Early prediction of endocrine responsiveness in ER+/HER2-negative metastatic breast cancer (MBC): pilot study with 18F-fluoroestradiol (18F-FES) CT/PET.

BACKGROUND: 18F-fluoroestradiol (FES) positron emission tomography (PET)/computed tomography (CT) is considered an accurate diagnostic tool to determine whole-body endocrine responsiveness. In the endocrine therapy (ET)-FES trial, we evaluated 18F-FES PET/CT as a predictive tool in estrogen receptor-positive (ER+)/human epidermal growth factor receptor 2-negative (HER2-) metastatic breast cancer (MBC). PATIENTS AND METHODS: Eligible patients underwent an 18F-FES PET/CT at baseline. Patients with standardized uptake value (SUV) ≥ 2 received single-agent ET until progressive disease; patients with SUV < 2 were randomized to single-agent ET (arm A) or chemotherapy (ChT) (arm B). The primary objective was to compare the activity of first-line ET versus ChT in patients with 18F-FES SUV < 2. RESULTS: Overall, 147 patients were enrolled; 117 presented with 18F-FES SUV ≥ 2 and received ET; 30 patients with SUV < 2 were randomized to ET or ChT. After a median follow-up of 62.4 months, 104 patients (73.2%) had disease progression and 53 died (37.3%). Median progression-free survival (PFS) was 12.4 months [95% confidence interval (CI) 3.1-59.6 months] in patients with SUV < 2 randomized to arm A versus 23.0 months (95% CI 7.7-30.0 months) in arm B, [hazard (HR) = 0.71, 95% CI 0.3-1.7 months]; median PFS was 18.0 months (95% CI 11.2-23.1 months) in patients with SUV ≥ 2 treated with ET. Median overall survival (OS) was 28.2 months (95% CI 14.2 months-not estimable) in patients with SUV < 2 randomized to ET (arm A) versus 52.8 months (95% CI 16.2 months-not estimable) in arm B (ChT). Median OS was not reached in patients with SUV ≥ 2. 60-month OS rate was 41.6% (95% CI 10.4% to 71.1%) in arm A, 42.0% (95% CI 14.0% to 68.2%) in arm B, and 59.6% (95% CI 48.6% to 69.0%) in patients with SUV ≥ 2. In patients with SUV ≥ 2, 60-month OS rate was 72.6% if treated with aromatase inhibitors (AIs) versus 40.6% in case of fulvestrant or tamoxifen (P < 0.005). CONCLUSIONS: The ET-FES trial demonstrated that ER+/HER2- MBC patients are a heterogeneous population, with different levels of endocrine responsiveness based on 18F-FES CT/PET SUV.

G45 and V386 in Transmembrane Domains 1 and 8 Are Critical for the Activation of OATP1B3-Mediated E17ßG Uptake by Clotrimazole.

Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are two highly homologous transport proteins. However, OATP1B1- and 1B3-mediated estradiol-17ß-glucuronide (E17ßG) uptake can be differentially affected by clotrimazole. In this study, by functional characterization on chimeric transporters and single mutants, we find that G45 in transmembrane domain 1 (TM1) and V386 in TM8 are critical for the activation of OATP1B3-mediated E17ßG uptake by clotrimazole. However, the effect of clotrimazole on the function of OATP1B3 is substrate-dependent as clotrimazole does not stimulate OATP1B3-mediated uptake of 4',5'-dibromofluorescein (DBF) and rosuvastatin. In addition, clotrimazole is not transported by OATP1B3, but it can efficiently permeate the plasma membrane due to its lipophilic properties. Homology modeling and molecular docking indicate that E17ßG binds in a substrate binding pocket of OATP1B3 through hydrogen bonding and hydrophobic interactions, among which its sterol scaffold forms hydrophobic contacts with V386. In addition, a flexible glycine residue at position 45 is essential for the activation of OATP1B3. Finally, clotrimazole is predicted to bind at an allosteric site, which mainly consists of hydrophobic residues located at the cytoplasmic halves of TMs 4, 5, 10, and 11.

Targeting an Old Foe for Cancer: A Molecular Dynamics Perspective to Unravel the Specific Binding Nature of 2-Methoxy Estradiol to Human ß-Tubulin Isotypes.

