An Investigation into the Effect of Fine Lactose Particles on the Fluidization Behaviour and Aerosolization Performance of Carrier-Based Dry Powder Inhaler Formulations

The effect of milled and micronized lactose fines on the fluidization and in vitro aerosolization properties of dry powder inhaler (DPI) formulations was investigated, and the suitability of static and dynamic methods for characterizing general powder flow properties of these blends was assessed. Lactose carrier pre-blends were prepared by adding different lactose fines (Lactohale® (LH) 300, 230 and 210) with coarse carrier lactose (Lactohale100) at 2.5, 5, 10 and 20 wt% concentrations. Powder flow properties of lactose pre-blends were characterized using the Freeman Technology FT4 and Schulze RST-XS ring shear tester. A strong correlation was found between the basic flow energy (BFE_Norm) measured using the Freeman FT4 Rheometer and the flowability number (ff_c) measured on Schulze RST-XS. These data indicate that both static and dynamic methods are suitable for characterizing general powder flow properties of lactose carriers. Increasing concentration of fines corresponded with an increase in the normalized fluidization energy (FE_Norm). The inclusion of fine particles of lactose resulted in a significant (p < 0.05) increase in fine particle delivery of budesonide and correlated with FE_Norm. This trend was strongest for lactose containing up to 10 wt% LH300. A similar trend was found for the milled lactose grades LH230 and LH210. However, the increase in FE_Norm upon addition of milled fines only corresponded to a very slight improvement in the performance. These data suggest that whilst the fluidization energy correlated with fine particle delivery, this relationship is specific to lactose grades of similar particle size.KEY WORDS: dry powder inhaler, fluidisation, lactose, powder flow     

Niosomal Gel of Lornoxicam for Topical Delivery: In vitro Assessment and Pharmacodynamic Activity

Lornoxicam is a potent oxicam class of non steroidal anti-inflammatory agent, prescribed for mild to moderate pain and inflammation. Niosomal gel of lornoxicam was developed for topical application. Lornoxicam niosomes (Lor-Nio) were fabricated by thin film hydration technique. Bilayer composition of niosomal vesicles was optimized. Lor-Nio dispersion was characterized by DSC, XRD, and FT-IR. Morphological evaluation was performed by scanning electron microscopy (SEM). Lor-Nio dispersion was incorporated into a gel using 2% w/w Carbopol 980 NF. Rheological and texture properties of Lor-Nio gel formulation showed suitability of the gel for topical application. The developed formulation was evaluated for in vitro skin permeation and skin deposition studies, occlusivity test and skin irritation studies. Pharmacodynamic activity of the Lor-Nio gel was performed by carragenan-induced rat paw model. Optimized Lor-Nio comprised of Span 60 and cholesterol in a molar ratio of 3:1 with 30 μM dicetyl palmitate as a stabilizer. It had particle size of 1.125 ± 0.212 μm (d₉₀), with entrapment efficiency of 52.38 ± 2.1%. DSC, XRD, and IR studies showed inclusion of Lor into niosomal vesicles. SEM studies showed spherical closed vesicular structure with particles in nanometer range. The in vitro skin permeation studies showed significant improvement in skin permeation and skin deposition for Lor-Nio gel (31.41 ± 2.24 μg/cm², 30.079 ± 1.2 μg/cm²) over plain lornoxicam gel (7.37 ± 1.27 μg/cm², 6.6 ± 2.52 μg/cm²). The Lor-Nio gel formulation showed enhanced anti-inflammatory activity by exhibiting mean edema inhibition (87.69 ± 1.43%) which was significantly more than the plain lornoxicam gel (53.84 ± 2.21%).KEY WORDS: anti-inflammatory activity, lornoxicam, niosomes, rheology, texture analysis     

Freeze-Dried Targeted Mannosylated Selenium-Loaded Nanoliposomes: Development and Evaluation

