New Andrastin-Type Meroterpenoids from the Marine-Derived Fungus Penicillium sp.

Three new andrastin-type meroterpenoids penimeroterpenoids A-C (1-3) together with two known analogs (4 and 5) were isolated from the cultures of the marine-derived Penicillium species (sp.). The structures of the new compounds were elucidated on the basis of 1- and 2-dimensional (1D/2D) Nuclear Magnetic Resonance (NMR) spectroscopic and mass spectrometric analysis. The absolute configurations of 1-3 were determined by comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Compound 1 showed moderate cytotoxicity against A549, HCT116, and SW480 cell lines.

The Pharmacokinetics of Fluticasone Furoate Given Intranasally in Healthy Subjects Using an Ultra-Sensitive Analytical Assay.

PURPOSE: It has been previously shown that the complete pharmacokinetic profile, in particular the elimination phase, of intranasal fluticasone furoate has not been fully characterized due to the inability to quantify concentrations at low enough levels. This study was designed to evaluate the pharmacokinetic profile of intranasal FF using a validated, ultra-sensitive analytical method in healthy subjects. METHODS: This was an open-label, single-dose, two-period, one-treatment, crossover study. A dose of 880 µg fluticasone furoate was administered intra nasally. Blood samples for pharmacokinetic analysis were collected at 23 time points up to 36 h and analyzed for FF plasma levels using a lower limit of quantitation (LLOQ) of 0.1 pg/mL. Medical and adverse events (AE) were monitored throughout the study. RESULTS: Eighteen subjects were enrolled in and 17 completed the study. The results showed that all 17 subjects had measurable fluticasone furoate plasma concentrations at all time points with a clearly defined elimination phase, thus allowing estimation of AUCinf and t1/2. Median Tmax was 1.33 h (range=0.75-6.00), mean Cmax was 13.05±7.59 pg/mL, mean AUCt was 148.48±77.76 pg/mL*h, mean AUCinf was 279.07±187.81 pg/mL*h, and mean t1/2 was 31.67±29.23 h. In total 4 subjects (22.2%) experienced 4 AEs. CONCLUSION: Using a lower LLOQ than what has been previously reported, a complete characterization of intranasal fluticasone furoate pharmacokinetics, including a clearly defined terminal elimination phase, was achieved. This method will allow for further investigations into the pharmacokinetics of fluticasone furoate.

Biotransformation of androstenedione and androstadienedione by selected Ascomycota and Zygomycota fungal strains.

Filamentous fungi is a huge phylum of lower eukaryotes with diverse activities towards various substrates, however, their biocatalytic potential towards steroids remains greatly underestimated. In this study, more than forty Ascomycota and Zygomycota fungal strains of 23 different genera were screened for the ability to catalyze structural modifications of 3-oxo-androstane steroids, - androst-4-ene-3,17-dione (AD) and androsta-1,4-diene-3,17-dione (ADD). Previously unexplored for these purposes strains of Absidia, Acremonium, Beauveria, Cunninghamella, Doratomyces, Drechslera, Fusarium, Gibberella genera were revealed capable of producing in a good yield valuable 7α-, 7ß-, 11α- and 14α-hydroxylated derivatives, as well as 17ß-reduced and 1(2)-dehydrogenated androstanes. The bioconversion routes of AD and ADD were proposed based on the key intermediates identification and time courses of the bioprocesses. Six ascomycete strains were discovered to provide effective 7ß-hydroxylation of ADD which has not been so far reported. The structures of major products and intermediates were confirmed by HPLC, mass-spectrometry (MS), 1H and 13C NMR analyses. The results contribute to the knowledge on the functional diversity of steroid-transforming filamentous fungi. Previously unexplored fungal biocatalysts capable of effective performing structural modification of AD and ADD can be applied for industrial bioprocesses of new generation.

Extraction and determination of trace amounts of three anticancer pharmaceuticals in urine by three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography.