Microtubules, composed of α- and ß-tubulin subunits are crucial for cell division with their dynamic tissue-specificity which is dictated by expression of isotypes. These isotypes differ in carboxy-terminal tails (CTTs), rich in negatively charged acidic residues in addition to the differences in the composition of active site residues. 2-Methoxy estradiol (2-ME) is the first antimicrotubule agent that showed less affinity toward hemopoietic-specific ß1 isotype consequently preventing myelosuppression toxicity. The present study focuses on the MD-directed conformational analysis of 2-ME and estimation of its binding affinity in the colchicine binding pocket of various ß-tubulin isotypes combined with the α-tubulin isotype, α1B. AlphaFold 2.0 was used to predict the 3D structure of phylogenetically divergent human ß-tubulin isotypes in dimer form with α1B. The dimeric complexes were subjected to induced-fit docking with 2-ME. The statistical analysis of docking showed differences in the binding characteristics of 2-ME with different isotypes. The replicas of atom-based molecular dynamic simulations of the best conformation of 2-ME provided insights into the molecular-level details of its binding pattern across the isotypes. Furthermore, the MM/GBSA analyses revealed the specific binding energy profile of 2-ME in ß-tubulin isotypes. It also highlighed, 2-ME exhibits the lowest binding affinity toward the ß1 isotype as supported by experimental study. The present study may offer useful information for designing next-generation antimicrotubule agents that are more specific and less toxic.

Bovine somatotropin increases the pregnancy rate in fixed-time artificial insemination in beef cattle.

This study evaluated the effect of bovine somatotropin (bST) on pregnancy rate (PR) and size of the dominant follicle (DF) on the day of intravaginal progesterone (P4) removal in protocols for fixed-time artificial insemination (FTAI). Bos indicus (Nellore) females (n = 392) were distributed into three groups. The control group (CG; n = 92) received an intravaginal P4 device + estradiol benzoate on day (d)0; prostaglandin F2α on d7 (first application); removal of P4 + estradiol cypionate (EC) + PGF2α (second application) + ultrasound (US) of the DF on d9; the FTAI was performed on d11; and pregnancy diagnosis (PD) was performed on d45. The bST group (bSTG; n = 142) underwent the same protocol as the CG, except that the animals received 125 mg of bST on d7. The equine chorionic gonadotropin (eCG) group (eCGG; n = 158) underwent the same protocol as the CG, except that the animals received 300 IU of eCG on d9. The PRs of the bSTG, eCGG, and CG were 48%, 48%, and 35%, respectively (p < .05); the bSTG and eCGG showed greater PRs, with follicles 6-7.9 mm (p < .05) and 8-8.9 mm in diameter, respectively. The bSTG exhibited a greater dimension of the DF on d9 of the protocol (p < .05). The eCGG had higher PRs with a body condition score (BCS) of 2.5, and the bSTG had a BCS of 3.0 (p < .05). It was concluded that bST increased PR, bST showed better performance in smaller DF and larger follicular diameter on d9 of the protocol, eCG acted better on animals with lower BCSs, and bST can be used in FTAI.

Effect of exogenous oestrogen treatment frequency on induction of artificial lactation in pseudopregnant sows.

In this study, we examined whether the frequency of exogenous oestrogen treatment affects the induction of artificial lactation and milk production. Furthermore, we analysed changes in milk components obtained from artificially lactating sows. Pseudopregnant induced by treatment with 30 mg of estradiol dipropionate (EDP) on Day 10 (Day 0 = the last day of estrus) were divided into three groups: those administered 5 mg of EDP once on Day 39 (n = 5), twice on Days 32 and 39 (n = 5) and three times on Days 25, 32 and 39 (n = 6). All animals were treated with prostaglandin F2α (PGF2α) on Day 46 for induced lactation. Artificial lactation was induced in 66.7%-80.0% of sows, and the EDP treatment frequency before PGF2α administration had no significant effect on either the induction rate of artificial lactation or the milk yield during the experimental period. The milk composition (levels of crude protein, crude fat, crude ash, lactose and immunoglobulin) did not differ among the groups. In conclusion, the number of EDP treatments prior to PGF2α administration had no effect on either the efficiency of artificial lactation induction or milk production.

The role of Ca2+ signalling and InsP3R in the pathogenesis of intrahepatic cholestasis of pregnancy.

