Three new phomalone derivatives from a deep-sea-derived fungus Alternaria sp. MCCC 3A00467.

Three new phomalone derivatives, phomalichenones E-G (1-3), and seven known analogues (4-10) were isolated from the cultures of a deep-sea-derived fungus Alternaria sp. MCCC 3A00467. Their structures were elucidated by spectroscopic methods, including the 1D and 2D NMR, and ECD spectrum. Among the compounds isolated, phomalichenone F (2) presented cytotoxic activity against human myeloma cancer U266 cells with IC50 value of 24.99 µg/mL. The most active compound, 10, showed cytotoxicity against U266, HepG2 and A549 cells with IC50 values of 13.26, 14.69 and 24.39 µg/mL, respectively.

Two new phenylspirodrimanes from the deep-sea derived fungus Stachybotrys sp. MCCC 3A00409.

Two new phenylspirodrimanes, stachybotrin H (1) and stachybotrysin H (9) together with eleven known analogues (2-8, 10-13) were isolated from deep-sea derived Stachybotrys sp. MCCC 3A00409. Their structures were determined by extensive NMR data and mass spectroscopic analysis. Compounds 9-12 showed weak cytotoxicity against three human cancer cell lines K562, Hela and HL60 with IC50 in the range of 18.5-52.8 µM.

A new indole-type alkaloid from the roots of Clematis florida var. plena.

One new indole-type alkaloid, α-L-rhamnopyranosyl-(1→6)-ß-D- glucopyranosyl 6-methoxy-3-indolecarbonate (1), together with three known alkaloids (2-4), one aromatic acid (5) and five known saponins (6-10), was isolated from the roots of Clematis florida var. plena. Their structures were established by NMR spectroscopic analysis and acid hydrolysis. In in vivo anti-inflammatory activity, n-butanol extract was found to be potent against ear edema in mice, with inhibition rate of 48.7% at a dose of 800 mg/kg. Furthermore, compounds 8 and 9 obtained from the n-butanol extract exhibited significant anti-inflammatory activities with inhibition rates of 50.9% and 54.7% at a dose of 200 mg/kg.

Discovery, gene modification, and optimization of fermentation of an enduracidin-producing strain.

Enduracidin significantly inhibits Gram-positive bacteria and had been widely used in many fields. However, as the poor technology for production of enduracidin and its scarcity, identification of novel strategies for production of enduracidin is important. Our group developed two methods to improve the yield of the production of enduracidin. The yield of enduracidin was increased by three- to fivefold. The highest yields of enduracidin A and enduracidin B achieved were 63.7 and 82.13 mg/ml. Thus, our results might provide a new reference method for the industrial production of enduracidin.

Four New Tirucallane Triterpenoids from the Fruits of Melia azedarach and Their Cytotoxic Activities.

Four new tirucallane triterpenoids, (21S,23R,24R)-21,23-epoxy-21,24-dihydroxy-25-methoxytirucall-7-en-3-one (2), (3S,21S,23R,24S)-21,23-epoxy-21,25-dimethoxytirucall-7-ene-3,24-diol (8), (21S,23R,24R)-21,23-epoxy-24-hydroxy-21-methoxytirucalla-7,25-dien-3-one (11), and (21S,23R,24R)-21,23-epoxy-21,24-dihydroxytirucalla-7,25-dien-3-one (12), along with 16 known analogues, 1, 3 - 7, 9 - 10, and 13 - 20, were isolated from the fruits of Melia azedarach. Their structures were elucidated by spectroscopic methods including 1D- and 2D-NMR techniques and mass spectrometry. These compounds were evaluated for their cytotoxicities against HepG2 (liver), SGC7901 (stomach), K562 (leukemia), and HL60 (leukemia) cancer cell lines. Compound 20 exhibited potent cytotoxicity against HepG2 and SGC7901 cancer cells with the IC50 values of 6.9 and 6.9 µm, respectively.