An automated three-phase hollow fiber liquid-phase microextraction based on two immiscible organic solvents followed by high-performance liquid chromatography with UV-Vis detection method was applied for the extraction and determination of exemestane, letrozole, and paclitaxel in water and urine samples. n-Dodecane was selected as the supported liquid membrane and its polarity was justified by trioctylphosphine oxide. Acetonitrile was used as an organic acceptor phase with desirable immiscibility having n-dodecane. All the effective parameters of the microextraction procedure such as type of the organic acceptor phase, the supported liquid membrane composition, extraction time, pH of the donor phase, hollow fiber length, stirring rate, and ionic strength were evaluated and optimized separately by a one variable at-a-time method. Under the optimal conditions, the linear dynamic ranges were 1.8-200 (R2  = 0.9991), 0.9-200 (R2  = 0.9987) and 1.2-200 µg/L (R2  = 0.9983), and the limits of detection were 0.6, 0.3, and 0.4 µg/L for exemestane, letrozole, and paclitaxel, respectively. To evaluate the capability of the proposed method in the analysis of biological samples, three different urinary samples were analyzed under the optimal conditions. The relative recoveries of the three pharmaceuticals were in the range of 91-107.3% for these three analytes.

Isolation and X-ray structure analysis of citreohybridonol from marine-derived Penicillium atrovenetum.

The anti-neuroinflammatory meroterpenoid citreohybridonol was isolated for the first time from a sponge-derived fungus Penicillium atrovenetum. In this study, in addition to isolation and structure featuring, its unambiguous absolute configuration was determined exclusively by single crystal X-ray diffraction. The C-17-keto tautomer was clearly observed in X-ray analysis. The substance crystallises in the monoclinic space group P21 with a = 10.7496(5) Å, b = 14.3286(7) Å, c = 17.4909(8) Å, ß = 103.235(2)°, V = 2622.5(2) Å3, Z = 2, Dcalcd = 1.280 g/cm3. The chirality of the asymmetric carbon atoms was as follows: C3 (S), C5 (R), C6 (S), C8 (S), C9 (R), C10 (R), C13 (R), C14 (R).

A new wortmannine derivative from a Tripterygium wilfordii endophytic fungus Talaromyces wortmannii LGT-4.

A new wortmannine derivative named wortmannine E (1) was isolated from Talaromyces wortmannii LGT-4, an endophytic fungus of Tripterygium wilfordii. Its structure was established by 1D and 2D NMR spectra.

Bioactive Constituents from an Endophytic Fungus, Penicillium polonicum NFW9, Associated with Taxus fauna.

BACKGROUND: Endophytic fungi are being recognized as vital and untapped sources of a variety of structurally novel and unique bioactive secondary metabolites in the field of natural products drug discovery. Herein, this study reports the isolation and characterization of secondary metabolites from an endophytic fungus Penicillium polonicum (NFW9) associated with Taxus fuana. METHOD: The extracts of the endophytic fungus cultured on potato dextrose agar were purified using several chromatographic techniques. Biological evaluation was performed based on their abilities to inhibit tumor necrosis factor-alpha (TNF-α)-induced nuclear factor-kappa B (NF-κB) and cytotoxicity assays. RESULTS: Bioactivity-directed fractionation of the ethyl acetate extract of a fermentation culture of an endophytic fungus, Penicillium polonicum led to the isolation of a dimeric anthraquinone, (R)- 1,1',3,3',5,5'-hexahydroxy-7,7'-dimethyl[2,2'-bianthracene]-9,9',10,10'-tetraone (1), a steroidal furanoid (-)-wortmannolone (2), along with three other compounds (3-4). Moreover, this is the first report on the isolation of compound 1 from an endophytic fungus. All purified metabolites were characterized by NMR and MS data analyses. The stereo structure of compound 1 was determined by the measurement of specific optical rotation and CD spectrum. The relative stereochemistry of 2 was confirmed by single-crystal X-ray diffraction. Compounds 2-3 showed inhibitory activities in the TNF-α-induced NF-κB assay with IC50 values in the range of 0.47-2.11 µM. Compounds 1, 4 and 5 showed moderate inhibition against NF-κB and cancer cell lines. CONCLUSION: The endophytic fungus Penicillium polonicum of Taxus fuana is capable of producing biologically active natural compounds. Our results provide a scientific rationale for further chemical investigations into endophyte-producing natural products, drug discovery and development.