BACKGROUND: In order to contribute new insights for future prevention and treatment of intrahepatic cholestasis of pregnancy (ICP), and to promote positive pregnancy outcomes, we evaluated serum Ca2+ levels and inositol 1,4,5-trisphosphate receptor (InsP3R) expression in the liver tissue of a rat ICP model. METHODS: After establishing the model by injection of oestradiol benzoate and progesterone into pregnant rats, animals were divided into normal control (n = 5) and ICP model groups (n = 5). The expression of InsP3R protein in the liver, and serum levels of Ca2+, glycocholic acid and bile acid were detected. RESULTS: InsP3R mRNA and protein were significantly lower in the ICP model group compared to the normal group, as determined by qPCR and immunohistochemistry, respectively. Serum enzyme-linked immunosorbent assay results revealed significantly higher levels of glycocholic acid and bile acid in the ICP model group compared to the normal group, while Ca2+ levels were significantly lower. The levers of Ca2+ were significantly and negatively correlated with the levels of glycocholic acid. The observed decrease in Ca2+ was associated with an increase in total bile acids, but there was no significant correlation. CONCLUSIONS: Our results revealed that the expression of InsP3R and serum Ca2+ levels was significantly decreased in the liver tissue of ICP model rats. Additionally, Ca2+ levels were found to be negatively correlated with the level of glycocholic acid.

This study investigated the relationship between serum Ca²⁺ levels, inositol 1,4,5-trisphosphate receptor (InsP3R) expression and intrahepatic cholestasis of pregnancy (ICP) in a rat model. The results indicated a significant decrease in InsP3R expression and Ca²⁺ in the disease group compared to the control group, alongside elevated levels of glycocholic acid and bile acid. The levels of Ca²⁺ exhibited a negative correlation with the levels of glycocholic acid. These findings indicated that the decrease of InsP3R expression and Ca²⁺ levels may be related to the pathogenesis of ICP. The study provides further insight into the treatment of this disease.

Efficacy of low-dose vaginal 17ß-estradiol versus vaginal promestriene for vulvovaginal atrophy.

OBJECTIVE: Vulvovaginal atrophy is frequent, can be bothersome and can impair quality of life in postmenopausal women. The main objective of this prospective, randomized study was to compare the acceptability of low-dose vaginal 17ß-estradiol (estradiol) tablets and vaginal promestriene cream in postmenopausal women with moderate-to-severe symptomatic vulvovaginal atrophy. METHODS: Overall, 120 patients were randomized to receive estradiol or promestriene (n = 60 per group). Acceptability was assessed with a specific questionnaire. Symptom intensity, the Vaginal Health Index (VHI), vaginal pH and the Vaginal Maturation Index were also evaluated. RESULTS: Acceptability was higher for estradiol tablets. Compared to promestriene cream, hygiene and ease of use were greater after 4 weeks (p = 0.011 and p = 0.001, respectively) and after 12 weeks (p = 0.009 and p = 0.011, respectively). Reduction of symptom intensity was greater with estradiol. Both treatments improved the VHI and decreased vaginal pH. However, superficial cell percentages increased significantly (p < 0.001) with estradiol but not with promestriene (p = 0.241), with a statistically significant difference between means (p = 0.004). CONCLUSION: Our results support the use of vaginal low-dose estradiol tablets as compared to vaginal promestriene cream for the management of moderate-to-severe symptomatic vulvovaginal atrophy in postmenopausal women. CLINICALTRIALS.GOV IDENTIFIER: NCT04232813.

Identification of Estradiol Benzoate as an Inhibitor of HBx Using Inducible Stably Transfected HepG2 Cells Expressing HiBiT Tagged HBx.

HBx plays a significant role in the cccDNA epigenetic modification regulating the hepatitis B virus (HBV) life cycle and in hepatocyte proliferation and carcinogenesis. By using the sleeping-beauty transposon system, we constructed a tetracycline-induced HBx-expressing stable cell line, SBHX21. HBx with a HiBiT tag can be quickly detected utilizing a NanoLuc-based HiBiT detection system. By screening a drug library using SBHX21 cells, we identified estradiol benzoate as a novel anti-HBx agent. Estradiol benzoate also markedly reduced the production of HBeAg, HBsAg, HBV pgRNA, and HBV DNA in a dose-dependent manner, suggesting that estradiol benzoate could be an anti-HBV agent. Docking model results revealed that estradiol benzoate binds to HBx at TRP87 and TRP107. Collectively, our results suggest that estradiol benzoate inhibits the HBx protein and HBV transcription and replication, which may serve as a novel anti-HBV molecular compound for investigating new treatment strategies for HBV infection.

