PIK3CA dependence and sensitivity to therapeutic targeting in urothelial carcinoma.

BACKGROUND: Many urothelial carcinomas (UC) contain activating PIK3CA mutations. In telomerase-immortalized normal urothelial cells (TERT-NHUC), ectopic expression of mutant PIK3CA induces PI3K pathway activation, cell proliferation and cell migration. However, it is not clear whether advanced UC tumors are PIK3CA-dependent and whether PI3K pathway inhibition is a good therapeutic option in such cases. METHODS: We used retrovirus-mediated delivery of shRNA to knock down mutant PIK3CA in UC cell lines and assessed effects on pathway activation, cell proliferation, migration and tumorigenicity. The effect of the class I PI3K inhibitor GDC-0941 was assessed in a panel of UC cell lines with a range of known molecular alterations in the PI3K pathway. RESULTS: Specific knockdown of PIK3CA inhibited proliferation, migration, anchorage-independent growth and in vivo tumor growth of cells with PIK3CA mutations. Sensitivity to GDC-0941 was dependent on hotspot PIK3CA mutation status. Cells with rare PIK3CA mutations and co-occurring TSC1 or PTEN mutations were less sensitive. Furthermore, downstream PI3K pathway alterations in TSC1 or PTEN or co-occurring AKT1 and RAS gene mutations were associated with GDC-0941 resistance. CONCLUSIONS: Mutant PIK3CA is a potent oncogenic driver in many UC cell lines and may represent a valuable therapeutic target in advanced bladder cancer.

Clot structure and fibrinolytic potential in patients with post thrombotic syndrome.

INTRODUCTION: Post-thrombotic syndrome (PTS) is a chronic sequel of deep vein thrombosis (DVT). The clot structure and fibrinolytic potential in PTS is currently unknown. OBJECTIVE: To assess the fibrinolytic potential and clot structure in patients with PTS. MATERIALS AND METHODS: Patients with a history of DVT were included in a case-control study: patients with PTS (cases n=30) and without PTS (controls n=30), and 30 apparently healthy individuals (HI) without venous thromboembolism (VTE) or venous insufficiency were enrolled. Fibrinolysis and clot structure were assessed by turbidimetric assays, permeation, and confocal microscopy. Fibrinogen was measured by Clauss and fibrinogen γ' by ELISA. RESULTS: We observed a significant trend of decreasing maximum turbidity from HI (median 0.52 [IQR 0.46-0.62]), to controls (0.49 [IQR 0.41-0.55]), to cases (0.46 [IQR 0.39-0.49]) p=0.020. Fibrinogen was lower in patients (cases and controls) (3.69g/L [IQR 3.31-4.26]) compared to HI (4.17 [IQR 3.69-4.65]) p=0.041. Patients with recurrent VTE had lower maximum turbidity and lower permeation than patients with one episode of VTE; (0.31 [IQR 0.25-0.39] versus 0.38 [IQR 0.34-0.44] p=0.008) and (6.0×10(-9)/cm(2) [IQR 5.1-7.9] versus 7.7×10(-9)/cm(2) [IQR 6.0-10.0] p=0.047) respectively, at equal fibrinogen levels. There were no differences in lysis time, confocal microscopy, or fibrinogen γ'. CONCLUSIONS: Lower maximum turbidity, indicating a tendency towards thinner fibres and denser clots, was found in patients with PTS as well as in patients with recurrent VTE. Fibrinogen levels did not explain these differences in clot structure. The abnormal clot structure may contribute to the increased thrombotic risk profile in patients with PTS.

Next-Gen phylogeography of rainforest trees: exploring landscape-level cpDNA variation from whole-genome sequencing.

Standardized phylogeographic studies across codistributed taxa can identify important refugia and biogeographic barriers, and potentially uncover how changes in adaptive constraints through space and time impact on the distribution of genetic diversity. The combination of next-generation sequencing and methodologies that enable uncomplicated analysis of the full chloroplast genome may provide an invaluable resource for such studies. Here, we assess the potential of a shotgun-based method across twelve nonmodel rainforest trees sampled from two evolutionary distinct regions. Whole genomic shotgun sequencing libraries consisting of pooled individuals were used to assemble species-specific chloroplast references (in silicio). For each species, the pooled libraries allowed for the detection of variation within and between data sets (each representing a geographic region). The potential use of nuclear rDNA as an additional marker from the NGS libraries was investigated by mapping reads against available references. We successfully obtained phylogeographically informative sequence data from a range of previously unstudied rainforest trees. Greater levels of diversity were found in northern refugial rainforests than in southern expansion areas. The genetic signatures of varying evolutionary histories were detected, and interesting associative patterns between functional characteristics and genetic diversity were identified. This approach can suit a wide range of landscape-level studies. As the key laboratory-based steps do not require prior species-specific knowledge and can be easily outsourced, the techniques described here are even suitable for researchers without access to wet-laboratory facilities, making evolutionary ecology questions increasingly accessible to the research community.