The aim of this investigation was to develop and evaluate freeze-dried mannosylated liposomes for the targeted delivery of selenium. Dipalmitoylphosphatidylcholine, distearoylphosphatidylglycerol, and cholesterol were dissolved in a chloroform and methanol mixture and allowed to form a thin film within a rotatory evaporator. This thin film was hydrated with a sodium selenite (5.8 μM) solution to form multilamellar vesicles and homogenized under high pressure to yield unilamellar nanoliposomes. Se-loaded nanoliposomes were mannosylated by 0.1% w/v mannosamine (Man-Lip-Se) prior to being lyophilized. Mannosamine concentration was optimized with cellular uptake studies in M receptor expressing cells. Non-lyophilized and lyophilized Man-Lip-Se were characterized for size, zeta potential, and entrapment efficiency. The influence of liposomal composition on the characteristics of Man-Lip-Se were evaluated using acidic and basic medium for 24 h. Thermal analysis and powder X-ray diffraction were used to determine the interaction of components within the Man-Lip-Se. The size, zeta potential and entrapment efficiency of the optimum Man-Lip-Se were observed to be 158 ± 28.9 nm, 33.21 ± 0.89 mV, and 77.27 ± 2.34%, respectively. An in vitro Se release of 70–75% up to 24 h in PBS pH 6.8 and <8% Se release in acidic media (0.1 N HCl) in 1 h was observed. The Man-Lip-Se were found to withstand gastric-like environments and showed sustained release. Stable freeze-dried Man-Lip-Se were successfully formulated with a size of <200 nm, ∼75% entrapment, and achieved controlled release of Se with stability under acidic media, which may be of importance in the targeted delivery of Se to the immune system.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-013-9988-3) contains supplementary material, which is available to authorized users.Key words: mannosylation, nanoliposome, selenium, thermal properties     

Increased In Situ Intestinal Absorption of Phytoestrogenic Diarylheptanoids from Curcuma comosa in Nanoemulsions

Curcuma comosa has long been used as a gynecological medicine. Several diarylheptanoids have been purified from this plant, and their pharmacological effects were proven. However, there is no information about the absorption of C. comosa components to support the formulation usage. In the present study, C. comosa hexane extract and the mixture of its two major compounds, (4E,6E)-1,7-diphenylhepta-4,6-dien-3-ol (DA1) and (6E)-1,7-diphenylhept-6-en-3-ol (DA2), were formulated into nanoemulsions. The physical properties of the nanoemulsions and the in situ intestinal absorptions of DA1 and DA2 were evaluated. The results demonstrated the mean particle sizes at 0.207 ± 0.001 and 0.408 ± 0.014 μm, and the zeta potential at −14.57 ± 0.85 and −10.47 ± 0.32 mV for C. comosa nanoemulsion (C.c-Nano) and mixture of diarlylheptanoid nanoemulsions (DA-Nano), respectively. The entrapments of DA1 and DA2 were 76.61% and 75.41%, and 71.91% and 71.63% for C.c-Nano and DA-Nano, respectively. The drug loading ratios of DA1 and DA2 were 351.47 and 614.53 μg/mg, and 59.48 and 126.72 μg/mg for C.c-Nano and DA-Nano. The intestinal absorption rates of DA1 and DA2 were 0.329 ± 0.015 and 0.519 ± 0.026 μg/min/cm² in C.c-Nano, and 0.380 ± 0.006 and 0.428 ± 0.036 μg/min/cm² in DA-Nano, which were five to ten times faster than those in oil. In conclusion, the formulation in nanoemulsion forms obviously increased the intestinal absorption rate of diarylheptanoids.KEY WORDS: Curcuma comosa, diarylheptanoids, intestinal absorption, nanoemulsion, phytoestrogen     

Mixed Polyethylene Glycol-Modified Breviscapine-Loaded Solid Lipid Nanoparticles for Improved Brain Bioavailability: Preparation,Characterization, and In Vivo Cerebral Microdialysis Evaluation in Adult Sprague Dawley Rats