2-Hydroxyestradiol Overcomes Mesenchymal Stem Cells-Mediated Platinum Chemoresistance in Ovarian Cancer Cells in an ERK-Independent Fashion.

Ovarian cancer (OC) is the second most common type of gynecological malignancy. Platinum (Pt)-based chemotherapy is the standard of care for OC, but toxicity and acquired chemoresistance has proven challenging. Recently, we reported that sensitivity to platinum was significantly reduced in a co-culture of OC cells with MSC. To discover compounds capable of restoring platinum sensitivity, we screened a number of candidates and monitored ability to induce PARP cleavage. Moreover, we monitored platinum uptake and expression of ABC transporters in OC cells. Our results showed that 2-hydroxyestradiol (2HE2), a metabolite of estradiol, and dasatinib, an Abl/Src kinase inhibitor, were significantly effective in overcoming MSC-mediated platinum drug resistance. Dasatinib activity was dependent on ERK1/2 activation, whereas 2HE2 was independent of the activation of ERK1/2. MSC-mediated platinum drug resistance was accompanied by reduced intracellular platinum concentrations in OC cells. Moreover, MSC co-cultured with OC cells resulted in downregulation of the expression of cellular transporters required for platinum uptake and efflux. Exposure to 2HE2 and other modulators resulted in an increase in intracellular platinum concentrations. Thus, 2HE2 and dasatinib might act as sensitizers to restore platinum drug sensitivity to OC cells and thus to limit TME-mediated chemoresistance in OC.

Efficacy of Yougui pill combined with Buzhong Yiqi decoction in alleviating the sexual dysfunction in female rats through modulation of the gut microbiota.

CONTEXT: Yougui pill combined with Buzhong Yiqi decoction (YPBYD) is used to relieve sexual dysfunction in clinical practice. OBJECTIVE: To investigate changes in microbial composition caused by sexual dysfunction and identify dominant bacteria related to YPBYD treatment. MATERIALS AND METHODS: Female Sprague-Dawley rats were randomly divided into four groups (n = 6): one group underwent Sham operation (Sham group), while three groups underwent ovariectomy (one model and two treatment groups). The ovariectomized (OVX) rats received oestradiol benzoate (250 µg/kg/week) or YPBYD (3.6 mL/d) via oral gavage for 4 weeks. Vaginal smear assay was performed; the serum levels of cyclic adenosine monophosphate (cAMP) and oestradiol (E2) were measured, followed by collection of stool samples for 16S rRNA sequencing. RESULTS: After YPBYD treatment, the levels of E2 and cAMP in OVX rats significantly increased (E2: from 20.45 ± 1.60 ng/L to 24.38 ± 1.70 ng/L; cAMP: from 261.41 ± 9.21 pg/mL to 373.75 ± 17.37 pg/mL). OVX treatment decreased diversity of gut microbiota and YPBYD treatment restored gut microbiota composition. Compared with Sham group, the abundance of Romboutsia significantly increased, while those of Proteobacteria and Staphylococcus markedly decreased in OVX group (all p < 0.05); meanwhile, the abundance of these microbes showed an opposite trend after YPBYD treatment. These microbiotas were involved in tyrosine and tryptophan biosynthesis and fatty acid metabolism. DISCUSSION AND CONCLUSIONS: These findings are the first to indicate YPBYD can alleviate female sexual dysfunction by modulating gut microbiota in OVX rats, which will help enhance the understanding on potential mechanism of YPBYD against sexual dysfunction.

Urinary oestrogen steroidome as an indicator of the risk of localised prostate cancer progression.

