Hepatic improvement within 27 days of avenciguat treatment in Child-Pugh a cirrhosis detected by an oral cholate challenge test.

New methods for measuring hepatic improvement in clinical trials and the clinic are needed. One new method, HepQuant SHUNT, detected dose-dependent improvements in hepatic function and portal physiology in the Phase 1b study (NCT03842761) of avenciguat, an activator of soluble guanylyl cyclase that is being developed for the treatment of portal hypertension. Herein we examined whether HepQuant Duo, an easy-to-administer test version, could similarly detect the effects of avenciguat. Twenty-three patients with Child-Pugh A cirrhosis and liver stiffness >15 kPa received either placebo (n=5) or a maximum twice-daily avenciguat dose of 1 mg, 2 mg, or 3 mg (n=6 per group) for 28 days. The DuO test was performed at baseline and days 11 and 27 in each subject. The test involved administering 40 mg of d4-cholate orally, measuring d4-cholate concentrations in serum at 20 and 60 minutes, and calculating portal hepatic filtration rate (HFR), disease severity index (DSI), portal-systemic shunting (SHUNT%), and hepatic reserve (HR%). Avenciguat demonstrated dose-dependent improvement in all test parameters. Changes from baseline in SHUNT% after 27 days' treatment were 0.1±9.0% for placebo, 1.7±5.5% for 1 mg twice-daily, -3.2±2.7% for 2 mg twice-daily, and -6.1±5.0% for 3 mg twice-daily (paired t-test for change from baseline p=0.98, 0.48, 0.04, and 0.03, respectively). The changes detected by HepQuant DuO were similar to those previously observed and reported for HepQuant SHUNT. The results support further study of avenciguat in treating portal hypertension and spotlight the utility of HepQuant DuO in the development of drug therapy for liver disease. HepQuant DuO facilitates use of function testing to measure hepatic improvement in clinical trials and the clinic.

Pharmacodynamics, pharmacokinetics and CYP3A4 interaction potential of the selective P2X3 receptor antagonist filapixant: A randomized multiple ascending-dose study in healthy young men.

AIMS: We report on investigations exploring the P2X3-receptor antagonist filapixant's effect on taste perception and cough-reflex sensitivity and describe its pharmacokinetics, including its CYP3A4-interaction potential. METHODS: In a randomized, placebo-controlled, double-blind study, 3 × 12 healthy men (18-45 years) were assigned (3:1) to filapixant (20, 80 or 250 mg by mouth) or placebo twice daily over 2 weeks. A single dose of midazolam (1 mg), a CYP3A4 substrate, was administered with and without filapixant. Assessments included a taste-strips test, a taste questionnaire, cough challenge with adenosine triphosphate, adverse event reports and standard safety assessments. RESULTS: Taste disturbances were observed mainly in the 250-mg group: six of nine participants (67%) in this group reported hypo- or dysgeusia in the questionnaire; eight participants (89%) reported taste-related adverse events. Five participants (56%) had a decrease in overall taste-strips-test scores ≥2 points (point estimate -1.1 points, 90% confidence interval [-3.3; 1.1]). Cough counts increased with adenosine triphosphate concentration but without major differences between treatments. Filapixant exposure increased proportionally to dose. Co-administration of filapixant had no clinically relevant effect on midazolam pharmacokinetics. Area under the concentration-time curve ratios and 90% confidence intervals were within 80-125%. No serious or severe adverse events were reported. CONCLUSIONS: Overall, filapixant was safe and well tolerated, apart from mild, transient taste disturbances. Such disturbances occurred more frequently than expected based on (in vitro) receptor-selectivity data, suggesting that other factors than P2X3:P2X2/3 selectivity might also play an important role in this context. The cough-challenge test showed no clear treatment effect. Filapixant has no clinically relevant CYP3A4 interaction potential.

The P2X3 receptor antagonist filapixant in patients with refractory chronic cough: a randomized controlled trial.

