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Abstract Background Sterile male rear-and-release programmes are of growing interest for controlling Aedes aegypti, including use an “incompatible insect technique” (IIT) to suppress transmission of dengue, Zika, and other viruses. Under IIT, males infected with Wolbachia are released into the suppression area to induce cytoplasmic incompatibility in uninfected populations. These and similar mosquito-release programmes require cost-effective field surveys of both sexes to optimize the locations, timing, and quantity of releases. Unfortunately, traps that sample male Ae. aegypti effectively are expensive and usually require mains power. Recently, an electronic lure was developed that attracts males using a 484 Hz sinusoidal tone mimicking the female wingbeat frequencies, broadcast in a 120 s on/off cycle. When deployed in commercially available gravid Aedes traps (GATs), the new combination, sound-GAT (SGAT), captures both males and females effectively. Given its success, there is interest in optimizing SGAT to reduce cost and power usage while maximizing catch rates. Methods Options considered in this study included use of a smaller, lower-power microcontroller (Tiny) with either the original or a lower-cost speaker (lcS). A 30 s on/off cycle was tested in addition to the original 120 s cycle to minimize the potential that the longer cycle induced habituation. The original SGAT was compared against other traps incorporating the Tiny-based lures for mosquito capture in a large semi-field cage. The catch rates in waterproofed versions of this trap were then compared with catch rates in standard [BG-Sentinel 2 (BGS 2); Biogents AG, Regensburg, Germany] traps during an IIT field study in the Innisfail region of Queensland, Australia in 2017. Results The system with a low-power microcontroller and low-cost speaker playing a 30 s tone (Tiny-lcS-30s) caught the highest proportion of males. The mean proportions of males caught in a semi-field cage were not significantly different among the original design and the four low-power, low-cost versions of the SGAT. During the IIT field study, the waterproofed version of the highest-rated, Tiny-lcS-30s SGAT captured male Ae. aegypti at similar rates as co-located BGS-2 traps. Conclusions Power- and cost-optimized, waterproofed versions of male Ae. aegypti acoustic lures in GATs are now available for field use in areas with sterile male mosquito rear-and-release programmes.

The Aedes aegypti mosquito is a primary vector of several serious arboviruses throughout the world and is therefore of great concern to many public health organizations. With vector control methodology pivoting towards rearing and releasing large numbers of genetically modified, sterilized, or Wolbachia-infected male mosquitoes to control vector populations, economical surveillance methods for release tracking becomes increasingly necessary. Previous work has identified that male Ae. aegypti are attracted to female wingbeat frequencies and can be captured through artificial playback of these frequencies, but the tested systems are cost-prohibitive for wide-scale monitoring. Thus, we have developed a simple, low-cost, battery-powered, microcontroller-based sound lure which mimics the wingbeat frequency of female Ae. aegypti, thereby attracting males. We then tested the efficacy of this lure in combination with a passive (non-powered) gravid Aedes trap (GAT) against the current gold-standard, the Biogents Sentinel (BGS) trap, which requires main power (household power) and costs several times what the GAT does. Capture rates of male Ae. aegypti in sound-baited GATs (Sound-GATs) in these field tests were comparable to that of the BGS with no inhibitory effects of sound playback on female capture. We conclude that the Sound-GAT is an effective replacement of the costly BGS for surveillance of male Ae. aegypti mosquitoes, particularly in the developing countries where funding is limited, and has the potential to be adapted to target males of other medically important species.

Grape root borer (GRB), Vitacea polistiformis, is a root-feeding pest of grapevines in the US southeast that causes underground damage well before vines show visible symptoms. A 269-d study was conducted at 31 sites in a Florida vineyard to record short bursts of insect movement and feeding vibrations in grapevine root systems and provide information that can improve timing and targeting of GRB management efforts. Characteristic spectral and temporal patterns in the subterranean vibrations facilitated discrimination of GRB from background noise and non-targeted arthropods. Infestation likelihood of GRB at each site was estimated from previous studies relating infestation to burst rate. In all, 39% of recordings indicated low infestation likelihood. Sites with medium or high infestation likelihood were confined to a small region of the vineyard where a vine with larval feeding damage was confirmed. The restricted area suggests that the biological control or chemical treatments could be reduced elsewhere. Acoustic activity was significantly greater in fall and winter than in spring, and greater in evening than afternoon; fall evenings seemed best for GRB acoustic surveys. The GRB seasonal and circadian acoustic variation reflected phenological variation in grape root growth and nutrients and was not significantly correlated with temperature.

Techniques for stable, rapid and repeatable small-animal immobilization are necessary for high-throughput in vivo genetic/drug screens using cellular and sub-cellular features in multi-cellular organisms. We demonstrate a method for non-invasive and high-throughput on-chip immobilization of physiologically active C. elegans without the use of anesthesia or cooling, but with comparable stability even for the most demanding purposes. We show observation and manipulation of sub-cellular features in immobilized animals using two-photon microscopy and femtosecond-laser microsurgery. [ABSTRACT FROM AUTHOR]

This study evaluated and characterized the pharmacological activity of the orally administered interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitors BAY1834845 (zabedosertib) and BAY1830839 in healthy male volunteers. Participants received one of either IRAK4 inhibitors or a control treatment (prednisolone 20 mg or placebo) twice daily for 7 days. Localized skin inflammation was induced by topical application of imiquimod (IMQ) cream for 3 days, starting at Day 3 of treatment. The inflammatory response was evaluated by laser speckle contrast imaging (skin perfusion) and multispectral imaging (erythema). At Day 7, participants received 1 ng/kg intravenous lipopolysaccharide (LPS). Circulating inflammatory proteins, leukocyte differentiation, acute phase proteins, and clinical parameters were evaluated before and after the systemic LPS challenge. Treatment with BAY1834845 significantly reduced the mean IMQ-induced skin perfusion response (geometric mean ratio [GMR] vs. placebo: 0.69 for BAY1834845, 0.70 for prednisolone; both p < 0.05). Treatment with BAY1834845 and BAY1830839 significantly reduced IMQ-induced erythema (GMR vs. placebo: 0.75 and 0.83, respectively, both p < 0.05; 0.86 for prednisolone, not significant). Both IRAK4 inhibitors significantly suppressed the serum TNF-α and IL-6 responses (≥80% suppression vs. placebo, p < 0.05) and inhibited C-reactive protein, procalcitonin, and IL-8 responses to intravenous LPS. This study demonstrated the pharmacological effectiveness of BAY1834845 and BAY1830839 in suppressing systemically and locally induced inflammatory responses in the same range as prednisolone, underlining the potential value of these IRAK4 inhibitors as future therapies for dermatological or other immune-mediated inflammatory diseases., (© 2024 Bayer AG and Centre for Human Drug Research. Clinical and Translational Science published by Wiley Periodicals LLC on behalf of American Society for Clinical Pharmacology and Therapeutics.)

Objective: Aldo-keto reductase 1C3 (AKR1C3) has been postulated to be involved in androgen, progesterone, and estrogen metabolism. Aldo-keto reductase 1C3 inhibition has been proposed for treatment of endometriosis and polycystic ovary syndrome. Clinical biomarkers of target engagement, which can greatly facilitate drug development, have not yet been described for AKR1C3 inhibitors. Here, we analyzed pharmacodynamic data from a phase 1 study with a new selective AKR1C3 inhibitor, BAY1128688, to identify response biomarkers and assess effects on ovarian function., Design: In a multiple-ascending-dose placebo-controlled study, 33 postmenopausal women received BAY1128688 (3, 30, or 90 mg once daily or 60 mg twice daily) or placebo for 14 days. Eighteen premenopausal women received 60 mg BAY1128688 once or twice daily for 28 days., Methods: We measured 17 serum steroids by liquid chromatography-tandem mass spectrometry, alongside analysis of pharmacokinetics, menstrual cyclicity, and safety parameters., Results: In both study populations, we observed substantial, dose-dependent increases in circulating concentrations of the inactive androgen metabolite androsterone and minor increases in circulating etiocholanolone and dihydrotestosterone concentrations. In premenopausal women, androsterone concentrations increased 2.95-fold on average (95% confidence interval: 0.35-3.55) during once- or twice-daily treatment. Note, no concomitant changes in serum 17β-estradiol and progesterone were observed, and menstrual cyclicity and ovarian function were not altered by the treatment., Conclusions: Serum androsterone was identified as a robust response biomarker for AKR1C3 inhibitor treatment in women. Aldo-keto reductase 1C3 inhibitor administration for 4 weeks did not affect ovarian function.ClinicalTrials.gov Identifier: NCT02434640; EudraCT Number: 2014-005298-36., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Society of Endocrinology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Vilaprisan is a novel selective progesterone receptor modulator for the long-term treatment of uterine fibroids and endometriosis. This study investigated the pharmacokinetics, safety, and tolerability of vilaprisan in healthy Chinese postmenopausal women. Twelve participants received multiple doses of vilaprisan once daily over 14 days as a 2-mg tablet. Plasma vilaprisan concentrations were determined using liquid chromatography-tandem mass spectrometry. The main pharmacokinetic parameters of vilaprisan were assessed with noncompartmental analysis, including maximum observed concentration (C max ), systemic exposure (area under the plasma concentration-time curve), time to reach C max and terminal half-life. Safety assessments include the documentation of adverse events, measurement of clinical/anthropometric parameters and vital signs, electrocardiogram, and physical and gynecologic examination. The participants had a mean age of 53.3 (± 4.2) years and a body mass index of 23.8 ± 2.8 kg/m 2 . Median time to reach C max was 1.5 hours after both single and multiple vilaprisan administration. Mean C max values obtained after multiple dosing (23.3 μg/L [standard deviation (SD) = 6.73]) were 1.92-fold (SD = 0.554) higher compared to single dosing (12.5 μg/L [SD = 3.04]). Mean area under the plasma concentration-time curve in the dosing interval increased with an accumulation factor of 2.98 (SD = 0.767) between single (91.3 μg · h/L [SD = 20.4]) and multiple dosing (276 μg · h/L [SD = 109]). The mean terminal half-life of vilaprisan was 44.5 hours (SD = 10.3) after multiple dosing. Mild to moderate adverse events were observed similar to previous studies. Overall, daily oral administration of the therapeutic dose of 2 mg of vilaprisan over 14 days was safe and well tolerated by all participants., (© 2020 The Authors. Clinical Pharmacology in Drug Development published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology.)