BACKGROUND: Prostate cancer (PCa) is the most common cancer in North American men. Beyond the established contribution of androgens to disease progression, growing evidence suggest that oestrogen-related pathways might also be of clinical importance. The aim of this study was to explore the association of urinary oestrogen levels with clinical outcomes. METHODS: Urine samples from the prospective multi-institutional PROCURE cohort were collected before RP for discovery (n = 259) and validation (n = 253). Urinary total oestrogens (unconjugated + conjugated), including oestrone and oestradiol, their bioactive and inactive catechol and methyl derivatives (n = 15), were measured using mass spectrometry (MS). RESULTS: The median follow-up time for the discovery and replication cohorts was 7.6 and 6.5 years, respectively. Highly significant correlations between urinary oestrogens were observed; however, correlations with circulating oestrogens were modest. Our findings indicate that higher levels of urinary oestriol and 16-ketoestradiol were associated with lower risk of BCR. In contrast, higher levels of 2-methoxyestrone were associated with an increased risk of development of metastasis/deaths. CONCLUSIONS: Our data suggest that urinary levels of oestriol and 16-ketoestradiol metabolites are associated with a more favourable outcome, whereas those of 2-methoxyestrone are associated with an elevated risk of metastasis after RP. Further studies are required to better understand the impact of oestrogens on disease biology and as easily accessible urine-based risk-stratification markers.

Overall Survival with Palbociclib and Fulvestrant in Advanced Breast Cancer.

BACKGROUND: The cyclin-dependent kinase 4 and 6 (CDK4/6) inhibitor palbociclib, in combination with fulvestrant therapy, prolongs progression-free survival among patients with hormone-receptor-positive, human epidermal growth factor receptor 2 (HER2)-negative advanced breast cancer. We report the results of a prespecified analysis of overall survival. METHODS: We randomly assigned patients with hormone-receptor-positive, HER2-negative advanced breast cancer who had progression or relapse during previous endocrine therapy to receive palbociclib plus fulvestrant or placebo plus fulvestrant. We analyzed overall survival; the effect of palbociclib according to the prespecified stratification factors of presence or absence of sensitivity to endocrine therapy, presence or absence of visceral metastatic disease, and menopausal status; the efficacy of subsequent therapies after disease progression; and safety. RESULTS: Among 521 patients who underwent randomization, the median overall survival was 34.9 months (95% confidence interval [CI], 28.8 to 40.0) in the palbociclib-fulvestrant group and 28.0 months (95% CI, 23.6 to 34.6) in the placebo-fulvestrant group (hazard ratio for death, 0.81; 95% CI, 0.64 to 1.03; P=0.09; absolute difference, 6.9 months). CDK4/6 inhibitor treatment after the completion of the trial regimen occurred in 16% of the patients in the placebo-fulvestrant group. Among 410 patients with sensitivity to previous endocrine therapy, the median overall survival was 39.7 months (95% CI, 34.8 to 45.7) in the palbociclib-fulvestrant group and 29.7 months (95% CI, 23.8 to 37.9) in the placebo-fulvestrant group (hazard ratio, 0.72; 95% CI, 0.55 to 0.94; absolute difference, 10.0 months). The median duration of subsequent therapy was similar in the two groups, and the median time to the receipt of chemotherapy was 17.6 months in the palbociclib-fulvestrant group, as compared with 8.8 months in the placebo-fulvestrant group (hazard ratio, 0.58; 95% CI, 0.47 to 0.73; P<0.001). No new safety signals were observed with 44.8 months of follow-up. CONCLUSIONS: Among patients with hormone-receptor-positive, HER2-negative advanced breast cancer who had sensitivity to previous endocrine therapy, treatment with palbociclib-fulvestrant resulted in longer overall survival than treatment with placebo-fulvestrant. The differences in overall survival in the entire trial group were not significant. (Funded by Pfizer; PALOMA-3 ClinicalTrials.gov number, NCT01942135 .).

Maternal systemic vascular dysfunction in a primate model of defective uterine spiral artery remodeling.