Anti-neuroinflammatory effects of citreohybridonol involving TLR4-MyD88-mediated inhibition of NF-кB and MAPK signaling pathways in lipopolysaccharide-stimulated BV2 cells.

In the course of searching for anti-neuroinflammatory metabolites from marine fungi, citreohybridonol was isolated from marine-derived fungal strain Toxicocladosporium sp. SF-5699. Citreohybridonol inhibited production of nitric oxide (NO) and prostaglandin E2 (PGE2) in BV2 cells stimulated by lipopolysaccharide (LPS). Citreohybridonol also suppressed the expression of inducible NO synthase (iNOS), cyclooxygenase-2 (COX-2), and other pro-inflammatory cytokines including interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the LPS-stimulated cells. In the further study, citreohybridonol disturbed nuclear translocation of nuclear factor-kappa B (NF-κB) in LPS-stimulated BV2 cells by inhibiting the phosphorylation of the inhibitor kappa B-α (IκB-α). Citreohybridonol also had inhibitory effect on the LPS-stimulated phosphorylation of p38 mitogen-activated protein kinase (MAPK). Finally, citreohybridonol suppressed the protein expression of Toll-like receptor 4 (TLR4) and myeloid differentiation factor 88 (MyD88) in LPS-induced BV2 cells. These results suggest that citreohybridonol has anti-neuroinflammatory effect in LPS-stimulated BV2 cells by modulating TLR4-mediated several inflammatory pathways such as NF-κB and p38 MAPK pathways.

Isolation, characterization and antifungal docking studies of wortmannin isolated from Penicillium radicum.

During the search for a potent antifungal drug, a cell-permeable metabolite was isolated from a soil isolate taxonomically identified as Penicillium radicum. The strain was found to be a potent antifungal agent. Production conditions of the active compound were optimized and the active compound was isolated, purified, characterized and identified as a phosphoinositide 3-kinase (PI3K) inhibitor, commonly known as wortmannin (Wtmn). This is very first time we are reporting the production of Wtmn from P. radicum. In addition to its previously discovered anticancer properties, the broad spectrum antifungal property of Wtmn was re-confirmed using various fungal strains. Virtual screening was performed through molecular docking studies against potential antifungal targets, and it was found that Wtmn was predicted to impede the actions of these targets more efficiently than known antifungal compounds such as voriconazole and nikkomycin i.e. 1) mevalonate-5-diphosphate decarboxylase (1FI4), responsible for sterol/isoprenoid biosynthesis; 2) exocyst complex component SEC3 (3A58) where Rho- and phosphoinositide-dependent localization is present and 3) Kre2p/Mnt1p a Golgi alpha1,2-mannosyltransferase (1S4N) involved in the biosynthesis of yeast cell wall glycoproteins). We conclude that Wtmn produced from P. radicum is a promising lead compound which could be potentially used as an efficient antifungal drug in the near future after appropriate structural modifications to reduce toxicity and improve stability.

Secondary metabolites from Penicillium corylophilum isolated from damp buildings.

Indoor exposure to the spores and mycelial fragments of fungi that grow on damp building materials can result in increased non-atopic asthma and upper respiratory disease. The mechanism appears to involve exposure to low doses of fungal metabolites. Penicillium corylophilum is surprisingly common in damp buildings in USA, Canada and western Europe. We examined isolates of P. corylophilum geographically distributed across Canada in the first comprehensive study of secondary metabolites of this fungus. The sesquiterpene phomenone, the meroterpenoids citreohybridonol and andrastin A, koninginin A, E and G, three new alpha pyrones and four new isochromans were identified from extracts of culture filtrates. This is the first report of koninginins, meroterpenoids and alpha pyrones from P. corylophilum. These secondary metabolite data support the removal of P. corylophilum from Penicillium section Citrina and suggest that further taxonomic studies are required on this species.