BACKGROUND: P2X3 receptor antagonists seem to have a promising potential for treating patients with refractory chronic cough. In this double-blind, randomized, placebo-controlled study, we investigated the efficacy, safety, and tolerability of the novel selective P2X3 receptor antagonist filapixant (BAY1902607) in patients with refractory chronic cough. METHODS: Following a crossover design, 23 patients with refractory chronic cough (age: 60.4 ± 9.1 years) received ascending doses of filapixant in one period (20, 80, 150, and 250 mg, twice daily, 4-days-on/3-days-off) and placebo in the other. The primary efficacy endpoint was the 24-h cough frequency on Day 4 of each dosing step. Further, subjective cough severity and health-related quality of life were assessed. RESULTS: Filapixant at doses ≥ 80 mg significantly reduced cough frequency and severity and improved cough health-related quality of life. Reductions in 24-h cough frequency over placebo ranged from 17% (80 mg dose) to 37% (250 mg dose), reductions over baseline from 23% (80 mg) to 41% (250 mg) (placebo: 6%). Reductions in cough severity ratings on a 100-mm visual analog scale ranged from 8 mm (80 mg) to 21 mm (250 mg). No serious or severe adverse events or adverse events leading to discontinuation of treatment were reported. Taste-related adverse events occurred in 4%, 13%, 43%, and 57% of patients treated with filapixant 20, 80, 150, and 250 mg, respectively, and in 12% treated with placebo. CONCLUSIONS: Filapixant proved to be efficacious, safe, and-apart from the occurrence of taste disturbances, especially at higher dosages-well tolerated during the short therapeutic intervention. Clinical trial registration EudraCT, eudract.ema.europa.eu, 2018-000129-29; ClinicalTrials.gov, NCT03535168.

First-in-human study of eliapixant (BAY 1817080), a highly selective P2X3 receptor antagonist: Tolerability, safety and pharmacokinetics.

AIMS: Neuronal hypersensitisation due to adenosine triphosphate-dependent P2X3 receptor signalling plays a significant role in several disorders including chronic cough and endometriosis. This first-in-human study of eliapixant (BAY 1817080) investigated the tolerability, safety and pharmacokinetics (PK) of single doses of eliapixant, including the effect of food and coadministration with a CYP3A inhibitor on eliapixant relative bioavailability. METHODS: In this randomised, double-blind phase I study (NCT02817100), 88 healthy male subjects received single ascending doses of immediate-release eliapixant (10-800 mg) tablets or placebo under fasted conditions, with food (low-fat continental or high-fat American breakfast) or with itraconazole (fasted state). PK parameters, dose proportionality, adverse events and taste assessments (taste strips; dysgeusia questionnaire) were evaluated. RESULTS: Eliapixant had a long half-life (23.5-58.9 h [fasted state]; 32.8-43.8 h [high-fat breakfast]; 38.9-46.0 h [low-fat breakfast]). Less than dose-proportional increases in maximum plasma concentrations (Cmax ) and area under the concentration-time curve from time 0 to infinity (AUC[0-inf] ) were observed with ascending eliapixant doses. We observed a pronounced food effect with the high-fat breakfast (4.1-fold increased Cmax ; 2.7-fold increased AUC[0-inf] ), a smaller food effect with the low-fat breakfast and a mild-to-moderate effect of itraconazole coadministration on eliapixant (1.1-1.2-fold increased Cmax ; 1.7-fold increased AUC from 0 to 72 h). Eliapixant was well tolerated with minimal impact on taste perception. CONCLUSION: The PK profile, particularly the long half-life, and favourable tolerability with no taste-related adverse events, supports the further development of eliapixant in disorders with underlying P2X3 receptor-mediated neuronal hypersensitisation.

Eliapixant (BAY 1817080), a P2X3 receptor antagonist, in refractory chronic cough: a randomised, placebo-controlled, crossover phase 2a study.