ProAir(®) HFA delivers warmer, lower-impact, longer-duration plumes containing higher fine particle dose than Ventolin(®) HFA.

BACKGROUND: Inhaler technique and spray characteristics are critical for adequate management of asthma symptoms with pressurized metered-dose inhalers (pMDIs). A lower spray force has been directly associated with a decrease in throat deposition of asthma medication, and a higher spray temperature may alleviate the "cold Freon effect" associated with pMDIs. The objective of the study was to characterize and compare the temperature, maximum spray force, and duration of the emitted plume from two pMDIs: ProAir(®) hydrofluoroalkane (HFA) and Ventolin(®) HFA. METHODS: A spray force tester model SFT1000 and thermocouple were used to test 10 units from three separate lots (total of 30 units) of each inhaler type. Three consecutive actuations were tested at a spray distance of 40 mm from the edge of the mouthpiece. Room temperature, humidity, and initial weight of the pMDI were recorded. Final weight of each pMDI was recorded to determine the spray weight of individual actuations. pMDIs were primed and operated according to instructions provided in the package insert. Aerodynamic particle size distribution (APSD) was also assessed using a next-generation impactor at a flow rate of 28.3 L/min. RESULTS: Measurements were obtained from three consecutive actuations for each of 30 units of ProAir(®) HFA and Ventolin(®) HFA (10 units from three separate lots), resulting in a total of 90 actuations tested for each pMDI. Minimum plume temperatures recorded were 7.2 ± 0.7°C and -35.9 ± 12.7°C, respectively, for ProAir(®) HFA and Ventolin(®) HFA. ProAir(®) HFA produced more than a twofold greater plume duration (385 ± 46 ms vs. 156 ± 58 ms; p<0.001) and a significantly lower mean maximum spray force (33.6 ± 11.4 mN vs. 75.9 ± 12.0 mN; p<0.0001) compared with Ventolin(®) HFA. APSD analysis demonstrated that ProAir(®) HFA produced almost twice as much fine particle (<5 µm) dose with lower geometric standard deviation, compared with Ventolin(®) HFA. Two inhalers produced similar mass median aerodynamic diameters, ranging from 2.3 to 2.4 µm. CONCLUSIONS: The ProAir(®) HFA delivers a warmer, lower-impact, and longer-lasting plume compared with Ventolin(®) HFA, which may provide a more consistent, comfortable experience for patients using a pMDI. ProAir(®) HFA produces higher fine particle dose than Ventolin(®) HFA.

The 'stage-by-stage' deposition of drugs from commercial single-active and combination dry powder inhaler formulations.

Inhalation of salmeterol xinafoate (SX) and fluticasone propionate (FP) from a combination product is reported to produce superior clinical outcomes in comparison to the concurrent administration of 'similar' doses via separate single-active inhalers. For bioequivalence determination across different products, emphasis is currently placed on the assessment of drug deposition within inertial impactors on a 'stage-by-stage' basis as stipulated in a recent European Medicines Agency guidance. The aim of this study was to compare the stage-by-stage deposition of drugs aerosolised from the single-active Accuhaler® products Serevent® (SX) and Flixotide® (FP) with the SX-FP combination product Seretide® Accuhaler® in vitro. Drug deposition on a stage-by-stage basis was assessed using the next generation impactor (NGI). Significant differences in drug deposition profiles were obtained following aerosolisation from the single-active versus combination products. The concurrent administration of the two single-active products: Serevent® and Flixotide® Accuhaler® may not be bioequivalent to inhalation of the combination product Seretide® Accuhaler®. The observed differences may have resulted from different characteristics of the active pharmaceutical ingredient (APIs) and the carrier alpha-lactose monohydrate between the single-active and combination inhalers and/or a change in the drug-carrier inter-particulate interaction as a consequence of the presence of a second active.

The production of 'aerodynamically equivalent' drug and excipient inhalable powders using a novel fractionation technique.

