The applicability of species sensitivity distributions to the development of generic doses for phytosanitary irradiation.

Ionizing radiation is used as a phytosanitary treatment to prevent the introduction of pests through trade. Generic doses are a valuable means to increase the number of pest-commodity combinations that can be treated using phytosanitary irradiation. Generic doses allow for the treatment of the entire taxa for which the dose has been approved, allowing for the treatment of untested species. As such, the approval of a generic dose requires substantial supporting data and careful consideration of the risks involved. We adopt the Species Sensitivity Distribution (SSD) framework, already in widespread use in the field of ecotoxicology and environmental risk assessment, to evaluate generic doses for phytosanitary irradiation treatments. Parametric SSDs for Curculionidae and Tephritidae were developed using existing data on efficacious phytosanitary irradiation treatments. The resulting SSDs provided estimates of the taxa coverage expected by the generic dose, along with the margin of uncertainty. The SSD analysis lends support to the existing 150 Gy generic dose for Tephritidae and a proposed 175 Gy generic dose for Curculionidae. The quantitative estimates of risk produced by the SSD approach can be a valuable tool for phytosanitary rule making, improving the process for generic dose development and approval.

Comparative Toxicity of Helicoverpa armigera and Helicoverpa zea (Lepidoptera: Noctuidae) to Selected Insecticides.

Until recently, the Old World bollworm (OWB) Helicoverpa armigera (Hübner) and the corn earworm Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae) were geographically isolated. Both species are major pests of agricultural commodities that are known to develop insecticide resistance, and they now coexist in areas where H. armigera invaded the Americas. This is the first study to compare the susceptibility of the two species to conventional insecticides. The susceptibility of third instar H. armigera and H. zea larvae to indoxacarb, methomyl, spinetoram, and spinosad was determined using a diet-overlay bioassay in a quarantine laboratory in Puerto Rico. Mortality was assessed at 48 h after exposure for up to eight concentrations per insecticide. Spinetoram exhibited the highest acute toxicity against H. armigera, with a median lethal concentration (LC50) of 0.11 µg a.i./cm2, followed by indoxacarb and spinosad (0.17 µg a.i./cm2 for both) and methomyl (0.32 µg a.i./cm2). Spinetoram was also the most toxic to H. zea (LC50 of 0.08 µg a.i./cm2), followed by spinosad (0.17 µg a.i./cm2) and methomyl (0.18 µg a.i./cm2). Indoxacarb was the least toxic to H. zea, with an LC50 of 0.21 µg a.i./cm2. These findings could serve as a comparative reference for monitoring the susceptibility of H. armigera and H. zea to indoxacarb, methomyl, spinetoram, and spinosad in Puerto Rico, and may facilitate the detection of field-selected resistance for these two species and their potential hybrids in areas recently invaded by H. armigera.

Critical PO2 as a diagnostic biomarker for the effects of low-oxygen modified and controlled atmospheres on phytosanitary irradiation treatments in the cabbage looper Trichoplusia ni (Hübner).

BACKGROUND: Phytosanitary irradiation is a sustainable alternative to chemical fumigants for disinfesting fresh commodities from insect pests. However, irradiating insects in modified atmospheres with very low oxygen (<1 kPa O2 ) has repeatedly been shown to increase radioprotective response. Thus, there is a concern that modified atmosphere packaging could reduce the efficacy of phytosanitary irradiation. One hurdle slowing the widespread application of phytosanitary irradiation is a lack of knowledge about how moderate levels of hypoxia relevant to the modified atmosphere packaging of most fresh commodities (3-10 kPa O2 ) may affect phytosanitary irradiation treatments. Therefore, we hypothesize that critical PO2 (Pcrit ), the level of oxygen at which an insect's metabolism becomes impaired, can be used as a diagnostic biomarker to predict the induction of a radioprotective response. RESULTS: Using the cabbage looper Trichoplusia ni (Hübner), we show that there is a substantial increase in radiation resistance when larvae are irradiated in atmospheres more hypoxic than their Pcrit (3.3 kPa O2 ). These data are consistent with our hypothesis that Pcrit could be used as a diagnostic biomarker for what levels of hypoxia may induce radioprotective effects that could impact phytosanitary irradiation treatments. CONCLUSION: We propose that the relationship between Pcrit and radioprotective effects could allow us to build a framework for predicting the effects of low-oxygen atmospheres on the efficacy of phytosanitary irradiation. However, more widespread studies across pest species are still needed to test the generality of this idea.

Effectiveness of Hot-Water Immersion Against Brevipalpus yothersi (Acari: Tenuipalpidae) as a Postharvest Treatment for Lemons.

