Evaluating the toxicity of estetrol, 17α-ethinylestradiol, and their combination with drospirenone on zebrafish larvae: A behavioural and proteomic study.

OBJECTIVE: To characterise and compare the toxicity of estetrol (E4) and 17α-ethinylestradiol (EE2), and their respective mixture with the progestin drospirenone (DRSP) in zebrafish (Danio rerio) embryos. METHODS: Zebrafish embryos were exposed to E4, EE2, DRSP, E4+DRSP, and EE2+DRSP in a fish embryo acute toxicity (FET) test. A second test examined behavioural responses and, using label-free proteomics, identified changes in protein expression in response to hormonal treatments, across a range of concentrations, including those that are considered to be environmentally relevant. RESULTS: In the FET test, no effects were found from E4 at concentrations ≤100 mg/L, while EE2 induced mortality and morphological abnormalities at concentrations of 1-2 mg/L. In the behavioural test, exposure to 30 ng/L EE2 (∼200 × predicted environmental concentration - PEC) resulted in hypoactivity in fish larvae and exposure to 0.3 ng/L EE2 (∼2 × PEC) led to quantitative changes in protein abundance, revealing potential impacts on RNA processing and protein synthesis machinery. Exposure to E4 did not alter behaviour, but several groups of proteins were modulated, mainly at 710 ng/L (∼200 × PEC), including proteins involved in oxidative phosphorylation. When combined with DRSP, EE2 induced reduced effects on behaviour and proteomic responses, suggesting an antagonistic effect of DRSP. E4+DRSP induced no significant effects on behaviour or proteomic profiles at tested concentrations. CONCLUSIONS: These findings suggest that E4-based combined oral contraceptives present a more favourable environmental profile than EE2-based contraceptives, particularly during the early developmental stages of fish.

Estetrol/drospirenone versus 17α-ethinylestradiol/drospirenone: An extended one generation test to evaluate the endocrine disruption potential on zebrafish (Danio rerio).

Combined oral contraceptives, comprising of both an oestrogen and a progestin component, are released in aquatic environments and potentially pose a risk to aquatic wildlife by their capacity to disrupt physiological mechanisms. In this study, the endocrine disruptive potential of two mixtures, 17α-ethinylestradiol (EE2), a synthetic oestrogen, or estetrol (E4), a natural oestrogen, with the progestin drospirenone (DRSP) have been characterised in three generations of zebrafish, according to an adapted Medaka Extended One Generation Reproduction Test. Zebrafish (Danio rerio) were exposed to a range of concentrations of EE2/DRSP and E4/DRSP (∼1×, ∼3×, ∼10× and ∼30× predicted environmental concentration, PEC). Survival, growth, hatching success, fecundity, fertilisation success, vitellogenin (VTG), gonad histopathology, sex differentiation, and transcriptional analysis of genes related to gonadal sex steroid hormones synthesis were assessed. In the F0 generation, exposure to EE2/DRSP at ∼10 and ∼30× PEC decreased fecundity and increased male VTG concentrations. The highest concentration of EE2/DRSP also affected VTG concentrations in female zebrafish and the expression of genes implicated in steroid hormones synthesis. In the F1 generation, sex determination was impaired in fish exposed to EE2/DRSP at concentrations as low as ∼3× PEC. Decreased fecundity and fertility, and abnormal gonadal histopathology were also observed. No effects were observed in the F2 generation. In contrast, E4/DRSP induced only minor histopathological changes and an increase in the proportion of males, at the highest concentration tested (∼30× PEC) in the F1 generation and had no effect on hatching success of F2 generation. Overall, this study suggests that the combination E4/DRSP has a more favourable environmental profile than EE2/DRSP.

Chronic simultaneous exposure of common carp (Cyprinus carpio) from embryonic to juvenile stage to drospirenone and gestodene at low ng/L level caused intersex.

