Reducing the rate of negative ureteroscopy: predictive factors and the role of preoperative imaging.

INTRODUCTION: The aim of this study was to investigate factors that may predict a negative ureteroscopy (URS) performed for ureteric calculi in prestented patients and to assess preoperative imaging in reducing the rate of negative URS. METHODS: Data were collected on emergency stent placement for a ureteric calculus from April 2011 to February 2016 (Group A) and October 2016 to October 2019 (Group B). Data included patient demographics, indication for a stent, stone characteristics, baseline bloods, urine culture, readmission, negative URS rate and the use of pre-URS imaging. Multivariate logistic regression was used for statistical analysis. RESULTS: Of 257 patients who underwent emergency stent insertion, 251 underwent deferred URS for a ureteric calculus and 6 avoided URS due to pre-URS imaging. Indications for stent were pain (42%), sepsis (39%) and acute kidney injury (19%). Mean stone size was 7.8mm, mean stone density was 699 Hounsfield units (HU) and the stone locations were upper (62%), mid (13%) and lower ureter (25%). The overall negative URS rate was 12%. The negative URS rate was lower in patients with pre-URS imaging compared with those with none, 6% and 14%, respectively (OR=2.33, 95% CI: 0.69-7.56, p=0.2214). Logistic regression analysis indicated stone size as the only significant predictor of a negative URS, where the greater the size of the stone the less likely URS would be negative (ß=0.75, 95% CI: 0.60-0.94 p=0.011). CONCLUSIONS: Utilising pre-URS imaging can lead to a reduction in negative URS rate. Stone size <5mm appears to be the subgroup most likely to benefit from imaging.

A Potential Mechanism for Immune Suppression by Beta-Adrenergic Receptor Stimulation following Traumatic Injury.

BACKGROUND: ß-Adrenergic agents suppress inflammation and may play an important role in posttraumatic infections. Mechanisms may include inhibition of MAP kinase signaling. We sought to determine whether MKP-1 contributed to catecholamine suppression of innate immunity and also wanted to know whether early catecholamine treatment after traumatic injury increases the risk of later nosocomial infection. METHODS: We performed experiments using THP-1 cells and peripheral blood mononuclear cells from healthy individuals. We exposed cells to epinephrine and/or LPS and measured inflammatory gene transcription and MAP kinase activation. We inhibited MKP-1 activity to determine its role in catecholamine-induced immune suppression. Finally, we studied injured subjects to determine whether early catecholamine treatment was associated with nosocomial infection. RESULTS: Epinephrine increases MKP-1 transcripts and protein and decreases LPS-induced p38 and JNK phosphorylation and TNF-α gene transcription. RNAi inhibition of MKP-1 at least partially restores LPS-induced TNF-α gene expression (p = 0.024). In the clinical cohort, subjects treated with ß-adrenergic agents had an increased risk of ventilator-associated pneumonia (aOR = 1.9; 95% CI = 1.3-2.6) and bacteremia (aOR = 1.5; 95% CI = 1.1-2.3). CONCLUSIONS: MKP-1 may have a role in catecholamine-induced suppression of innate immunity, and exogenous catecholamines might contribute to nosocomial infection risk.

From ionic to cellular variability in human atrial myocytes: an integrative computational and experimental study.

Variability refers to differences in physiological function between individuals, which may translate into different disease susceptibility and treatment efficacy. Experiments in human cardiomyocytes face wide variability and restricted tissue access; under these conditions, computational models are a useful complementary tool. We conducted a computational and experimental investigation in cardiomyocytes isolated from samples of the right atrial appendage of patients undergoing cardiac surgery to evaluate the impact of variability in action potentials (APs) and subcellular ionic densities on Ca2+ transient dynamics. Results showed that 1) variability in APs and ionic densities is large, even within an apparently homogenous patient cohort, and translates into ±100% variation in ionic conductances; 2) experimentally calibrated populations of models with wide variations in ionic densities yield APs overlapping with those obtained experimentally, even if AP characteristics of the original generic model differed significantly from experimental APs; 3) model calibration with AP recordings restricts the variability in ionic densities affecting upstroke and resting potential, but redundancy in repolarization currents admits substantial variability in ionic densities; and 4) model populations constrained with experimental APs and ionic densities exhibit three Ca2+ transient phenotypes, differing in intracellular Ca2+ handling and Na+/Ca2+ membrane extrusion. These findings advance our understanding of the impact of variability in human atrial electrophysiology. NEW & NOTEWORTHY Variability in human atrial electrophysiology is investigated by integrating for the first time cellular-level and ion channel recordings in computational electrophysiological models. Ion channel calibration restricts current densities but not cellular phenotypic variability. Reduced Na+/Ca2+ exchanger is identified as a primary mechanism underlying diastolic Ca2+ fluctuations in human atrial myocytes.