Breviscapine is used in the treatment of ischemic cerebrovascular diseases, but it has a low bioavailability in the brain due to its poor physicochemical properties and the activity of P-glycoprotein efflux pumps located at the blood–brain barrier. In the present study, breviscapine-loaded solid lipid nanoparticles (SLN) coated with polyethylene glycol (PEG) derivatives were formulated and evaluated for their ability to enhance brain bioavailability. The SLNs were either coated with polyethylene glycol (40) (PEG-40) stearate alone (Bre-GBSLN-PS) or a mixture of PEG-40 stearate and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-PEG2000 (DSPE-PEG₂₀₀₀) (Bre-GBSLN-PS-DSPE) and were characterized both in vitro and in vivo. The mean particle size, polydispersity index, and entrapment efficiency for Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE were 21.60 ± 0.10 and 22.60 ± 0.70 nm, 0.27 ± 0.01 and 0.26 ± 0.04, and 46.89 ± 0.73% and 47.62 ± 1.86%, respectively. The brain pharmacokinetic parameters revealed that the brain bioavailability of breviscapine from the Bre-GBSLN-PS and Bre-GBSLN-PS-DSPE was significantly enhanced (p < 0.01) with the area under concentration–time curve (AUC) of 1.59 ± 0.39 and 1.42 ± 0.58 μg h/mL of breviscapine, respectively, in comparison to 0.11 ± 0.02 μg h/mL from the commercial breviscapine injection. The ratios of the brain AUC for scutellarin in comparison with the plasma scutellarin AUC for commercial breviscapine injection, Bre-GBSLN-PS, and Bre-GBSLN-PS-DSPE were 0.66%, 2.82%, and 4.51%, respectively. These results showed that though both SLN formulations increased brain uptake of breviscapine, Bre-GBSLN-PS-DSPE which was coated with a binary combination of PEG-40 stearate and DSPE-PEG₂₀₀₀ had a better brain bioavailability than Bre-GBSLN-PS. Thus, the coating of SLNs with the appropriate PEG derivative combination could improve brain bioavailability of breviscapine and can be a promising tool for brain drug delivery.KEY WORDS: breviscapine, microdialysis, mixed PEGylation, P-glycoprotein (P-gp), solid lipid nanoparticles     

Effect of Grewia Gum as a Suspending Agent on Ibuprofen Pediatric Formulation

The purpose of this work was to evaluate the potential of grewia gum (GG) as a suspending agent in pharmaceutical oral formulation using ibuprofen as model drug. Ibuprofen pediatric suspension (25 mg/5 mL) was formulated with grewia gum (0.5% w/v) as the suspending agent. Similar suspensions of Ibuprofen containing either sodium carboxymethylcellulose (Na-CMC) or hydroxymethylpropylcellulose (HPMC) were also produced. The suspensions were evaluated for ease of redispersion, sedimentation, rheological properties, and the effect of aging on the rheological properties at 25°C. The particle size and particle size distributions of the dispersed solute were determined. The redispersion time was 19, 11, and 0.5 min, respectively, for formulation containing Na-CMC, HPMC, and GG .The sedimentation volumes were 0.05, 0.05, and 0.125 mL, respectively, for Na-CMC, HPMC, and GG . Viscosities of suspensions at spindle speed of 25 rpm were of the order: GG > HPMC > Na-CMC when freshly prepared and of the order: HPMC > GG > Na-CMC within 6 months of storage. The particles size was 72.72, 73.82, 81.93, and 83.41 μm, respectively, for suspensions containing Na-CMC, ibuprofen alone, HPMC, and GG. Greatest hysteresis was observed in formulation containing HPMC. All the formulations were stable. It was our conclusion that the difference in the physicochemical properties of ibuprofen pediatric formulations was influenced more by the suspending agent used in the formulations than the drug. GG combined better redispersion with minimal changes in viscosity on storage compared to Na-CMC and HPMC as suspending agent. Thus GG may serve as a good suspending agent requiring no further aid in suspension redispersibility.KEY WORDS: grewia gum, oral pharmaceutical formulations, physicochemical properties, potential suspending agent     

Factors Affecting the Stability and Performance of Ipratropium Bromide; Fenoterol Hydrobromide Pressurized-Metered Dose Inhalers

The aim of the study was to investigate the factors affecting the stability and performance of ipratropium bromide and fenoterol hydrobromide in a pressurized-metered dose inhaler (pMDI). A factorial design was applied to investigate the effects of three parameters (propellant, water, and ethanol) on the performance of 27 designed formulations of a solution-based pMDI. The formulations that contained a hydrofluoroalkane (HFA) propellant lower than 72% v/v and an ethanol concentration higher than 27% v/v remained as clear solutions. Nine formulations that contained the HFA propellant higher than 74% v/v precipitated. The results indicated that it was not only the HFA propellant content of the formulations that was related to the formulation instability but also ethanol content. Only six formulations from the 18 formulations, that did not precipitate, produced drug contents that were within the acceptable range (80–120%). These six formulations generated aerosols with mass median aerodynamic diameters (MMAD) of approximately 2 μm with a fine particle fraction (FPF; particle size, <6.4 μm) between 45% and 52%. The MMAD and FPF did not change significantly after 6 months of storage (P > 0.05).KEY WORDS: fenoterol hydrobromide, ipratropium bromide, pressurized metered dose inhaler, stability     