Synthetic progestins are emerging contaminants of the aquatic environment with endocrine disrupting potential. The main aim of the present study was to investigate the effects of the synthetic progestins gestodene, and drospirenone on sex differentiation in common carp (Cyprinus carpio) by histological analysis. To gain insights into the mechanisms behind the observations from the in vivo experiment on sex differentiation, we analyzed expression of genes involved in hypothalamus-pituitary-gonad (HPG) and hypothalamus-pituitary-thyroid (HPT) axes, histology of hepatopancreas, and in vitro bioassays. Carp were continuously exposed to concentrations of 2 ng/L of single progestins (gestodene or drospirenone) or to their mixture at concentration 2 ng/L of each. The exposure started 24 h after fertilization of eggs and concluded 160 days post-hatching. Our results showed that exposure of common carp to a binary mixture of drospirenone and gestodene caused increased incidence of intersex (32%) when compared to clean water and solvent control groups (both 3%). Intersex most probably was induced by a combination of multiple modes of action of the studied substances, namely anti-gonadotropic activity, interference with androgen receptor, and potentially also with HPT axis or estrogen receptor.

Sex steroids and metabolic responses in mussels Mytilus galloprovincialis exposed to drospirenone.

Drospirenone (DRO) is a synthetic progestin derived from 17α-spironolactone with a pharmacological mechanism of action similar to progesterone. Despite its wide use as pharmaceutical and consequent continuous release into the aquatic environment, DRO effects have been poorly investigated on aquatic biota. In order to unravel the toxicity mechanisms of DRO, mussels Mytilus galloprovincialis were exposed for 7 days to different concentrations of DRO, namely 20ng/L (Low; L), 200ng/L (Medium; M), 2000ng/L (High; H) and 10µg/L (Super High; SH) nominal doses. Following exposure, no significant effect was observed on gonad maturation of treated and untreated mussels. The levels of progesterone (P4) and testosterone (T) were measured in mantle/gonad tissues and no significant alteration detected after exposure. However, the application of a protonic nuclear magnetic resonance (1H NMR)-based metabolomics approach enabled a comprehensive assessment of DRO effects in mussels. Specifically, 1H NMR metabolic fingerprints of digestive glands of DRO treated mussel groups were clearly separated from each other and from controls through a principal component analysis (PCA). Moreover, a number of metabolites involved in different metabolic pathways were found to significantly change in DRO-exposed mussels compared to control, suggesting the occurrence of alterations in energy metabolism, amino acids metabolism, and glycerophospholipid metabolism. Overall, despite no changes in gonad maturation and steroids levels were recorded in mussels after DRO exposure, the metabolomics approach demonstrated its effectiveness and high sensitivity in elucidating DRO-induced metabolic disturbances in marine mussels, and thus its usefulness in the environmental risk assessment of pharmaceuticals.

Synthesis of androstene oxime-nitrogen mustard bioconjugates as potent antineoplastic agents.

In the present study, synthesis and antineoplastic activity of phenylacetic acid and benzoic acid nitrogen mustard conjugates of various steroidal oximes are reported for the first time. The conjugation was achieved through a more stable oxime-ester linkage and the resulting newly synthesized conjugates were evaluated in vitro on various human cancer cell lines for cytotoxicity. The extent of their alkylating activity was investigated by the in vitro colorimetric 4-(p-nitrobenzyl)pyridine (NBP) assay. The 17E-steroidal oxime-benzoic acid mustard ester 3ß-acetoxy-17E-[p-(N,N-bis(2-chloroethyl)amino)]benzoyloxyimino-androst-5-ene (8) emerged as the most potent conjugate having significant cytotoxicity on most of the NCI 60-cell lines. Outstanding growth inhibition was observed on the IGROV1 ovarian cancer cell line with GI50=0.937µM. In general, the D-ring derived androstene oxime-nitrogen mustard conjugates were found to possess better antineoplastic activity over a variety of cancer cells in comparison to those derived from other rings of the steroid skeleton.

Drospirenone intake alters plasmatic steroid levels and cyp17a1 expression in gonads of juvenile sea bass.

