Inhibition of phosphatidylinositol-3-kinase by the furanosesquiterpenoid hibiscone C.

The phosphatidylinositol-3-kinase (PI3K) pathway regulates cellular metabolism and is upregulated in many cancers, making it an attractive chemotherapeutic target. Wortmannin is a potent inhibitor of PI3K; however, its potential as a chemotherapeutic is limited due to its instability, lack of selectivity, and lengthy chemical synthesis. In contrast, hibiscone C, a structurally simpler and less studied member of the furanosteroid family, has been expediently prepared by total synthesis. We demonstrate that hibiscone C competitively inhibits PI3K activity in intact cells, slows proliferation, and induces cell death. Hibiscone C may therefore serve as a productive scaffold for the development of therapeutically relevant PI3K inhibitors.

Differential profile of letrozole and exemestane on bone turnover markers in vinylcyclohexene diepoxide treated ovotoxic female mice.

The third generation aromatase inhibitors are currently the drugs of choice for the treatment of early and advanced breast cancer in postmenopausal women. One of the significant limiting factor during therapy is their negative impact on bone health. In this study, we compared the effects of a nonsteroidal (letrozole) and a steroidal aromatase inhibitor (exemestane) on bone mineral density and markers of bone turnover including alkaline phosphatase (ALP), tartrate-resistant acid phosphatase (TRAP), hydroxyproline (HxP), receptor activator of nuclear factor kappa B ligand (RANKL), sclerostin, and dickkopf-1 (DKK-1) in vinylcyclohexene diepoxide (VCD)-induced ovotoxic female mice. VCD administration for 15 days mimicked a postmenopausal state with reduced serum estradiol levels. Ovotoxicity was accompanied by reduced ALP, HxP and enhanced TRAP, sclerostin and DKK-1 activity in femoral epiphysis and lumbar vertebrae of mice. While letrozole (1 mg/kg) administration for 1 month enhanced bone turnover in ovotoxic mice, exemestane (3.25 mg/kg) was devoid of such effects in both normal and ovotoxic mice. The latter, however, reduced ALP in femoral epiphysis of ovotoxic mice. Letrozole depleted estradiol levels in ovotoxic mice and enhanced RANKL activity while exemestane neither affected estradiol nor RANKL in both normal and ovotoxic mice, and enhanced sclerostin and DKK-1 in femoral epiphysis only. The study indicates that the two aromatase inhibitors possesses differential profile in terms of their effects on bone and that exemestane could be a better option for the treatment of breast cancer in postmenopausal women at least in terms of its effects on bone.

Differential response to abiraterone acetate and di-n-butyl phthalate in an androgen-sensitive human fetal testis xenograft bioassay.

In utero exposure to antiandrogenic xenobiotics such as di-n-butyl phthalate (DBP) has been linked to congenital defects of the male reproductive tract, including cryptorchidism and hypospadias, as well as later life effects such as testicular cancer and decreased sperm counts. Experimental evidence indicates that DBP has in utero antiandrogenic effects in the rat. However, it is unclear whether DBP has similar effects on androgen biosynthesis in human fetal testis. To address this issue, we developed a xenograft bioassay with multiple androgen-sensitive physiological endpoints, similar to the rodent Hershberger assay. Adult male athymic nude mice were castrated, and human fetal testis was xenografted into the renal subcapsular space. Hosts were treated with human chorionic gonadotropin for 4 weeks to stimulate testosterone production. During weeks 3 and 4, hosts were exposed to DBP or abiraterone acetate, a CYP17A1 inhibitor. Although abiraterone acetate (14 d, 75 mg/kg/d po) dramatically reduced testosterone and the weights of androgen-sensitive host organs, DBP (14 d, 500 mg/kg/d po) had no effect on androgenic endpoints. DBP did produce a near-significant trend toward increased multinucleated germ cells in the xenografts. Gene expression analysis showed that abiraterone decreased expression of genes related to transcription and cell differentiation while increasing expression of genes involved in epigenetic control of gene expression. DBP induced expression of oxidative stress response genes and altered expression of actin cytoskeleton genes.

Inhaled fluticasone propionate impairs pulmonary clearance of Klebsiella pneumoniae in mice.