Citrus leprosis is a destructive disease of citrus caused by several viruses (CiLVs) that are quarantine pests in the United States. Brevipalpus yothersi Baker (Acari: Tenuipalpidae) vectors the most virulent strain of CiLV. This mite is present in the United States and could facilitate the spread of the disease if CiLV reaches the country. Postharvest treatments could mitigate B. yothersi on imported commodities from areas where CiLV exists. The current study explores the effectiveness of hot-water immersion as a postharvest treatment against B. yothersi. Lemons were immersed in water at 21, 48, 53, or 63°C for 5, 10, and 15 min. Immersions at 53 and 63°C for all time schedules dislodged over 99% of adult mites. Lemon fruit quality and B. yothersi egg viability after hot-water immersion were also evaluated. Fruit quality significantly decreased in lemons treated at 63°C resulting in decay (grade 3, rejection), while at 53°C there was a quality reduction (grade 2, minimum acceptable market level) compared to lemons immersed at 21°C or nontreated controls (grade 1). None of the eggs hatched when the lemons were immersed in water at 63°C and an average of 1.5% hatched at 53°C for all time schedules. Immersion in water at 53°C for 5 min dislodged 99.71% and 57.14% of adult and immature mites, respectively, and resulted in 98.11% unhatched eggs without significant fruit quality reduction. Hot-water immersion could be a key component in a systems approach to control B. yothersi on imported citrus fruits from countries where citrus leprosis is present.

Deleterious effects of electron beam irradiation on development and reproduction of tomato/potato psyllids, Bactericera cockerelli.

The potato/tomato psyllid Bactericera cockerelli causes serious damage to several solanaceous crops by direct feeding and vectoring Candidatus Liberibacter solanacearum, a bacterial pathogen. Electron beam (eBeam) irradiation is an environmentally friendly, chemical-free alternative method that is increasing in use for disinfestation of insect pests. We hypothesize that this irradiation technology will have detrimental effects on potato psyllid and thus impede its disease vectoring. To this end, we explored the effects of eBeam treatment ranging from 50 to 500 Gy on survival, development and reproduction of this pest. Impact on psyllids was apparently dose-dependent. When irradiated at 350 Gy, eggs could not hatch, 1st instar nymphs failed to emerge, and although a small portion of irradiated 5th instar nymphs survived, the emerged adults were mostly deformed. Abnormality in eclosed adults suggests harmful effects of eBeam on metamorphosis. Reproduction was seriously impaired when female psyllids were exposed to eBeam at the 5th instar nymphal or young adult stage, presumably due to inability to form oocytes. In addition, reciprocal crosses between irradiated and untreated psyllids indicated that female psyllids were more radiosensitive than males to eBeam. Taken together, these findings indicate that eBeam negatively impacted potato psyllid development and reproduction, which would inevitably compromise its disease transmission capacity. A dose of 350 Gy can be considered as a reference dose for effective control of potato psyllids.

The affinity, intrinsic activity and selectivity of a structurally novel EP2 receptor agonist at human prostanoid receptors.

BACKGROUND AND PURPOSE: Prostanoid EP2 receptor agonists exhibit several activities including ocular hypotension, tocolysis and anti-inflammatory activity. This report describes the affinity and selectivity of a structurally novel, non-prostanoid EP2 receptor agonist, PGN-9856, and its therapeutic potential. EXPERIMENTAL APPROACH: The pharmacology of a series of non-prostanoid EP2 receptor agonists was determined according to functional and radioligand binding studies, mostly using human recombinant prostanoid receptor transfectants. The selectivity of PGN-9856, as the preferred compound, was subsequently determined by using a diverse variety of non-prostanoid target proteins. The therapeutic potential of PGN-9856 was addressed by determining its activity in relevant primate cell, tissue and disease models. KEY RESULTS: PGN-9856 was a selective and high affinity (pKi ≥ 8.3) ligand at human recombinant EP2 receptors. In addition to high affinity binding, it was a potent and full EP2 receptor agonist with a high level of selectivity at EP1 , EP3 , EP4 , DP, FP, IP and TP receptors. In cells overexpressing human recombinant EP2 receptors, PGN-9856 displayed a potency (pEC50 ≥ 8.5) and a maximal response (increase in cAMP) comparable to that of the endogenous agonist PGE2 . PGN-9856 exhibited no appreciable affinity (up 10 µM) for a range of 53 other receptors, ion channels and enzymes. Finally, PGN-9856 exhibited tocolytic, anti-inflammatory and long-acting ocular hypotensive properties consistent with its potent EP2 receptor agonist properties. CONCLUSIONS AND IMPLICATIONS: PGN-9856 is a potent, selective and efficacious prostanoid EP2 receptor agonist with diverse potential therapeutic applications: tocolytic, anti-inflammatory and notably anti-glaucoma.

Dulaglutide versus insulin glargine in patients with type 2 diabetes and moderate-to-severe chronic kidney disease (AWARD-7): a multicentre, open-label, randomised trial.