Drospirenone (DRO) is one of the most widely used progestins in contraceptive treatments and hormone replacement therapies. The pharmacokinetics and potential toxicological effects of DRO were investigated in juvenile sea bass (Dicentrarchus labrax) exposed through the diet (0.01-10 µg DRO/g) for up to 31 days. DRO was detected in the blood (4-27 ng/mL) of fish exposed to the highest concentration, with no significant bioaccumulation over time and no alteration of hepatic metabolizing enzymes, namely, CYP1A and CYP3A-catalysed activities and UDP-glucuronyltransferase (UGT). Pregnenolone (P5), progesterone (P4), 17α-hydroxyprogesterone (17P4), 17α-hydroxypregnenolone (17P5), androstenedione (AD) and testosterone (T) were determined in plasma and gene expression of cyp17a1, cyp19a1a and cyp11ß analysed by qRT-PCR in gonads. The significant increase in plasmatic levels of 17P5, 17P4 and AD detected after 31 days exposure to 10 ng DRO/g together with the increased expression of cyp17a1 in females evidence the ability of DRO to alter steroid synthesis at low intake concentrations (7 ng DRO/day). However, the potential consequences of this steroid shift for female reproduction remain to be investigated.

Activity of binary mixtures of drospirenone with progesterone and 17α-ethinylestradiol in vitro and in vivo.

Despite potential exposure of aquatic organisms to mixtures of steroid hormones, very little is known on their joint activity in fish. Drospirenone (DRS) is a new synthetic progestin used in contraceptive pills in combination with 17α-ethinylestradiol (EE2). Here we systematically analyzed effects of DRS in binary mixtures with progesterone (P4) and EE2. First, we determined the in vitro activity of single compounds in recombinant yeast assays that express the human progesterone, androgen, or estrogen receptor, followed by determination of mixture activities of DRS and P4, DRS and EE2, as well as medroxyprogesterone acetate (MPA) and dydrogesterone (DDG). Mixtures of DRS and P4, as well as of DRS and EE2 showed additive progestogenic and androgenic activities. However, DDG and MPA showed non-additive progestogenic and androgenic activities. We then analyzed the in vivo activity of single compounds and mixtures of DRS and P4, as well as DRS and EE2, by assessing transcriptional changes of up to 14 selected target genes in zebrafish embryos at 48h post fertilization (hpf), and in eleuthero-embryos at 96hpf and 144hpf. DRS, P4, and EE2 led to significant transcriptional alteration of genes, including those encoding hormone receptors (pgr, esr1), a steroidogenic enzyme (hsd17b3), and estrogenic markers (vtg1, cyp19b), in particular at 144 hpf. In general, DRS showed stronger transcriptional changes than P4. In mixtures of DRS and P4, they were mainly non-additive (antagonistic interaction). In mixtures of DRS and EE2, transcriptional responses of esr1, vtg1 and cyp19b were dominated by EE2, suggesting an antagonistic interaction or independent action. Equi-effective mixtures of DRS and EE2, based on progesterone receptor transcripts, showed antagonistic interactions. Our data suggest that interactions in mixtures assessed in vitro in recombinant yeast cannot be translated to the in vivo situation. The receptor-based responses did not correspond well to the transcriptional responses in embryos which are much more complex due to the interplay between hormonal pathways, receptor crosstalk, and hormonal feedback loops.

The Aminosteroid Derivative RM-133 Shows In Vitro and In Vivo Antitumor Activity in Human Ovarian and Pancreatic Cancers.

Ovarian and pancreatic cancers are two of the most aggressive and lethal cancers, whose management faces only limited therapeutic options. Typically, these tumors spread insidiously accompanied first with atypical symptoms, and usually shift to a drug resistance phenotype with the current pharmaceutical armamentarium. Thus, the development of new drugs acting via a different mechanism of action represents a clear priority. Herein, we are reporting for the first time that the aminosteroid derivative RM-133, developed in our laboratory, displays promising activity on two models of aggressive cancers, namely ovarian (OVCAR-3) and pancreatic (PANC-1) cancers. The IC50 value of RM-133 was 0.8 µM and 0.3 µM for OVCAR-3 and PANC-1 cell lines in culture, respectively. Based on pharmacokinetic studies on RM-133 using 11 different vehicles, we selected two main vehicles: aqueous 0.4% methylcellulose:ethanol (92:8) and sunflower oil:ethanol (92:8) for in vivo studies. Using subcutaneous injection of RM-133 with the methylcellulose-based vehicle, growth of PANC-1 tumors xenografted to nude mice was inhibited by 63%. Quite interestingly, RM-133 injected subcutaneously with the methylcellulose-based or sunflower-based vehicles reduced OVCAR-3 xenograft growth by 122% and 100%, respectively. After the end of RM-133 treatment using the methylcellulose-based vehicle, OVCAR-3 tumor growth inhibition was maintained for ≥ 1 week. RM-133 was also well tolerated in the whole animal, no apparent sign of toxicity having been detected in the xenograft studies.