Torcetrapib-induced blood pressure elevation is independent of CETP inhibition and is accompanied by increased circulating levels of aldosterone.

BACKGROUND AND PURPOSE: Inhibition of cholesteryl ester transfer protein (CETP) with torcetrapib in humans increases plasma high density lipoprotein (HDL) cholesterol levels but is associated with increased blood pressure. In a phase 3 clinical study, evaluating the effects of torcetrapib in atherosclerosis, there was an excess of deaths and adverse cardiovascular events in patients taking torcetrapib. The studies reported herein sought to evaluate off-target effects of torcetrapib. EXPERIMENTAL APPROACH: Cardiovascular effects of the CETP inhibitors torcetrapib and anacetrapib were evaluated in animal models. KEY RESULTS: Torcetrapib evoked an acute increase in blood pressure in all species evaluated whereas no increase was observed with anacetrapib. The pressor effect of torcetrapib was not diminished in the presence of adrenoceptor, angiotensin II or endothelin receptor antagonists. Torcetrapib did not have a contractile effect on vascular smooth muscle suggesting its effects in vivo are via the release of a secondary mediator. Treatment with torcetrapib was associated with an increase in plasma levels of aldosterone and corticosterone and, in vitro, was shown to release aldosterone from adrenocortical cells. Increased adrenal steroid levels were not observed with anacetrapib. Inhibition of adrenal steroid synthesis did not inhibit the pressor response to torcetrapib whereas adrenalectomy prevented the ability of torcetrapib to increase blood pressure in rats. CONCLUSIONS AND IMPLICATIONS: Torcetrapib evoked an acute increase in blood pressure and an acute increase in plasma adrenal steroids. The acute pressor response to torcetrapib was not mediated by adrenal steroids but was dependent on intact adrenal glands.

Cardiovascular monkey telemetry: sensitivity to detect QT interval prolongation.

INTRODUCTION: Preclinical evaluation of delayed ventricular repolarization manifests electrocardiographically as QT interval prolongation and is routinely used as an indicator of potential risk for pro-arrhythmia (potential to cause Torsades de Pointes) of novel human pharmaceuticals. In accordance with ICH S7A and S7B guidelines we evaluated the sensitivity and validity of the monkey telemetry model as a preclinical predictor of QT interval prolongation in humans. METHODS: Cardiovascular monitoring was conducted for 2 h pre-dose and 24 h post-dosing with Moxifloxacin (MOX), with a toxicokinetic (TK) evaluation in a separate group of monkeys. In both studies, MOX was administered orally by gavage in 0.5% methylcellulose at 0, 10, 30, 100, 175 mg/kg. Each monkey received all 5 doses using a dose-escalation paradigm. Inherent variability of the model was assessed with administration of vehicle alone for 4 days in all 4 monkeys (0.5% methylcellulose in deionized water). RESULTS: MOX had no significant effect on mean arterial pressure, heart rate, PR or QRS intervals. MOX produced significant dose-related increases in QTc at doses of 30 (Cmax=5.5+/-0.6 microM), 100 (Cmax=16.5+/-1.6 microM), and 175 (Cmax=17.3+/-0.7 microM) mg/kg with peak increases of 22 (8%), 27 (10%), and 47 (18%) ms, respectively (p

Providing culturally sensitive care to Hispanic patients in Arkansas.

Providing culturally sensitive care to Hispanic patients can lead to a better patient-physician relationship. Greater compliance, improved health care delivery and treatment efficacy also are enhanced when health care providers understand the Hispanic patient's expectation of care and health paradigm. An inability to understand the Hispanic patient's health beliefs, social system and barriers to health care may lead to a communication breakdown and compromise maximal delivery of care.

How effective is lithium in the prevention of relapse in bipolar disorder? A prospective naturalistic follow-up study.