Uterine spiral artery remodeling (UAR) is essential for placental perfusion and fetal development. A defect in UAR underpins placental ischemia disorders, e.g., preeclampsia, that result in maternal systemic vascular endothelial dysfunction and hypertension. We have established a model of impaired UAR by prematurely elevating maternal serum estradiol levels during the first trimester of baboon pregnancy. However, it is unknown whether this experimental paradigm is associated with maternal vascular endothelial dysfunction. Therefore, in the present study baboons were administered estradiol on days 25-59 of gestation to suppress UAR and maternal vascular function determined on day 165 (term = 184 days) peripherally and in skeletal muscle, which accounts for over 40% of body mass and 25% of resting systemic vascular resistance. Maternal serum sFlt-1 levels were 2.5-fold higher (P < 0.05), and skeletal muscle arteriolar endothelial nitric oxide synthase (eNOS) protein expression and luminal area, and skeletal muscle capillary density were 30-50% lower (P < 0.05) in UAR suppressed baboons. Coinciding with these changes in eNOS expression, luminal area, and capillary density, maternal brachial artery flow-mediated dilation and volume flow were 70% and 55% lower (P < 0.05), respectively, and mean arterial blood pressure 29% higher (P < 0.01) in UAR defective baboons. In summary, maternal vascular function was disrupted in a baboon model of impaired UAR. These results highlight the translational impact of this primate model and relevance to adverse conditions of human pregnancy underpinned by improper uterine artery transformation.NEW & NOTEWORTHY Maternal vascular dysfunction is a hallmark of abnormal human pregnancy, particularly early-onset preeclampsia, elicited by impaired UAR. The present study makes the novel discovery that maternal systemic vascular dysfunction was induced in a baboon experimental model of impaired UAR. This study highlights the translational relevance of this nonhuman primate model to adverse conditions of human pregnancy underpinned by defective UAR.

Insulin-like growth factor 2 is produced by antral follicles and promotes preantral follicle development in macaques†.

Insulin-like growth factors (IGFs) are known for their involvement in endocrine and paracrine regulation of ovarian function. Although IGF2 is the predominant circulating and intraovarian form of IGFs in primate species, the stage-specific follicular expression, action, and regulation of IGF2 are not well defined. Therefore, experiments were conducted to investigate the follicular IGF production in response to steroid hormone regulation and the direct IGF actions on follicular development and function in vitro. Preantral follicles were isolated from rhesus macaque ovaries and cultured to the antral stage in media supplemented with follicle-stimulating hormone and insulin. Follicles were randomly assigned to treatment groups: (a) control, (b) trilostane (a steroid synthesis inhibitor), (c) trilostane + estradiol, (d) trilostane + progesterone, and (e) trilostane + dihydrotestosterone. Media was analyzed for IGF concentrations, which were correlated to follicle growth. Follicles produced IGF2, but not IGF1, at the antral stage. Steroid depletion decreased, whereas steroid replacement increased, IGF2 production by antral follicles. Media IGF2 levels correlated positively with antral follicle diameters. Macaque preantral follicles and granulosa cells were subsequently cultured without (control) and with recombinant human IGF2 supplementation. Follicle survival, growth, and paracrine factor production, as well as granulosa cell proliferation and gonadotropin receptor gene expression, were assessed. IGF2 addition increased follicle survival rates, diameters and inhibin B production, as well as granulosa cell proliferation. These data demonstrate that IGF2 produced by antral follicles, in response to steroid hormone regulation, could act as a paracrine factor that positively impacts preantral follicle development and function in primates.

Broad diversity of bacteria degrading 17ß-estradiol-3-sulfate isolated from river sediment and biofilm at a wastewater treatment plant discharge.

Conjugated estrogens, such as 17ß-estradiol-3-sulfate (E2-3S), can be released into aquatic environments through wastewater treatment plants (WWTP). There, they are microbiologically degraded into free estrogens, which can have harmful effects on aquatic wildlife. Here, the degradation of E2-3S in environmental samples taken upstream, downstream and at the effluent of a WWTP was assessed. Sediment and biofilm samples were enriched for E2-3S-degrading microorganisms, yielding a broad diversity of bacterial isolates, including known and novel degraders of estrogens. Since E2-3S-degrading bacteria were also isolated in the sample upstream of the WWTP, the WWTP does not influence the ability of the microbial community to degrade E2-3S.

17α-Ethinyl estradiol-3-sulfate increases survival and hemodynamic functioning in a large animal model of combined traumatic brain injury and hemorrhagic shock: a randomized control trial.