Environmental Progestins Progesterone and Drospirenone Alter the Circadian Rhythm Network in Zebrafish (Danio rerio).

Progestins alter hormone homeostasis and may result in reproductive effects in humans and animals. Thus far, studies in fish have focused on the hypothalamic-pituitary-gonadal (HPG)-axis and reproduction, but other effects have little been investigated. Here we report that progesterone (P4) and drospirenone (DRS) interfere with regulation of the circadian rhythm in fish. Breeding pairs of adult zebrafish were exposed to P4 and DRS at concentrations between 7 and 13 650 ng/L for 21 days. Transcriptional analysis revealed significant and dose-dependent alterations of the circadian rhythm network in the brain with little effects in the gonads. Significant alterations of many target transcripts occurred even at environmental relevant concentrations of 7 ng/L P4 and at 99 ng/L DRS. They were fully consistent with the well-described circadian rhythm negative/positive feedback loops. Transcriptional alterations of the circadian rhythm network were correlated with those in the HPG-Liver-axis. Fecundity was decreased at 742 (P4) and 2763 (DRS) ng/L. Dose-dependent alterations in the circadian rhythm network were also observed in F1 eleuthero-embryos. Our results suggest a potential target of environmental progestins, the circadian rhythm network, in addition to the adverse reproductive effects. Forthcoming studies should show whether the transcriptional alterations in circadian rhythm translate into physiological effects.

Chemical synthesis, cytotoxicity, selectivity and bioavailability of 5α-androstane-3α,17ß-diol derivatives.

Aminosteroid derivatives represent a new family of compounds with promising antiproliferative activity over different cancer cell lines. Among all the aminosteroid derivatives synthesised in our laboratory, we have identified E-37P as one of the more potent when tested in vitro. Unfortunately, the pharmacokinetic properties of E-37P decrease its effectiveness when tested in vivo. To improve the bioavailability and increase the efficiency of aminosteroid E-37P, two series of analog compounds were synthesised by classic chemical synthesis, they were then characterized, and the concentration that inhibits 50% of cell proliferation (IC50) was determined on different cell lines. RM-133, a 5α-androstane-3α,17ß-diol derivative with a quinoline nucleus at the end of the piperazine-proline side-chain at position 2ß and an ethinyl at position 17α, showed very good antiproliferative activity among the five cancer cell lines studied (IC50=0.1, 0.1, 0.1, 2.0 and 1.1 µM for HL-60, MCF-7, T-47D, LNCaP and WEHI-3, respectively). Moreover, the plasmatic concentration of RM-133 at 3h, when injected subcutaneously in rats, was 2.3-fold higher than that of E-37P (151 vs 64.8 ng/mL). Furthermore, RM-133 weakly inhibited the two representative liver enzymes, CYP3A4 and CYP2D6, indicating a very low risk of drug-drug interactions. The cytotoxicity of RM-133 against normal cells was tested on peripheral blood lymphocytes (PBL) obtained from different donors and previously activated with phytohemagglutinin-L. PBL responded differently to treatment with RM-133, we observed a stimulation of cell proliferation and/or cytotoxicity in a dose-dependent manner. Based on these results, additional studies are currently underway to evaluate the selectivity of our lead compound against normal cell lines in a more detailed fashion.

Toxicity and bioconcentration of the pharmaceuticals moxifloxacin, rosuvastatin, and drospirenone to the unionid mussel Lampsilis siliquoidea.