Aims: The primary objective was to explore whether the suppression of ovarian activity induced by a combined oral contraceptive (COC) is influenced by the simultaneous intake of the selective progesterone receptor modulator (SPRM) vilaprisan (VPR)., Methods: In this exploratory randomized, double-blind, parallel-group study, 71 healthy premenopausal women were randomized (1:1) to receive either 2 mg/d VPR or placebo for 3 months. Concomitantly, a COC (0.15 mg levonorgestrel, 0.03 mg ethinyloestradiol) was administered in a cyclic regimen. Ovarian activity (Hoogland score based on follicle size and hormone concentrations), cervical function (Insler score), bleeding pattern and endometrial thickness/histology were assessed before treatment, in treatment cycle 3 and during follow-up., Results: The known COC-driven suppression of ovarian activity was mildly affected by VPR. COC+VPR group: 22, 0 and 6% of the subjects had Hoogland scores of 4 (active follicle-like structures), 5 or 6 (ovulation). COC+placebo group: 14% of the subjects had a score of 4 and none a score of 5 or 6 (Bayesian analysis for Hoogland score = 4, median difference in response rate: 7.5%; 90% credible interval [-8.5; 23.5%]). COC effects on cervical function were moderately affected (mucus more sperm permeable under COC+VPR). COC withdrawal bleeding, in contrast, was absent in 81% of the subjects receiving COC+VPR vs 0% receiving COC+placebo., Conclusion: The SPRM VPR interfered with the pharmacodynamic effects of the COC. Therefore, full contraceptive effectiveness cannot be assumed without final judgement by a Pearl index study. Women on SPRMs should be advised to use nonhormonal contraception methods., (© 2020 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

Reduction of postharvest losses is gaining increased priority in warm regions where insect infestation may cause rapid deterioration of staple commodities. Acoustic detection can be used to assess the likelihood of insect infestations in bags of grain, flour, and other commodities that are stored in small holdings in developing countries, enabling rapid targeting of treatments. A portable postharvest insect detection system was developed with the goal to provide low-cost capability to acoustically assess infestations in small-scale storage facilities. Electret microphones input pest insect sounds to a 32-bit microcontroller platform that digitized and stored the signals on a digital memory card transferable to a portable laptop computer. The insect sounds then were analyzed by custom-written software that matched their spectra to those of known pests. Infestations of Sitophilus oryzae (L) in 2.6-kg bags could be detected down to densities of 1.9 adults/kg in grain and Tribolium castaneum (Herbst) down to 3.8 adults/kg in flour in laboratory settings. Also, differences in the rates of sounds per insect in treatments with different numbers ranging from 5 to 50 insects suggested that the sound rates of adults of different species at different population densities may be noticeably affected by aggregation pheromones or other behaviorally active semiochemicals. Further testing is needed but previous experience with acoustic detection systems suggests that the prototype has potential for use in small storage facilities where early detection of infestations is difficult to provide., (Published by Oxford University Press on behalf of Entomological Society of America 2020.)

Grain production is an important component of food security in Kenya but due to environmental conditions that favor rapid growth of insect populations, farmers and other agricultural stakeholders face ongoing and novel challenges from crop and stored product pest insects. To assist development of methods to reduce economic losses from stored product insect pests in Kenya, acoustic, visual, and pitfall trap surveys were conducted in five grain storage warehouses. Two commercially available acoustic systems successfully detected the pests of greatest economic importance, Sitophilus zeamais (Motschulsky) and Prostephanus truncatus (Horn). Other insects of lesser economic importance also were observed in the visual surveys, including Sitotroga cerealella (Olivier) (Lepidoptera: Gelechiidae), and Tribolium castaneum (Herbst). This study demonstrated that the use of acoustic technology with visual surveys and pitfall traps can help managers to identify and target infestations within their warehouses, enabling them to reduce postharvest losses. With most warehouses being located in relatively noisy urban or peri-urban areas, background noise considerations are being incorporated into the design of future acoustic detectors for stored pest infestations. Kenya must import grain yearly to meet consumption needs; however, if the current yearly postharvest losses of 20-30% in warehouses decreased, import costs could be reduced considerably.

BAY 1158061 is a potent monoclonal prolactin (PRL) receptor antibody, blocking PRL receptor (PRLR)-mediated signaling in a noncompetitive manner, which was tested in a randomized, placebo-controlled multiple dose study in postmenopausal women. The objective was to investigate safety, tolerability, pharmacokinetic characteristics, and effects of BAY 1158061 on serum PRL level. The study consisted of 4 parallel groups receiving up to 3 subcutaneous (sc) administrations of BAY 1158061 or placebo in 2 different dosing regimens. Twenty-nine healthy postmenopausal women were randomized and treated with BAY 1158061 or placebo: 30 mg at 14-day interval (7 participants), 60 mg at 28-day interval (8 participants), 90 mg at 14-day interval (7 participants), and placebo (7 participants). To keep the blinding, all randomized participants received sc injections biweekly (14-day interval) on 3 occasions in the lower abdomen. The PRLR antibody showed a favorable safety and tolerability profile in postmenopausal women with no distinct differences in occurrence of adverse events in BAY 1158061 or placebo-treated participants. BAY 1158061 displayed low immunogenicity with low titers of antidrug antibodies and absence of neutralizing antidrug antibodies. Pharmacokinetics were characterized by slow absorption after sc administration with median peak plasma concentrations 7 to 11 days after first dose and about 2-fold accumulation after repeated dosing every 2 weeks. The apparent mean elimination half-life was 9 to 16 days. The PRL concentration-time profiles over 24 hours showed no differences between verum- and placebo-treated participants. Based on the data obtained, BAY 1158061 is considered a good candidate for further development in endometriosis or other PRL-mediated disease conditions.

It is known that a small fraction of orally administered norethisterone is metabolically converted to ethinylestradiol. This exploratory, open-label, nonrandomized study was conducted to investigate the systemic exposure to ethinylestradiol after intramuscular administration of norethisterone enantate in comparison with the exposure to ethinylestradiol after administration of a standard combined oral contraceptive. Sixteen healthy premenopausal women received an oral contraceptive (ethinylestradiol 30 μg/levonorgestrel 150 μg) once daily for 21 days and-after a 1-week washout period-a single intramuscular dose of 200 mg norethisterone enantate. Blood samples to determine ethinylestradiol in serum were taken over 24 hours after the last dose of ethinylestradiol/levonorgestrel and over 8 weeks after administration of norethisterone enantate. Oral equivalent doses of ethinylestradiol were estimated based on area under the concentration-time curves. The ethinylestradiol serum concentrations observed after administration of norethisterone enantate were relatively low: The mean maximum concentration was only 32% of the maximum observed after ethinylestradiol/levonorgestrel (90% confidence interval, 22.5%-44.7%). The maximum oral equivalent dose of ethinylestradiol was markedly lower than 30 μg ethinylestradiol per day (20.3 μg/day; 90% confidence interval, 14.8-28.0 μg/day). The same applied to the average oral equivalent dose of ethinylestradiol for the 8-week postdose interval (4.41 μg/day; 90% confidence interval, 3.57-5.46 μg/day). To conclude, the study results indicate that metabolic conversion of norethisterone to ethinylestradiol also occurs after intramuscular administration of 200 mg norethisterone enantate, but is associated with a lower exposure to ethinylestradiol than the use of a combined oral contraceptive containing 30 μg ethinylestradiol (plus 150 μg levonorgestrel)., (© 2018, The American College of Clinical Pharmacology.)