BACKGROUND: Many antihyperglycaemic drugs, including insulin, are primarily cleared by the kidneys, restricting treatment options for patients with kidney disease. Dulaglutide is a long-acting glucagon-like peptide-1 receptor agonist that is not cleared by the kidneys, and confers a lower risk of hypoglycaemia than does insulin. We assessed the efficacy and safety of dulaglutide in patients with type 2 diabetes and moderate-to-severe chronic kidney disease. METHODS: AWARD-7 was a multicentre, open-label trial done at 99 sites in nine countries. Eligible patients were adults with type 2 diabetes and moderate-to-severe chronic kidney disease (stages 3-4), with an HbA1c of 7·5-10·5%, and who were being treated with insulin or insulin plus an oral antihyperglycaemic drug and were taking a maximum tolerated dose of an angiotensin-converting enzyme inhibitor or an angiotensin receptor blocker. Participants were randomly assigned (1:1:1) by use of a computer-generated random sequence with an interactive response system to once-weekly injectable dulaglutide 1·5 mg, once-weekly dulaglutide 0·75 mg, or daily insulin glargine as basal therapy, all in combination with insulin lispro, for 52 weeks. Insulin glargine and lispro doses were titrated as per an adjustment algorithm; dulaglutide doses were masked to participants and investigators. The primary outcome was HbA1c at 26 weeks, with a 0·4% non-inferiority margin. Secondary outcomes included estimated glomerular filtration rate (eGFR) and urine albumin-to-creatinine ratio (UACR). The primary analysis population was all randomly assigned patients who received at least one dose of study treatment and had at least one post-randomisation HbA1c measurement. The safety population was all patients who received at least one dose of study treatment and had any post-dose data. This study is registered with ClinicalTrials.gov, number NCT01621178. FINDINGS: Between Aug 15, 2012, and Nov 30, 2015, 577 patients were randomly assigned, 193 to dulaglutide 1·5 mg, 190 to dulaglutide 0·75 mg, and 194 to insulin glargine. The effects on HbA1c change at 26 weeks of dulaglutide 1·5 mg and 0·75 mg were non-inferior to those of insulin glargine (least squares mean [LSM] -1·2% [SE 0·1] with dulaglutide 1·5 mg [183 patients]; -1·1% [0·1] with dulaglutide 0·75 mg [180 patients]; -1·1% [0·1] with insulin glargine [186 patients]; one-sided p≤0·0001 for both dulaglutide doses vs insulin glargine). The differences in HbA1c concentration at 26 weeks between dulaglutide and insulin glargine treatments were LSM difference -0·05% (95% CI -0·26 to 0·15, p<0·0001) with dulaglutide 1·5 mg and 0·02% (-0·18 to -0·22, p=0·0001) with dulaglutide 0·75 mg. HbA1c-lowering effects persisted to 52 weeks (LSM -1·1% [SE 0·1] with dulaglutide 1·5 mg; -1·1% [0·1] with dulaglutide 0·75 mg; -1·0% [0·1] with insulin glargine). At 52 weeks, eGFR was higher with dulaglutide 1·5 mg (Chronic Kidney Disease Epidemiology Collaboration equation by cystatin C geometric LSM 34·0 mL/min per 1·73 m2 [SE 0·7]; p=0·005 vs insulin glargine) and dulaglutide 0·75 mg (33·8 mL/min per 1·73 m2 [0·7]; p=0·009 vs insulin glargine) than with insulin glargine (31·3 mL/min per 1·73 m2 [0·7]). At 52 weeks, the effects of dulaglutide 1·5 mg and 0·75 mg on UACR reduction were not significantly different from that of insulin glargine (LSM -22·5% [95% CI -35·1 to -7·5] with dulaglutide 1·5 mg; -20·1% [-33·1 to -4·6] with dulaglutide 0·75 mg; -13·0% [-27·1 to 3·9] with insulin glargine). Proportions of patients with any serious adverse events were similar across groups (20% [38 of 192] with dulaglutide 1·5 mg, 24% [45 of 190] with dulaglutide 0·75 mg, and 27% [52 of 194] with insulin glargine). Dulaglutide was associated with higher rates of nausea (20% [38 of 192] with dulaglutide 1·5 mg and 14% [27 of 190] with 0·75 mg, vs 5% [nine of 194] with insulin glargine) and diarrhoea (17% [33 of 192] with dulaglutide 1·5 mg and 16% [30 of 190] with 0·75 mg, vs 7% [14 of 194] with insulin glargine) and lower rates of symptomatic hypoglycaemia (4·4 events per patient per year with dulaglutide 1·5 mg and 4·3 with dulaglutide 0·75 mg, vs 9·6 with insulin glargine). End-stage renal disease occurred in 38 participants: eight (4%) of 192 with dulaglutide 1·5 mg, 14 (7%) of 190 with dulaglutide 0·75 mg, and 16 (8%) of 194 with insulin glargine. INTERPRETATION: In patients with type 2 diabetes and moderate-to-severe chronic kidney disease, once-weekly dulaglutide produced glycaemic control similar to that achieved with insulin glargine, with reduced decline in eGFR. Dulaglutide seems to be safe to use to achieve glycaemic control in patients with moderate-to-severe chronic kidney disease. FUNDING: Eli Lilly and Company.