Pharmaceuticals and personal care products (PPCPs) and their metabolites are continually released from wastewater treatment plants into the aquatic environment; however, their impact on aquatic biota is poorly understood. This study examined the toxicity and bioconcentration of three pharmaceuticals: moxifloxacin, rosuvastatin, and drospirenone to the unionid mussel Lampsilis siliquoidea. Effects of moxifloxacin and rosuvastatin were assessed through aqueous 21-d static-renewal tests using 2-year-old mussels, at 0.01, 0.1, 1, 10 and 100mg/L (nominal concentrations). Following exposure, survival, behavior, algal clearance rate, hemocyte viability and density, and glutathione S-transferase (GST) activity were assessed. In addition, the acute (48 h) toxicity of moxifloxacin (0-100mg/L) and drospirenone (0-3mg/L) to glochidia (larval mussels) were examined. In 21 day exposures (2-yr old mussels), there were no differences in survival, oxygen consumption, hemocyte density, or GST activity over the range of concentrations examined; however, the proportion of time mussels spent filtering, and consequently the algal clearance rate, decreased at the higher moxifloxacin and rosuvastatin concentrations. Bioconcentration factors (BCFs) ranged between 0.03 and 70 for moxifloxacin, and between 0 and 0.05 for rosuvastatin for exposures up to 100mg/L. The BCF for moxifloxacin at the highest exposure concentration was lower than that at the mid-level concentrations, likely due to decreased filtering activity at the higher exposure levels. The feeding rates declined and the amount of time the subadult mussels spent with their valves closed increased at the higher moxifloxacin and rosuvastatin exposures. Glochidia viability did not vary with exposure to drospirenone, but declined at the highest moxifloxacin concentration, resulting in an EC50 of 120 mg/L. Overall, observed sublethal and lethal effects occurred at concentrations which exceed expected environmental concentrations through aqueous exposure, suggesting a low risk to freshwater mussels from these particular PPCPs.

Transcriptional and physiological responses induced by binary mixtures of drospirenone and progesterone in zebrafish (Danio rerio).

Drospirenone (DRS) is a synthetic progestin increasingly used in oral contraceptives with similar effects to progesterone (P4). Wild fish are exposed to DRS and P4 through wastewater. However, the effects of DRS on fish, both as an individual compound and in mixtures, have not been extensively studied. Therefore, in this study, global gene expression profiles of ovary and brain of female zebrafish (Danio rerio) were characterized after exposure to 55, 553, and 5442 ng/L DRS for 14 days. The effects were then compared to the observed responses after exposure to mixtures of DRS and P4 (DRS+P4: 27 + 0.8, 277 + 8 and 3118 + 123 ng/L). Transcriptomics findings were related to the changes in vitellogenin protein concentrations in the blood, morphology, and histology of gonads. Multivariate analysis indicated tissue-, dose-, and treatment-dependent expression profiles. Genes involved in steroid hormone receptor activity and circadian rhythm were enriched in DRS and mixture groups, among other pathways. In mixtures, the magnitude of response was dose- and transcript-dependent, both at the molecular and physiological levels. Effects of DRS and P4 were additive for most of the investigated parameters and occurred at environmentally relevant concentrations. They may translate to adverse reproductive effects in fish.

Role of cathepsin D in U18666A-induced neuronal cell death: potential implication in Niemann-Pick type C disease pathogenesis.

Cathepsin D is an aspartyl protease that plays a crucial role in normal cellular functions and in a variety of neurodegenerative disorders, including Niemann-Pick type C (NPC) disease, which is characterized by intracellular accumulation of cholesterol and glycosphingolipids in many tissues, including the brain. There is evidence that the level and activity of cathepsin D increased markedly in vulnerable neurons in NPC pathology, but its involvement in neurodegeneration remains unclear. In the present study, using mouse hippocampal cultured neurons, we evaluated the significance of cathepsin D in toxicity induced by U18666A, a class II amphiphile, which triggers cell death by impairing the trafficking of cholesterol, as observed in NPC pathology. Our results showed that U18666A-mediated toxicity is accompanied by an increase in cathepsin D mRNA and enzyme activity but a decrease in the total peptide content. The cytosolic level of cathepsin D, on the other hand, was increased along with cytochrome c and activated caspase-3 in U18666A-treated neurons. The cathepsin D inhibitor, pepstatin A, partially protected neurons against toxicity by attenuating these signaling mechanisms. Additionally, down-regulation of cathepsin D level prevented, whereas overexpression of the protease increased, vulnerability of cultured N2a cells to U18666A-induced toxicity. We also showed that extracellular cathepsin D from U18666A-treated neurons or application of exogenous enzyme can induce neurotoxicity by activating the autophagic pathway. These results suggest that increased release/activation of cathepsin D can trigger neurodegeneration and possibly development of NPC pathology. Thus, targeting cathepsin D level/activity may provide a new therapeutic opportunity for the treatment of NPC pathology.