Parenteral and enteral nutrition in surgical critical care: Plasma metabolomics demonstrates divergent effects on nitrogen, fatty-acid, ribonucleotide, and oxidative metabolism.

BACKGROUND: Artificial nutrition support is central to the care of critically ill patients and is primarily provided enterally (EN). There are circumstances when parenteral nutrition (PN) is considered necessary. We are uncertain how each of these approaches confer clinical benefits beyond simply providing calories. We sought to better understand how each of these techniques influence metabolism in critically ill patients using a broad-based metabolomics approach. Metabolic responses to EN and PN may differ in ways that could help us understand how to optimize use of these therapies. METHODS: We prospectively enrolled subjects over 7 months in 2015 at an urban, Level I trauma center. Subjects were included before starting either EN or PN during their inpatient admission. Plasma samples were obtained between 1 and 12 hours before initiation of artificial nutrition, and 3 and 7 days later. All samples were analyzed with liquid chromatography/mass spectrometry-based metabolomics. Differences in metabolite concentrations were assessed via principal component analyses and multiple linear regression. RESULTS: We enrolled 30 subjects. Among the critically ill subjects, 10 received EN and 10 received PN. In subjects receiving EN, amino acid and urea cycle metabolites (citrulline, p = 0.04; ornithine, p = 0.05) increased, as did ribonucleic acid metabolites (uridine, p = 0.04; cysteine, 0 = 0.05; oxypurinol, p = 0.04). Oxidative stress decreased over time (increased betaine, p = 0.05; decreased 4-pyridoxic acid, p = 0.04). In subjects receiving PN, amino acid concentrations increased over time (taurine, p = 0.04; phenylalanine, p = 0.05); omega 6 and omega 3 fatty acid concentrations decreased over time (p = 0.05 and 0.03, respectively). CONCLUSION: EN was associated with amino acid repletion, urea cycle upregulation, restoration of antioxidants, and increasing ribonucleic acid synthesis. Parenteral nutrition was associated with increased amino acid concentrations, but did not influence protein metabolism or antioxidant repletion. This suggests that parenteral amino acids are used less effectively than those given enterally. The biomarkers reported in this study may be useful in guiding nutrition therapy for critically ill patients. LEVEL OF EVIDENCE: Therapeutic study, level III; prognostic study, level II.

Use of Metabolomics to Trend Recovery and Therapy After Injury in Critically Ill Trauma Patients.

IMPORTANCE: Metabolomics is the broad and parallel study of metabolites within an organism and provides a contemporaneous snapshot of physiologic state. Use of metabolomics in the clinical setting may help achieve precision medicine for those who have experienced trauma, where diagnosis and treatment are tailored to the individual patient. OBJECTIVE: To examine whether metabolomics can (1) distinguish healthy volunteers from trauma patients and (2) quantify changes in catabolic metabolites over time after injury. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study with enrollment from September 2014 to May 2015 at an urban, level 1 trauma center. Included in the study were 10 patients with severe blunt trauma admitted within 12 hours of injury with systolic blood pressure less than 90 mm Hg or base deficit greater than 6 mEq/L and 5 healthy volunteers. Plasma samples (n = 35) were obtained on days 1, 3, and 7, and they were analyzed using mass spectrometry. MAIN OUTCOMES AND MEASURES: Principal component analyses, multiple linear regression, and paired t tests were used to select biomarkers of interest. A broad-based metabolite profile comparison between trauma patients and healthy volunteers was performed. Specific biomarkers of interest were oxidative catabolites. RESULTS: Trauma patients had a median age of 45 years and a median injury severity score of 43 (interquartile range, 34-50). Healthy fasting volunteers had a median age of 33 years. Compared with healthy volunteers, trauma patients showed oxidative stress on day 1: niacinamide concentrations were a mean (interquartile range) of 0.95 (0.30-1.45) relative units for trauma patients vs 1.06 (0.96-1.09) relative units for healthy volunteers (P = .02), biotin concentrations, 0.43 (0.27-0.58) relative units for trauma patients vs 1.21 (0.93-1.56) relative units for healthy volunteers (P = .049); and choline concentrations, 0.17 (0.09-0.22) relative units for trauma patients vs 0.21 (0.18-0.22) relative units for healthy volunteers (P = .004). Trauma patients showed lower nucleotide synthesis on day 1: adenylosuccinate concentrations were 0.08 (0.04-0.12) relative units for trauma patients vs 0.15 (0.14-0.17) relative units for healthy volunteers (P = .02) and cytidine concentrations were 1.44 (0.95-1.73) relative units for trauma patients vs 1.74 (1.62-1.98) relative units for healthy volunteers (P = .05). From trauma day 1 to day 7, trauma patients showed increasing muscle catabolism: serine levels increased from 42.03 (31.20-54.95) µM to 79.37 (50.29-106.37) µM (P = .002), leucine levels increased from 69.21 (48.36-99.89) µM to 114.16 (92.89-143.52) µM (P = .004), isoleucine levels increased from 20.43 (10.92-27.41) µM to 48.72 (36.28-64.84) µM (P < .001), and valine levels increased from 122.56 (95.63-140.61) µM to 190.52 (136.68-226.07) µM (P = .004). There was an incomplete reversal of oxidative stress. CONCLUSIONS AND RELEVANCE: Metabolomics can function as a serial, comprehensive, and potentially personalized tool to characterize metabolism after injury. A targeted metabolomics approach was associated with ongoing oxidative stress, impaired nucleotide synthesis, and initial suppression of protein metabolism followed by increased nitrogen turnover. This technique may provide new therapeutic and nutrition targets in critically injured patients.