Aim: Regorafenib is a multikinase inhibitor under investigation for use in neovascular age-related macular degeneration. In this phase I study, regorafenib eye drops were administered to healthy volunteers to provide information on safety, tolerability and systemic exposure., Methods: This was a single-centre, randomized, double-masked, parallel-group, dose-escalation, placebo-controlled study. Subjects received regorafenib eye drops (30 mg ml -1 , 25 μl) as a 0.75 mg single dose (Cohort 1), 0.75 mg twice daily (bid) or thrice daily (tid) over 14 days (Cohorts 2 and 3, respectively), 1.5 mg tid unilaterally for 3 days, then bilaterally for up to 14 days (Cohort 4), or placebo. Plasma samples were taken to estimate systemic exposure. Safety and functional assessments were performed throughout the study., Results: Thirty-six subjects received regorafenib and 12 received placebo. Regorafenib was safe and well tolerated over the dose range. No pathological changes occurred in the anterior, vitreous or posterior eye compartments. Mild eyelid redness, oedema and conjunctival hyperaemia were observed across all regorafenib cohorts; these were comparable with the effects seen with placebo. Predominant symptoms were blurred vision in the active and placebo groups. Systemic safety evaluations showed no clinically relevant findings. Absolute systemic exposure after multiple administrations of regorafenib eye drops at a dose of 0.75 mg was 600-700-fold lower than after multiple oral administration of 160 mg day -1 , the dose approved in cancer indications., Conclusion: These results indicate a favourable safety and tolerability profile of regorafenib eye drops up to 30 mg ml -1 tid for use in clinical studies., (© 2018 The Authors. British Journal of Clinical Pharmacology published by John Wiley & Sons Ltd on behalf of British Pharmacological Society.)

This randomized, double-blind, parallel-group study in healthy young women investigated the effect of treatment with vilaprisan (0.5, 1, 2, or 4 mg/day for 12 weeks) on ovarian function by assessing the Hoogland score, which is based on the size of follicle-like structures as determined by transvaginal ultrasound and on estradiol and progesterone serum concentrations. Ovulation inhibition (ie, Hoogland score <6 in treatment weeks 1-4 and 8-12) was observed in >80% of the subjects receiving vilaprisan ≥1 mg/day. The effect was dose dependent. With a Bayesian approach, the percentage of subjects with ovulation inhibition was estimated to increase from 37% in subjects receiving 0.5 mg/day vilaprisan to 76%, 86%, and 88% in subjects receiving 1, 2, and 4 mg/day, respectively. Follicle growth was not suppressed during treatment. The majority of subjects receiving ≥1 mg/day had a Hoogland score of 4 (active follicle-like structures, ie, follicle diameter >13 mm, estradiol >27.2 pg/mL, no progesterone increase) both at beginning and end of treatment. Mean average estradiol as well as mean maximum progesterone concentrations were noticeably decreased during treatment with vilaprisan ≥1 mg/day compared to pretreatment, but estradiol concentrations remained >80 pg/mL. Both hormones returned to pretreatment levels after the end of treatment, indicating a rapid resumption of normal ovarian activity. Amenorrhea occurred in the majority of subjects during treatment at dosages ≥1 mg/day. The adverse events observed in this study confirm the known safety profile of vilaprisan. All in all, the results of this study support the development of vilaprisan for the long-term treatment of uterine fibroids., (© 2017, The Authors. The Journal of Clinical Pharmacology Published by Wiley Periodicals, Inc. on behalf of American College of Clinical Pharmacology.)

The BREELIB nebulizer was developed for iloprost to reduce inhalation times for patients with pulmonary arterial hypertension (PAH). This multicenter, randomized, unblinded, four-part study compared inhalation time, pharmacokinetics, and acute tolerability of iloprost 5 µg at mouthpiece delivered via BREELIB versus the standard I-Neb nebulizer in 27 patients with PAH. The primary safety outcome was the proportion of patients with a maximum increase in heart rate (HR) ≥ 25% and/or a maximum decrease in systolic blood pressure ≥ 20% within 30 min after inhalation. Other safety outcomes included systolic, diastolic, and mean blood pressure, HR, oxygen saturation, and adverse events (AEs). Median inhalation times were considerably shorter with BREELIB versus I-Neb (2.6 versus 10.9 min; n = 24). Maximum iloprost plasma concentration and systemic exposure (area under the plasma concentration-time curve) were 77% and 42% higher, respectively, with BREELIB versus I-Neb. Five patients experienced a maximum systolic blood pressure decrease ≥ 20%, four with BREELIB (one mildly and transiently symptomatic), and one with I-Neb; none required medical intervention. AEs reported during the study were consistent with the known safety profile of iloprost. The BREELIB nebulizer offers reduced inhalation time, good tolerability, and may improve iloprost aerosol therapy convenience and thus compliance for patients with PAH.