Viridin analogs derived from steroidal building blocks.

Naturally occurring furanosteroids such as viridin and wortmannin have long been known as potent inhibitors of the lipid kinase PI-3K. We have been interested in directly accessing analogs of these complex natural products from abundant steroid feedstock materials. In this communication, we describe the synthesis of viridin/wortmannin hybrid molecules from readily available building blocks that function as PI-3K inhibitors and maintain their electrophilic properties. The compounds also show anti-proliferative effects against a breast cancer line.

Effects of synthetic gestagens on fish reproduction.

Although it is well known that estrogenic steroidal hormones are able to affect the sexual development and reproduction of fish at low concentrations, no data on environmental effects of the class of progestogenic hormones are available yet. Synthetic gestagens (progestins) are a component in oral contraceptives. Upon their use, a fraction of the progestins will be excreted via urine into the aquatic environment. On the basis of their pharmacological action in mammals, it is supposed that fish reproduction is the most sensitive endpoint for the progestin treatment. In order to test this assumption, the effects of two progestins currently marketed in contraceptive formulations, levonorgestrel (LNG) and drospirenone (DRSP), were investigated in adult fathead minnows (Pimephales promelas) following an Organization for Economic Cooperation and Development 21-d fish reproduction screening assay draft protocol with additional end points. Levonorgestrel was tested at measured concentrations of 0.8, 3.3, and 29.6 ng/L, and DRSP at concentrations of 0.66, 6.5, and 70 microg/L. Both tested progestins caused an inhibition of reproduction. For LNG, this occurred at concentrations of >or=0.8 ng/L, no no-observed-effect concentration (NOEC) could be defined. Higher concentrations resulted in masculinization of females with de novo synthesis of nuptial tubercles. Drospirenone treatment, however, affected the reproductive success of fathead minnow at concentrations of 6.5 microg/L and higher with a clear dose-response relationship and a NOEC of 0.66 microg/L, which is above environmentally relevant concentrations.

New steroidal derivatives synthesized using 3beta-hydroxyandrosten-17-one as starting material.

In this study, we synthesized some new substituted steroidal derivatives using 3beta-hydroxyandrosten-17-one (dehydroepiandrosterone) as starting material. The synthesized steroidal derivatives 1-11 were evaluated for their androgenic-anabolic activities compared to testosterone as positive control. Details of the synthesis, spectroscopic data and toxicity (LD50) of synthesized compounds are reported.

Investigating cytoskeletal alterations as a potential marker of retinal and lens drug-related toxicity.

Actin filaments play a critical role in the normal physiology of lenticular and retinal cells in the eye. Disruption of the actin cytoskeleton has been associated with retinal pathology and lens cataract formation. Ocular toxicity is an infrequent observation in drug safety studies, yet its impact to the drug development process is significant. Recognizing compounds through screening with a potential ocular safety liability is one way to prioritize development candidates while reducing development attrition. Lens epithelial cells from human, dog, and rat origins and retinal pigmented epithelium cells from human, monkey, and rat origins were cultured and investigated with immunocytochemical techniques. Cells were treated using noncytotoxic doses of the compound, fixed, stained for actin with rhodamine phalloidin, and counterstained for nuclei with TOTO-3, followed by confocal imaging. Tamoxifen and several experimental compounds known to be in vivo lens and retinal toxicants caused a reduction in F-actin fluorescence at noncytotoxic concentrations in all cells tested as observed by confocal microscopy. Developing an assay that predicts ocular toxicity helps the development process by prioritizing compounds for further investigation. Drug-induced cytoskeletal alterations may be useful as a potential safety-screening marker of retinal and lens toxicity. The knowledge of actin molecular biology and the application of other mechanistic screens to toxicology are discussed. Reducing this work to a high-throughput platform will enable chemists to select compounds with a reduced risk of ocular toxicity.