OBJECTIVE: The effectiveness of lithium in preventing recurrences of bipolar disorder was examined prospectively for 2 years in two representative samples of bipolar I patients being treated in a comprehensive program following recommended guidelines. METHOD: One hundred and twenty patients were recruited from consecutive admissions to two catchment area psychiatric services (one in the United Kingdom, the other in New Zealand). They were seen at 3-monthly intervals by a member of the research team. Treatment was adjusted according to clinical needs. RESULTS: Overall, two-thirds of the patients had a recurrence. Of the 57 on lithium as sole treatment, 39 (68%) had a further episode; 17 after stopping lithium. The 42 on other mood stabilisers and/or an antipsychotic, with or without lithium, did no better. By contrast, only eight (38%) of the 21 who were taking no prophylactic medication had a recurrence. CONCLUSIONS: Lithium is much less effective in clinical practice than would be expected from clinical trial results. A major reason for this is poor compliance. Alternative treatment strategies are needed to improve the outcome for bipolar disorder patients.

Is there a seasonal pattern of relapse in bipolar affective disorders? A dual northern and southern hemisphere cohort study.

BACKGROUND: Admission statistics for mania frequently show an increase in the summer. The present two-centre study was designed to test the hypothesis, in a representative sample of bipolar patients, that manic and depressive relapses show a seasonal pattern. METHOD: Two cohorts of bipolar I patients, one in London, England (n = 86), the other in Dunedin, New Zealand (n = 58), were tracked retrospectively during 1985-88 and prospectively during 1989-91, with the onset of all relapses being carefully dated. RESULTS: In the London cohort there were 221 episodes of mania and 76 of depression; in the Dunedin cohort there were 201 of mania and 61 of depression. No consistent seasonal pattern of mania was detected in either centre. There was an autumn preponderance of depressive episodes in both centres. CONCLUSIONS: Relapse of bipolar depression, but not of mania, appears to be determined in part by seasonal factors.

Life events and relapse in established bipolar affective disorder.

A New Zealand cohort of 58 patients with bipolar affective disorder was studied prospectively with three-monthly interviews in order to determine the relationship between life events and their relapses. Careful attention was paid to dating life events and the earliest signs of relapse and to assessing the independence of life events from the illness. No statistically significant association was found between life events and the likelihood of relapses, either mania or depression, for the 71% of patients who experienced at least one relapse during the two-year study. This finding is at variance with a companion study, with identical methodology, which found a small increase of life events before relapse. These data add further weight to the previous reports that life events are significant precipitants of bipolar illness only for earlier episodes in the course of this chronic disorder.

The social networks of bipolar affective disorder patients.

The social interaction parameters of a carefully delineated group of bipolar patients were compared to those of a random New Zealand community sample. The bipolar subjects had significantly lower scores for perceived availability and adequacy of both intimate and diffuse social relationships. Their mean scores did not differ from those of the subgroup in the random community survey who were classified as showing psychiatric morbidity, most of whom were depressed. Social interaction scores were negatively correlated with the bipolar patients' age and duration of illness. Those bipolar subjects with a predominance of manic episodes had lower mean values for their social interaction indices than those with more depressions. The results may suggest that the longer the illness continues, the greater is the impoverishment of the sufferer's social interaction patterns. Overall, manic episodes appeared to have a more deleterious effect on social relationships than depressive episodes.

Socioeconomic characteristics of a Dunedin sample of bipolar patients.

In order to determine the sociodemographic characteristics of people suffering from bipolar affective disorder, a cohort of hospitalised adult patients was compared with the general New Zealand population. A review of the casenotes of all those treated in Dunedin's psychiatric inpatient facilities between 1 January 1985 and 31 December 1987 identified 50 women and 41 men. Although their age structure did not differ from the general population, more bipolar patients were unmarried, either never married or divorced. These hospitalised bipolar patients held more secondary school qualifications than the average New Zealander but their subsequent employment record was statistically poorer. A majority were social welfare beneficiaries. Often thought to be a relatively benign condition, bipolar affective disorder has serious and destructive influences on marital and occupational function.

Synthesis of 3-deazaneplanocin A, a powerful inhibitor of S-adenosylhomocysteine hydrolase with potent and selective in vitro and in vivo antiviral activities.