BACKGROUND: Traumatic brain injury (TBI) and severe blood loss resulting in hemorrhagic shock (HS) represent leading causes of trauma-induced mortality, especially when co-occurring in pre-hospital settings where standard therapies are not readily available. The primary objective of this study was to determine if 17α-ethinyl estradiol-3-sulfate (EE-3-SO4) increases survival, promotes more rapid cardiovascular recovery, or confers neuroprotection relative to Placebo following TBI + HS. METHODS: All methods were approved by required regulatory agencies prior to study initiation. In this fully randomized, blinded preclinical study, eighty (50% females) sexually mature (190.64 ± 21.04 days old; 28.18 ± 2.72 kg) Yucatan swine were used. Sixty-eight animals received a closed-head, accelerative TBI followed by removal of approximately 40% of circulating blood volume. Animals were then intravenously administered EE-3-SO4 formulated in the vehicle at 5.0 mg/mL (dosed at 0.2 mL/kg) or Placebo (0.45% sodium chloride solution) via a continuous pump (0.2 mL/kg over 5 min). Twelve swine were included as uninjured Shams to further characterize model pathology and replicate previous findings. All animals were monitored for up to 5 h in the absence of any other life-saving measures (e.g., mechanical ventilation, fluid resuscitation). RESULTS: A comparison of Placebo-treated relative to Sham animals indicated evidence of acidosis, decreased arterial pressure, increased heart rate, diffuse axonal injury and blood-brain barrier breach. The percentage of animals surviving to 295 min post-injury was significantly higher for the EE-3-SO4 (28/31; 90.3%) relative to Placebo (24/33; 72.7%) cohort. EE-3-SO4 also restored pulse pressure more rapidly post-drug administration, but did not confer any benefits in terms of shock index. Primary blood-based measurements of neuroinflammation and blood brain breach were also null, whereas secondary measurements of diffuse axonal injury suggested a more rapid return to baseline for the EE-3-SO4 group. Survival status was associated with biological sex (female > male), as well as evidence of increased acidosis and neurotrauma independent of EE-3-SO4 or Placebo administration. CONCLUSIONS: EE-3-SO4 is efficacious in promoting survival and more rapidly restoring cardiovascular homeostasis following polytraumatic injuries in pre-hospital environments (rural and military) in the absence of standard therapies. Poly-therapeutic approaches targeting additional mechanisms (increased hemostasis, oxygen-carrying capacity, etc.) should be considered in future studies.

The role of MAPK11/12/13/14 (p38 MAPK) protein in dopamine agonist-resistant prolactinomas.

BACKGROUND: Prolactinoma is a functional pituitary adenoma that secretes excessive prolactin. Dopamine agonists (DAs) such as bromocriptine (BRC) are the first-line treatment for prolactinomas, but the resistance rate is increasing year by year, creating a clinical challenge. Therefore, it is urgent to explore the molecular mechanism of bromocriptine resistance in prolactinomas. Activation of the P38 MAPK pathway affects multidrug resistance in tumours. Our previous studies have demonstrated that inhibiting MAPK14 can suppress the occurrence of prolactinoma, but the role of MAPK11/12/13/14 (p38 MAPK) signalling in dopamine agonist-resistant prolactinomas is still unclear. METHODS: A prolactinoma rat model was established to determine the effect of bromocriptine on MAPK11/12/13/14 signalling. DA-resistant GH3 cells and DA-sensitive MMQ cells were used, and the role of MAPK11/12/13/14 in bromocriptine-resistant prolactinomas was preliminarily verified by western blot, RT-qPCR, ELISA, flow cytometry and CCK-8 experiments. The effects of MAPK11 or MAPK14 on bromocriptine-resistant prolactinomas were further verified by siRNA transfection experiments. RESULTS: Bromocriptine was used to treat rat prolactinoma by upregulating DRD2 expression and downregulating the expression level of MAPK11/12/13/14 in vivo experiments. The in vitro experiments showed that GH3 cells are resistant to bromocriptine and that MMQ cells are sensitive to bromocriptine. Bromocriptine could significantly reduce the expression of MAPK12 and MAPK13 in GH3 cells and MMQ cells. Bromocriptine could significantly reduce the expression of MAPK11, MAPK14, NF-κB p65 and Bcl2 in MMQ but had no effect on MAPK11, MAPK14, NF-κB p65 and Bcl2 in GH3 cells. In addition, knockdown of MAPK11 and MAPK14 in GH3 cells by siRNA transfection reversed the resistance of GH3 cells to bromocriptine, and haloperidol (HAL) blocked the inhibitory effect of bromocriptine on MAPK14, MAPK11, and PRL in MMQ cells. Our findings show that MAPK11 and MAPK14 proteins are involved in bromocriptine resistance in prolactinomas. CONCLUSION: Bromocriptine reduces the expression of MAPK11/12/13/14 in prolactinomas, and MAPK11 and MAPK14 are involved in bromocriptine resistance in prolactinomas by regulating apoptosis. Reducing the expression of MAPK11 or MAPK14 can reverse bromocriptine resistance in prolactinomas.