Dulaglutide as add-on therapy to SGLT2 inhibitors in patients with inadequately controlled type 2 diabetes (AWARD-10): a 24-week, randomised, double-blind, placebo-controlled trial.

BACKGROUND: Glucagon-like peptide-1 (GLP-1) receptor agonists and sodium-glucose co-transporter-2 (SGLT2) inhibitors improve glycaemic control and reduce bodyweight in patients with type 2 diabetes through different mechanisms. We assessed the safety and efficacy of the addition of the once-weekly GLP-1 receptor agonist dulaglutide to the ongoing treatment regimen in patients whose diabetes is inadequately controlled with SGLT2 inhibitors, with or without metformin. METHODS: AWARD-10 was a phase 3b, double-blind, parallel-arm, placebo-controlled, 24-week study done at 40 clinical sites in Austria, Czech Republic, Germany, Hungary, Israel, Mexico, Spain, and the USA. Eligible adult patients (≥18 years) with inadequately controlled type 2 diabetes (HbA1c concentration ≥7·0% [53 mmol/mol] and ≤9·5% [80 mmol/mol]), a BMI of 45 kg/m2 or less, and taking stable doses (>3 months) of an SGLT2 inhibitor (with or without metformin) were randomly assigned (1:1:1) via an interactive web-response system to subcutaneous injections of either dulaglutide 1·5 mg, dulaglutide 0·75 mg, or placebo once per week for 24 weeks. Patients and investigators were masked to dulaglutide and placebo assignment, and those assessing outcomes were masked to study drug assignment. The primary objective was to test for the superiority of dulaglutide (1·5 mg or 0·75 mg) versus placebo for change in HbA1c concentration from baseline at 24 weeks. All analyses were done in the intention-to-treat population, defined as all randomly assigned patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov, number NCT02597049. FINDINGS: Between Dec 7, 2015, and Feb 3, 2017, 424 patients were randomly assigned to dulaglutide 1·5 mg (n=142), dulaglutide 0·75 mg (n=142), and placebo (n=140). One patient in the dulaglutide 0·75 mg group was excluded from the analysis because they did not receive any dose of the study drug. The reduction in HbA1c concentration at 24 weeks was larger in patients receiving dulaglutide (least squares mean [LSM] for dulaglutide 1·5 mg -1·34% [SE 0·06] or -14·7 mmol/mol [0·6]; dulaglutide 0·75 mg -1·21% [0·06] or -13·2 mmol/mol [0·6]) than in patients receiving placebo (-0·54% [0·06] or -5·9 mmol/mol [0·6]; p<0·0001 for both groups vs placebo). The LSM differences were -0·79% (95% CI -0·97 to -0·61) or -8·6 mmol/mol (-10·6 to -6·7) for dulaglutide 1·5 mg and -0·66% (-0·84 to -0·49) or -7·2 mmol/mol (-9·2 to -5·4) for dulaglutide 0·75 mg (p<0·0001 for both). Serious adverse events were reported for five (4%) patients in the dulaglutide 1·5 mg group, three (2%) patients in the dulaglutide 0·75 mg group, and five (4%) patients in the placebo group. Treatment-emergent adverse events were more common in patients treated with dulaglutide than in patients who received placebo, mainly because of an increased incidence of gastrointestinal adverse events. Nausea (21 [15%] patients in the dulaglutide 1·5 mg group vs seven [5%] in the dulaglutide 0·75 mg group vs five [4%] in the placebo group), diarrhoea (eight [6%] vs 14 [10%] vs four [3%]), and vomiting (five [4%] vs four [3%] vs one [1%]) were more common with dulaglutide than with placebo. One episode of severe hypoglycaemia was reported in the dulaglutide 0·75 mg group. Two (1%) patients receiving dulaglutide 1·5 mg died, but these deaths were not considered to be related to study drug; no deaths occurred in the other groups. INTERPRETATION: Dulaglutide as add-on treatment to SGLT2 inhibitors (with or without metformin) resulted in significant and clinically relevant improvements in glycaemic control, with acceptable tolerability that is consistent with the established safety profile of dulaglutide. FUNDING: Eli Lilly and Company.

Placebo-controlled, randomized trial of the addition of once-weekly glucagon-like peptide-1 receptor agonist dulaglutide to titrated daily insulin glargine in patients with type 2 diabetes (AWARD-9).