BACKGROUND: Recent trials demonstrate increased pneumonia risk in chronic obstructive pulmonary disease patients treated with the inhaled corticosteroid (ICS) fluticasone propionate (FP). There is limited work describing FP effects on host defenses against bacterial pneumonia. METHODS: C57BL/6 mice received daily, nose-only exposure to nebulized FP or vehicle for 8 days, followed by pulmonary challenge with Klebsiella pneumoniae. Bacterial burden, phagocytosis, leukocyte recruitment, cytokine expression, nitric oxide release, and survival were measured. RESULTS: Inhaled FP increased bacterial burden in lungs and blood 48 h after infection but affected neither in vivo phagocytosis of bacteria by alveolar macrophages (AM) nor alveolar neutrophil recruitment. AM from FP-treated mice showed impaired expression of infection induced TNF-alpha, IP-10 (CXCL-10), and interleukin 6 (IL-6), and AM also showed a trend towards impaired intracellular pathogen control following in vivo infection. In vitro FP treatment resulted in a dose-dependent impairment of cytokine expression by AM. Furthermore, infection-induced nitric oxide (but not hydrogen peroxide) production was impaired by FP in vivo and in vitro. FP decreased survival in this model. CONCLUSIONS: Exposure to inhaled FP impairs pulmonary clearance of K. pneumoniae in mice, an effect associated with greater systemic bacteremia and death. Decreased AM cytokine and nitric oxide expression parallel the failure to control infection. These results support the study of ICS effects on human pulmonary host defenses.

Revival of the abandoned therapeutic wortmannin by nanoparticle drug delivery.

One of the promises of nanoparticle (NP) carriers is the reformulation of promising therapeutics that have failed clinical development due to pharmacologic challenges. However, current nanomedicine research has been focused on the delivery of established and novel therapeutics. Here we demonstrate proof of the principle of using NPs to revive the clinical potential of abandoned compounds using wortmannin (Wtmn) as a model drug. Wtmn is a potent inhibitor of phosphatidylinositol 3' kinase-related kinases but failed clinical translation due to drug-delivery challenges. We engineered a NP formulation of Wtmn and demonstrated that NP Wtmn has higher solubility and lower toxicity compared with Wtmn. To establish the clinical translation potential of NP Wtmn, we evaluated the therapeutic as a radiosensitizer in vitro and in vivo. NP Wtmn was found to be a potent radiosensitizer and was significantly more effective than the commonly used radiosensitizer cisplatin in vitro in three cancer cell lines. The mechanism of action of NP Wtmn radiosensitization was found to be through the inhibition of DNA-dependent protein kinase phosphorylation. Finally, NP Wtmn was shown to be an effective radiosensitizer in vivo using two murine xenograft models of cancer. Our results demonstrate that NP drug-delivery systems can promote the readoption of abandoned drugs such as Wtmn by overcoming drug-delivery challenges.

A rabbit model of age-dependant ocular hypertensive response to topical corticosteroids.

OBJECTIVE: To investigate the ocular hypertensive response to topical dexamethasone (DEX), rimexolone (RIM), loteprednol etabonate (LOT) and fluorometholone (FML) in rabbits of different ages. METHODS: Seventy-five rabbits of three age groups (7 weeks, 6 months and 1-year old) received topical administration of 0.1% DEX, 1% RIM, 0.5% LOT, 0.1% FML or balanced salt solution four times daily for 1 month. Intraocular pressure (IOP) was monitored at regular time intervals. After a month, eyes were harvested for histological study with haematoxylin and eosin (H&E), periodic acid Schiff and Masson trichrome staining. Trabecular meshwork changes were graded by masked ocular pathologists. RESULTS: Topical DEX caused the greatest increase in IOP, followed by RIM and FML. LOT caused the least IOP increase. Similar pattern of IOP response to the four corticosteroids was observed in the three studied age groups. Young rabbits (7 week) were the most responsive to corticosteroids among the age groups. Extracellular matrix thickening in the trabecular meshwork region and loss of trabecular meshwork cells were observed after DEX, FML or RIM treatments. CONCLUSION: Young rabbits are more susceptible to steroid induced increase in IOP, even for milder steroids such as fluorometholone and rimexolone.

Impaired reproduction in adult male, but not female, rats following juvenile treatment with the aromatase inhibitor, exemestane.