Comparison on Metabolism of Estradiol and Its 17-Sulfate by Recombinant Human CYP Isoforms.

To identify the CYP isoforms involved in the production of 2-hydroxyestradiol 17-sulfate (2-OH-ES), which we assume to be an antioxidant in vivo, the 2-hydroxylation reaction of estradiol 17-sulfate (ES) by human liver microsome was investigated. As a result, it was estimated that CYP2C8 and 2C9 were largely involved in the production of 2-OH-ES. Therefore, the 2-hydroxylation kinetic analysis of ES was performed for both CYPs, and the metabolic clearance Vmax/Km (µL/nmol CYP/min) was determined. On comparing the results of ES with those of estradiol (E2), it was found that CYP2C8 was about 2.5 times higher and CYP2C9 was about 3 times higher, and ES was more likely to be a substrate for the 2-hydroxylation reaction by both CYPs. The CYP isoforms involved in A-ring hydroxylation of E2 and ES differed. From this, it was speculated that 2-OH-ES plays a different role to 2-hydroxyestradiol (2-OH-E2), which is recognized as an antioxidant in the body.

Potential Estrogenic Properties of 17ß-Hydroxy-ethylimine Estradiol Derivative Targeted to Menopause Stage.

BACKGROUND/AIM: Hormone replacement therapy during menopause increases the risk of thromboembolic diseases and cancer, so safety alternative therapeutic strategies are needed. 17ß-Aminoestrogens are a synthetic estrogens group that possess mild anticoagulant activity that contrasts with the pro-coagulant effects showed by estradiol's (E2) in rodents. Being considered an alternative to conventional hormone replacement therapy during menopause without thrombogenic risks producing. The present study aimed to determine the estrogenic profile and anxiolytic activity of 17ß-[hydroxy-ethylimine]-1,3,5(10)-estratrien-3-ol (IE2), a related compound unknown until now. METHODS: IE2 was assessed in immature rats by uterotrophic assay administering IE2, E2, or vehicle. In ovariectomized adult Wistar rats (Ovx) to facilitating the lordotic behavior compared with E2, estradiol benzoate, or vehicle. The effect of IE2 on anxiety was estimated in Ovx animals treated with IE2, E2, or vehicle group and evaluated in the elevated plus-maze model. RESULTS AND CONCLUSION: IE2 produced an uterotrophic effect, lordotic behavior, and anxiolytic effect in a dose-dependent manner, similar to E2. IE2 depicted estrogenicity, indicating potential clinical use as hormone replacement therapy during menopause.

Novel testing strategy for prediction of rat biliary excretion of intravenously administered estradiol-17ß glucuronide.

The aim of the present study was to develop a generic rat physiologically based kinetic (PBK) model that includes a novel testing strategy where active biliary excretion is incorporated using estradiol-17ß glucuronide (E217ßG) as the model substance. A major challenge was the definition of the scaling factor for the in vitro to in vivo conversion of the PBK-model parameter Vmax. In vitro values for the Vmax and Km for transport of E217ßG were found in the literature in four different studies based on experiments with primary rat hepatocytes. The required scaling factor was defined based on fitting the PBK model-based predicted values to reported experimental data on E217ßG blood levels and cumulative biliary E217ßG excretion. This resulted in a scaling factor of 129 mg protein/g liver. With this scaling factor the PBK model predicted the in vivo data for blood and cumulative biliary E217ßG levels with on average of less than 1.8-fold deviation. The study provides a proof of principle on how biliary excretion can be included in a generic PBK model using primary hepatocytes to define the kinetic parameters that describe the biliary excretion.