AIM: To compare the addition of weekly dulaglutide vs the addition of placebo to titrated glargine in patients with type 2 diabetes (T2D) with sub-optimum glycated haemoglobin (HbA1c) concentration. MATERIALS AND METHODS: Patients (N = 300) from this phase III, double-blind, parallel-arm, placebo-controlled study were randomized to weekly subcutaneous injections of dulaglutide 1.5 mg or placebo with titrated daily glargine (mean ± standard deviation baseline dose: 39 ± 22 U), with or without metformin (≥1500 mg/d). The primary endpoint was superiority of dulaglutide/glargine to placebo/glargine with regard to change from baseline in HbA1c level at 28 weeks. RESULTS: Least squares (LS) mean ± standard error (s.e.) HbA1c changes from baseline were -1.44 ± 0.09% (-15.74 ± 0.98 mmol/mol) with dulaglutide/glargine and -0.67 ± 0.09% (-7.32 ± 0.98 mmol/mol) with placebo/glargine at 28 weeks (LS mean difference [95% confidence interval] -0.77% [-0.97, -0.56]; P < .001). Body weight decreased with dulaglutide/glargine and increased with placebo/glargine (LS mean difference: -2.41 ± 0.39 kg; P < .001). Increases from baseline in mean glargine dose were significantly smaller with dulaglutide/glargine vs placebo/glargine (13 ± 2 U [0.1 ± 0.02 U/kg] vs 26 ± 2 U [0.3 ± 0.02 U/kg], respectively; P < .001; LS mean ± s.e. final dose: dulaglutide/glargine, 51 ± 2 U; placebo/glargine, 65 ± 2 U). The hypoglycaemia rate (≤3.9 mmol/L threshold) was 7.69 ± 15.15 and 8.56 ± 16.13 events/patient/year, respectively (P = .488). One episode of severe hypoglycaemia occurred in the dulaglutide/glargine group. Common gastrointestinal adverse events with dulaglutide were nausea (12.0%), diarrhoea (11.3%) and vomiting (6.0%). CONCLUSIONS: Weekly dulaglutide 1.5 mg added to basal insulin is an efficacious and well tolerated treatment option for patients with T2D.

Effects of Low-Oxygen Environments on the Radiation Tolerance of the Cabbage Looper Moth (Lepidoptera: Noctuidae).

Ionizing radiation is used as a phytosanitary treatment to mitigate risks from invasive species associated with trade of fresh fruits and vegetables. Commodity producers prefer to irradiate fresh product stored in modified atmosphere packaging that increases shelf life and delays ripening. However, irradiating insects in low oxygen may increase radiation tolerance, and regulatory agencies are concerned modified atmosphere packaging will decrease efficacy of radiation doses. Here, we examined how irradiation in a series of oxygen conditions (0.1-20.9 kPa O2) alters radiotolerance of larvae and pupae of a model lepidopteran Trichoplusia ni (Hubner) (Diptera: Noctuidae). Irradiating in severe hypoxia (0.1 kPa O2) increased radiation tolerance of insects compared with irradiating in atmospheric oxygen (20.9 kPa O2). Our data show irradiating pharate adult pupae at 600 Gy in moderately severe hypoxia (5 kPa O2) increased adult emergence compared with irradiation in atmospheric oxygen (20.9 kPa O2). Our data also show that in one of the three temporal replicates, irradiating T. ni larvae in moderately severe hypoxia (5 kPa O2) can also increase radiotolerance at an intermediate radiation dose of 100 Gy compared with irradiating in atmospheric oxygen conditions, but not at higher or lower doses. We discuss implications of our results in this model insect for the current generic doses for phytosanitary irradiation, including the recently proposed 250 Gy generic dose for lepidioptera larvae, and temporary restriction on irradiating commodities in modified atmosphere packaging that reduces the atmosphere to < 18 kPa O2.

Endocannabinoids and their oxygenation by cyclo-oxygenases, lipoxygenases and other oxygenases.

The naturally occurring mammalian endocannabinoids possess biological attributes that extend beyond interaction with cannabinoid receptors. These extended biological properties are the result of oxidative metabolism of the principal mammalian endocannabinoids arachidonoyl ethanolamide (anandamide; A-EA) and 2-arachidonoylglycerol (2-AG). Both endocannabinoids are oxidized by cyclo-oxygenase-2 (COX-2), but not by COX-1, to a series of prostaglandin derivatives (PGs) with quite different biological properties from those of the parent substrates. PG ethanolamides (prostamides, PG-EAs) and PG glyceryl esters (PG-Gs) are not only pharmacologically distinct from their parent endocannabinoids, they are distinct from the corresponding acidic PGs, and are differentiated from each other. Ethanolamides and glyceryl esters of the major prostanoids PGD2, PGE2, PGF2α, and PGI2 are formed by the various PG synthases, and thromboxane ethanolamides and glyceryl esters are not similarly produced. COX-2 is also of interest by virtue of its corollary central role in modulating endocannabinoid tone, providing a new therapeutic approach for treating pain and anxiety. Other major oxidative conversion pathways are provided for both A-EA and 2-AG by several lipoxygenases (LOXs), resulting in the formation of numerous hydroxyl metabolites. These do not necessarily represent inactivation pathways for endocannabinoids but may mimic or modulate the endocannabinoids or even display alternative pharmacology. Similarly, A-EA and 2-AG may be oxidized by P450 enzymes. Again a very diverse number of metabolites are formed, with either cannabinoid-like biological properties or an introduction of disparate pharmacology. The biological activity of epoxy and hydroxyl derivatives of the endocannabinoids remains to be fully elucidated. This review attempts to consolidate and compare the findings obtained to date in an increasingly important research area. This article is part of a Special Issue entitled "Oxygenated metabolism of PUFA: analysis and biological relevance".