BACKGROUND: ATP acting via P2X3 receptors is an important mediator of refractory chronic cough (RCC). This phase 2a double-blinded crossover study assessed the safety, tolerability and efficacy of eliapixant (BAY 1817080), a selective P2X3 receptor antagonist, in adults with RCC attending specialist centres. METHODS: In period A, patients received placebo for 2 weeks then eliapixant 10 mg for 1 week. In period B, patients received eliapixant 50, 200 and 750 mg twice daily for 1 week per dose level. Patients were randomised 1:1 to period A-B (n=20) or B-A (n=20). The primary efficacy end-point was change in cough frequency assessed over 24 h. The primary safety end-point was frequency and severity of adverse events (AEs). RESULTS: 37 patients completed randomised therapy. Mean cough frequency fell by 17.4% versus baseline with placebo. Eliapixant reduced cough frequency at doses ≥50 mg (reduction versus placebo at 750 mg: 25% (90% CI 11.5-36.5%); p=0.002). Doses ≥50 mg also significantly reduced cough severity. AEs, mostly mild or moderate, were reported in 65% of patients with placebo and 41-49% receiving eliapixant. Cumulative rates of taste-related AEs were 3% with placebo and 5-21% with eliapixant; all were mild. CONCLUSIONS: Selective P2X3 antagonism with eliapixant significantly reduced cough frequency and severity, confirming this as a viable therapeutic pathway for RCC. Taste-related side-effects were lower at therapeutic doses than with the less selective P2X3 antagonist gefapixant. Selective P2X3 antagonism appears to be a novel therapeutic approach for RCC.

Non-invasive biomarkers prognostic of decompensation events in NASH cirrhosis: a systematic literature review.

Liver cirrhosis due to nonalcoholic steatohepatitis (NASH) is a life-threatening condition with increasing incidence world-wide. Although its symptoms are unspecific, it can lead to decompensation events such as ascites, hepatic encephalopathy, variceal hemorrhage, and hepatocellular carcinoma (HCC). In addition, an increased risk for cardiovascular events has been demonstrated in patients with NASH. Pharmacological treatments for NASH cirrhosis are not yet available, one of the reasons being the lack in surrogate endpoints available in clinical trials of NASH cirrhosis. The feasibility of non-invasive prognostic biomarkers makes them interesting candidates as possible surrogate endpoints if their change following treatment would result in better outcomes for patients in future clinical trials of NASH cirrhosis. In this systematic literature review, a summary of the available literature on the prognostic performance of non-invasive biomarkers in terms of cardiovascular events, liver-related events, and mortality is outlined. Due to the scarcity of data specific for NASH cirrhosis, this review includes studies on NAFLD whose evaluation focuses on cirrhosis. Our search strategy identified the following non-invasive biomarkers with prognostic value in studies of NASH patients: NAFLD fibrosis score (NFS), Fibrosis-4 (FIB-4), aspartate aminotransferase (AST) to platelet ratio index (APRI), enhanced liver fibrosis (ELF™), BARD (BMI, AST/ALT (alanine aminotransferase) ratio, diabetes), Hepamet Fibrosis Score (HFS), liver enzymes (AST + ALT), alpha-fetoprotein, platelet count, neutrophil to lymphocyte ratio (NLR), Lysyl oxidase-like (LOXL) 2, miR-122, liver stiffness, MEFIB (liver stiffness measured with magnetic resonance elastography (MRE) + FIB-4), and PNPLA3 GG genotype. The aim of the present systematic literature review is to provide the reader with a summary of the non-invasive biomarkers with prognostic value in NASH cirrhosis and give an evaluation of their utility as treatment monitoring biomarkers in future clinical trials.

The rationale and study design of two phase II trials examining the effects of BI 685,509, a soluble guanylyl cyclase activator, on clinically significant portal hypertension in patients with compensated cirrhosis.