A liquid chromatography-tandem mass spectrometry method for the simultaneous determination of exemestane and its metabolite 17-dihydroexemestane in human plasma.

A simple and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method has been developed and validated for the quantitation of exemestane (Exe) and its main metabolite 17-dihydroexemestane (DhExe) in human plasma. The analytes were extracted by protein precipitation with acetonitrile, containing stable 13C-labelled Exe (13C3-Exe) as internal standard, and measured by LC-MS/MS. The best chromatographic separation of the analytes from the interferences was achieved by using a Phenyl column operating under isocratic regime conditions. The total chromatographic runtime was 5.0 min and the elution of Exe and DhExe occurred at 2.5 min and 2.9 min, respectively. Quantitation was performed by employing the positive electrospray ionization (ESI) technique and multiple reaction monitoring mode (MRM). The monitored precursor to product-ion transitions for Exe, DhExe and 13C3-Exe internal standard were m/z 297.0 --> 120.8, m/z 299.1 --> 134.9 and m/z 300.0 --> 123.2, respectively. The lower limit of quantitation (LLOQ) was 0.1 ng/ml for DhExe and 0.2 ng/ml for Exe. The method was linear up to 36-51 ng/ml with r2 > or = 0.998. The intra- and inter-assay precision were < or = 7.7% and 5.1% for Exe and < or = 8.1 and 4.9% for DhExe while deviations from nominal values were in the 1.5-13.2% and - 9.0-5.8% ranges for Exe and DhExe, respectively. The analytical method resulted robust and suitable for pharmacokinetic monitoring of Exe and its main metabolite during adjuvant therapy in patients with breast cancer.

A test of rats' tolerance for 3beta-acetoxyandrosta-1,5-dien-17-one ethylene ketal (ADEK), a new anti-androgen.

Following the demonstration that the androgen activity of androsta-5-ene-3beta,17beta-diol (Adiol) is not inhibited by the anti-androgens currently used to treat prostate cancer, we sought agents that would inhibit the androgenic function of Adiol as well as of dihydrotestosterone. The steroid 3beta-acetoxyandrosta-1,5-dien-17-one ethylene ketal (ADEK) met this criterion. Its tolerance was assessed in rats by oral and by subcutaneous administration for four weeks. Neither route of ADEK administration resulted in any behavioral changes. There was no effect on weight gain during the 28 days of steroid intake and no effect on the weight of the kidneys, heart, liver, testes, adrenals or the ventral lobe of the prostate glands. The seminal vesicles of the treated rats were 23-29% and the weights of the anterior prostates of the respective groups were 17-26% smaller than the controls. In contrast, the dorsolateral prostates were increased 26-55% as compared with the controls. There were no detectable changes in the histology of the kidneys, hearts, livers, testes and adrenals of any of the rats, but both groups of ADEK-treated rats had mild atrophic changes in their seminal vesicles and in the ventral lobe of their prostate glands. Both ADEK-treated groups showed focal glandular epithelial hyperplasia in the dorsolateral lobes in comparison with the control group. Orally administered ADEK was rapidly converted to several metabolites, which were nearly completely cleared from the blood within 4h.

Sensitive simultaneous determination of ciclesonide, ciclesonide-M1-metabolite and fluticasone propionate in human serum by HPLC-MS/MS with APPI.