BACKGROUND: Exemestane is an irreversible steroidal inhibitor of cytochrome-P450 aromatase required for estrogen synthesis. The safety of the drug in the pediatric population, particularly in males, has not previously been evaluated. Given the increased interest in treating children with aromatase inhibitors, we undertook a study in rats to assess the potential for exemestane to alter reproductive development and function when administered to juveniles. METHODS: Male and female rats were treated with exemestane at doses anticipated to produce exposures approximately 2- and 35-fold the expected clinical plasma exposure in young adult males during the period of reproductive maturation. After maturation, treated rats were mated to evaluate the potential impact on reproductive function. RESULTS AND CONCLUSION: There were no effects on sexual maturation in either sex or on female reproductive function. Treatment of juvenile male rats caused increased cohabitation time and decreased copulation rates; pregnancy rates and litter size were not affected in rats that mated. Decreased testis (10-15%) and epididymis (20-30%) weights, and decreased Sertoli cell numbers were noted at all doses. This indicates that exemestane can reduce Sertoli cell proliferation during maturation. The sensitive window for this effect is expected to be limited to the period of Sertoli cell proliferation, which is completed by around postnatal day 15 in rats and before puberty in humans. Treatment beginning at a later time relative to the window for Sertoli cell proliferation or for a longer duration is not expected to have additional adverse effect as the effect was not shown to be degenerative.

Histologic and cytologic detection of endocrine and reproductive tract effects of exemestane in female rats treated for up to twenty-eight days.

The objective of this study was to determine the shortest period of time necessary to detect histologic evidence of estrous cycle disruption in Sprague-Dawley rats treated for up to 28 days with the aromatase inhibitor exemestane at 1,000 mg/kg. Rats were evaluated on day 5, 8, 15, or 29. Vaginal mucification, uterine and cervical epithelial atrophy, uterine luminal epithelial vacuolation, decreased uterine granulocytes, and hypertrophy/hyperplasia of mammary ducts and alveoli were noted by day 5 and persisted throughout the study. From day 8 to day 29, absence of recent basophilic corpora lutea, increased atresia of antral follicles, interstitial cell hyperplasia, and increased luteinized follicles were present in the ovaries of treated rats. Vaginal smears detected persistent diestrus, confirming estrous cycle disruption between days 5 and 8. Ovary and uterine weights were largely unaffected. Serum hormone levels were not useful due to the study design employed. Other effects of exemestane included decreased adrenal weights and decreased cell size in both the adrenal zona fasciculata and the pituitary pars distalis. While early histologic changes were evident on day 5, only after 8 days of treatment were findings considered sufficient to clearly identify exemestane-induced estrous cycle disruption using microscopy alone.

Enhanced bioavailability of exemestane via proliposomes based transdermal delivery.

Exemestane, a novel steroidal aromatase inactivator used in the treatment of advanced breast cancer has limited bioavailability (42%) due to poor solubility, extensive first-pass metabolism, and also the absorption is dependent on formulation type and food. The present study is aimed to evaluate the feasibility of proliposomes for transdermal delivery of exemestane. The prepared proliposomes were characterized for size, zeta potential, and entrapment efficiency. The size of the vesicles was found to be between 440 and 700 nm with high entrapment efficiency for the formulation containing greater amounts of phosphatidylcholine. Differential scanning calorimetry and Fourier transform infrared studies were performed to understand the phase transition behavior and mechanism for skin permeation, respectively. The drug release across cellophane membrane follows zero-order kinetics by diffusion. Ex vivo permeation enhancement assessed from flux, permeability coefficient, and enhancement ratio were significantly higher for proliposome gels compared with control. A significant improvement in the bioavailability (2.4-fold) was observed from optimized proliposome gel compared with control (oral suspension). The stability data reveal that the formulations are more stable when stored at 4°C. In conclusion, proliposomal gels offer potential and prove to be efficient carriers for improved and sustained transdermal delivery of exemestane.

The inhaled glucocorticoid fluticasone propionate efficiently inactivates cytochrome P450 3A5, a predominant lung P450 enzyme.