BACKGROUND: Clinically significant portal hypertension (CSPH) drives cirrhosis-related complications (i.e. hepatic decompensation). Impaired nitric oxide (NO) bioavailability promotes sinusoidal vasoconstriction, which is the initial pathomechanism of CSPH development. Activation of soluble guanylyl cyclase (sGC), a key downstream effector of NO, facilitates sinusoidal vasodilation, which in turn may improve CSPH. Two phase II studies are being conducted to assess the efficacy of the NO-independent sGC activator BI 685,509 in patients with CSPH due to various cirrhosis aetiologies. METHODS: The 1366.0021 trial (NCT05161481) is a randomised, placebo-controlled, exploratory study that will assess BI 685,509 (moderate or high dose) for 24 weeks in patients with CSPH due to alcohol-related liver disease. The 1366.0029 trial (NCT05282121) is a randomised, open-label, parallel-group, exploratory study that will assess BI 685,509 (high dose) alone in patients with hepatitis B or C virus infection or non-alcoholic steatohepatitis (NASH) and in combination with 10 mg empagliflozin in patients with NASH and type 2 diabetes mellitus for 8 weeks. The 1366.0021 trial will enrol 105 patients, and the 1366.0029 trial will enrol 80 patients. In both studies, the primary endpoint is the change from baseline in hepatic venous pressure gradient (HVPG) until the end of treatment (24 or 8 weeks, respectively). Secondary endpoints include the proportion of patients with an HVPG reduction of > 10% from baseline, the development of decompensation events and the change from baseline in HVPG after 8 weeks in the 1366.0021 trial. In addition, the trials will assess changes in liver and spleen stiffness by transient elastography, changes in hepatic and renal function and the tolerability of BI 685,509. DISCUSSION: These trials will enable the assessment of the short-term (8 weeks) and longer-term (24 weeks) effects and safety of sGC activation by BI 685,509 on CSPH due to various cirrhosis aetiologies. The trials will use central readings of the diagnostic gold standard HVPG for the primary endpoint, as well as changes in established non-invasive biomarkers, such as liver and spleen stiffness. Ultimately, these trials will provide key information for developing future phase III trials. TRIAL REGISTRATION: 1366.0021: EudraCT no. 2021-001,285-38; ClinicalTrials.gov NCT05161481. Registered on 17 December 2021, https://www. CLINICALTRIALS: gov/ct2/show/NCT05161481 . 1366.0029: EudraCT no. 2021-005,171-40; ClinicalTrials.gov NCT05282121. Registered on 16 March 2022, https://www. CLINICALTRIALS: gov/ct2/show/NCT05282121 .

Hepatotoxicity of AKR1C3 Inhibitor BAY1128688: Findings from an Early Terminated Phase IIa Trial for the Treatment of Endometriosis.

INTRODUCTION: BAY1128688 is a selective inhibitor of aldo-keto reductase family 1 member C3 (AKR1C3), an enzyme implicated in the pathology of endometriosis and other disorders. In vivo animal studies suggested a potential therapeutic application of BAY1128688 in treating endometriosis. Early clinical studies in healthy volunteers supported the start of phase IIa. OBJECTIVE: This manuscript reports the results of a clinical trial (AKRENDO1) assessing the effects of BAY1128688 in adult premenopausal women with endometriosis-related pain symptoms over a 12-week treatment period. METHODS: Participants in this placebo-controlled, multicenter phase IIa clinical trial (NCT03373422) were randomized into one of five BAY1128688 treatment groups: 3 mg once daily (OD), 10 mg OD, 30 mg OD, 30 mg twice daily (BID), 60 mg BID; or a placebo group. The efficacy, safety, and tolerability of BAY1128688 were investigated. RESULTS: Dose-/exposure-dependent hepatotoxicity was observed following BAY1128688 treatment, characterized by elevations in serum alanine transferase (ALT) occurring at around 12 weeks of treatment and prompting premature trial termination. The reduced number of valid trial completers precludes conclusions regarding treatment efficacy. The pharmacokinetics and pharmacodynamics of BAY1128688 among participants with endometriosis were comparable with those previously found in healthy volunteers and were not predictive of the subsequent ALT elevations observed. CONCLUSIONS: The hepatotoxicity of BAY1128688 observed in AKRENDO1 was not predicted by animal studies nor by studies in healthy volunteers. However, in vitro interactions of BAY1128688 with bile salt transporters indicated a potential risk factor for hepatotoxicity at higher doses. This highlights the importance of in vitro mechanistic and transporter interaction studies in the assessment of hepatoxicity risk and suggests further mechanistic understanding is required. CLINICAL TRIAL REGISTRATION: NCT03373422 (date registered: November 23, 2017).

Novel aldo-keto reductase 1C3 inhibitor affects androgen metabolism but not ovarian function in healthy women: a phase 1 study.

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 17ß-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.

Variability of transient elastography-based spleen stiffness performed at 100 Hz.

