1) antagonist, p.o., 0.3 mg kg(-1)) or seratrodast (a TP antagonist, p.o., 3 mg kg(-1)). KP-496 (1%) and oral co-administration of montelukast (10 mg kg(-1)) and seratrodast (20 mg kg(-1)) significantly inhibited antigen-induced bronchoconstriction, whereas administration of montelukast or seratrodast separately did not inhibit antigen-induced bronchoconstriction. KP-496 exhibited dose-dependent and significant inhibitory effects on the immediate and late airway responses and airway hyperresponsiveness following antigen challenge.CONCLUSIONS AND IMPLICATIONS: KP-496 exerts effects in guinea pigs which could be beneficial in asthma. These effects of KP-496 were greater than those of a CysLT(1) antagonist or a TP antagonist, in preventing antigen-induced airway obstruction.     

Epithelium-dependent modulation of responsiveness of airways from caveolin-1 knockout mice is mediated through cyclooxygenase-2 and 5-lipoxygenase

BACKGROUND AND PURPOSE Acute silencing of caveolin-1 (Cav-1) modulates receptor-mediated contraction of airway smooth muscle. Moreover, COX-2- and 5-lipoxygenase (5-LO)-derived prostaglandin and leukotriene biosynthesis can influence smooth muscle reactivity. COX-2 half-life can be prolonged through association with Cav-1. We suggested that lack of Cav-1 modulated levels of COX-2 which in turn modulated tracheal contraction, when arachidonic acid signalling was disturbed by inhibition of COX-2. EXPERIMENTAL APPROACH Using tracheal rings from Cav-1 knockout (KO) and wild-type mice (B6129SF2/J), we measured isometric contractions to methacholine and used PCR, immunoblotting and immunohistology to monitor expression of relevant proteins. KEY RESULTS Tracheal rings from Cav-1 KO and wild-type mice exhibited similar responses, but the COX-2 inhibitor, indomethacin, increased responses of tracheal rings from Cav-1 KO mice to methacholine. The phospholipase A(2) inhibitor, eicosatetraynoic acid, which inhibits formation of both COX-2 and 5-LO metabolites, had no effect on wild-type or Cav-1 KO tissues. Indomethacin-mediated hyperreactivity was ablated by the LTD(4) receptor antagonist (montelukast) and 5-LO inhibitor (zileuton). The potentiating effect of indomethacin on Cav-1 KO responses to methacholine was blocked by epithelial denudation. Immunoprecipitation showed that COX-2 binds Cav-1 in wild-type lungs. Immunoblotting and qPCR revealed elevated levels of COX-2 and 5-LO protein, but not COX-1, in Cav-1 KO tracheas, a feature that was prevented by removal of the epithelium. CONCLUSION AND IMPLICATIONS The indomethacin-induced hypercontractility observed in Cav-1 KO tracheas was linked to increased expression of COX-2 and 5-LO, which probably enhanced arachidonic acid shunting and generation of pro-contractile leukotrienes when COX-2 was inhibited.     

Cyclooxygenase-2 (COX-2)-dependent and -independent anticarcinogenic effects of celecoxib in human colon carcinoma cells

Celecoxib, a selective cyclooxygenase-2 (COX-2) inhibitor, is the only non-steroidal anti-inflammatory drug so far which has been approved by the FDA for adjuvant treatment of patients with familial adenomatous polyposis. The molecular mechanism responsible for the anticarcinogenic effects of celecoxib is still not fully understood. To investigate the extent to which the anticarcinogenic effect of celecoxib depends on COX-2 expression, we transfected human colon carcinoma cells (Caco-2) with the human COX-2 cDNA, in both sense and in antisense orientation, to generate cells which either overexpress COX-2 (human COX-2-sense, hCOX-2-s), express no COX-2 (human COX-2-antisense, hCOX-2-as) or express only very small amounts of COX-2 (control cells). Treatment of these cells with celecoxib dose-dependently (0-100microM) reduced cell survival which was accompanied by an induction of a G(0)/G(1) phase block and apoptosis. The effect of celecoxib treatment on both, cell survival and induction of apoptosis in hCOX-2-as cells was less marked than in the COX-2-expressing cells. Apoptosis was accompanied by an activation of caspase-3 and caspase-9 and cytochrome c release. In contrast, we observed no difference in sensitivity with regard to the induction of a cell cycle block between the different cell clones. The G(0)/G(1) phase block caused by celecoxib correlated with a decrease in expression levels of cyclin A and cyclin B1 and an increase in the expression of the cell cycle inhibitory proteins p21(Waf1) and p27(Kip1) irrespective of the type of cell used. These data indicate that apoptosis-inducing effects of celecoxib partly depend on COX-2 expression of the cells, whereas induction of a cell cycle block occurred COX-2 independently. Thus, the anticarinogenic effects of celecoxib can be explained by both COX-2-dependent and -independent mechanisms.     

Selectivity profiling of the novel EP2 receptor antagonist,PF-04418948, in functional bioassay systems: atypical affinity at the guinea pig EP2 receptor

BACKGROUND AND PURPOSE

Understanding the role of the EP₂ receptor has been hampered by the lack of a selective antagonist. Recently, a selective EP₂ receptor antagonist, PF-04418948, has been discovered. The aim of this study was to demonstrate the selectivity profile of PF-04418948 for the EP₂ receptor over other EP receptors using a range of isolated tissue systems.

EXPERIMENTAL APPROACH

PF-04418948 was profiled on a range of isolated tissues to assess its EP receptor potency and selectivity: ONO-DI-004-induced contraction of guinea pig trachea (EP₁); ONO-AE1-259 and PGE₂- induced relaxation of mouse and guinea pig trachea (EP₂); PGE₂-induced depolarization of guinea pig isolated vagus (EP₃); PGE₂-induced relaxation of human and rat trachea (EP₄). PF-04418948 was also profiled in functional murine TP, IP, DP and FP receptor assays.

KEY RESULTS

In bioassay systems, where assessment of potency/selectivity is made against the ‘native’ receptor, PF-04418948 only acted as an antagonist of EP₂ receptor-mediated events. PF-04418948 competitively inhibited relaxations of murine and guinea pig trachea induced by ONO-AE1-259 and PGE₂ respectively. However, the affinity of PF-04418948 was not equal in the two preparations.

CONCLUSIONS AND IMPLICATIONS

Using a wide range of bioassay systems, we have demonstrated that PF-04418948 is a selective EP₂-receptor antagonist. Interestingly, an atypically low affinity was found on the guinea pig trachea, questioning its utility as an EP₂ receptor assay system. Nevertheless, this compound should be an invaluable tool for investigating the biological activity of PGE₂ and the role of EP₂ receptors in health and disease.     

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro

BACKGROUND AND PURPOSE

Both CB₁ and CB₂ cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro.

EXPERIMENTAL APPROACH

Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing α_vβ₃ or with F-actin rings, or measurement of resorption area.

KEY RESULTS

Human osteoclasts express both CB₁ and CB₂ receptors. CB₂ expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB₁ and/or CB₂ antagonists.

CONCLUSIONS AND IMPLICATIONS

Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB₂ receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease.

LINKED ARTICLES

This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7