Inhaled glucocorticoid (GC) therapy is a vital part of the management of chronic asthma. GCs are metabolized by members of the cytochrome P450 3A family in both liver and lung, but the enzymes are differentially expressed. Selective inhibition of one or more P450 3A enzymes could substantially modify target and systemic concentrations of GCs. In this study, we have evaluated the mechanism-based inactivation of P450 3A4, 3A5, and 3A7 enzymes by GCs. Among the five major inhaled GCs approved for clinical use in the United States, fluticasone propionate (FLT) was the most potent mechanism-based inactivator of P450 3A5, the predominant P450 enzyme in the lung. FLT inactivated P450 3A5 in a time- and concentration-dependent manner with K(I), k(inact), and partition ratio of 16 muM, 0.027 min(-1), and 3, respectively. In contrast, FLT minimally inactivated P450 3A4 and did not inactivate 3A7, even with a concentration of 100 muM. The inactivation of P450 3A5 by FLT was irreversible because dialysis did not restore enzyme activity. In addition, the exogenous nucleophilic scavenger GSH did not attenuate inactivation. The prosthetic heme of P450 3A5 was not modified by FLT. The loss of P450 3A5 activity in lung cells could substantially decrease the metabolism of FLT, which would increase the effective FLT concentration at its target site, the respiratory epithelium. Also, inactivation of lung P450 3A5 could increase the absorption of inhaled FLT, which could lead to high systemic concentrations and adverse effects, such as life-threatening adrenal crises or cataracts that have been documented in children receiving high doses of inhaled GCs.

Efficacy of exemestane, a new generation of aromatase inhibitor, on sex differentiation in a gonochoristic fish.

We report the first use of exemestane (EM), a steroidal aromatase inhibitor (AI) commercially known as aromasin, in studies of sex differentiation in fish. The effectiveness of EM was examined in two different age groups of the gonochoristic fish, Nile tilapia (Oreochromis niloticus). Untreated control fish (all female) showed normal ovarian differentiation through 120 days after hatching (dah), whereas fish treated with EM at 1000 and 2000 microg/g of feed from 9 dah through 35 dah, the critical period for sex differentiation, exhibited complete testicular differentiation; all stages of spermatogenic germ cells were evident and well developed efferent ducts were present. Fish treated with EM at 1000 microg/g of feed from 70 dah through 100 dah significantly suppressed plasma estradiol-17beta level and increased level of 11-ketotestosterone. Furthermore, untreated control fish showed strong gonadal expression of the steroidogenic enzymes P450 cholesterol-side chain-cleavage enzyme (P450scc), 3beta-hydroxysteroid dehydrogenase (3beta-HSD), and cytochrome P450 aromatase (P450arom). In contrast, EM-treated fish showed immunopositive reactions against P450scc and 3beta-HSD but not against P450arom in interstitial Leydig cells. These results indicate that treatment of tilapia juveniles with EM during sex differentiation leads to the development of testes, apparently by a complete suppression of aromatase activity.

Dexmedetomidine provides cortical neuroprotection: impact on anaesthetic-induced neuroapoptosis in the rat developing brain.

BACKGROUND: Recent evidence has demonstrated the anti-apoptotic of dexmedetomidine in different brain injury models. Herein, we investigated whether dexmedetomidine could directly protect against cortical injury in vitro and in vivo. METHODS: Apoptosis was induced by staurosporine or wortmannin treatment in cortical neuronal cultures in vitro or by 6 h of isoflurane (0.75%) administration to post-natal day 7 rat pups in vivo. Dexmedetomidine was then applied in escalating doses to assess the neuroprotective potential of this agent. Cell survival was quantified using an MTT assay in vitro and in vivo apoptosis was assessed using cleaved caspase-3 immunohistochemistry. Cortical Western blots were conducted for the cellular survival proteins Bcl-2 and phosphorylated extracellular signal-regulated protein kinase (pERK)1 and 2. RESULTS: In vitro dexmedetomidine dose-dependently prevented both staurosporine- and wortmannin-induced injury in cortical neuronal cultures, indicating that dexmedetomidine can prevent apoptosis when applied directly. In vivo isoflurane induced cortical neuroapoptosis compared with air (327+/-80 vs. 34+/-9 caspase-3-positive neurons; P<0.05). Dexmedetomidine inhibited isoflurane-induced caspase-3 expression (P<0.05), although the protection achieved did not completely attenuate the isoflurane injury (P<0.05 vs. air). Isoflurane treatment decreased Bcl-2 and pERK protein expression relative to air, an effect reversed by dexmedetomidine treatment. CONCLUSIONS: Dexmedetomidine prevents cortical apoptosis in vitro and in vivo. However, using higher doses of dexmedetomidine does not further increase protection against isoflurane injury in the cortex than previously observed.

Verapamil toxicity dysregulates the phosphatidylinositol 3-kinase pathway.