Safety of minimizing preoperative starvation in critically ill and intubated trauma patients.

BACKGROUND: Cessation of enteral nutrition prior to an operation/procedure is the most common reason for feeding interruption in critically ill trauma patients and contributes to substantial calorie deficits. This study reports on a strategy to increase calorie intake by continuing feeds until transfer for operations/procedures. METHODS: Nutrition guidelines were modified in 2006 to allow continuation of feeding in intubated patients up until transfer to the operating room. Prior to 2006, enteral feeding was stopped at least 6 hours prior to surgery. A retrospective cohort design from 2003 to 2010 compared clinical outcomes in groups of adult trauma subjects before and after guideline changes and in subjects at other centers without guideline changes. RESULTS: During the first week, subjects in the preimplementation cohort (n = 245) received a median of 3,787 kcal per person per week, while subjects in the postimplementation cohort (n = 368) received a median of 6,662 kcal per person per week (p < 0.001). There was no change in calorie intake for subjects at other centers (n = 1,002). The risks of acute respiratory distress syndrome, pneumonia, and mortality were decreased after implementation relative to the preimplementation cohort (acute respiratory distress syndrome: relative risk ratio [RR], 0.69; 95% confidence interval [CI], 0.59-0.81; pneumonia: RR, 0.82; 95% CI, 0.65-1.00; mortality: RR, 0.67; 95% CI, 0.46-0.99). Ventilator-free days increased by 1.4 days (95% CI, 0.1-2.7), while intensive care unit stay and hospital length of stay were unchanged. These outcomes showed similar trends over time at other participating centers. CONCLUSIONS: Allowing intubated trauma patients to continue enteral nutrition until transfer for operations or procedures was associated with increased caloric intake without evidence of increased pulmonary complications. This represents an important strategy to reduce calorie deficits in the trauma intensive care unit. LEVEL OF EVIDENCE: Therapeutic study/care management, level III.

Characterizing the Morbidity of Postchemotherapy Retroperitoneal Lymph Node Dissection for Testis Cancer in a National Cohort of Privately Insured Patients.