Recent progress in prostaglandin F2α ethanolamide (prostamide F2α) research and therapeutics.

Prostamide (prostaglandin ethanolamide) research emerged from two distinct lines of research: 1) the unique pharmacology of the antiglaucoma drug bimatoprost and 2) the discovery that endocannabinoid anandamide was converted by COX-2 to a series of electrochemically neutral prostaglandin (PG) ethanolamides. Bimatoprost pharmacology was found to be virtually identical to that of prostamide F2α. The earliest studies relied on comparison of agonist potencies compared with PGF2α and synthetic prostaglandin F2α (FP) receptor agonists. The subsequent discovery of selective and potent prostamide receptor antagonists (AGN 211334-6, as shown in Fig. 3) was critical for distinguishing between prostamide and FP receptor-mediated effects. The prostamide F2α receptor was then modeled by cotransfecting the wild-type FP receptor with an mRNA splicing variant (altFP4).Bimatoprost is now used therapeutically for treating both glaucoma and eyelash hypotrichosis. Bimatoprost also stimulates hair growth in isolated human scalp hair follicles. A strong effect is also seen in mouse pelage hair, where bimatoprost essentially halves the onset of hair regrowth and the time to achieve full hair regrowth in shaved mice. Beyond glaucoma and hair growth, bimatoprost has potential for reducing fat deposition. Studies to date suggest that preadipocytes are the cellular target for bimatoprost. The discovery of the enzyme prostamide/PGF synthase was invaluable in elucidating the anatomic distribution of prostamide F2α. High expression in the central nervous system provided the impetus for later studies that described prostamide F2α as a nociceptive mediator in the spinal cord. At the translational level, bimatoprost has already provided therapeutics in two distinct areas and the use of both prostamide agonists and antagonists may provide other useful medicaments.

Biomarker response to drotrecogin alfa (activated) in children with severe sepsis: results from the RESOLVE clinical trial*.

OBJECTIVE: REsearching severe Sepsis and Organ dysfunction in children: A gLobal perspective (RESOLVE), a phase III trial of drotrecogin alfa (activated) in pediatric severe sepsis, examined biomarker changes in inflammation and coagulation. This report describes biomarker profiles in early severe sepsis and the pharmacodynamic assessment of drotrecogin alfa (activated) in RESOLVE. DESIGN: Serial measurements of interleukin-1ß, interleukin-6, interleukin-8, interleukin-10, tissue necrosis factor-α, procalcitonin, D-dimer, and thrombin-antithrombin complex were performed at baseline and daily over the first five study days. Protein C levels were performed at baseline and at the end of the 96-hr study drug infusion. Analysis of variance-based log-transformed data compared the treatment groups for each measured variable. SETTING: : One hundred four pediatric intensive care units in 18 countries. PATIENTS: Four hundred seventy-seven children between 38 wks corrected gestational age and 17 yrs with sepsis-induced cardiovascular and respiratory dysfunction. INTERVENTIONS: Drotrecogin alfa (activated). MEASUREMENTS AND MAIN RESULTS: Pharmacodynamic activity of drotrecogin alfa (activated) compared with placebo was observed with reduction of D-dimer on day 1 (p < .01) and thrombin-antithrombin complex on days 1-4 (p < .05). There were no significant changes by treatment in multiple cytokines or procalcitonin. In the overall population, a median protein C difference was not observed (p > .05) with drotrecogin alfa (activated) administration compared with placebo, although a difference (median percentage change from baseline) in favor of drotrecogin alfa (activated) was observed in patients >1 yr old (p = .0449). CONCLUSIONS: While children in the RESOLVE trial were similar to adults in that they showed a relationship between severity of coagulation and inflammation abnormalities and mortality, their pharmacodynamic response to drotrecogin alfa (activated) differed with respect to changes in protein C activity and systemic inflammation.

Immunohistochemical demonstration of increased prostaglandin F2α levels in the rat hippocampus following kainic acid-induced seizures.