BACKGROUND: Spleen stiffness measurement (SSM) performed by transient elastography at 100 Hz is a novel technology for the evaluation of portal hypertension in advanced chronic liver disease, but technical aspects are lacking. We aimed to evaluate the intraexamination variability of SSM and to determine the best transient elastography protocol for obtaining robust measurements to be used in clinical practice. METHODS: We analyzed 253 SSM exams with up to 20 scans for each examination, performed between April 2021 and June 2022. All SSM results were evaluated according to different protocols by dividing data into groups of n measurements (from 2 to 19). Considering as reference the median SSM values across all the 20 measurements, we calculated the distribution of the absolute deviations of each protocol from the reference median. This analysis was repeated 1,000 times by resampling the data. Distributions were also stratified by etiology (chronic liver disease versus clinically significant portal hypertension) and different SSM ranges: < 25 kPa, 25-75, and > 75 kPa. RESULTS: Overall, we observed that the spleen stiffness exam had less variability if it exceeded 12 measurements, i.e., absolute deviations ≤ 5 kPa at 95% confidence. For exams with higher SSM values (> 75 kPa), as seen in clinically significant portal hypertension, at least 15 measurements are highly recommendable. CONCLUSIONS: Fifteen scans per examination should be considered for each SSM exam performed at 100 Hz to achieve a low intraexamination variability within a reasonable time in clinical practice. RELEVANCE STATEMENT: Performing at least 15 scans per examination is recommended for 100 Hz SSM in order to achieve a low intraexamination variability, in particular for values > 75 kPa compatible with clinically significant portal hypertension. KEY POINTS: • Spleen stiffness measurement by transient elastography is used for stratification in patients with portal hypertension. • At 100 Hz, this method may have intraexamination variability. • A minimum of 15 scans per examination achieves a low intraexamination variability.

Epidermal growth factor upregulates endometrial CYR61 expression via activation of the JAK2/STAT3 pathway.

Endometrial cysteine-rich protein 61 (CYR61, CCN1) is a growth factor-inducible gene whose expression is elevated during the proliferative phase of the menstrual cycle and which has been implicated in the pathogenesis of endometriosis. This study aimed to define the mediators of epidermal growth factor (EGF) signalling on CYR61 expression in spontaneously immortalised human endometrial epithelial cells (HES) as a model system. After 30 min of EGF treatment, the receptor was phosphorylated and internalised as well as mRNA CYR61 increased in HES cells. However, neither inhibition of C-terminal EGF receptor (EGFR)-phosphorylation nor blockage of the mitogen-activated proteinkinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was able to reduce CYR61 levels. Surprisingly, the HES cells showed upregulation of CYR61 mRNA expression after inhibition of the MAPK/ERK pathway when treated with EGF. Specific inhibitor studies identified the contribution of Janus kinase 2 (JAK2) and the signal transducer and activator of transcription protein STAT3 to the regulation of CYR61 expression. The JAK2/STAT3 interaction contributed to the basal expression of CYR61 and mediated EGF-driven regulation of CYR61 after 30 and 120 min of treatment. In summary, EGF-mediated CYR61 upregulation in HES cells involves STAT3 and is counter-regulated by the EGFR/MAPK/ERK pathway.

Safety, Pharmacodynamics, and Pharmacokinetics of P2X3 Receptor Antagonist Eliapixant (BAY 1817080) in Healthy Subjects: Double-Blind Randomized Study.

BACKGROUND AND OBJECTIVE: There is no licensed treatment for refractory chronic cough; off-label therapies have limited efficacy and can produce adverse effects. Excessive adenosine triphosphate signaling via P2X3 receptors is implicated in refractory chronic cough, and selective P2X3 receptor antagonists such as eliapixant (BAY 1817080) are under investigation. The objective of the study was to investigate the safety and tolerability of ascending repeated oral doses of eliapixant in healthy volunteers. METHODS: We conducted a repeated-dose, double-blind, randomized, placebo-controlled study in 47 healthy male individuals. Subjects received repeated twice-daily ascending oral doses of eliapixant (10, 50, 200, and 750 mg) or placebo for 2 weeks. The primary outcome was frequency and severity of adverse events. Other outcomes included pharmacokinetics and evaluation of taste disturbances, which have occurred with the less selective P2X3 receptor antagonist gefapixant. RESULTS: Peak plasma concentrations of eliapixant were reached 3-4 h after administration of the first and subsequent doses. With multiple dosing, steady-state plasma concentrations were reached after ~ 6 days, and plasma concentrations predicted to achieve ≥ 80% P2X3 receptor occupancy (the level required for efficacy) were reached at 200 and 750 mg. Increases in plasma concentrations with increasing doses were less than dose proportional. After multiple dosing, mean plasma concentrations of eliapixant showed low peak-trough fluctuations and were similar for 200- and 750-mg doses. Eliapixant was well tolerated with a low incidence of taste-related adverse events. CONCLUSIONS: Eliapixant (200 and 750 mg) produced plasma concentrations that cover the predicted therapeutic threshold over 24 h, with good safety and tolerability. These results enabled eliapixant to progress to clinical trials in patients with refractory chronic cough. CLINICAL TRIAL REGISTRATION: Clinicaltrials.gov: NCT03310645 (initial registration: 16 October, 2017).