Objectives: Vilaprisan is a novel, potent, and highly selective progesterone receptor modulator, which might offer a promising option for the treatment of uterine fibroids., Methods and Materials: In this randomized, placebo-controlled, parallel-group phase 1 study, the pharmacokinetics and safety of vilaprisan were investigated in healthy postmenopausal women. Subjects received a single oral dose of vilaprisan (1, 5, 15, or 30 mg) or placebo and - after a wash-out period - daily doses of the same strength over 28 days. Safety assessments included vital signs, ECGs, clinical laboratory tests, and adverse events. Blood samples for pharmacokinetic (PK) profiles were collected over 14 days after single dose (sd) and multiple dose (md; day 28)., Results: Vilaprisan was well tolerated. Mild to moderate adverse events occurred with similar frequency at all dose levels. Following single dose, maximum vilaprisan concentrations were observed 1 - 2 hours post-dose. Terminal half-lives ranged from 31 to 38 hours. Maximum concentrations of vilaprisan (Cmax) and exposure to vilaprisan (AUC) increased roughly dose-proportionally from 3.74 µg/L (1 mg) to 68.6 µg/L (30 mg) and 58.5 µg×h/L to 1,590 µg×h/L, respectively. With daily dosing, accumulation consistent with the long terminal half-life was observed (AUC(0-24)md/AUC(0-24)sd ratios: 1.9 to 3.2). The ratio AUC(0-24)md/AUCsd increased with dose from ~ 1 (1 mg) to 1.5 (30 mg)., Conclusions: Exposure to vilaprisan increased roughly dose-proportionally in the dose range studied and accumulated after multiple dosing as expected based on t1/2, indicating linear pharmacokinetics of vilaprisan in the expected therapeutic dose range.
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Study Question: Does administration of vilaprisan (VPR) to healthy women for 12 weeks reduce menstrual bleeding?, Summary Answer: In this 12-week proof-of-concept phase 1 trial, most women (30/33, 90%) who received VPR at daily doses of 1-5 mg reported the absence of menstrual bleeding., What Is Known Already: Vilaprisan (BAY 1002670) is a novel, highly potent selective progesterone receptor modulator that markedly reduces the growth of human leiomyoma tissue in a preclinical model of uterine fibroids (UFs)., Study Design, Size, Duration: In this double-blind, parallel-group study, of the 163 healthy women enrolled 73 were randomized to daily VPR 0.1 mg (n = 12), 0.5 mg (n = 12), 1 mg (n = 13), 2 mg (n = 12), 5 mg (n = 12) or placebo tablets (n = 12) for 12 weeks. Participants were followed up until the start of the second menstrual bleeding after the end of treatment. Trial simulations were used to determine the minimum sample size required to estimate the non-bleeding rate (i.e. self-assessed bleeding intensity of 'none' or 'spotting') using Bayesian dose-response estimation with incorporated prior information. It was estimated that 48 participants in the per-protocol analysis population would be sufficient., Participants/materials, Setting, Methods: Women aged 18-45 years who had been sterilized by tubal ligation were enrolled between November 2011 and May 2012. Participants kept a daily diary of bleeding intensity. Blood and urine samples were taken, and transvaginal ultrasound was performed before treatment, during treatment and follow-up. Endometrial biopsies were obtained during the pretreatment cycle, at the end of the treatment period and during the follow-up phase. The primary outcome was the estimated dose-response curve of the observed non-bleeding rate during Days 10-84 of treatment, excluding the endometrial biopsy day and 2 days after biopsy. Secondary outcomes included return of bleeding during follow-up, size of follicle-like structures and serum hormone levels. Safety assessments included adverse events (AEs), endometrial thickness and histology, laboratory parameters, vital signs and 12-lead electrocardiography., Main Results and the Role of Chance: All 73 randomized participants received at least one dose of study medication and were included in safety analyses; six participants were excluded from the per-protocol analyses. A total of 69 completed the study. Observed non-bleeding rates increased with VPR dose: 0.1 mg (0%; 90% confidence interval [CI]: 0-23.8), 0.5 mg (27.3%; 90% CI: 7.9-56.4), 1 mg (80.0%; 90% CI: 49.3-96.3), 2 mg (100%; 90% CI: 77.9-100), 5 mg (90.9%; 90% CI: 63.6-99.5), compared with 0% (90% CI: 0-22.1) in the placebo group. Maximal non-bleeding rates were reached at doses of 2 mg and higher. Return of menstrual bleeding was observed in all women ≤52 days after VPR discontinuation. No treatment-emergent critical endometrial findings occurred. Follicular growth was not suppressed and minimum average estradiol levels remained above 40 pg/ml. No serious treatment-emergent AEs or study discontinuations due to AEs were reported. Clinically relevant changes in laboratory parameters or vital signs were not evident., Limitations, Reasons for Caution: The results of this small proof-of-concept study will need to be confirmed in larger trials in patients with UFs to establish the potential therapeutic benefits and safety of VPR., Wider Implications of the Findings: The high rates of non-bleeding (80-100% at VPR doses of 1-5 mg) support further evaluation of VPR in patients with UFs and heavy menstrual bleeding., Study Funding/competing Interests: This study was funded by Bayer Pharma AG. B.S., A.K., M.-H.S.M., C.S. and B.R. are employees of Bayer Pharma AG. B.S., A.K. and M.-H.S.M. are listed as inventors of an issued patent related to this work, and received payment for this from Bayer Pharma AG. D.B. is the founder of Biokinetic Europe Ltd, UK, which received funding for this study from Bayer Pharma AG. M.K. is an employee of Nuvisan GmbH, Germany, which received funding for this study from Bayer Pharma AG., Trial Registration Number: Clinicaltrials.gov identifier: NCT01816815., Trial Registration Date: 20 March 2013., Date of First Patient's Enrolment: 28 November 2011., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Study Question: What are suitable doses of the aromatase inhibitor anastrozole (ATZ) and the progestin levonorgestrel (LNG), when delivered to the systemic circulation by an intravaginal ring (IVR), for further clinical development as a potential new therapy for the treatment of endometriosis?, Summary Answer: Anticipated targets for pharmacokinetics, pharmacodynamics and safety/tolerability were achieved for both drug components of the IVR at the doses investigated, supporting selection of the doses to be investigated in Phase 2 studies., What Is Known Already: Aromatase is a key enzyme in the biosynthesis of estrogens and is known to increase local levels of estradiol (E2) at extragonadal sites. Up-regulation of aromatase expression has been demonstrated in endometriotic lesions and the use of oral aromatase inhibitors has been shown to reduce endometriosis-associated pelvic pain in small-scale clinical trials., Study Design, Size, Duration: This Phase 1, randomized, multicentre, parallel-group, three-arm, open-label study assessed the pharmacokinetics, pharmacodynamics, safety and tolerability of various IVRs intended for systemic drug delivery. After screening, healthy, ovulating women aged 18-35 years were randomized to use IVRs releasing one of the three ATZ/LNG dose combinations (in vitro nominal daily drug release rates on Day 29: ATZ/LNG 500 µg/20 µg [low dose], ATZ/LNG 1000 µg/30 µg [mid dose] or ATZ/LNG 1500 µg/40 µg [high dose]) for two consecutive 28-day wearing periods without a treatment break., Participants/materials, Setting, Methods: Sixty women were included in the per protocol set. The primary variables were plasma concentrations of ATZ and LNG at the end of each treatment period and the mean size of largest follicle-like structures (FLSs) over 56 days. Serum concentrations of several hormones were also evaluated, with emphasis on E2 levels., Main Results and the Role of Chance: At the end of the first treatment period, geometric mean plasma concentrations of LNG and ATZ, respectively, were 0.228 and 12.5 µg/l for the low dose, 0.269 and 19.8 µg/l for the mid dose and 0.384 and 37.3 µg/l for the high dose; results were similar at the end of the second treatment period. Over the entire treatment period, mean FLS sizes were higher in all three treatment groups than during the pretreatment cycle; more women in the mid- and high-dose groups had FLSs of at least 30 mm (32-45%) than those in the low-dose group (14-24%). Changes in the mean size of FLSs were similar to those reported for low-dose progestin-only oral contraceptives and generally resolved during the 2-month treatment period. Serum E2 levels were decreased, but only one woman in each of the mid- and high-dose groups, and no woman in the low-dose group, had a serum E2 level below 20 pg/ml in both cycles. All ATZ and LNG combinations showed good tolerability., Limitations, Reasons for Caution: This was an exploratory study; no formal power calculation was performed., Wider Implications of the Findings: The results of this first-in-human study of the ATZ/LNG IVR facilitated the selection of ATZ and LNG doses to be investigated in the Phase 2 studies of patients with endometriosis., Study Funding/competing Interest: The study was funded by Bayer Pharma AG. T.R. is an employee of DINOX GmbH, which received funding from Bayer Pharma AG to perform this study. M.-H.S.-M., K.W., R.N., S.K., J.K., H.S. and B.R. are or have been employees of Bayer Pharma AG. H.S. is a named inventor on EP 2 552 404 B1, a patent application relating to this work., Trial Registration Number: EudraCT number: 2011-005620-18., Trial Registration Date: 16 November 2011., Date of First Patient's Enrolment: 14 March 2012., (© The Author 2016. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Rhynchophorus ferrugineus (Olivier) (Coleoptera: Dryophthoridae) larvae are cryptic, internal tissue-feeding pests of palm trees that are difficult to detect; consequently, infestations may remain hidden until they are widespread in an orchard. Infested trees and propagable offshoots that develop from axillary buds on the trunk frequently are transported inadvertently to previously uninfested areas. Acoustic methods can be used for scouting and early detection of R. ferrugineus, but until now have not been tested on multiple trees and offshoots in commercial date palm orchard environments. For this report, the acoustic detectability of R. ferrugineus was assessed in Saudi Arabian date palm orchards in the presence of commonly occurring wind, bird noise, machinery noise, and nontarget insects. Signal analyses were developed to detect R. ferrugineus and another insect pest, Oryctes elegans Prell (Coleoptera: Scarabaeidae), frequently co-occurring in the orchards, and discriminate both from background noise. In addition, it was possible to distinguish R. ferrugineus from O. elegans in offshoots by differences in the temporal patterns of their sound impulses. As has been observed often with other insect pests, populations of the two species appeared clumped rather than uniform or random. The results are discussed in relation to development of automated methods that could assist orchard managers in quickly identifying infested trees and offshoots so that R. ferrugineus infestations can be targeted and the likelihood of transferring infested offshoots to uninfested areas can be reduced.

Here we report the findings of a two-centre, open-label, randomised, Phase IIa study designed to investigate whether an ethinyl estradiol (EE)/gestodene (GSD) patch that has been developed (referred to herein as the 'EE/GSD patch') reliably inhibits ovulation in comparison with patches delivering lower doses of these hormones. The study rationale was to provide justification of the doses of EE and GSD selected for the EE/GSD patch. Healthy women, aged 18-35 years, were randomised to receive treatment with either the EE/GSD patch, a 'reduced-GSD patch' (delivering similar amounts of EE and approximately half the amount of GSD) or a 'reduced-EE/GSD patch' (delivering half the amount of EE and GSD). Treatment was administered for three 28-day cycles (three × 7 patch-wearing days, plus a 7-day patch-free interval). The primary pharmacodynamic variable was the percentage of women with ovulation in at least one of Cycles 2 and/or 3, as indicated by Hoogland score. Pharmacokinetic parameters for EE and GSD were also measured. Results indicated that the EE/GSD patch effectively suppressed ovulation, while patches delivering lower doses of EE and GSD were less effective for this purpose. All three patches showed comparable tolerability.

Pharmacokinetic (PK) interactions between the cytochrome P450 3A4 (CYP3A4) pathway and transdermally administered ethinyl estradiol (EE) and gestodene (GSD) were investigated. This paper reports the findings of three open-label, intra-individual, one-way crossover, Phase I trials. In two studies, women used a novel contraceptive patch for 3 weeks during two 4-week study periods; in the second period, the CYP3A4 inhibitors erythromycin (Study 1) or ketoconazole (Study 2) were administered concurrently. In a third study, women received single doses of the CYP3A4 model substrate midazolam, alone and after 3 weeks of concurrent patch application. In each period, the EE/GSD patch (delivering low EE and GSD doses resulting in the same systemic exposure as a combined oral contraceptive containing 0.02 mg EE and 0.06 mg GSD) was applied once weekly for 3 weeks, with one patch-free week. Erythromycin, ketoconazole, and midazolam were administered orally. Main outcome measures were area under the curves (AUCs) and maximum plasma concentration (C max) of EE, and total and unbound GSD (Studies 1 and 2). AUC and C max of midazolam (Study 3). Co-administration of CYP3A4 inhibitors did not affect EE metabolism, and had only weak effects on the PK of total and unbound GSD. The patch had no clinically relevant effect on metabolism of the CYP3A4 substrate midazolam.

The prolactin receptor (PRLR) has been implicated in a variety of physiological processes (lactation, reproduction) and diseases (breast cancer, autoimmune diseases). Prolactin synthesis in the pituitary and extrapituitary sites is regulated by different promoters. Dopamine receptor agonists such as bromocriptine can only interfere with pituitary prolactin synthesis and thus do not induce a complete blockade of PRLR signaling. Here we describe the identification of a human monoclonal antibody 005-C04 that blocks PRLR-mediated signaling at nanomolar concentrations in vitro. In contrast to a negative control antibody, the neutralizing PRLR antibody 005-C04 inhibits signal transducer and activator of transcription 5 phosphorylation in T47D cells and proliferation of BaF3 cells stably expressing murine or human PRLRs in a dose-dependent manner. In vivo application of this new function-blocking PRLR antibody reflects the phenotype of PRLR-deficient mice. After antibody administration female mice become infertile in a reversible manner. In lactating dams, the antibody induces mammary gland involution and negatively interferes with lactation capacity as evidenced by reduced milk protein expression in mammary glands and impaired litter weight gain. Antibody-mediated blockade of the PRLR in vivo stimulates hair regrowth in female mice. Compared with peptide-derived PRLR antagonists, the PRLR antibody 005-C04 exhibits several advantages such as higher potency, noncompetitive inhibition of PRLR signaling, and a longer half-life, which allows its use as a tool compound also in long-term in vivo studies. Therefore, we suggest that this antibody will help to further our understanding of the role of auto- and paracrine PRLR signaling in health and disease.