OBJECTIVE: To characterize morbidity of postchemotherapy retroperitoneal lymph node dissection (PC-RPLND) for testis cancer, we analyze a contemporary national database. PC-RPLND is the standard for residual radiographic masses ≥1 cm (nonseminoma) and positron emission tomography-avid masses ≥3 cm (seminoma). Morbidity for PC-RPLND is greater than primary RPLND, which may be mitigated by performing surgery at a high-volume cancer center. METHODS: Current Procedural Terminology and International Classification of Diseases, Ninth Edition codes identified men with testis cancer undergoing PC- or primary RPLND in MarketScan (2007-2012). Multivariable logistic regression assessed factors associated with receiving adjunctive procedures (ie, nephrectomy, vascular reconstruction), prolonged hospitalization, and 90-day readmission. Geographic variables assessed regionalization of PC-RPLND. RESULTS: Of 559 men with claims for PC- or primary RPLND (206, 37% PC-RPLND), 19% of PC-RPLND underwent adjunctive procedures (vs 1% among RPLND, P < .01). For PC-RPLND, the nephrectomy rate was 10% and the vascular reconstruction rate was 8%. On multivariable analysis, PC-RPLND was associated with undergoing adjunctive procedures (odds ratio 41.9; 95% confidence interval 11.7, 150) and prolonged hospitalization (odds ratio 3.75; 95% confidence interval 1.68, 8.42) compared to primary RPLND. PC-RPLND was not associated with 90-day readmission. Up to 29% of PC-RPLNDs are performed in centers, billing just a single case through MarketScan in the 6 years studied. CONCLUSION: PC-RPLND is associated with adjunctive procedures and longer hospitalizations. Given the morbidity of PC-RPLND in this young patient population, efforts are needed to establish quality benchmarks for, reduce the morbidity of, and to accurately discriminate risk during patient discussions prior to this complex, specialized surgery.

The relationship between ambient ultraviolet radiation (UVR) and objectively measured personal UVR exposure dose is modified by season and latitude.

Despite the widespread use of ambient ultraviolet radiation (UVR) as a proxy measure of personal exposure to UVR, the relationship between the two is not well-defined. This paper examines the effects of season and latitude on the relationship between ambient UVR and personal UVR exposure. We used data from the AusD Study, a multi-centre cross-sectional study among Australian adults (18-75 years), where personal UVR exposure was objectively measured using polysulphone dosimeters. Data were analysed for 991 participants from 4 Australian cities of different latitude: Townsville (19.3°S), Brisbane (27.5°S), Canberra (35.3°S) and Hobart (42.8°S). Daily personal UVR exposure varied from 0.01 to 21 Standard Erythemal Doses (median = 1.1, IQR: 0.5-2.1), on average accounting for 5% of the total available ambient dose. There was an overall positive correlation between ambient UVR and personal UVR exposure (r = 0.23, p < 0.001). However, the correlations varied according to season and study location: from strong correlations in winter (r = 0.50) and at high latitudes (Hobart, r = 0.50; Canberra, r = 0.39), to null or even slightly negative correlations, in summer (r = 0.01) and at low latitudes (Townsville, r = -0.06; Brisbane, r = -0.16). Multiple regression models showed significant effect modification by season and location. Personal exposure fraction of total available ambient dose was highest in winter (7%) and amongst Hobart participants (7%) and lowest in summer (1%) and in Townsville (4%). These results suggest season and latitude modify the relationship between ambient UVR and personal UVR exposure. Ambient UVR may not be a good indicator for personal exposure dose under some circumstances.

The AusD Study: a population-based study of the determinants of serum 25-hydroxyvitamin D concentration across a broad latitude range.

Observational studies suggest that people with a high serum 25-hydroxyvitamin D (25(OH)D) concentration may have reduced risk of chronic diseases such as osteoporosis, multiple sclerosis, type 1 diabetes, cardiovascular disease, and some cancers. The AusD Study (A Quantitative Assessment of Solar UV Exposure for Vitamin D Synthesis in Australian Adults) was conducted to clarify the relationships between ultraviolet (UV) radiation exposure, dietary intake of vitamin D, and serum 25(OH)D concentration among Australian adults residing in Townsville (19.3°S), Brisbane (27.5°S), Canberra (35.3°S), and Hobart (42.8°S). Participants aged 18-75 years were recruited from the Australian Electoral Roll between 2009 and 2010. Measurements were made of height, weight, waist:hip ratio, skin, hair, and eye color, blood pressure, and grip strength. Participants completed a questionnaire on sun exposure and vitamin D intake, together with 10 days of personal UV dosimetry and an associated sun-exposure and physical-activity diary that was temporally linked to a blood test for measurement of 25(OH)D concentration. Ambient solar UV radiation was also monitored at all study sites. We collected comprehensive, high-quality data from 1,002 participants (459 males, 543 females) assessed simultaneously across a range of latitudes and through all seasons. Here we describe the scientific and methodological issues considered in designing the AusD Study.

Prolonging hormone sensitivity in prostate cancer xenografts through dual inhibition of AR and mTOR.