Inhalation particles can be produced by various techniques such as milling, controlled crystallisation and spray-drying, but current methods cannot, to-date, precisely control the aerodynamic size distribution of produced powders. The aim of this study was to develop and validate a novel preparative technique whereby the efficient and reproducible aerodynamic fractionation of drug and excipient powders could be achieved. Salmeterol xinafoate (SX), fluticasone propionate (FP) and fine α-lactose monohydrate (FL) were chosen as model compounds. Powders were aerosolised using a dry powder feeder into a Next Generation Impactor operated at 60 L min(-1). Powders deposited on NGI stages were then collected and analysed. The fractionation process was successful for all powders producing significant linear correlations between the pre-set aerodynamic cut-off limits and geometric size measurements. For each of SX, FP and FL, sufficient powder quantities were recovered from NGI stages 1-6 producing six fractions with sequential aerodynamic and geometric particle size distributions. The fractionation technique was efficient and reproducible for all powders studied. The method can be equally applied to various drugs and excipients regardless of their previous production/processing history. Therefore, the aerodynamic fractionation technique may be used to compare and contrast samples produced by different processes.

Aerodynamic deposition of combination dry powder inhaler formulations in vitro: a comparison of three impactors.

Inertial impaction is generally regarded as the 'gold standard' for the in vitro assessment of aerodynamic deposition of inhaled formulations. Despite the availability of several impactors, few studies have compared measurements of aerodynamic deposition using multiple impactors and none employed a combination formulation. The aerodynamic deposition of the combination dry powder inhaler (DPI) Seretide Accuhaler, which contains salmeterol xinafoate (SX) and fluticasone propionate (FP), was assessed using the Andersen cascade impactor (ACI), multi-stage liquid impinger (MSLI) and next generation impactor (NGI) and the results were compared. Two Seretide products were tested at flow rates of 30 and QLmin(-1), the latter corresponding to a pressure drop of 4kPa across the device. Significant differences in the particle size distributions were observed when the same formulation was tested using various impactors. The ACI was found to be less suitable for DPI testing at flow rates considerably higher than 28.3Lmin(-1) due to the significant overlap in the cut-off curves of the pre-separator and stage 0. This was not the case with the MSLI but the data derived were limited by the relatively small number of stages. Deposition data determined by the three impactors were significantly different. The NGI produced good resolution and minimal inter-stage overlap and was regarded as the impactor of choice for DPI testing.

[Application of skin and soft tissue distraction technique for defect of skin and soft tissue].

OBJECTIVE: To investigate the repair of skin and soft tissue defect by skin and soft tissue distraction tecnique and evaluate clinical results. METHODS: Twenty-five patients with skin and soft tissue defect were repaired by skin and soft tissue distraction technique including 16 male and 9 female. The age ranged from 8 to 66 years with an average age of 29.3 years. Parallel Kirschner wires were inserted through the defect skin margin at the interval of every 0.5 to 1.5 cm, the Kirschner wire was gradually drawed to the close by thick silk thread or tiny steel-wire, the defect of skin and soft tiuuse was closed from two sides and the wound disappeared. RESULTS: The wounds of 15 patients were anastomosed dircetly through skin and soft tissue distraction for 20 to 60 minutes, other ten wounds were anastomosed from 3 to 26 days, all healed well. By the follow up from 2 to 26 months, the wound skin looked as line scar, corresponding to the result of common debridement anastomosis. CONCLUSION: Skin and soft tissue distraction tecnique can not replace skin grafting, flap transferring and flap grafting. But it may avoid damage to the supply area and the risk of flap necrosis. Skin and soft tissue distraction tecnique is a good method to repair skin and soft tissue defect.

HPLC method for the pharmacokinetics and tissue distribution of taspine solution and taspine liposome after intravenous administrations to mice.