HspA1A facilitates DNA repair in human bronchial epithelial cells exposed to Benzo[a]pyrene and interacts with casein kinase 2

Benzo[a]pyrene (BaP) is a ubiquitously distributed environmental pollutant that induces deoxyribonucleic acid (DNA) damage. The inducible heat shock protein (HspA1A) can function as a molecular chaperone; however, its role in DNA repair remains largely unknown. In the present study, human bronchial epithelial cells (16HBE) stably transfected with plasmids carrying HspA1A gene or shRNAs against HspA1A were treated with BaP. DNA damage levels of the cells were evaluated by comet assay. Results suggest that HspA1A could protect cells against DNA damage and facilitate the decrease of DNA damage levels during the first 2 h of DNA repair. DNA repair capacity (DRC) of Benzo(a)pyrene diol epoxide (BPDE)-DNA adducts was evaluated by host cell reactivation assay in the stable 16HBE cells transfected with luciferase reporter vector PCMVluc pretreated with BPDE. Compared with control cells, cells overexpressing HspA1A showed higher DRC (p < 0.01 at 10 μM BPDE and p < 0.05 at 20 μM BPDE, respectively), while knockdown of HspA1A inhibited DNA repair (p < 0.05 at 10 μM BPDE). Moreover, casein kinase 2 (CK2) was shown to interact with HspA1A by mass spectrometry and co-immunoprecipitation assays. The two proteins were co-localized in the cell nucleus and perinuclear region during DNA repair, and were identified by confocal laser scanning microscope. In addition, cells overexpressing HspA1A showed an increased CK2 activity after BaP treatment compared with control cells (p < 0.01). Our results suggest that HspA1A facilitates DNA repair after BaP treatment. HspA1A also interacts with CK2 and enhances the kinase activities of CK2 during DNA repair.

Electronic supplementary material

The online version of this article (doi:10.1007/s12192-013-0454-7) contains supplementary material, which is available to authorized users.     

Development of Controlled-Release Matrix Tablet of Risperidone: Influence of Methocel®- and Ethocel®-Based Novel Polymeric Blend on <Emphasis Type="Italic">In Vitro</Emphasis> Drug Release and Bioavailability

Controlled-release (CR) matrix tablet of 4 mg risperidone was developed using flow bound dry granulation–slugging method to improve its safety profile and compliance. Model formulations F1, F2, and F3, consisting of distinct blends of Methocel® K100 LV-CR and Ethocel® standard 7FP premium, were slugged. Each batch of granules (250–1,000 μm), obtained by crushing the slugs, was divided into three portions after lubrication and then compressed to 9-, 12-, and 15-kg hard tablets. In vitro drug release studies were carried out in 0.1 N HCl (pH 1.2) and phosphate buffer (pH 6.8) using a paddle dissolution apparatus run at 50 rpm. The CR test tablet, containing 30% Methocel® and 60% Ethocel® (F3) with 12-kg hardness, exhibited pH-independent zero-order release kinetics for 24 h. The drug release rate was inversely proportional to the content of Ethocel®, while the gel layer formed of Methocel® helped in maintaining the integrity of the matrix. Changes in the hardness of tablet did not affect the release kinetics. The tablets were reproducible and stable for 6 months at 40 ± 2°C/75 ± 5% relative humidity. Risperidone and its active metabolite, 9-hydroxyrisperidone, present in the pooled rabbit’s serum, were analyzed with HPLC-UV at λ_max 280 nm. The CR test tablet exhibited bioequivalence to reference conventional tablet in addition to the significantly (p < 0.05) optimized peak concentration, C_max, and extended peak time, T_max, of the active moiety. There was a good association between drug absorption in vivo and drug release in vitro (R² = 0.7293). The successfully developed CR test tablet may be used for better therapeutic outcomes of risperidone.KEY WORDS: controlled release, dry granulation slugging method, risperidone     

An efficient in vitro shoot regeneration from immature inflorescence and ex vitro rooting of Arnebia hispidissima (Lehm). DC. - A red dye (Alkannin) yielding plant