BACKGROUND: To determine the mechanisms associated with loss of androgen dependency and disease progression in prostate cancer (PCa), we investigated the relationship between the androgen receptor (AR) and mTOR pathways and the impact of inhibiting both pathways in androgen-dependent and castration-resistant PCa models. EXPERIMENTAL DESIGN: Androgen-dependent (LNCaP) and castration-resistant PCa (HP-LNCaP) cells were grown as tumours in SCID mice. Once tumours reached 500 mm(3), animals were grouped and injected subcutaneous with vehicle, our novel anti-androgen/androgen synthesis inhibitor, VN/124-1, bicalutamide, and everolimus. Tumour volumes were measured biweekly. The PSA and protein analyses were performed after completion of the treatment. RESULTS: The addition of everolimus to bicalutamide treatment of resistant tumours significantly reduced tumour growth rates and tumour volumes. Anti-androgen treatment also increased protein expression of multiple signal transduction pathways earlier than vehicle-treated control xenografts. VN/124-1 plus everolimus acted in concert to reduce tumour growth rates in our castration-resistant xenograft model. CONCLUSIONS: This study suggests that dual inhibition of AR and mTOR in castration-resistant xenograft models can restore sensitivity of tumours to anti-androgen therapy. Furthermore, after bicalutamide failure, dual inhibition with VN/124-1 and everolimus was the most effective treatment.

Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling.

BACKGROUND: The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers. METHODS: The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs. RESULTS: The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab. INTERPRETATION: Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.

History of aromatase: saga of an important biological mediator and therapeutic target.

Aromatase is the enzyme that catalyzes the conversion of androgens to estrogens. Initial studies of its enzymatic activity and function took place in an environment focused on estrogen as a component of the birth control pill. At an early stage, investigators recognized that inhibition of this enzyme could have major practical applications for treatment of hormone-dependent breast cancer, alterations of ovarian and endometrial function, and treatment of benign disorders such as gynecomastia. Two general approaches ultimately led to the development of potent and selective aromatase inhibitors. One targeted the enzyme using analogs of natural steroidal substrates to work out the relationships between structure and function. The other approach initially sought to block adrenal function as a treatment for breast cancer but led to the serendipitous finding that a nonsteroidal P450 steroidogenesis inhibitor, aminoglutethimide, served as a potent but nonselective aromatase inhibitor. Proof of the therapeutic concept of aromatase inhibition involved a variety of studies with aminoglutethimide and the selective steroidal inhibitor, formestane. The requirement for even more potent and selective inhibitors led to intensive molecular studies to identify the structure of aromatase, to development of high-sensitivity estrogen assays, and to "mega" clinical trials of the third-generation aromatase inhibitors, letrozole, anastrozole, and exemestane, which are now in clinical use in breast cancer. During these studies, unexpected findings led investigators to appreciate the important role of estrogens in males as well as in females and in multiple organs, particularly the bone and brain. These studies identified the important regulatory properties of aromatase acting in an autocrine, paracrine, intracrine, neurocrine, and juxtacrine fashion and the organ-specific enhancers and promoters controlling its transcription. The saga of these studies of aromatase and the ultimate utilization of inhibitors as highly effective treatments of breast cancer and for use in reproductive disorders serves as the basis for this first Endocrine Reviews history manuscript.

Xenograft models for aromatase inhibitor studies.

As several aromatase inhibitors are now available for treating breast cancer, we developed a model system to compare their antitumor efficacy and to explore strategies for their optimal use. Tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca) and can therefore synthesize as well as respond to estrogen. Results from this model have been predictive of clinical outcome. Thus, inhibiting estrogen action and estrogen synthesis by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen in combination did not result in synergy. Moreover, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in response to sequential letrozole treatment. However, our findings indicate that letrozole alone was better than all other treatments. Although letrozole resulted in long sustained growth inhibition, tumors eventually grew despite continued treatment. Mechanisms of resistance to letrozole were investigated during the course of treatment. ER was initially upregulated in responding tumors, but subsequently decreased below control levels in tumors no longer responsive to letrozole. Her-2 as well as adapter proteins (p-Shc and Grb-2) and signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), were all increased in letrozole resistant tumors. In LTLT cells, isolated from the letrozole resistant tumors and treated with inhibitors of the MAPKinase pathway, MAPK activity was decreased and ER expression restored to control levels. Inhibitors of EGFR/Her-2 also restored the sensitivity of LTLT cells to letrozole. These results suggest that crosstalk occurs between ER and tyrosine kinase receptor signaling. Therefore, to investigate whether down-regulating ER would prevent activation of MAPK and resistance to letrozole, xenografts were treated with letrozole and faslodex in combination. Her-2 and MAPK were not increased and tumor growth was inhibited throughout 29 weeks of treatment. These results suggest that blocking both ER and growth factor mediated transcription may delay development of resistance to letrozole and maintain its growth inhibition of breast cancer.