The neplanocin A analogue 3-deazaneplanocin A (2b) has been synthesized. A direct SN2 displacement on the cyclopentenyl mesylate 3 by the sodium salt of 6-chloro-3-deazapurine afforded the desired regioisomer 4 as the major product. After deprotection, this material was converted to 3-deazaneplanocin A in two steps. X-ray crystallographic analysis confirmed the assigned structure. Consistent with its potent inhibition of S-adenosylhomocysteine hydrolase, 3-deazaneplanocin A displayed excellent antiviral activity in cell culture against vesicular stomatitis, parainfluenza type 3, yellow fever, and vaccinia viruses. Antiviral activity was also displayed in vivo against vaccinia virus by using a mouse tailpox assay. The significantly lower cytotoxicity of 3-deazaneplanocin A, relative to its parent compound neplanocin A, may be due to its lack of conversion to 5'-triphosphate and S-adenosylmethionine metabolites.

Synthesis and murine antineoplastic activity of bis[(carbamoyloxy)methyl] derivatives of pyrrolo[2,1-a]isoquinoline.

The synthesis of 4,5-dihydropyrrolo[2,1-a]isoquinolines is reported. A key intermediate in the synthesis of 8-methoxy-4,5-dihydropyrrolo[2,1-a]isoquinolines, 6-hydroxy-1,2,3,4-tetrahydroisoquinoline-1-carboxylic acid (6), was prepared by using a regiospecific phenolic cyclization reaction. The P388 lymphocytic activity is reported for 1,2-bis(hydroxymethyl)-5,6-dihydro-8-methoxy-3-methylpyrrolo [2,1-a]isoquinoline bis(isopropylcarbamate) (11a), 1,2-bis(hydroxymethyl)-5,6-dihydro-8-methoxy-3-methylpyrrolo[2,1-a ]isoquinoline bis(cyclohexylcarbamate) (11b), 1,2-bis(hydroxymethyl)-5,6-dihydro-3-methylpyrrolo[2,1-a]isoqui nol ine bis(methylcarbamate) (13a), 1,2-bis(hydroxymethyl)-5,6-dihydro-3-methylpyrrolo [2,1-a]isoquinoline bis(ethylcarbamate) (13b), and 1,2-bis(hydroxymethyl)-5,6-dihydroxy-3-methylpyrrolo[2,1-a]isoq uin oline bis(cyclohexylcarbamate) (13c); all of the compounds were active. Compound 11a was tested in an expanded tumor panel and was shown to be active against B16 melanocarcinoma, CD8F1 mammary, L1210 lymphoid leukemia, colon 38, and MX-1 human tumor breast xenograft systems.

Synthesis, evaluation of chemical reactivity, and murine antineoplastic activity of 2-hydroxy-5-(3,4-dichlorophenyl)-6,7-bis(hydroxymethyl)-2,3-dihydro-1H- pyrrolizine bis(2-propylcarbamate) and 2-acyloxy derivatives as potential water-soluble prodrugs.

2-Hydroxy-5-(3,4-dichlorophenyl)-6,7-bis(hydroxymethyl)-2,3-dihydro-1H- pyrrolizine bis(2-propylcarbamate) (11) was prepared in a multistep synthesis. The 2-hydroxy group was used to prepare ester prodrugs 14 and 15, and the antineoplastic activities of 11, 14, and 15a were compared to 1 (the 2-deoxy analogue of 11) in murine P388 lymphocytic leukemia and B16 melanocarcinoma. The alcohol 11 showed comparable activity to 1, while 14 was less active and 15a showed very low activity. The hydrolytic rates of 1, 11, 14, 15a, and 15b were compared, and it was found that the two carbamate moieties were much more susceptible toward hydrolysis than the C-2 esters. The salts 15a and 15b exhibited good water solubility, 3.0 X 10(-2) and 3.88 X 10(-2) M, respectively.

Vinylogous carbinolamine tumor inhibitors. 8. Activity of bis(acyloxymethyl) derivatives of pyrroles and pyrrolizines against a panel of murine leukemias and solid tumors.

The activity of three pyrroles and four pyrrolizines is compared in several different experimental leukemias and solid tumors in mice. Two compounds were particularly noteworthy, the bis(N-cyclohexylcarbamate) and the bis[N-(2-propyl)] derivatives of 2,3-dihydro-5-(3,4-dichlorophenyl)-6,7-bis(hydroxymethyl)-1H-pyrrolizine. These two compounds showed a very high level of activity against B16 melanocarcinoma, CD8F1 mammary tumor, colon tumor 26, and colon tumor 38, and a significant number of "cures" were recorded. The isopropyl compound was more potent than the cyclohexyl compound, but both showed a similar profile of activity.