OBJECTIVES: Recent animal research and clinical case reports suggest benefit from high-dose insulin therapy (HDIT) for the treatment of calcium channel blocker (CCB) toxicity. One molecular signaling pathway, the phosphatidylinositol 3-kinase (PI3K) pathway, that contributes to CCB toxicity and the efficacy of HDIT, was examined for a role in this phenomenon. METHODS: A differentiated 3T3-L1 adipocyte model system was utilized to characterize metabolic and molecular signaling events dysregulated in response to acute CCB toxicity. Glucose uptake assays were performed in the presence of representatives of three classes of CCB drugs, and the ability of HDIT to reverse observed inhibition was assessed. Western blot analyses were utilized to probe which insulin-dependent signaling pathway was inhibited by CCB toxicity. RESULTS: Representative compounds from the dihydropyridine and phenylalkylamine classes of CCBs were more effective at inhibiting glucose uptake in differentiated 3T3-L1 adipocytes than a representative from the benzothiazepine class. Phosphorylation at serine 473 of the Akt protein (P-Akt), a protein representing a common pathway for insulin receptors (IR), insulinlike growth factor receptors (IGFR), and hybrid receptors formed by IR and IGFR subunits, was abolished in the presence of toxic doses of the phenylalkylamine CCB verapamil. Phosphorylation at serine 473 of Akt was rescued in the presence high concentrations of insulin. CONCLUSIONS: These data suggest that dysregulation of the insulin-dependent PI3K pathway is partially responsible for insulin resistance and the hyperglycemic state observed in response to acute CCB toxicity.

Effect of anabolics on bovine granulosa-luteal cell primary cultures.

Granulosa cell tumours are observed with increased frequency among calves slaughtered in Northern Italy. The use of illegal anabolics in breeding was taken into account as a cause of this pathology. An in vitro approach was used to detect the possible alterations of cell proliferation induced by anabolics on primary cultures of bovine granulosa-luteal cells. Cultures were treated with different concentrations of substances illegally used in cattle (17beta-estradiol, clenbuterol and boldione). Cytotoxicity was determined by means of MTT test, to exclude toxic effects induced by anabolics and to determine the highest concentration to be tested. Morphological changes were evaluated by means of routine cytology, while PCNA expression was quantified in order to estimate cell proliferation. Cytotoxic effects were revealed at the highest concentrations. The only stimulating effect on cell proliferation was detected in boldione treated cultures: after 48 h treated cells, compared to controls, showed a doubled expression of PCNA. In clenbuterol and 17beta-estradiol treated cells PCNA expression was similar to controls or even decreased. As the data suggest an alteration in cell proliferation, boldione could have a role in the early stage of pathogenesis of granulosa cell tumour in cattle.

Determination of the androgenic potency of whole effluents using mosquitofish and trout bioassays.

This study combined bioassay-derived and direct chemical analysis of steroidal compounds in pulp and paper and municipal sewage effluent in order to determine the cause of masculinization of female mosquitofish. The bioassays used in this study consisted of androgen and estrogen receptor binding, and aromatase inhibition using tissues from rainbow trout. This study observed no masculinization of female mosquitofish from a pulp and paper mill effluent that was previously observed to cause this effect. Mosquitofish sampled from the receiving environment of the same mill verified that masculinization was not occurring in the wild. The municipal sewage effluent also had no masculinizing effect. In vitro bioassays indicated significant sources of both androgens and estrogens in the effluents tested with sewage effluent having both the highest estradiol (41 ng/L) and testosterone (182 ng/L) equivalent concentration. These results could not be attributed to any particular compounds measured in the effluents. Two compounds implicated in the masculinization of mosquitofish by pulp and paper effluent, androstenedione and androstadienedione required relatively large (10-100 microg/L) waterborne concentrations to elicit a masculinizing effect and neither of these compounds are likely to occur at levels this high in the natural environment. The potent aromatase inhibitor, 4-hydroxyandrostenedione also did not cause masculinization at 100 microg/L indicating that masculinization did not occur through this mechanism. The mammalian anti-androgen, cyproterone acetate was only partially effective in mosquitofish and reduced the severity of masculinization in the presence of methyl testosterone. While neither effluent masculinized mosquitofish, there was a significant reduction of in vitro ovarian steroid production with the most severe effects observed with the sewage effluent. Overall, this study found the disappearance of a masculinizing effect that had been previously observed; concluded that based on 21 days aqueous exposures androstenedione and androstadienedione are not likely candidates for mosquitofish masculinization, and showed that masculinization and in vitro steroid production are unrelated biological endpoints.

In vitro effects of epristeride on sperm in rats, dogs and man.