Taspine is a bioactive aporphine alkaloid, which has many potent pharmacological effects. A simple, rapid HPLC method to quantify taspine in mouse plasma and tissue homogenates containing either taspine solution or liposome was developed and validated. Sample preparation was achieved by liquid-liquid extraction with acetoacetate. Taspine was separated on a C(18) reversed phase HPLC column, and quantified by its absorbance at 245 nm. The pharmacokinetics and tissue distribution after intravenous administrations of taspine liposome (L-Ta) and taspine solution (Ta) to ICR mice were then compared. The area under the plasma concentration-time curve (AUC) was higher for L-Ta than for Ta. In contrast, the total body clearance (CL), apparent volume of distribution V(c) and plasma half-life for the distribution (t(1/2 alpha)) and elimination phase (t(1/2 beta)) were lower for L-Ta, in comparison to the respective parameter of Ta. The AUC values were higher in the lung than in other organs for both L-Ta and Ta. The AUC in the spleen, kidney and liver of L-Ta were higher than those of Ta. However, the heart and brain AUC of Ta was higher than that of L-Ta. It can thus be concluded that incorporation into liposomes prolonged taspine retention within the systemic circulation, increased its distribution to the spleen and liver but reduced its distribution to the heart and brain.

[Study on association of BAFF receptors gene expression and primary biliary cirrhosis].

OBJECTIVE: To explore the relationship between expression level of B cell-activating factor belonging to the TNF family (BAFF) receptor (BAFF-R) gene family and primary biliary cirrhosis (PBC). METHODS: With 18S rRNA as control, real-time fluorescent semi-quantification reverse transcriptional PCR was established, according to the difference of threshold cycles (DeltaCt) of target genes and 18S rRNA. B cell maturation antigen (BCMA), transmembrane activator and CAMI-interactor (TACI) and BAFF-R mRNA of peripheral blood mononuclear cells (PBMCs) in 30 healthy people and 30 PBC patients were measured. The correlation between their gene expression levels and the concentrations of IgM, ALP and GGT was analyzed. RESULTS: The relative expression level of BCMA mRNA in patients with PBC was 8.6 folds higher than that of control (P < 0.05). In contrast, PBC patients showed relatively lower TACI mRNA level than control. For BAFF-R mRNA, there was no significant difference between patients and control. The gene expression of BCMA showed a close correlation with IgM, GGT and ALP (all P < 0.01), while BAFF-R showed no significant correlation with them (all P > 0.05). The correlation between TACI and IgM was significant (P < 0.05), but not for GGT or ALP (both P > 0.05). CONCLUSION: The gene expression of PBMCs in PBC patients is significantly elevated for BCMA and decreased for TACI, indicating that the pathogenesis of PBC is closely related to enhanced humoral immunity and broken tolerance.

Influence of realistic inspiratory flow profiles on fine particle fractions of dry powder aerosol formulations.

PURPOSE: The purpose of the study was to determine how air flow profiles affect fine particle fractions (FPF) (<5 microm) from dry powder aerosol formulations and whether laser diffraction (LD) could be used to measure FPF of aerosols generated by variable flows. MATERIALS AND METHODS: Carrier-based formulations containing 1.5% w/w micronized salbutamol base blended with the 63-90 microm fraction of alpha-lactose monohydrate or sorbitol or maltose were aerosolised from a model glass device using either a constant flow rate or a predetermined flow profile. The FPFs of the same aerosolised particles were first measured by LD and then by a liquid impinger. Volunteer inhalation airflow profiles and 3-phase (acceleration, constant flow rate and deceleration) square wave airflow profiles were generated using the Electronic Lung and an Inhalation Profile Recorder. Similar experiments were conducted for a carrier-free formulation from the Bricanyl Turbohaler. RESULTS: Salbutamol FPFs of all carrier-based formulations were found to increase by increasing the initial flow increase rate (FIR) from 200 to 600 l min(-1) s(-1) although they could be placed in an increasing order of maltose blend < sorbitol blend < lactose blend. A significant linear correlation was found between FPFs measured by LD and by inertial impaction (R (2) = 0.95, p < 0.01, ANOVA). For the Bricanyl Turbohaler, increasing FIR from 120 to 600 l min(-1) s(-1) for a constant peak flow rate (PFR) of 60 l min(-1) increased the mean Terbutaline FPF from 18.2% to 45.5%. For the volunteer inhalation profiles, a higher FIR tended to be associated with higher PFR, leading to a marked increase in drug FPF due to the combined effect of FIR and PFR. CONCLUSION: Drug FPF from either carrier-free or carrier-based formulations is determined by both FIR and PFR. LD is a viable technique to measure the performance of dry powder aerosol formulations at realistic inspiratory flow profiles.