Background: We assessed the clinical utility of β-amyloid (Aβ) imaging with (18)F-florbetaben (FBB) in mild cognitive impairment (MCI) by evaluating its prognostic accuracy for progression to Alzheimer's disease (AD), comparing semiquantitative with visual scan assessment, and exploring the relationships among Aβ, hippocampal volume (HV) and memory over time., Methods: 45 MCI underwent FBB positron emission tomography, MRI and neuropsychological assessment at baseline and 2 years and clinical follow-up at 4 years. Positive FBB (FBB+), defined by a cortical to cerebellar cortex standardised uptake value ratio (SUVR) ≥ 1.45, was compared with visual assessment by five readers. Amnestic MCI (aMCI) was defined by a composite episodic memory (EM) Z-score of <-1.5., Results: At baseline, 24 (53%) MCI were FBB+. Majority reads agreed with SUVR classification (κ 0.96). In 2 years, 18 (75%) FBB+ progressed to AD compared with 2 (9.5%) FBB-, yielding a predictive accuracy of 83% (95% CI 61% to 94%). Four FBB- developed non-AD dementia. Predictive accuracies of HV (58% (95% CI 42% to 73%)) and aMCI status (73% (95% CI 58% to 81%)) were lower. Combinations did not improve accuracy. By 4 years, 21 (87.5%) FBB+ had AD whereas 5 (24%) FBB- had non-AD dementia yielding a predictive accuracy of 94% (95% CI 74% to 99%). While the strong baseline association between FBB SUVR and EM declined over 2 years, the association between EM and HV became stronger. FBB SUVR increased 2.2%/year in FBB+ with no change in FBB-., Conclusions: (18)F-florbetaben Aβ imaging facilitates accurate detection of prodromal AD. As neurodegeneration progresses, and in contrast with the early stages of the disease, hippocampal atrophy and not Aβ, seems to drive memory decline., Trial Registration Number: NCT01138111., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Unlabelled: (18)F-florbetaben is a novel (18)F-labeled tracer for PET imaging of β-amyloid deposits in the human brain. We evaluated the kinetic model-based approaches to the quantification of β-amyloid binding in the brain from dynamic PET data. The validity of the practically useful tissue ratio was also evaluated against the model-based parameters., Methods: (18)F-florbetaben PET imaging was performed with concurrent multiple arterial sampling after tracer injection (300 MBq) in 10 Alzheimer disease (AD) patients and 10 age-matched healthy controls. Regional brain-tissue time-activity curves for 90 min were analyzed by a 1-tissue-compartment model and a 2-tissue-compartment model (2TCM) with metabolite-corrected plasma data estimating the specific distribution volume (VS) and distribution volume ratio (DVR [2TCM]) and a multilinear reference tissue model estimating DVR (DVR [MRTM]) using the cerebellar cortex as the reference tissue. Target-to-reference tissue standardized uptake value ratios (SUVRs) at 70-90 min were also calculated., Results: All brain regions required 2TCM to describe the time-activity curves. All β-amyloid binding parameters in the cerebral cortex (VS, DVR [2TCM], DVR [MRTM], and SUVR) were significantly increased in AD patients (P < 0.05), and there were significant linear correlations among these parameters (r(2) > 0.83). Effect sizes in group discrimination between 8 β-amyloid-positive AD scans and 9 β-amyloid-negative healthy control scans for all binding parameters were excellent, being largest for DVR (2TCM) (4.22) and smallest for VS (3.25) and intermediate and the same for DVR (MRTM) and SUVR (4.03)., Conclusion: These results suggest that compartment kinetic model-based quantification of β-amyloid binding from (18)F-florbetaben PET data is feasible and that all β-amyloid binding parameters including SUVR are excellent in discriminating between β-amyloid-positive and -negative scans.

Background: Adequate folate supplementation in the periconceptional phase is recommended to reduce the risk of neural tube defects. Oral contraceptives may provide a reasonable delivery vehicle for folate supplementation before conception in women of childbearing potential. This study aimed to demonstrate that a fixed-dose combination of an oral contraceptive and levomefolate calcium leads to sustainable improvements in folate status compared with an oral contraceptive + folic acid., Methods: This was a double-blind, randomized, parallel-group study in which 172 healthy women aged 18-40 years received ethinylestradiol (EE)-drospirenone-levomefolate calcium or EE-drospirenone + folic acid for 24 weeks (invasion phase), and EE-drospirenone for an additional 20 weeks (folate elimination phase). The main objective of the invasion phase was to examine the area under the folate concentration time-curve for plasma and red blood cell (RBC) folate, while the main objective of the elimination phase was to determine the duration of time for which RBC folate concentration remained ≥ 906 nmol/L after cessation of EE-drospirenone-levomefolate calcium., Results: Mean concentration-time curves for plasma folate, RBC folate, and homocysteine were comparable between treatment groups during both study phases. During the invasion phase, plasma and RBC folate concentrations increased and approached steady-state after about 8 weeks (plasma) or 24 weeks (RBC). After cessation of treatment with levomefolate calcium, folate concentrations decreased slowly. The median time to RBC folate concentrations falling below 906 nmol/L was 10 weeks (95% confidence interval 8-12 weeks) after cessation of EE-drospirenone-levomefolate calcium treatment. Plasma and RBC folate levels remained above baseline values in 41.3% and 89.3% of women, respectively, at the end of the 20-week elimination phase., Conclusion: Improvements in folate status were comparable between EE-drospirenone-levomefolate calcium and EE-drospirenone + folic acid. Plasma and RBC folate levels remained elevated for several months following cessation of treatment with EE-drospirenone-levomefolate calcium.

Background: We evaluated the effects of cytochrome P450 3A4 (CYP3A4) induction and inhibition on steady-state pharmacokinetics of the components of a novel oral contraceptive (OC) containing estradiol valerate (E₂V) and dienogest (DNG)., Study Design: CYP3A4 induction was assessed in an open-label, one-arm study. Sixteen healthy postmenopausal women received E₂V 2 mg/DNG 3 mg (days 1-17) and concomitant rifampicin (600 mg, days 12-16). Ratios of the area under the serum concentration-time curve between 0 and 24 h [AUC(0-24 h)] and maximum serum concentration (C(max)) of E₂ and DNG on days 17 and 11 (after and before rifampicin intervention) are presented. CYP3A4 inhibition was investigated in an open-label, parallel-group study in 24 healthy postmenopausal women receiving E₂V 2 mg/DNG 3 mg (days 1-14) and concomitant ketoconazole (400 mg, n=12) or erythromycin (500 mg three times daily, n=12) on days 8-14. Mean ratios of AUC(0-24 h) and C(max) of E₂ and DNG on days 7 and 14 are presented., Results: Concomitant administration of rifampicin decreased systemic drug exposure and yielded geometric mean ratios for E₂C(max) and AUC(0-24 h) of 75% and 56%, respectively. Corresponding mean ratios for DNG were 48% and 17%, respectively. Ketoconazole coadministration increased systemic drug exposure and yielded ratios of E₂ of 165% and 157%, respectively, and ratios of DNG of 194% and 286%, respectively. Erythromycin coadministration also resulted in increased mean C(max) and AUC(0-24 h) of both E₂ and DNG. Geometric mean ratios of C(max) and AUC(0-24 h) for E₂ were 151% and 133%, respectively. Corresponding ratios for DNG were 133% and 162%, respectively., Conclusions: Significant drug-drug interactions are apparent when CYP3A4 modulators are coadministered with the components of a novel OC containing E₂V/DNG. Coadministration of CYP3A4 modulators should be avoided where possible, and another type of contraception should be used when coadministration of CYP3A4 inducers like rifampicin is unavoidable., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Neural tube defects (NTDs) are congenital malformations that occur during early embryonic development. Suboptimal maternal folate status is a well-known risk factor for the occurrence of NTDs, and periconceptional folic acid supplementation has been shown to reduce the risk of NTDs. Folate-supplemented oral contraceptives (OCs) offer a means of improving folate status in women of childbearing potential by increasing their likelihood of having raised folate levels at the time of conception., Objective: This study aimed to demonstrate bioequivalence of ethinylestradiol (EE), drospirenone and L-5-methyl-tetrahydrofolate (L-5-methyl-THF; active moiety of levomefolate calcium) when taken as a new folate-supplemented OC containing EE/drospirenone/levomefolate calcium, with the respective OC containing EE/drospirenone and a tablet containing levomefolate calcium only., Methods: This was a randomized, open-label, three-period crossover study carried out at a single centre in Germany. The study included 45 healthy women (age range 18-38 years). The women were randomly assigned to single doses of (i) EE 0.03 mg/drospirenone 3 mg/levomefolate calcium 0.451 mg (SAFYRAL®), (ii) EE 0.03 mg/drospirenone 3 mg (Yasmin®), and (iii) levomefolate calcium 0.451 mg, administered using a crossover design, with one or more menstrual cycle washout between doses. The primary variables were maximum concentrations (C(max)) and area under the concentration versus time curve (AUC) values for EE, drospirenone and L-5-methyl-THF., Results: The bioavailability of EE and drospirenone was similar after administration of EE/drospirenone/levomefolate calcium and EE/drospirenone. The geometric mean ratios (GMRs) and its 90% confidence intervals (CIs) for AUC values and C(max) were within the pre-specified range (80.00-125.00%) for bioequivalence for EE and drospirenone in both formulations. The bioavailability of L-5-methyl-THF was similar after administration of EE/drospirenone/levomefolate calcium and levomefolate calcium. The respective GMRs and 90% CIs of baseline-uncorrected and -corrected AUC(last) (AUC from time zero to time of last measurable concentration) and C(max) were also within the 80.00-125.00% range., Conclusion: The novel folate-supplemented OC EE/drospirenone/levomefolate calcium is bioequivalent to the established OC Yasmin® (EE/drospirenone components) and to levomefolate calcium (folate component).