Novel retinoic acid metabolism blocking agents have potent inhibitory activities on human breast cancer cells and tumour growth.

Antitumour effects of retinoids are attributed to their influence on cell proliferation, differentiation, apoptosis and angiogenesis. In our effort to develop useful agents for breast cancer therapy, we evaluated the effects of four representative retinoic acid metabolism blocking agents (RAMBAs, VN/14-1, VN/50-1, VN/66-1 and VN/69-1) on growth inhibition of oestrogen receptor positive (ER +ve, MCF-7 and T-47D) and oestrogen receptor negative (ER -ve, MDA-MB-231) human breast cancer cells. Additionally, we investigated the biological effects/molecular mechanism(s) underlying their growth inhibitory properties as well as their antitumour efficacies against MCF-7 and MCF-7Ca tumour xenografts in nude mice. We also assessed the effect of combining VN/14-1 and all-trans-retinoic acid (ATRA) on MCF-7 tumour xenografts. The ER +ve cell lines were more sensitive (IC(50) values between 3.0 and 609 nM) to the RAMBAs than the ER -ve MDA-MB-231 cell line (IC(50)=5.6-24.0 microM). Retinoic acid metabolism blocking agents induced cell differentiation as determined by increased expression of cytokeratin 8/18 and oestrogen receptor-alpha (ER-alpha). Similar to ATRA, they also induced apoptosis via activation of caspase 9. Cell cycle analysis indicated that RAMBAs arrested cells in the G1 and G2/M phases and caused significant downregulation (>80%) of cyclin D1 protein. In vivo, the growth of MCF-7 mammary tumours was dose-dependently and significantly inhibited (92.6%, P<0.0005) by VN/14-1. The combination of VN/14-1 and ATRA also inhibited MCF-7 breast tumour growth in vivo (up to 120%) as compared with single agents (P<0.025). VN/14-1 was also very effective in preventing the formation of MCF-7Ca tumours and it significantly inhibited the growth of established MCF-7Ca tumours, being as effective as the clinically used aromatase inhibitors, anastrozole and letrozole. Decrease in cyclin D1 and upregulation of cytokeratins, Bad and Bax with VN/14-1 may be responsible for the efficacy of this compound in inhibiting breast cancer cell growth in vitro and in vivo. Our results suggest that our RAMBAs, especially VN/14-1 may be useful novel therapy for breast cancer.

Aromatase and breast cancer.

Several aromatase inhibitors and also new antiestrogens are now available for treating breast cancer. We have developed a model to compare the antitumor efficacy of these agents and to explore strategies for their optimal use. Results from the model have been predictive of clinical outcome. In this model, tumors are grown in ovariectomized, immunodeficient mice from MCF-7 human breast cancer cells transfected with the aromatase gene (MCF-7Ca). The possibility that blockade of estrogen action and estrogen synthesis may be synergistic was explored by treating mice with the aromatase inhibitor letrozole and the antiestrogen tamoxifen alone and in combination. The results indicated that letrozole alone was better than all other treatments. In addition, when tamoxifen treatment was no longer effective, tumor growth was significantly reduced in mice switched to letrozole treatment. However, tumors ultimately began to grow during continued treatment. To investigate the mechanisms by which tumors eventually adapt and grow during letrozole treatment, we determined the expression of signaling proteins in tumors during the course of letrozole treatment compared to the tumors of control mice. Tumors initially up-regulated the ER while responding to treatment, but subsequently receptor levels decreased in tumors unresponsive to letrozole. Also, Her-2 and adapter proteins (p-Shc and Grb-2) as well as all of the signaling proteins in the MAPK cascade (p-Raf, p-Mekl/2, and p-MAPK), but not in the Pl3/Akt pathway, were increased in tumors no longer responsive to letrozole. To investigate whether sensitivity to letrozole could be regained, cells were isolated from the letrozole resistant tumors (LTLT) and treated with inhibitors of the MAPKinase pathway (PD98059 and UO126). These compounds reduced MAPK activity and increased ER expression. EGFR/Her-2 inhibitors, gefitinib and AEE78S although not effective in the parental MCF-70a cells, restored the sensitivity of LTLT cells to letrozole. In xenografts, beginning treatment with letrozole and faslodex to down regulate the ER prevented increases in Her-2 and activation of MAPK and was highly effective in inhibiting tumor growth throughout 29 weeks of treatment. These results suggest that blocking both ER- and growth factor-mediated transcription may delay development of resistance and maintain growth inhibition of ER+ breast cancer.