A new and very sensitive analytical method has been developed and validated to jointly determine the anti-inflammatory drug ciclesonide (CIC), its active principle metabolite M1 (CIC-M1) and fluticasone propionate (FP) in human serum, in the low concentration range from 10 to 1000 pg/mL. This was accomplished by high-performance liquid chromatography and tandem mass spectrometry using atmospheric pressure photo ionisation (HPLC-MS/MS with APPI) using 0.5 mL of serum. Serum was mixed with the internal standards (IS) D11-CIC and D11-CIC-M1 and extracted with diisopropylether. A gradient with acetonitrile (containing 10 mM of acetic acid and 10% of acetone) was used. HPLC-MS/MS of the acetic acid adducts of the analytes was performed in negative mode. The novel aspect of this method is that instead of the dopant being introduced directly into the source by means of an external HPLC pump, it was added to the mobile phase. This provided significantly better sensitivity than the usual method of in-source addition of the dopant, and with no loss in HPLC performance. Sensitivity for the analytes was about four times greater than with either APCI or ESI. Validation was performed in three batches. The inter-batch precision (CV) of the quality control samples in human serum ranged from 4.08% to 6.78% for CIC, from 2.57% to 7.74% for CIC-M1, and from 2.38% to 9.61% for FP. The inter-batch accuracy (with reference to the mean value) of the quality control samples in human serum ranged from 99.3% to 110.0% for CIC, from 101.8% to 104.7% for CIC-M1, and from 100.4% to 101.8% for FP. Calibration data and LLOQ data are also presented in this paper. The analytes were stable in human serum over three freeze/thaw cycles, or for 4h at room temperature, or for at least 18 months when stored at below -20 degrees C. This method was used for quantifying the analytes after inhalation of low-mug amounts of the drugs by patients.

Synthesis of exemestane labelled with (13)C.

The synthesis of exemestane Aromasin, an irreversible steroidal aromatase inhibitor, specifically labelled with (13)C is reported. The preparation of [(13)C(3)]exemestane was achieved according to an eight-step procedure starting from the commercially available testosterone.

Influence of mouth washing procedures on the removal of drug residues following inhalation of corticosteroids.

Mouth washing after inhalation of corticosteroids is effective for prevention of local adverse effects such as hoarseness and oropharyngeal candidiasis. To establish an optimal procedure for such mouth washing, we investigated the removal rates of drug residues remaining on the oropharyngeal mucosa using various mouth washing methods following inhalation. A beclomethasone dipropionate metered dose inhaler (BDP-MDI) (100 microg) and a fluticasone propionate dry powder inhaler (FP-DPI) (100 microg) were used. The effects of different mouth washing methods were evaluated by quantification of drugs in the expectorated rinse solution using an HPLC method. The amounts of BDP recovered in the rinse after gargling and rinsing for 5 s each were 47.1+/-13.6 microg, while they were 42.9+/-9.4 microg after rinsing alone for 10 s and 38.7+/-9.2 microg after gargling alone for 10 s. Under the same conditions, FP amounts were 32.9+/-7.3 microg, 28.9+/-2.4 microg, and 27.1+/-7.9 microg, respectively. In a comparison of washing time, the amounts of BDP recovered were 49.8+/-9.7 microg after gargling and rinsing for 2 s each, 53.5+/-10.2 microg after those for 3 s each, and 47.1+/-13.6 microg after those for 5 s each, while the amounts of FP under the same conditions were 36.4+/-2.4 microg, 33.3+/-6.4 microg, and 32.9+/-7.4 microg, respectively. As for the effect of time lag before mouth washing, the amount of BDP recovered decreased by 65.7% with a lag time of 1 min and by 5.6% after 10 min, while that of FP decreased by 51.1% with a lag time of 1 min and by 7.7% after 10 min. Our results suggest that the amount of drugs removed by mouth washing is significantly associated with the time lag between inhalation and mouth washing. We concluded that immediate gargling and rinsing after inhalation is most useful for the removal of drugs following inhalation of corticosteroids.

Separación de los epímeros de la 17-cianhidrina-4-androsten-3-ona por cromatografía de capa fina (comunicación corta) / Separation of epimeres in 17-ciandrine-4-andiosten-3-ona by thin-layer chromatography

Se describe una técnica para la separación de los epímeros 17a-ciano-17b-hidroxi- y 17b-ciano-17a–hidroxi-4-androsten-3-ona, mediante cromatografía de capa fina con doble desarrollo en una misma fase móvil.Se reportan los valores de Rf en cada sistema de solventes utilizados, así como los por ciento de androstendiona (AD) y a-epímero en los productos crudos obtenidos por el método de síntesis descrito(AU)

Use of reduced sorbent bed and disk membrane solid-phase extraction for the analysis of pharmaceutical compounds in biological fluids, with applications in the 96-well format.