Background: Analysis and visualization of large collections of molecules is one of the most frequent challenges cheminformatics experts in pharmaceutical industry are facing. Various sophisticated methods are available to perform this task, including clustering, dimensionality reduction or scaffold frequency analysis. In any case, however, viewing and analyzing large tables with molecular structures is necessary. We present a new visualization technique, providing basic information about the composition of molecular data sets at a single glance., Summary: A method is presented here allowing visual representation of the most common structural features of chemical databases in a form of a cloud diagram. The frequency of molecules containing particular substructure is indicated by the size of respective structural image. The method is useful to quickly perceive the most prominent structural features present in the data set. This approach was inspired by popular word cloud diagrams that are used to visualize textual information in a compact form. Therefore we call this approach "Molecule Cloud". The method also supports visualization of additional information, for example biological activity of molecules containing this scaffold or the protein target class typical for particular scaffolds, by color coding. Detailed description of the algorithm is provided, allowing easy implementation of the method by any cheminformatics toolkit. The layout algorithm is available as open source Java code., Conclusions: Visualization of large molecular data sets using the Molecule Cloud approach allows scientists to get information about the composition of molecular databases and their most frequent structural features easily. The method may be used in the areas where analysis of large molecular collections is needed, for example processing of high throughput screening results, virtual screening or compound purchasing. Several example visualizations of large data sets, including PubChem, ChEMBL and ZINC databases using the Molecule Cloud diagrams are provided.

Background: A new tablet formulation containing 0.02 mg ethinylestradiol/3 mg drospirenone/0.451 mg levomefolate calcium (calcium salt containing 0.416 mg L-5-methyltetrahydrofolate) was assessed for bioequivalence compared to the approved oral contraceptive (OC) tablet containing identical amounts of ethinylestradiol and drospirenone and to a tablet containing 0.451 mg levomefolate calcium., Study Design: Forty-four subjects received in an intraindividual crossover design single doses of the new tablet formulation or the established ethinylestradiol/drospirenone tablet or the levomefolate calcium tablet., Results: Bioequivalence was demonstrated for ethinylestradiol, drospirenone and L-5-methyltetrahydrofolate (active moiety of levomefolate calcium) between the investigated tablet formulations. The geometric mean ratios of the AUC((0-tlast)) and C(max) values for all three compounds and their 90% confidence intervals were well within the 80%-125% range generally accepted to demonstrate bioequivalence., Conclusion: The rate and extent of absorption of ethinylestradiol and drospirenone were not affected by the concomitant administration of levomefolate calcium and vice versa., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Purpose: Complementing clinical findings with those generated by biomarkers--such as β-amyloid-targeted positron emission tomography (PET) imaging--has been proposed as a means of increasing overall accuracy in the diagnosis of Alzheimer's disease (AD). Florbetaben ([(18)F]BAY 94-9172) is a novel β-amyloid PET tracer currently in global clinical development. We present the results of a proof of mechanism study in which the diagnostic efficacy, pharmacokinetics, safety and tolerability of florbetaben were assessed. The value of various quantitative parameters derived from the PET scans as potential surrogate markers of cognitive decline was also investigated., Methods: Ten patients with mild-moderate probable AD (DSM-IV and NINCDS-ADRDA criteria) and ten age-matched (≥ 55 years) healthy controls (HCs) were administered a single dose of 300 MBq florbetaben, which contained a tracer mass dose of < 5 μg. The 70-90 min post-injection brain PET data were visually analysed by three blinded experts. Quantitative assessment was also performed via MRI-based, anatomical sampling of predefined volumes of interest (VOI) and subsequent calculation of standardized uptake value (SUV) ratios (SUVRs, cerebellar cortex as reference region). Furthermore, single-case, voxelwise analysis was used to calculate individual "whole brain β-amyloid load"., Results: Visual analysis of the PET data revealed nine of the ten AD, but only one of the ten HC brains to be β-amyloid positive (p = 0.001), with high inter-reader agreement (weighted kappa ≥ 0.88). When compared to HCs, the neocortical SUVRs were significantly higher in the ADs (with descending order of effect size) in frontal cortex, lateral temporal cortex, occipital cortex, anterior and posterior cingulate cortices, and parietal cortex (p = 0.003-0.010). Voxel-based group comparison confirmed these differences. Amongst the PET-derived parameters, the Statistical Parametric Mapping-based whole brain β-amyloid load yielded the closest correlation with the Mini-Mental State Examination scores (r = -0.736, p < 0.001), following a nonlinear regression curve. No serious adverse events or other safety concerns were seen., Conclusion: These results indicate florbetaben to be a safe and efficacious β-amyloid-targeted tracer with favourable brain kinetics. Subjects with AD could be easily differentiated from HCs by both visual and quantitative assessment of the PET data. The operator-independent, voxel-based analysis yielded whole brain β-amyloid load which appeared valuable as a surrogate marker of disease severity.

Unlabelled: Amyloid imaging with (18)F-labeled radiotracers will allow widespread use, facilitating research, diagnosis, and therapeutic development for Alzheimer disease. The purpose of the study program was to compare cortical amyloid deposition using (18)F-florbetaben and PET in controls and subjects with mild cognitive impairment (MCI), frontotemporal lobar degeneration (FTLD), dementia with Lewy bodies (DLB), vascular dementia (VaD), Parkinson disease (PD), and Alzheimer disease (AD)., Methods: One hundred nine subjects in 3 clinical studies at Austin Health were reviewed: 32 controls, 20 subjects with MCI, and 30 patients with AD, 11 with FTLD, 7 with DLB, 5 with PD, and 4 with VaD underwent PET after intravenous injection of 300 MBq of (18)F-florbetaben. Standardized uptake value ratios (SUVR) using the cerebellar cortex as a reference region were calculated between 90 and 110 min after injection., Results: When compared with the other groups, AD patients demonstrated significantly higher SUVRs (P < 0.0001) in neocortical areas. Most AD patients (96%) and 60% of MCI subjects showed diffuse cortical (18)F-florbetaben retention. In contrast, only 9% of FTLD, 25% of VaD, 29% of DLB, and no PD patients and 16% of controls showed cortical binding. Although there was a correlation between Mini Mental State Examination and β-amyloid burden in the MCI group, no correlation was observed in controls, FTLD or AD., Conclusion: (18)F-florbetaben had high sensitivity for AD, clearly distinguished patients with FTLD from AD, and provided results comparable to those reported with (11)C-Pittsburgh Compound B in a variety of neurodegenerative diseases.

Novel therapies, e.g., cell and gene therapy or tissue engineering, are summarized in the European Union as advanced therapy medicinal products (ATMPs). In terms of composition and product properties, ATMPs are highly complex, and given their multiple potential actions they are subject to continuously developing regulatory requirements. Due to promising basic research findings, there are high expectations by the society toward the therapeutic potential of ATMPs. It is of utmost importance to develop a scientifically sound preclinical and clinical development plan before entering into the first clinical trial. Due to the complex features of ATMPs, this development plan should be discussed early with the regulatory authorities to define the specifics and challenges of each individual product. For planning as well as operational realization of the initial clinical trial involving ATMPs, specific requirements that need to be addressed are discussed in this paper.

Differential gene expression profiling has become of central importance for the analysis of cellular systems at the transcriptional level. By now, many platform technologies including DNA-microarrays, serial analysis of gene expression or RNA-seq have been established in order to facilitate transcriptional profiling. However, these technologies are all subjected to specific limitations, as they require a priori knowledge of annotated genome sequences or are based on substantial bioinformatic infrastructure, for example. As an unbiased alternative we describe here a multidimensional electrophoretic system of separation for the analysis of gene expression for the global transcriptional profiling in any eukaryotic organism. This approach is compatible with standard laboratory equipment comprising high-resolution separation of complex cDNA-probes using two-dimensional DNA-gel electrophoresis. In this context cDNA fragments are separated using non-denaturing PAGE in the first dimension with subsequent denaturing gradient gel electrophoresis in the second dimension. Two-dimensional spot patterns are quantified by well-established bioinformatic algorithms and selected spots are identified using DNA sequencing. Neither does this method necessarily depend on annotated genome sequences, nor does it require sophisticated instrumentation. Strikingly, quantitative data on differential gene expression derived from multidimensional electrophoretic system of separation for the analysis of gene expression highly correlate with corresponding data from quantitative RT-PCR even for transcriptional profiles of limited amounts of total RNA.