Humidity-induced changes of the aerodynamic properties of dry powder aerosol formulations containing different carriers.

This paper presents the findings of two related studies. The aim of the first was to study any changes in the aerodynamic properties of salbutamol base powder formulations when different sugars were used as the carriers, after storage at an elevated humidity (75% RH), and whether any such changes (if any) were related to the physical properties of the carriers. The aim of the second was to investigate whether "ageing", i.e. storage of the carrier, drug and blends under desiccation for more than 2 years, affected the aerodynamic properties of salbutamol sulphate powder formulations. Different formulations were prepared, each containing 1.5% (w/w) micronised salbutamol base or sulphate blended with the sieved fraction (63-90 microm) of one of the following sugars: alpha lactose monohydrate, sorbitol, maltose and dextrose. The salbutamol base blends were then stored unprotected at 75% RH (ambient temperature) and salbutamol fine particle fractions (FPFs) were measured by laser diffraction (LD) (% < 5.2 microm) and a multistage liquid impinger (MSLI) (% < 5.3 microm), following aerosolisation at 100 l min(-1) from a model glass inhaler, after storage of each formulation at the elevated conditions for 0, 1 and 6 days. Particle morphology and equilibrium moisture content (EMC) of each formulation prior to and after storage were also evaluated. However, the salbutamol sulphate blends containing either "fresh" or "aged" components were only characterized using LD at 60 l min(-1). Prior to exposure to 75% RH, the lactose blend was found to give the highest FPF of salbutamol (30% by LD and 37% by MSLI), followed by the sorbitol blend (17% by LD and 29% by MSLI), then by the dextrose blend (15% by LD and 25% by MSLI) and finally by the maltose blend (13% by LD and 13% by MSLI). Exposure to 75% RH for 6 days resulted in a small reduction of salbutamol FPF from the lactose blend but drastic diminution of salbutamol FPFs from other blends. After exposure to the high RH, the lactose blend adsorbed ca. 0.4% whilst each of the other sugars took up larger quantities of water (15-40%) and underwent a marked change in the surface texture of the particles. "Ageing" of the carriers and/or formulations did not seem to alter the aerodynamic properties of the drug. "Ageing" of micronised salbutamol sulphate prior to blending, however, was found to improve the FPF of drug. LD was capable of detecting subtle differences between the various formulations and generated FPF results that correlated with those measured by MSLI.

Development of a laser diffraction method for the determination of the particle size of aerosolised powder formulations.