Molecular pharmacology and antitumor activity of PX-866, a novel inhibitor of phosphoinositide-3-kinase signaling.

We have developed biologically stable semisynthetic viridins as inhibitors of phosphoinositide (PtdIns)-3-kinases. The most active compound was PX-866 (acetic acid (1S,4E,10R,11R,13S,14R)-[4-diallylaminomethylene-6-hydroxy-1-methoxymethyl-10,13-dimethyl-3,7,17-trioxo-1,3,4,7,10,11,12,13,14,15,16,17-dodecahydro-2-oxa-cyclopenta[a]phenanthren-11-yl ester), which inhibited purified PtdIns-3-kinase with an IC50 of 0.1 nmol/L and PtdIns-3-kinase signaling measured by phospho-Ser473-Akt levels in HT-29 colon cancer cells with an IC50 of 20 nmol/L. PX-866 administered to mice at 10 mg/kg inhibited phospho-Ser473-Akt in HT-29 colon tumor xenografts up to 80% with recovery taking >48 hours after p.o. administration but more rapidly after i.v. or i.p. administration. PX-866 was eliminated from mouse plasma with a half-life of 18 minutes and a clearance of 360 mL/min/kg following i.v. administration and, when administered i.p. or p.o., showed first-pass metabolism with sequential N-deallylation. Synthetic standards of the N-deallylated metabolites of PX-866 inhibited PtdIns-3-kinase at low nanomolar per liter concentrations. PX-866 exhibited in vivo antitumor activity against s.c. OvCar-3 human ovarian cancer and A-549 human lung cancer xenografts in immunodeficient mice with log cell kills up to 1.2. PX-866 also increased the antitumor activity of cisplatin against A-549 xenografts and radiation treatment against OvCar-3 xenografts. The results show that PX-866 is a biologically stable broad-spectrum PtdIns-3-kinase inhibitor with good pharmacokinetics that causes prolonged inhibition of PtdIns-3-kinase signaling in human tumor xenografts. PX-866 exhibits single agent in vivo antitumor activity and increases the antitumor effects of cisplatin and radiation treatment.

Synthesis and biological evaluation of synthetic viridins derived from C(20)-heteroalkylation of the steroidal PI-3-kinase inhibitor wortmannin.

A series of viridin analogs was prepared from wortmannin by nucleophilic ring opening at C(20) and evaluated against the signaling kinases PI-3-kinase and mTOR. Several subnanomolar enzyme inhibitors with orders of magnitude selectivity for PI-3-kinase and strong cytotoxic activity against four cancer cell lines were identified. Among the ten most promising derivatives, six demonstrated lower liver toxicity and greater promise for inhibition of tumor cell growth than the lead structure wortmannin.

Mutagenicity of bay-region amino-substituted cyclopenta[a]phenanthrenes and 2- and 5-aminochrysene.

The relative mutagenic potentials of 11-amino-16,17-dihydro-15H-cyclopenta[a]phenanthrene, its 17-keto derivative, and 2- and 5-aminochrysene have been compared in Salmonella typhimurium TA98 and TA100 in the presence of a postmitochondrial liver preparation from Aroclor 1254 induced rats. The 11-amino hydrocarbon is a very weak mutagen (0.27 revertants/nmol), whereas the 11-amino-17-ketone is much more active (129 revertants/nmol). 2-Aminochrysene is the most mutagenic arylamine ( approximately 500 revertants/nmol) among these compounds, but its 5-amino isomer is much less active (0.9 revertants/nmol). Possible reasons for these marked differences are suggested. Use of TA98 with over-expressing O-acetyltransferase (YG 1024) and deficient in this enzyme (TA98/l,8-DNP(6)) with the 11-amino-17-ketone and with 5-aminochrysene clearly indicates the importance of this enzyme in their bioactivation, implying oxidation of the amino group to the hydroxylamine in both these compounds.