The effect of tamoxifen on the pharmacokinetics of letrozole in female rats.

The effects of single doses of tamoxifen (TAM; 0.5-5 mg/kg, i.v.) and chronic pretreatment with TAM (0.1-5.0 mg/kg/day, i.p. for 7 consecutive days) on letrozole (0.5 mg/kg, i.v.) pharmacokinetics were evaluated in female Sprague-Dawley rats. The plasma concentration-time profiles of letrozole (0.1-2.0 mg/kg) after single i.v. doses were analysed by the non-compartment model with terminal half-lives (t(1/2,lambdaz)) ranging from 34.3 to 37.5 h. The volume of distribution at the terminal phases (Vd(lambdaz)) ranged from 1.9 to 2.1 l/kg and clearance (CL) varied from 0.036 to 0.042 l/(h.kg). After co-administration of TAM and letrozole intravenously, the t1/2, Vd(lambdaz) and CL of letrozole were not significantly altered. Chronic pretreatment with TAM significantly decreased the t1/2 of letrozole by about 33%, and increased its clearance by an average of 40%. However, TAM pretreatment did not significantly affect the Vd(lambdaz) of letrozole in female rats. Co-administration of letrozole and TAM orally increased the absorption half-life of letrozole threefold although the absolute bioavailability remained unchanged. These observations suggest that single oral doses of TAM delay the absorption of letrozole while chronic pretreatment with TAM accelerates the elimination of letrozole, probably due to induction of cytochrome P450 enzymes in rats.

Inhibitory effects of retinoic acid metabolism blocking agents (RAMBAs) on the growth of human prostate cancer cells and LNCaP prostate tumour xenografts in SCID mice.

In recent studies, we have identified several highly potent all-trans-retinoic acid (ATRA) metabolism blocking agents (RAMBAs). On the basis of previous effects of liarozole (a first-generation RAMBA) on the catabolism of ATRA and on growth of rat Dunning R3227G prostate tumours, we assessed the effects of our novel RAMBAs on human prostate tumour (PCA) cell lines. We examined three different PCA cell lines to determine their capacity to induce P450-mediated oxidation of ATRA. Among the three different cell lines, enhanced catabolism was detected in LNCaP, whereas it was not found in PC-3 and DU-145. This catabolism was strongly inhibited by our RAMBAs, the most potent being VN/14-1, VN/50-1, VN/66-1, and VN/69-1 with IC50 values of 6.5, 90.0, 62.5, and 90.0 nM, respectively. The RAMBAs inhibited the growth of LNCaP cells with IC50 values in the microM-range. In LNCaP cell proliferation assays, VN/14-1, VN/50-1, VN/66-1, and VN/69-1 also enhanced by 47-, 60-, 70-, and 65-fold, respectively, the ATRA-mediated antiproliferative activity. We then examined the molecular mechanism underlying the growth inhibitory properties of ATRA alone and in combination with RAMBAs. The mechanism appeared to involve the induction of differentiation, cell-cycle arrest, and induction of apoptosis (TUNEL), involving increase in Bad expression and decrease in Bcl-2 expression. Treatment of LNCaP tumours growing in SCID mice with VN/66-1 and VN/69-1 resulted in modest but statistically significant tumour growth inhibition of 44 and 47%, respectively, while treatment with VN/14-1 was unexpectedly ineffective. These results suggest that some of our novel RAMBAs may be useful agents for the treatment of prostate cancer.

Biochemical assessment of limits to estrogen synthesis in porcine follicles.