Arnebia hispidissima, which belongs to the family Boraginaceae, is an important medicinal and dye yielding plant. The alkannin, a red dye, are root-specific secondary metabolites of A. hispidissima. Shoots were regenerated from callus derived from immature inflorescence explants obtained from field grown plants. MS medium containing 4.52 μM 2, 4-D and 3.33 μM BAP was found to be most effective for the proliferation of callus, induced on medium containing 4.52 μM 2, 4-D. Maximum number (43.1 ± 0.25) with average length (5.2 ± 0.23) of shoots regenerated when callus was transferred to MS medium supplemented with 1.11 μM BAP, 1.16 μM Kin and 0.57 μM IAA. About 75.5 % of in vitro regenerated shoots were rooted on half-strength MS medium supplemented with 9.84 μM of IBA and 200 mg l⁻¹ of activated charcoal. In comparison to in vitro, higher percent (90.2 %) of shoots were rooted under ex vitro conditions when treated with IBA (0.98 mM) for 5 min. Plantlets rooted in vitro as well as ex vitro were acclimatized successfully under the green house conditions. Ex vitro rooted plants exhibited higher survival percentage (75 %) as compared to in vitro rooted plantlets (60 %). Present study may be applicable in the large-scale root-specific red dye (alkannin) production via root induction under ex vitro condition.     

Formulation and In Vitro Evaluation of Salbutamol Sulphate In Situ Gelling Nasal Inserts

The aim of this study was to formulate salbutamol sulfate (SS), a model drug, as mucoadhesive in situ gelling inserts having a high potential as nasal drug delivery system bypassing the first-pass metabolism. In situ gelling inserts, each containing 1.4% SS and 2% gel-forming polymer, hydroxypropyl methylcellulose (HPMC), carboxymethylcellulose sodium (CMC Na), sodium alginate (AL), and chitosan (CH) were prepared. The inserts were investigated for their different physicochemical properties. The weight of inserts was 16–27 mg, drug content was 3.9–4.2 mg, thickness ranged between 15 and 28 μm and surface pH was 5–7. Cumulative drug released from the inserts exhibited extended release for more than 10 h following the decreasing order: CH > AL > CMC Na > HPMC. The drug release from CMC Na and AL inserts followed zero-order kinetics while HPMC and CH inserts exhibited non-Fickian diffusion mechanism. The inserts exhibited different water uptake (7–23%) with the smallest values for CH. Differential scanning calorimetry study pointed out possible interaction of SS and oppositely charged anionic polymers (CMC Na and AL). The mucoadhesive in situ gelling inserts exhibited satisfactory mucoadhesive and extended drug release characteristics. The inserts could be used for nasal delivery of SS over about 12 h; bypassing the hepatic first-pass metabolism without potential irritation.KEY WORDS: in situ gelling inserts, mucoadhesion, nasal delivery, salbutamol sulfate     

Development and Characterization of Lyophilized Diazepam-Loaded Polymeric Micelles

Polymeric micelles were studied as delivery carriers of diazepam, a practically insoluble drug in water, for rectal administration. The diazepam-loaded polymeric micelles were developed by using poloxamer 407 (P407), poloxamer 188, and d-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS). Among the used polymers, TPGS resulted in polymeric micelles with good characteristics for encapsulation of diazepam which had the small particle size of 8–12 nm and narrow size distribution (PI 0.053–0.275). Additionally, 7.5% w/v of TPGS could entirely entrap the desired concentration of diazepam (5 mg/mL). To improve the physical stability upon lyophilization, an addition of P407 of 1% w/v prevented aggregation, increased physical stability, and maintained chemical stability of the lyophilized powders of diazepam-loaded polymeric micelles for 3 months storage at 4°C. The rate and amount of diazepam release from TPGS polymeric micelles mainly depended on the concentration of TPGS. The release data were fitted to Higuchi''s model suggesting that the drug release mechanism was controlled by Fickian diffusion. In conclusion, 10% w/v TPGS and 1% w/v P407 were the optimum formulation of lyophilized diazepam-loaded polymeric micelles.Key words: diazepam, lyophilization, poloxamer 407, polymeric micelles, d-α-tocopheryl poly(ethylene glycol) 1000 succinate (TPGS)     

Development and Clinical Evaluation of Clotrimazole–β-Cyclodextrin Eyedrops for the Treatment of Fungal Keratitis