The study was conducted to evaluate in vitro effects of epristeride on sperm in rats, beagle dogs and man. Semen samples were divided into 4 groups and treated with vehicle and epristeride. Motility and motile rate of sperm were videotaped and analyzed with CASA system after 1 h and 2 h incubation periods. Percentage of motile sperm (MOT) of rat sperm decreased after the treatment with epristeride (final concentrations were 0.6, 6 and 60 micromol/L) for 1 h, and MOT of rat sperm treated with middle dose and high dose levels of epristeride also decreased after 2 h, while MOT of dog sperm that treated with three dose levels of epristeride decreased after 2 h. Amplitude of lateral head displacement (ALH) and MOT of human sperm decreased after 2 h with 4.8 micromol/L epristeride treatment. Curvilinear velocity (VCL) and straight-line velocity (VSL) of rat sperm and human sperm changed after 2 h, but there were no significant differences. Therefore, epristeride had a toxic effect on sperm, and the effect varied in different species.

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.

A human and mouse pregnane X receptor reporter gene assay in combination with cytotoxicity measurements as a tool to evaluate species-specific CYP3A induction.

Recent studies have demonstrated that a member of the nuclear receptor family, pregnane X receptor (PXR) is a key regulator of the expression of cytochrome P450 3A (CYP3A) in humans and rodents. It is also known that species specificity in the induction of CYP3A by xenobiotics is likely a consequence of differences at the level of PXR activation. Because of the importance of CYP3A4 in drug metabolism, the development of rapid and accurate in vitro assays for predicting the effects of compounds on CYP3A4 expression or activity in humans has been a long-standing goal within pharmaceutical industries. PXR activation measurements using an in vitro reporter gene approach appears to provide a rapid and relatively inexpensive means for predicting whether compounds will induce CYP3A levels in vivo. In this study, using an HepG2 cell based human and mouse PXR reporter gene assay, 23 compounds were tested for their potential to activate hPXR or mPXR. Data demonstrated that potent activators of hPXR had virtually no activity on mPXR and efficient activators of mPXR had weak activity on hPXR. In addition, a third category of moderate/weak activators of both hPXR and mPXR was identified. Exemestane was a strong activator of mPXR ( approximately 22-fold activation) with only minor effect on hPXR ( approximately 5-fold activation). The importance of cell viability measurements as part of the PXR reporter gene assay was demonstrated as significant cytotoxicity or inhibition of cell proliferation might underestimate the potential for PXR activation.

Influence of preservatives and topical steroids on ciliary beat frequency in vitro.

OBJECTIVE: To measure the influence of topical steroids and the preservative potassium sorbate on the ciliary beat frequency (CBF) of human nasal mucosa in vitro. DESIGN: In vitro study of cultured ciliated cells of human nasal mucosa. METHODS: Human nasal mucosa was removed endoscopically and cultured for 10 days. Cell cultures with ciliated cells grown on an object slide were exposed to benzalkonium chloride and topical steroids in an exposure chamber. The CBF was measured with a photometer. RESULTS: The preservative potassium sorbate did not influence CBF in different concentrations. The glucocorticoid budesonide spray containing potassium sorbate did not affect CBF at 10% dilution and showed moderate reversible decrease of CBF at 50% dilution. The glucocorticoid sprays fluticasone propionate and mometasone fuorate containing the preservative benzalkonium chloride caused a reversible decrease of CBF at 10% dilution and a complete irreversible standstill at 50% dilution. CONCLUSIONS: In vitro, the steroid sprays containing fluticasone or mometasone, both with benzalkonium chloride, caused slowing or standstill of CBF depending on the concentration. The isolated preservative potassium sorbate and the budesonide nasal spray containing this preservative did not have negative influence on CBF in vitro. Potassium sorbate can therefore be considered harmless to the motility of ciliated cells.

Resistance to androstanes as an approach for androstandienedione yield enhancement in industrial mycobacteria.

The resistance to androstandienedione (ADD) of industrial mycobacteria was demonstrated as a valuable approach to increasing ADD yield in sterol fermentations. Colonies growing at 1 mg/ml ADD in culture medium after nitrosoguanidine mutagenesis showed a differential behavior in respect to parentals in cholesterol biotransformation. In the presence of exogenous ADD, a substantial depletion of ADD production was observed in parental strains B3683 and Ex4, whereas it was unaffected, and even increased, in resistant colonies. An apparent reduction from ADD to androstandione and testosterone was also noticed. Furthermore, the ADD resistance phenotype may be related to the increase in steroid 1,2 dehydrogenase activity.