Prostaglandin (PG) F(2α) is one of the major prostanoids biosynthesized by cyclooxygenases (COXs) from arachidonic acid. Although it has been reported that there is a selective surge in PGF(2α) production in the hippocampus during kainic acid (KA)-induced seizure activity, the precise intra-hippocampal distribution of PGF(2α) has not been elucidated due to the paucity of effective histological techniques for detecting PGs in tissues. We investigated the tissue distribution of PGF(2α) in the rat hippocampus 30 min after KA injection by developing fixation and immunohistological-staining methods. To detect PGF(2α) directly on histological sections, we used systemic perfusion fixation with water-soluble carbodiimide fixative, followed by immersion of the brains in Zamboni's fixative. We then performed immunofluorescence staining with anti-PGF(2α) antibody, with negative control experiments used to confirm the staining specificity. Definitive immunolabeling for PGF(2α) was evident most markedly in pyramidal cells of the hippocampal cornu Ammonis (CA) 3 sector and neurons of the hilus in KA-treated rats. Immunolabeling for PGF(2α) was also evident in granule cells of the dentate gyrus. Double immunfluorescence staining revealed that PGF(2α)-immunopositive neurons expressed cytosolic phospholipases A(2), COX-2, and FP receptor. These results suggest that the major source of PGF(2α) production immediately after KA injection was neurons of the hippocampal CA3 sector, hilus and dentate gyrus. These neurons exert PGF(2α)-mediated functions via FP receptors in an autocrine/paracrine manner and may play pathophysiological roles in the acute phase (30 min) of excitotoxicity.

Prostanoid EP1 receptors mediate up-regulation of the orphan nuclear receptor Nurr1 by cAMP-independent activation of protein kinase A, CREB and NF-κB.

BACKGROUND AND PURPOSE: Prostaglandin E(2) (PGE(2)) stimulation of the G protein-coupled prostanoid EP(1) receptor was found to up-regulate the expression of Nur-related factor 1 (Nurr1) (NR4A2), a transcription factor in the NR4A subfamily of nuclear receptors. The present studies characterize the molecular mechanism of this up-regulation. EXPERIMENTAL APPROACH: The expression of Nurr1 was examined by immunoblot analysis, the polymerase chain reaction and reporter gene assays in human embryonic kidney (HEK) cells stably expressing the recombinant EP(1) receptor and in SH-SY5Y neuroblastoma cells expressing endogenous EP(1) receptors. Signalling pathway inhibitors were used to examine the roles of Rho, PKA, the cAMP response element binding protein (CREB) and NF-κB on the PGE(2) stimulated up-regulation of Nurr1. CREB and NF-κB signalling were also examined by immunoblot analysis and reporter gene assays. KEY RESULTS: The EP(1) receptor mediated up-regulation of Nurr1 was blocked with inhibitors of Rho, PKA, NF-κB and CREB; but PGE(2) failed to significantly stimulate intracellular cAMP formation. PGE(2) stimulation of the EP1 receptor induced the phosphorylation and activation of CREB and NF-κB, which could be blocked by inhibition of PKA. CONCLUSIONS AND IMPLICATIONS: PGE(2) stimulation of the human EP(1) receptor up-regulates the expression of Nurr1 by a mechanism involving the sequential activation of the Rho, PKA, CREB and NF-κB signalling pathways. EP(1) receptors are implicated in tumorigenesis and the up-regulation of Nurr1 may underlie the anti-apoptotic effects of PGE(2) .

International Union of Basic and Clinical Pharmacology. LXXXIII: classification of prostanoid receptors, updating 15 years of progress.

It is now more than 15 years since the molecular structures of the major prostanoid receptors were elucidated. Since then, substantial progress has been achieved with respect to distribution and function, signal transduction mechanisms, and the design of agonists and antagonists (http://www.iuphar-db.org/DATABASE/FamilyIntroductionForward?familyId=58). This review systematically details these advances. More recent developments in prostanoid receptor research are included. The DP(2) receptor, also termed CRTH2, has little structural resemblance to DP(1) and other receptors described in the original prostanoid receptor classification. DP(2) receptors are more closely related to chemoattractant receptors. Prostanoid receptors have also been found to heterodimerize with other prostanoid receptor subtypes and nonprostanoids. This may extend signal transduction pathways and create new ligand recognition sites: prostacyclin/thromboxane A(2) heterodimeric receptors for 8-epi-prostaglandin E(2), wild-type/alternative (alt4) heterodimers for the prostaglandin FP receptor for bimatoprost and the prostamides. It is anticipated that the 15 years of research progress described herein will lead to novel therapeutic entities.

Interaction of prostanoid EP3 and TP receptors in guinea-pig isolated aorta: contractile self-synergism of 11-deoxy-16,16-dimethyl PGE2.