Limits to estrogen production by early and late preovulatory porcine follicles were assessed by comparing enzymatic capacities for androgen (17,20-lyase) and estrogen (aromatase) synthesis in theca interna and granulosa, support of enzyme activities by the redox partner proteins NADPH-cytochrome P450 oxidoreductase (reductase) and cytochrome b5, and tissue-specific expression and regulation of these proteins. Parameters included follicular fluid (FF) estradiol and progesterone levels, theca and granulosa aromatase and reductase activities, and theca 17,20-lyase activity. Expression of proteins responsible for these activities, aromatase (P450arom) and 17 alpha-hydroxylase/17,20-lyase (P450c17) cytochromes P450, reductase, and for the first time in ovarian tissues cytochrome b5, were examined by Western immunoblot and immunocytochemistry. Theca and granulosa aromatase activities were as much as 100-fold lower than theca 17,20-lyase activity, but aromatase was correlated with only the log of FF estradiol. Granulosa reductase activity was twice that of the theca, and cytochrome b5 expression was clearly identified in both the theca and granulosa layers, as was P450arom, but was not highly correlated with either 17,20-lyase or aromatase activities. Reductase expression did not change with stage of follicular development, but cytochrome b5, P450c17, and P450arom were markedly lower in post-LH tissues. These data indicate that aromatase and not 17,20-lyase must limit porcine follicular estradiol synthesis, but this limitation is not reflected acutely in FF steroid concentrations. Neither reductase nor cytochrome b5 appear to regulate P450 activities, but the expression of cytochrome b5 in granulosa and theca suggests possible alternative roles for this protein in follicular development or function.

Regulation of microsomal P450, redox partner proteins, and steroidogenesis in the developing testes of the neonatal pig.

Testicular growth and plasma androgen concentrations increase markedly in the first weeks of neonatal life of pigs. The regulation of steroidogenesis through this period was examined by measuring total microsomal cytochromes P450 (P450), 17alpha-hydroxylase/17,20-lyase P450 (P450c17) and aromatase P450 (P450arom) enzyme activities, and the redox partner proteins nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-cytochrome P450 reductase (reductase) and cytochrome b(5) in testicular microsomes. Testes were collected from 1-24 d of age, and testicular development was suppressed by a GnRH antagonist in some animals from d 1-14. Both 17/20-lyase and aromatase activities increased from d 1-7 but not thereafter, and 17-20-lyase activity was always at least 200-fold higher than aromatase activity. Reductase decreased in wk 1, then increased to d 24. No changes were seen in cytochrome b(5) expression. GnRH antagonist treatment suppressed plasma LH, testosterone and testes growth to d 14. 17,20-Lyase and aromatase activities in testicular microsomes were reduced by 20% and 50%, respectively. Total microsomal P450 concentration was reduced by 50% on d 7, but there was no effect of treatment on reductase or cytochrome b(5) expression. These data support the hypothesis that the rise in neonatal testicular androgen secretion is more likely due to gonadotropin-stimulated gonadal growth, rather than specific P450c17 expression. Neither P450c17 nor P450arom can account for the decline in total microsomal P450. Reductase and cytochrome b(5) expression appears to be constitutive, but reductase levels saturate both P450c17 and P450arom.

Pharmacokinetic profile of 3beta-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (VN/87-1), a potent androgen synthesis inhibitor, in mice.

The objective of this study was to assess the pharmacokinetics and bioavailability of 3beta-hydroxy-17-(1H-1,2,3-triazol-1-yl)androsta-5,16-diene (VN/87-1) in normal male mice and in SCID mice bearing human LNCaP tumor xenografts. VN/87-1 is a novel potent steroidal inhibitor of human testicular 17-alpha-hydroxylase/C(17,20)-lyase. The steroid also shows anti-androgenic activity and inhibits the growth of human prostate cancer cell lines (LNCaP) in vitro and in vivo. Male Balb/c mice were given a single oral, subcutaneous (s.c.) or intravenous (i.v.) bolus dose of VN/87-1 (25, 50 or 100 mg/kg). Male SCID mice bearing LNCaP tumor xenografts were injected with a single s.c. dose of VN/87-1 (50 mg/kg). The animals were sacrificed at various times up to 24 h after drug administration and blood was collected. The plasma samples were prepared and analyzed by a reversed phase HPLC system equipped with a diode array detector. A non-compartmental pharmacokinetic approach was used to evaluate the plasma level versus time data. Following i.v. administration of VN/87-1, the plasma levels declined exponentially with an elimination half-life of 1.2+/-0.03 h. The absolute bioavailability of the 50 mg/kg dose after oral or s.c. administration was 12.08+/-2 or 57.2+/-4.5%, respectively. VN/87-1 is a high clearance (5.0+/-1.3 l/h per kg) compound in mice and its volume of distribution was relatively large (6.5+/-1.2 l/kg). The pharmacokinetic parameters of VN/87-1 were not significantly altered in SCID mice bearing human LNCaP tumor xenografts. VN/87-1 is well absorbed from the subcutaneous site compared with absorption from the gastrointestinal tract and shows linear kinetics at doses up to 100 mg/kg.