Azole resistance in Candida albicans can be mediated by the upregulation of the ATP binding cassette transporter genes CDR1 and CDR2. Both genes are regulated by a cis-acting element called the drug-responsive element (DRE), with the consensus sequence 5'-CGGAWATCGGATATTTTTTT-3', and the transcription factor Tac1p. In order to analyze in detail the DRE sequence necessary for the regulation of CDR1 and CDR2 and properties of TAC1 alleles, a one-hybrid system was designed. This system is based on a P((CDR2))-HIS3 reporter system in which complementation of histidine auxotrophy can be monitored by activation of the reporter system by CDR2-inducing drugs such as estradiol. Our results show that most of the modifications within the DRE, but especially at the level of CGG triplets, strongly reduce CDR2 expression. The CDR2 DRE was replaced by putative DREs deduced from promoters of coregulated genes (CDR1, RTA3, and IFU5). Surprisingly, even if Tac1p was able to bind these putative DREs, as shown by chromatin immunoprecipitation, those from RTA3 and IFU5 did not functionally replace the CDR2 DRE. The one-hybrid system was also used for the identification of gain-of-function (GOF) mutations either in TAC1 alleles from clinical C. albicans isolates or inserted in TAC1 wild-type alleles by random mutagenesis. In all, 17 different GOF mutations were identified at 13 distinct positions. Five of them (G980E, N972D, A736V, T225A, and N977D) have already been described in clinical isolates, and four others (G980W, A736T, N972S, and N972I) occurred at already-described positions, thus suggesting that GOF mutations can occur in a limited number of positions in Tac1p. In conclusion, the one-hybrid system developed here is rapid and powerful and can be used for characterization of cis- and trans-acting elements in C. albicans.

Although Western blots are frequently quantified, densitometry is not documented and appears to be based merely on traditions and guesswork. Confirming previous experience, none of 100 randomly selected and systematically scanned most recent papers provided sufficient information on how Western blot results were translated into statistical values. The importance of such information, however, becomes evident from our correlations of plasma erythropoietin values of various mammals determined using RIA and Western blot densitometry. Different common densitometry procedures applied to the identical Western blot revealed p-values of these correlations ranging from 0.000013 to 0.76 reflecting the necessity of a scientifically sound basis for densitometry of Western blots. At present, the current lack of any definitions in densitometry opens the door to uncontrollable acquisition of any desired p-value. Here we provide data that define what should be considered, what avoided and what documented when quantifying Western blots.

The modification of a previously described technique to generate venous conduits in a lamb model from a decellularised matrix and autologous cells and its application to human tissue is described. A 49-year-old woman underwent surgery for a large malignant pelvic tumour (carcinoma of unknown primary) involving the right iliac artery and vein. The right iliac artery was reconstructed with a cryopreserved human arterial allograft. For iliac vein reconstruction a tissue-engineered neo-vein was developed utilising a decellularised cryopreserved vein allograft that was reseeded in a bioreactor with autologous endothelial cells derived from the recipient's great saphenous vein. Both interposition grafts were patent initially, after 3, 6, 12, and 24 months, but the tissue-engineered neo-vein had become obstructed due to evolving disease four month postoperatively. Tissue engineered neo-veins may be a therapeutic option in selected cases with symptomatic vein stenosis or obstruction not curable with interventional methods or standard prosthetic replacement.

Objective: The aim of the study was to evaluate the use of a decellularised scaffold and its re-endothelialisation in vitro in order to create human vascular substitutes containing venous valves. This research is clinically relevant particularly with regard to the development of venous (valve containing) transplants to replace a diseased femoral vein valve and/or obstructed veins. This technique may enable causal treatment of venous reflux and obstructions., Materials and Methods: Valve-bearing segments of human allogeneic great saphenous veins (GSVs) were decellularised using sodium deoxycholic acid (SD) and treated with DNase I. Human venous endothelial cells (ECs) were enzymatically harvested from the GSV, expanded up to the 3rd passage using FCS (n=20) or human AB serum (hABS; n=8) supplemented media before used for re-seeding. In special bioreactors, 3D re-seeding of 28 decellularised GSV was performed with constant perfusion (A; n=8), bidirectional perfusion (B; n=8), bidirectional perfusion/reduced flow (C; n=2), static conditions (D; n=2), and bidirectional perfusion/reduced flow using hABS (E; n=8) instead of FCS. Decellularised GSV, scaled-up EC and 3D-seeded tissue-engineered valve containing neo-veins underwent immunohistochemical and PCR characterisation., Results: Intact collagen and elastin networks as well as complete acellularity were shown after GSV decellularisation. In EC culture, supplementation with hABS led to a significantly higher expression of vWF compared to FCS (p=0.025). Additional EC markers such as CD 31, FLK-1 and VE-Cadherin were not altered. EC re-seeding using hABS supplemented medium (E) led to a confluent monolayer of cells that were immunohistochemically positive for FLK-1, CD 31, vWF and VE-Cadherin and by means of PCR after RNA preparation in 7 of 8 cases but was unsuccessful if FCS was used (A-D). In A-D cells presented as conglomerates positive for CD 31 and VE-Cadherin, suggesting sufficient intercellular contact but not cell-matrix contact., Conclusions: Treatment with SD and DNase enables complete decellularisation of human valve containing veins whereas 3D matrix components such as collagen and elastin remain preserved. The lumen of the scaffold including the valves can be successfully re-seeded with a human EC monolayer in a 3D bioreactor. There is substantial evidence that hABS and not FCS is essential for the completion of cell-matrix contacts in human veins.

CgPdr1p is a Candida glabrata Zn(2)-Cys(6) transcription factor involved in the regulation of the ABC-transporter genes CgCDR1, CgCDR2, and CgSNQ2, which are mediators of azole resistance. Single-point mutations in CgPDR1 are known to increase the expression of at least CgCDR1 and CgCDR2 and thus to contribute to azole resistance of clinical isolates. In this study, we investigated the incidence of CgPDR1 mutations in a large collection of clinical isolates and tested their relevance, not only to azole resistance in vitro and in vivo, but also to virulence. The comparison of CgPDR1 alleles from azole-susceptible and azole-resistant matched isolates enabled the identification of 57 amino acid substitutions, each positioned in distinct CgPDR1 alleles. These substitutions, which could be grouped into three different "hot spots," were gain of function (GOF) mutations since they conferred hyperactivity to CgPdr1p revealed by constitutive high expression of ABC-transporter genes. Interestingly, the major transporters involved in azole resistance (CgCDR1, CgCDR2, and CgSNQ2) were not always coordinately expressed in presence of specific CgPDR1 GOF mutations, thus suggesting that these are rather trans-acting elements (GOF in CgPDR1) than cis-acting elements (promoters) that lead to azole resistance by upregulating specific combinations of ABC-transporter genes. Moreover, C. glabrata isolates complemented with CgPDR1 hyperactive alleles were not only more virulent in mice than those with wild type alleles, but they also gained fitness in the same animal model. The presence of CgPDR1 hyperactive alleles also contributed to fluconazole treatment failure in the mouse model. In conclusion, this study shows for the first time that CgPDR1 mutations are not only responsible for in vitro/in vivo azole resistance but that they can also confer a selective advantage under host conditions.

The classical estrogen receptor (ER) mediates genomic as well as rapid nongenomic estradiol responses. In case of genomic responses, the ER acts as a ligand-dependent transcription factor that regulates gene expression in estrogen target tissues. In contrast, nongenomic effects are initiated at the plasma membrane and lead to rapid activation of cytoplasmic signal transduction pathways. Recently, an orphan G protein-coupled receptor, GPR30, has been claimed to bind to and to signal in response to estradiol. GPR30 therefore might mediate some of the nongenomic estradiol effects. The present study was performed to clarify the controversy about the subcellular localization of GPR30 and to gain insight into the in vivo function of this receptor. In transiently transfected cells as well as cells endogenously expressing GPR30, we confirmed that the receptor localized to the endoplasmic reticulum. However, using radioactive estradiol, we observed only saturable, specific binding to the classical ER but not to GPR30. Estradiol stimulation of cells expressing GPR30 had no impact on intracellular cAMP or calcium levels. To elucidate the physiological role of GPR30, we performed in vivo experiments with estradiol and G1, a compound that has been claimed to act as selective GPR30 agonist. In two classical estrogen target organs, the uterus and the mammary gland, G1 did not show any estrogenic effect. Taken together, we draw the conclusion that GPR30 is still an orphan receptor.

Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N-methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli, and shown to encode S-adenosyl-l-methionine-dependent anthranilate N-methyltransferase. SDS-PAGE of the purified enzyme revealed a mass of 40 +/- 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O-methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity ( approximately 20%) to N-methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O-methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3- and 4-aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N-methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT-PCR amplifications and Western blotting revealed expression of the N-methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.