There are few effective treatments for patients with a long-term (chronic) cough. It is thought that chronic cough is caused by nerves becoming oversensitive, wrongly causing a cough when there is no need. We tested a new drug called eliapixant in 47 healthy men. Eliapixant reduces the excessive nerve signaling responsible for chronic cough. We looked for side effects of eliapixant and measured how it behaves in the body. In particular we looked for side effects relating to the sense of taste because gefapixant, a similar drug to eliapixant, can affect taste. Participants took one of four eliapixant doses or a placebo twice daily for 2 weeks. The highest levels of eliapixant in the blood were seen 3­4 h after taking the drug, and stable concentrations were seen after about 6 days. At the two highest doses, eliapixant reached concentrations in the body that should be high enough to work in patients with chronic cough. Side effects were generally similar between eliapixant and placebo. Taste-related side effects were mild and went away without needing treatment. The positive results of this study meant that eliapixant could be tested in patients with chronic cough.

High-resolution ultrasound imaging: a novel technique for the noninvasive in vivo analysis of endometriotic lesion and cyst formation in small animal models.

Endometriosis, the presence of endometrial tissue at ectopic sites, is a highly prevalent gynecological disease severely affecting a patient's quality of life. To analyze the mechanisms involved in the disease and to identify new molecular targets for effective therapies, small animal models are an important approach. Herein, we report the first use of high-resolution ultrasound imaging for the in vivo analysis of intraperitoneal endometriotic lesions in mice. This noninvasive technology allows for the repetitive quantitative analysis of growth, cyst development, and adhesion formation of endometriotic lesions with a low intra- and interobserver variability. Moreover, it enables one to easily differentiate between endometrial cysts and stroma. Accordingly, volume measurements of both endometrial cysts and stroma indicated that the initial establishment of endometriotic lesions is associated with enhanced cellular proliferation, followed by a phase of increased secretory activity of endometrial glands. Results of ultrasound analysis correlated well with measurements of lesion volumes by caliper and histology. Importantly, ultrasound imaging could be performed repetitively and noninvasively and reflected best the in vivo situation. The technique could further be demonstrated to successfully monitor the significant inhibition of growth of endometriotic lesions after specific estrogen receptor destabilizator treatment. Thus, high-resolution ultrasound imaging represents an important tool for future preclinical small animal studies, which address the pathophysiology of endometriosis and the development of new treatment strategies.

The Effects of Weak and Strong CYP3A Induction by Rifampicin on the Pharmacokinetics of Five Progestins and Ethinylestradiol Compared to Midazolam.

It is known that co-administration of CYP3A inducers may decrease the effectiveness of oral contraceptives containing progestins as mono-preparations or combined with ethinylestradiol. In a randomized clinical drug-drug interaction study, we investigated the effects of CYP3A induction on the pharmacokinetics of commonly used progestins and ethinylestradiol. Rifampicin was used to induce CYP3A. The progestins chosen as victim drugs were levonorgestrel, norethindrone, desogestrel, and dienogest as mono-products, and drospirenone combined with ethinylestradiol. Postmenopausal women (n = 12-14 per treatment group) received, in fixed sequence, a single dose of the victim drug plus midazolam without rifampicin, with rifampicin 10 mg/day (weak induction), and with rifampicin 600 mg/day (strong induction). The effects on progestin exposure were compared with the effects on midazolam exposure (as a benchmark). Unbound concentrations were evaluated for drugs binding to sex hormone binding globulin. Weak CYP3A induction, as confirmed by a mean decrease in midazolam exposure by 46%, resulted in minor changes in progestin exposure (mean decreases: 15-37%). Strong CYP3A induction, in contrast, resulted in mean decreases by 57-90% (mean decrease in midazolam exposure: 86%). Namely, the magnitude of the observed induction effects varied from weak to strong. Our data might provide an impetus to revisit the currently applied clinical recommendations for oral contraceptives, especially for levonorgestrel and norethindrone-containing products, and they might give an indication as to which progestin could be used, if requested, by women taking weak CYP3A inducers-although it is acknowledged that the exact exposure-response relationship for contraceptive efficacy is currently unclear for most progestins.