BACKGROUND AND PURPOSE: Surprisingly high contractile activity was reported for 11-deoxy-16,16-dimethyl prostaglandin E2 (DX-DM PGE2) on pig cerebral artery when used as a selective EP3 receptor agonist. This study investigated the selectivity profile of DX-DM PGE2, focusing on the interaction between its EP3 and TP (thromboxane A2-like) agonist activities. EXPERIMENTAL APPROACH: Contraction of guinea-pig trachea (EP1 system) and aorta (EP3 and TP systems) was measured in conventional organ baths. KEY RESULTS: Strong contraction of guinea-pig aorta to sulprostone and 17-phenyl PGE2 (EP3 agonists) was only seen under priming with a second contractile agent such as phenylephrine, histamine or U-46619 (TP agonist). In contrast, DX-DM PGE2 induced strong contraction, which on the basis of treatment with (DG)-3ap (EP3 antagonist) and/or BMS-180291 (TP antagonist) was attributed to self-synergism arising from co-activation of EP3 and TP receptors. EP3/TP self-synergism also accounted for contraction induced by PGF(2α) and its analogues (+)-cloprostenol and latanoprost-FA. DX-DM PGE2 also showed significant EP1 agonism on guinea-pig trachea as defined by the EP1 antagonists SC-51322, (ONO)-5-methyl-1 and AH-6809, although AH-6809 exhibited poor specificity at concentrations ≥3 µM. CONCLUSIONS AND IMPLICATIONS: EP3/TP self-synergism, as seen with PGE/PGF analogues in this study, may confound EP3 agonist potency comparisons and the characterization of prostanoid receptor systems. The competitive profile of a TP antagonist may be distorted by variation in the silent/overt contraction profile of the EP3 system in different studies. The relevance of self-synergism to in vivo actions of natural prostanoid receptor agonists is discussed.

Roles of affinity and lipophilicity in the slow kinetics of prostanoid receptor antagonists on isolated smooth muscle preparations.

BACKGROUND AND PURPOSE: The highly lipophilic acyl-sulphonamides L-798106 and L-826266 showed surprisingly slow antagonism of the prostanoid EP3 receptor system in guinea-pig aorta. Roles of affinity and lipophilicity in the onset kinetics of these and other prostanoid ligands were investigated. EXPERIMENTAL APPROACH: Antagonist selectivity was assessed using a panel of human recombinant prostanoid receptor-fluorimetric imaging plate reader assays. Potencies/affinities and onset half-times of agonists and antagonists were obtained on guinea-pig-isolated aorta and vas deferens. n-Octanol-water partition coefficients were predicted. KEY RESULTS: L-798106, L-826266 and the less lipophilic congener (DG)-3ap appear to behave as selective, competitive-reversible EP3 antagonists. For ligands of low to moderate lipophilicity, potency increments for EP3 and TP (thromboxane-like) agonism on guinea-pig aorta (above pEC50 of 8.0) were associated with progressively longer onset half-times; similar trends were found for TP and histamine H1 antagonism above a pA2 limit of 8.0. In contrast, L-798106 (EP3), L-826266 (EP3, TP) and the lipophilic H1 antagonists astemizole and terfenadine exhibited very slow onset rates despite their moderate affinities; (DG)-3ap (EP3) had a faster onset. Agonism and antagonism on the vas deferens EP3 system were overall much faster, although trends were similar. CONCLUSIONS AND IMPLICATIONS: High affinity and high liphophilicity may contribute to the slow onsets of prostanoid ligands in some isolated smooth muscle preparations. Both relationships are explicable by tissue disposition under the limited diffusion model. EP3 antagonists used as research tools should have moderate lipophilicity. The influence of lipophilicity on the potential clinical use of EP3 antagonists is discussed.

Characterization of an in vivo model for the study of eyelash biology and trichomegaly: mouse eyelash morphology, development, growth cycle, and anagen prolongation by bimatoprost.

BACKGROUND: Hypertrichosis or alopecia of the eyelashes is associated with various diseases or may be drug induced. Although neither increase nor loss of eyelashes is life threatening, eyelash disorders can be psychologically disturbing. However, as control of eyelash growth and the underlying mechanisms of eyelash hypo- or hypertrichosis are largely obscure, available therapy is limited. OBJECTIVES: To improve this situation, we sought to establish a pragmatic, well-defined mouse model for the study and pharmacological investigation of eyelash follicle biology. METHODS: We took a morphometric approach to establish an eyelash model using female C57BL/6J mice by comparing with pelage hairs and highlighting the differences. We next applied a hypertrichosis-triggering agent and investigated its effect using the model. RESULTS: In eyelashes, a synchronized growth cycle was observed after morphogenesis but was completed earlier than pelage hairs. Exogen was strictly regulated and occurred in every cycle in the eyelash. Otherwise, general morphological features of mouse eyelashes (shafts, follicles, morphogenesis and growth cycle) were comparable with those of pelage hairs. The eyelash growth-stimulatory agent in humans, bimatoprost, significantly extended the duration of anagen, resulting in more and longer eyelashes, but there was no evidence of follicle neogenesis. CONCLUSIONS: This study shows that mouse eyelashes offer an excellent in vivo model for the quantitative and qualitative analysis of eyelash morphology, development, growth cycle, exogen and pharmacological modulation. This model will help to elucidate the unknown molecular controls of eyelash growth, and to develop novel drugs to treat eyelash disorders.