Background: Lentiviral vectors are attractive delivery tools for gene therapy, especially in terminally differentiated target cells. While restriction of gene expression to specific cell populations is of particular importance, highly efficient cell-type-specific gene expression after viral gene transfer so far has been hampered by low levels of transgene expression., Methods: Addressing this problem, we have integrated the human cytomegalovirus (CMV) immediate-early enhancer into an 'advanced' generation lentiviral vector. Expression cassettes with the reporter gene green fluorescent protein (GFP), combined with the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) under control of a ubiquitous phosphoglycerate kinase (mouse PGK), cardiomyocyte- (human atrial natriuretic factor (ANF), human ventricular myosin light chain (MLC2v)), or type II alveolar epithelial cell (AT-2)-specific human surfactant protein C (SP-C) promoter, were introduced. As insertion of an enhancing element can interfere with the promoter's specificity, expression levels conferred by our enhancer/promoter constructs were evaluated in target and non-target cells., Results: Transduction of target cells with human CMV enhancer containing lentiviral vectors resulted in a multiple-log increase in GFP expression compared to corresponding vectors lacking the human CMV enhancer. In the case of the ANF, the MLC2v, and the SP-C promoters, tissue-specific reporter gene expression in cardiomyocytes and in lung AT-2 cells was maintained, as expression in non-target cells increased only up to 7-fold., Conclusions: The results of this study indicate that lentiviral vectors with the human CMV enhancer conferring efficient cell-type-specific gene expression may be useful tools for gene therapy purposes or cell tracing, e.g. to analyze stem cell differentiation in transplantation and co-culture settings., ((c) 2007 John Wiley & Sons, Ltd.)

Purpose: This study evaluated the thyroidal kinetics of radioiodine in Graves' disease under continued thiamazole medication and after discontinuation of thiamazole for 1-2 days, with a view to keeping the period of discontinuation as short as possible and to exploring the underlying mechanism of a postulated radioprotective effect of antithyroid drugs., Methods: In 316 patients, diagnostic and therapeutic radioiodine kinetics were followed up for 2 days by ten uptake measurements each and were defined mathematically by a two-compartment model., Results: Without thiamazole or when thiamazole was discontinued for at least 2 days, all uptake curves could be fitted perfectly by a simple in- and output function; the mean square error (mse) was 0.38 (test) and 0.28 (therapy). Under continued thiamazole medication (11.0+/-7.0 mg/day), the energy dose delivered to the thyroid was lowered by factor of 2.5. Uptake curves were deformed (mse: 1.06, test and 0.86, therapy) and appeared two peaked, suggesting coexistence of follicles with blocked and follicles with intact hormone synthesis and hence heterogeneous radioiodine uptake in the thyroid. In patients with maximally altered uptake curves, the success rate was as low as 31%. One day after discontinuation of thiamazole, mse was still increased (0.78, test), while 2 days afterwards it had normalised (0.36, test) and 3 days afterwards (mse: 0.24, therapy) the success rate was 87%., Conclusion: Efficacy of radioiodine therapy under continued thiamazole medication is reduced not only by a lower uptake and shorter half-life of radioiodine, but also by a heterogeneous energy dose distribution in the thyroid. Discontinuation of thiamazole (but probably not of propylthiouracil) for at least 2 days is required to restore the efficacy of radioiodine.

Postmenopausal women that still have an uterus and suffer from hot flushes are treated with combinations of estrogens and progestins. Whereas estrogens are indispensable for treating postmenopausal symptoms, progestins are added to counteract the proliferative activity of estrogens on uterine epithelial cells. However, in the mammary gland, progestins, given together with estrogens, stimulate the proliferation of mammary epithelial cells. Therefore, progestins with reduced proliferative activity in the mammary gland would be of advantage for hormone therapy of postmenopausal women. In order to identify progestins with better tissue-selectivity, we exploited the activation of different signal transduction pathways by the classical progesterone receptor. We demonstrated that progestins with reduced non-genomic versus genomic activity in vitro show a better dissociation of uterine versus mammary gland effects in vivo than medroxyprogesterone acetate (MPA), a synthetic progestin that is widely used in hormone therapy.

The quality of QSAR (Quantitative Structure-Activity Relationships) predictions depends on a large number of factors including the descriptor set, the statistical method, and the data sets used. Here we study the quality of QSAR predictions mainly as a function of the data set and descriptor type using partial least squares as the statistical modeling method. The study makes use of the fact that we have access to a large number of data sets and to a variety of different QSAR descriptors. The main conclusions are that the quality of the predictions depends both on the data set and the descriptor used. The quality of the predictions correlates positively with the size of the data set and the range of biological activities. There is no clear dependence of the quality of the predictions on the complexity of the data set. All of the descriptors tested produced useful predictions for some of the data sets. None of the descriptors is best for all data sets; it is therefore necessary to test in each individual case, which descriptor produces the best model. In our tests, 2D fragment based descriptors usually performed better than simpler descriptors based on augmented atom types. Possible reasons for these observations are discussed.

Web-based tools offer many advantages for processing chemical information, most notably ease of use and high interactivity. Therefore more and more pharmaceutical companies are using web technology to deliver sophisticated molecular processing tools directly to the desks of their chemists, to assist them in the process of designing and developing new drugs. In this paper, the web-based cheminformatics system developed at Novartis and currently used by more than thousand users is described. The system allows various molecular modeling and molecular processing tasks, including the calculation of molecular and substituent properties, property-based virtual screening, visualization of molecules, bioisosteric design, diversity analysis, and support of combinatorial chemistry. The methodology to calculate various molecular properties relevant to drug design is described, including the prediction of intestinal absorption, blood-brain barrier penetration, efflux, and water solubility. Information about the web technology used is also provided.

Background: Inhalation of iloprost, a stable prostacyclin analog, is an effective therapy for pulmonary hypertension with few side effects. This approach may, however, be handicapped by limitations of currently available nebulization devices. We assessed whether the physical characterization of a device is sufficient to predict drug deposition and pharmacologic effects., Methods: We investigated the effects of a standardized iloprost aerosol dose (5 micro g; inhaled within approximately 10 min) in 12 patients with severe pulmonary hypertension in a crossover design employing three well-characterized nebulizers. The nebulizers use different techniques to increase efficiency and alveolar targeting (Ilo-Neb/Aerotrap [Nebu-Tec; Elsenfeld, Germany], Ventstream [MedicAid; Bognor Regis, UK], and HaloLite [Profile Therapeutics; Bognor Regis, UK]). Measurements were performed using a Swan-Ganz catheter and determination of arterial iloprost plasma levels., Results: During inhalation of iloprost, the pulmonary vascular resistance decreased substantially (baseline, approximately 1,250 dyne.s.cm(-5); decrease, - 35.5 to - 38.0%) and pulmonary artery pressure decreased substantially (baseline, approximately 58 mm Hg; decline, - 18.4 to -21.8%), whereas the systemic arterial pressure was largely unaffected. Cardiac output and mixed venous and arterial oxygen saturation displayed a marked increase. The pharmacodynamic profiles with the three devices were superimposable. Moreover, rapid entry of iloprost into the systemic circulation was noted, peaking immediately after termination of the inhalation maneuver, with very similar maximum serum concentrations (158 pg/mL, 155 pg/mL, and 157 pg/mL), and half-lives of serum levels (6.5 min, 9.4 min, and 7.7 min) for the three nebulizers, respectively. Interestingly, the "half-life" of the pharmacodynamic effects in the pulmonary vasculature (eg, decrease in pulmonary vascular resistance, ranging between 21 and 25 min) clearly outlasted this serum level-based pharmacokinetic half-life., Conclusions: A standardized dose of aerosolized iloprost delivered by different nebulizer types induces comparable pharmacodynamic and pharmacokinetic responses. Pulmonary vasodilation, persisting after disappearance of the drug from the systemic circulation, supports the hypothesis that local drug deposition largely contributes to the preferential pulmonary vasodilation in response to inhaled iloprost.

The phytopathogen Pseudomonas syringae produces the phytotoxin coronatine (COR) as a major virulence factor. COR and its precursor, coronafacic acid, function as molecular mimics of the plant signaling molecule jasmonate. A 32.8-kb plasmid-borne gene cluster mediates COR biosynthesis, which is optimal at 18 degrees C and non-detectable at 28 degrees C, the optimal growth temperature for P. syringae. The thermoregulation is mediated at the transcriptional level by an unconventional two-component regulatory system consisting of a histidine protein kinase, CorS, and two transcriptional activators, CorR and CorP. Dissection of this regulatory triad revealed that CorR binds to its target sequences in a thermoresponsive manner and that its DNA-binding activity is controlled by CorS. A Preliminary model for thermo-sensing by CorS is proposed based on its membrane topology and the analysis of translational fusions of CorS to reporter enzymes at different temperatures. CorP lacks a typical helix-turn-helix motif but possibly functions as a modulator of CorR or CorS activity. The thermoregulation of COR biosynthetic genes is widespread among various COR-producing P. syringae strains. Post-translational processes also contribute to the thermo-responsiveness of COR production. Additionally, COR synthesis in P. syringae is influenced by nutrient availability, rpoN encoding the alternative sigma factor sigma54, and HrpV, a negative regulator of hrp gene expression, suggesting a complex regulatory network governing phytotoxin synthesis.