Monoclonal Antibody Against Prolactin Receptor: A Randomized Placebo-Controlled Study Evaluating Safety, Tolerability, and Pharmacokinetics of Repeated Subcutaneous Administrations in Postmenopausal Women.

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.

Premenstrual regulation of the pro-angiogenic factor CYR61 in human endometrium.

The pro-angiogenic factor cysteine-rich protein 61 (CYR61/CCN1) mediates different signals in tumorigenesis, angiogenesis and is involved in the pathogenesis of endometriosis. In this study we investigated the temporal and spatial expression pattern in human endometrium during the menstrual cycle and its possible regulation mechanisms in the premenstrual phase. CYR61 transcript expression showed two distinct periods of elevated levels in the proliferative phase and in menstrual effluents. Because the menstrual breakdown of the functionalis is triggered by cytokines, prostaglandins (PGs), as well as hypoxia, we used a benign endometrial cell line to investigate if CYR61 is regulated by these factors. Hypoxic conditions transiently induced CYR61 mRNA levels and enhanced the secretion of the CYR61 protein into the medium. The hypoxia-inducible factor (HIF) 1alpha mediated this effect on CYR61 as evidenced by dimethyloxalylglycine treatment and by HIF1alpha short interfering RNA. CYR61 mRNA expression was further regulated by IL-1, TNFalpha, PGE2, and PGF2alpha. In addition, TNFalpha and PGE2 elevated significantly CYR61 cellular protein levels in well-oxygenated cells but had only a slight effect on the quantity of secreted protein. Moreover, PGE2 combined with hypoxic conditions increased CYR61 mRNA and protein levels synergistically, whereas the combination with TNFalpha abolished the CYR61 levels induced by hypoxia. Together, the up-regulation of CYR61 by hypoxia via HIF1alpha, TNFalpha, and PGE2 could represent possible mechanisms for the CYR61 increase at the onset of menstruation. The opposite effect of TNFalpha combined with hypoxia on CYR61 up-regulation could contribute to a balanced expression level of this angiogenic factor in the endometrium.

TLR3 and TLR4 expression in healthy and diseased human endometrium.

BACKGROUND: Toll-like receptors (TLRs) play an essential role in the innate immune system by initiating and directing immune response to pathogens. TLRs are expressed in the human endometrium and their regulation might be crucial for the pathogenesis of endometrial diseases. METHODS: TLR3 and TLR4 expression was investigated during the menstrual cycle and in postmenopausal endometrium considering peritoneal endometriosis, hyperplasia, and endometrial adenocarcinoma specimens (grade 1 to 3). The expression studies applied quantitative RT-PCR and immunolabelling of both proteins. RESULTS: TLR3 and TLR4 proteins were mostly localised to the glandular and luminal epithelium. In addition, TLR4 was present on endometrial dendritic cells, monocytes and macrophages. TLR3 and TLR4 mRNA levels did not show significant changes during the menstrual cycle. In patients with peritoneal endometriosis, TLR3 and TLR4 mRNA expression decreased significantly in proliferative diseased endometrium compared to controls. Interestingly, ectopic endometriotic lesions showed a significant increase of TLR3 und TLR4 mRNA expression compared to corresponding eutopic tissues, indicating a local gain of TLR expression. Endometrial hyperplasia and adenocarcinoma revealed significantly reduced receptor levels when compared with postmenopausal controls. The lowest TLR expression levels were determined in poor differentiated carcinoma (grade 3). CONCLUSION: Our data suggest an involvement of TLR3 and TLR4 in endometrial diseases as demonstrated by altered expression levels in endometriosis and endometrial cancer.

Endometriosis Leads to an Increased Trefoil Factor 3 Concentration in the Peritoneal Cavity but Does Not Alter Systemic Levels.