Impactor data are an essential component of marketing authorisation for new dry powder aerosol formulations. However such data are time-consuming to obtain and therefore impede the rapid screening of pilot formulations. In this phase of development it would be of considerable benefit to employ a technique where data acquisition was more rapid, such as laser diffraction, to predict the fine particle fraction. It was the aim of this study to investigate whether this is a feasible premise. Five different formulations were prepared, each containing 1.5% (w/w) micronised salbutamol base (volume median diameter: 2.42 microm) blended with the sieved fraction (63-90 microm) of one of the following sugars: regular crystalline lactose, spray dried lactose "Zeparox", sorbitol, maltose and dextrose monohydrate. A Perspex box was constructed to contain particles released from a glass inhaler and allow the particles to be measured by laser diffraction at different flow rates. After being validated using monodisperse aerosols, this assembly was then employed to measure the particle size distributions of each powder formulation and its respective sugar carrier at flow rates ranging from 28.3 to 100 l min(-1). Aerodynamic particle size distribution of salbutamol base from each formulation was also measured after aerosolisation at 28.3 l min(-1) from the glass inhaler into an Andersen cascade impactor. The flight of monodisperse particles with diameters (2-6 microm) in the desired size range of dry powders for inhalation could be contained and the size distribution determined by laser diffraction using the assembly at all flow rates investigated. Treatment of the particle size distributions measured by laser diffraction, i.e. examining only the aerosol particles with diameter <60 microm, highlighted the fine fraction (<5 microm) and enabled the aerosolisation of different blends to be feasibly compared at a range of different flow rates. The blends containing the following excipients could be placed in the following order of increasing fine fraction: spray-dried lactose

Characterisation of a carrier-free dry powder aerosol formulation using inertial impaction and laser diffraction.

PURPOSE: The purpose of the study was to examine the suitability of using laser diffraction to measure the fine particle fraction (FPF) of drugs emitted from carrier-free dry powder aerosol formulations. MATERIALS AND METHODS: Particle size distribution of terbutaline sulphate from Bricanyl Turbohaler, which contained loose agglomerates of drug particles only, was measured separately by laser diffraction apparatus equipped with a metal throat and a twin-stage, multi-stage liquid impingers, or Andersen cascade impactor at flow rates ranging from 28.3 to 100 l min(-1). In-line measurements were then conducted which allowed the same aerosolised particles to be measured first by laser diffraction then captured by an impactor or impinger for subsequent chemical analysis. RESULTS: A significant linear correlation (p < 0.001, R2 = 0.96, ANOVA) existed between the results obtained from two techniques when measurements were conducted independently. There was little difference in FPFs measured by inertial impaction and laser diffraction at the same flow rate. When in-line measurements were conducted, the FPFs measured by inertial impaction were approximately 0.7-0.9 times the aerosol FPFs measured by laser diffraction. This linear relationship was statistically significant and had a statistically insignificant y-intercept, regardless of inhaler batches, impinger types and measuring position of the laser beam. CONCLUSION: Laser diffraction could prove to be a reliable technique for development, evaluation and quality control of carrier-free, dry powder aerosol formulations.

Correlation between inertial impaction and laser diffraction sizing data for aerosolized carrier-based dry powder formulations.

PURPOSE: The purpose of the study was to determine whether the drug fine particle fraction (FPF) from different dry powder aerosol formulations measured by laser diffraction at a range of flow rates correlated with that measured by inertial impaction. MATERIALS AND METHODS: Ten binary formulations were prepared containing 1.5% w/w salbutamol base or sulphate, blended with the sieved (63-90 microm) fraction of different sugars (regular lactose, spray-dried lactose, sorbitol, dextrose or maltose). A further six ternary formulations were prepared containing 1.5% w/w salbutamol sulphate, 97% coarse lactose (63-90 microm) and 1.5% micronised or intermediate-sized lactose (1-50 microm). The FPF particles (< 5 microm) of these formulations were measured by laser diffraction and inertial impaction at flow rates between 28.3 and 100 l min(-1). RESULTS: When only the particles with diameter < 60 microm obtained by laser diffraction were considered the FPF (< 5 microm) could be determined and this enabled the aerosolisation of all 16 blends to be feasibly compared at flow rates ranging from 28.3 to 100 l min(-1). A significant linear correlation was found between the fine fractions measured by laser diffraction and the salbutamol fine fractions determined by inertial impaction (r2 = 0.934). Such correlation was also confirmed for formulations containing added fine lactose. CONCLUSION: Particle size measured by laser diffraction under the employed conditions reflected the aerodynamic properties of the drug. Laser diffraction can be used as on-, in- and/or at-line measurements and controls for dry powder aerosol formulations.