Significant improvements in the isolation of pharmaceutical compounds from plasma, serum and urine, have been achieved using ultra low mass sorbent bed and thin disk solid-phase extraction (SPE) material. The use of low sorbent masses or disk SPE material has allowed a significant reduction in solvent usage and extraction times. The reduction in solvent volumes required has allowed elution volumes to be reduced to as low as 30 microl with high and consistent analyte recovery. Several SPE RP-HPLC methods have been developed using these materials, including LC-MS methods. When the chromatographic conditions allow the eluent to be injected directly or injected after dilution with distilled water Empore disks are the extraction media of choice due to the materials low elution volume requirements. When operated in the 96-well microtitre format this micro-extraction provides a very efficient throughput and requires little sample manipulation.

Characterisation of impurities in bulk drug batches of fluticasone propionate using directly coupled HPLC-NMR spectroscopy and HPLC-MS.

Directly coupled HPLC NMR spectroscopic and HPLC-MS approaches have been used to confirm the identity of four known dimeric impurities in a partially purified batch of fluticasone propionate each at levels of 0.06-0.9% of parent compound based on UV absorption. It is also shown that HPLC NMR spectroscopy of the main drug peak in the 'time-slice' mode of operation, in which the elution of the HPLC peak is sampled a short time intervals, can be used to investigate the purity profile of the single HPLC peak detected by UV absorption. These studies show that HPLC-NMR is of considerable value in rapidly assessing HPLC peak purity and hence will be of benefit in providing additional information to support submission for drug registration to regulatory agencies.

Andrastins A-C, new protein farnesyltransferase inhibitors produced by Penicillium sp. FO-3929. I. Producing strain, fermentation, isolation, and biological activities.

New protein farnesyltransferase inhibitors, andrastins A-C, have been discovered in the cultured broth of Penicillium sp. FO-3929. Andrastins extracted from broth supernatant were purified by silica gel chromatography, ODS chromatography and HPLC. The IC50 of andrastins A, B, and C against protein farnesyltransferase were 24.9, 47.1, and 13.3 microM, respectively.

Wortmannin binds specifically to 1-phosphatidylinositol 3-kinase while inhibiting guanine nucleotide-binding protein-coupled receptor signaling in neutrophil leukocytes.

Wortmannin (WT) and its derivative 17-hydroxywortmannin (HWT) inhibit at nanomolar concentrations superoxide formation and exocytosis in neutrophils stimulated with chemotactic agonists. Treatment of neutrophils with radiolabeled [3H]HWT resulted in specific and saturable binding that paralleled the inhibition of the respiratory burst. Both half-maximal binding and half-maximal inhibition were observed at 5 nM, and > 90% of maximal binding and inhibition was observed at 20 nM HWT. Fluorography of subcellular fractions that were separated on NaDodSO4/PAGE showed that [3H]HWT binds covalently to a 110-kDa cytosolic protein. The WT-binding protein was purified from human neutrophils and bovine brain homogenates by column chromatography. The pure protein was eluted from gel filtration columns with an apparent molecular mass of 200 kDa and showed a heterodimeric structure on Coomassie-stained NaDodSO4/PAGE. In addition to the 110 kDa wortmannin binding protein an equally intense band was seen migrating at 85 kDa. This band was identified on Western blots as p85 alpha, the regulatory subunit of phosphatidylinositol (PI) 3-kinase (ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137). The purified protein contained PI 3-kinase activity that was enriched > 20,000-fold from human neutrophil cytosol during preparation. The data impose a key role for PI 3-kinase-mediated signal transduction through guanine nucleotide-binding protein-coupled receptors and suggest that 3-phosphorylated inositol phospholipids are important second messengers for immediate responses in neutrophils. Furthermore, the results show that WT is a powerful and selective tool to study the function of PI 3-kinase.