This study analyzed whether trefoil factor 3 (TFF3) is locally elevated and correlated with common biomarkers and inflammatory processes in endometriosis. Peritoneal fluid (PF) was obtained from 50 women and serum from 124 women with or without endometriosis. Experimental endometriosis was induced in female C57BL/6 mice by syngeneic transplantation of uterine tissue to the abdominal wall. Levels of TFF3 in PF of women with endometriosis were significantly increased ( P < .05) and correlated with local levels of known biomarkers for endometriosis: cancer antigen (CA) 125, CA-19-9, interleukin 8, monocyte chemotactic protein 1, and matrix metalloproteinase 7. Serum levels of TFF3 in women were significantly influenced by the menstrual cycle but were independent from disease state. In mice, local TFF3 levels were significantly elevated in early endometriosis (up to 4 weeks after transplantation, P < .001) and corresponded to increases in spleen weight as marker for systemic inflammation. This study provides the first evidence that TFF3 is locally elevated in the peritoneal cavity in endometriosis and might play a role in disease pathogenesis and its associated inflammatory processes. Furthermore, the results show that TFF3 is regulated through the menstrual cycle. With respect to animal models, syngeneic mouse model does reflect local TFF3 upregulation in the peritoneal cavity affected by endometriosis.

Differential expression of IGF components and insulin receptor isoforms in human seminoma versus normal testicular tissue.

Insulin-like growth factors (IGF) have mitogenic and antiapoptotic functions, and may be involved in tumor growth. The purpose of the study was to investigate the role of IGF components in seminoma compared to normal testis. Normal testicular tissues from autopsy cases and seminoma from surgery cases were obtained for microarray and real-time reverse transcription polymerase chain reaction (RT-PCR) analysis of IGF-1, IGF-2, IGF receptor type 1 (IGF-R1), IGF-R2, insulin receptor isoforms A (IR-A) and B (IR-B), and IGF-binding proteins (IGFBP) 1-6. IGF-2 was localized by immunohistochemistry. IGFBP-5 protein expression was evaluated by Western blot analysis. mRNA expression in microarray and real-time RT-PCR showed similar tendencies: IGF-1, IGF-R1, IGF-R2, IR-A, and IGFBP-2 were not different in both groups. IGF-2, IR-B, IGFBP-1, IGFBP-4, and IGFBP-6 mRNA were downregulated in seminoma. IGFBP-3 tended to be upregulated in pT1 seminoma, but downregulated in pT2 stages. IGFBP-5 and IGF-2 protein expression correlated with mRNA expression. In conclusion, downregulation of mainly inhibiting IGFBPs may allow a stimulated tumor growth. The downregulated IGF-2 does not seem to be involved in the growth regulation of seminoma. Constantly expressed genes (e.g., IGF-1, IGF-R1, IR-A, and IGFBP-2) may reflect an involvement in spermatogenesis, but may also play a major role in tumor growth as their expression is not downregulated despite the lack of spermatogenesis in tumor tissue.

Standardization strategy for quantitative PCR in human seminoma and normal testis.

Housekeeping genes are commonly used as endogenous references in quantitative RT-PCR. Ideally these genes are constitutionally expressed by all cell types and do not vary under experimental conditions. Tissues of 9 normal testes and 22 classical pure seminoma were obtained for RNA-extraction. Real-time RT-PCR was used to examine the mRNA-expression of ubiquitin C, beta-actin, GAPDH, 18S ribosomal RNA (18S rRNA) and porphobilinogen-deaminase (PBGD). Additionally, 3 normal testicular tissues and 39 seminoma, including 1 normal testis and 17 seminoma of the RT-PCR group, were utilized for microarray analyses. Ubiquitin C (protein degradation) was down-regulated, GAPDH (carbohydrate metabolism), beta-actin (cytoskeleton), 18S rRNA (ribosome) and PBGD (porphyrin metabolism) were up-regulated in seminoma. A normalization of the target gene data with up-regulated housekeeping genes would equalize or underestimate up-regulated data and overestimate down-regulated data. We demonstrate that none of the investigated housekeeping genes is suitable for normalization of the target gene RT-PCR data, but may be essential for tumor metabolism in human seminoma. Further, we developed a standardization strategy, which is applicable to many experimental investigations.