Fungal keratitis is a serious corneal disease that may result in loss of vision. There are limited treatment options available in Iraqi eye hospitals which might be the main reason behind the poor prognosis of many cases. The purpose of this study was to prepare and pharmaceutically evaluate clotrimazole–β-cyclodextrin (CTZ–β-CD) eyedrops then clinically assess its therapeutic efficacy on fungal keratitis compared with extemporaneous amphotericin B eyedrops (0.5% w/v). A CTZ–β-CD ophthalmic solution was prepared and evaluated by various physicochemical, microbiological, and biological tests. The prepared formula was stable in 0.05 M phosphate buffer pH 7.0 at 40 ± 2°C and 75 ± 5% RH for a period of 6 months. Light has no significant effect on the formula’s stability. The CTZ–β-CD eyedrops efficiently complied with the isotonicity, sterility, and antimicrobiological preservative effectiveness tests. Results of the clinical study revealed that 20 (80%) patients showed a favorable response to the CTZ–β-CD eyedrops, while 16 patients (64%) exhibited a favorable response to amphotericin B (P > 0.05). The mean course of treatment was significantly (P < 0.05) less in the CTZ treatment group than in the amphotericin group (21.5 ± 5.2 vs. 28.3 ± 6.4 days, respectively). The CTZ formulation was significantly (P < 0.05) more effective in the management of severe cases and also against Candida sp. than amphotericin B. There was no significant difference (P < 0.05) between both therapies against filamentous fungi. The CTZ–β-CD formulation can be used alternatively to other ophthalmic antimycotic treatment options in developing countries where stability, cost, or efficacy is a limiting factor.Key words: clotrimazole, β-cyclodextrin, eyedrops, fungal keratitis, Iraq     

Micropropagtion of Terminalia bellerica from nodal explants of mature tree and assessment of genetic fidelity using ISSR and RAPD markers

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l⁻¹) + ascorbic acid (100 mg l⁻¹) + citric acid (10 mg l⁻¹)] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.     

Treatment with atorvastatin is associated with a better prognosis in chronic heart failure with systolic dysfunction: results from The Daunia Heart Failure Registry

Background

Few works have evaluated the effect of statins on left ventricular dysfunction in patients with chronic heart failure (CHF), by using tissue Doppler imaging (TDI). We therefore aimed to investigate whether atorvastatin treatment may influence prognosis and myocardial performance evaluated by TDI in subjects with CHF.

Methods

Five hundred thirty-two consecutive CHF outpatients enrolled in a local registry, the Daunia Heart Failure Registry, were prospectively analysed. 195 patients with CHF and left ventricular ejection fraction (LVEF) ≤40 %, either in treatment with atorvastatin (N: 114) or without statins (N: 81), underwent TDI examination. Adverse events were evaluated during follow-up.

Results

The atorvastatin group showed a lower incidence of adverse events (cardiac death: 0 % vs 7 %, p < 0.01), and better TDI performance (E/E’ 15 ± 5.7 vs 18 ± 8.3, p < 001) than controls. Ischaemic CHF patients in treatment with atorvastatin also showed a lower incidence of adverse events (death: 10 % vs 26 %, p < 0.05; sustained ventricular arrhythmias: 5 % vs 19 %, p < 0.05, cardiac death: 0 vs 8 %, p < 0.05) and better TDI performance (E/E’ ratio: 15.00 ± 5.68 vs 19.72 ± 9.14, p < 0.01; St: 353.70 ± 48.96 vs 303.33 ± 68.52 msec, p < 0.01) than controls. The association between atorvastatin and lower rates of cardiac death remained statistically significant even after correction in a multivariable analysis (RR 0.83, 95 % CI 0.71–0.96, p < 0.05 in CHF with LVEF ≤40 %; RR 0.77, 95 % CI 0.62–0.95, p < 0.05 in ischaemic CHF with LVEF ≤40 %).

Conclusions

Treatment with atorvastatin in outpatients with systolic CHF is associated with fewer cardiac deaths, and a better left ventricular performance, as assessed by TDI.     

Salivary extracellular heat shock protein 70 (eHSP70) levels increase after 59 min of intense exercise and correlate with resting salivary secretory immunoglobulin A (SIgA) levels at rest

This study aimed to identify the response of a salivary stress protein, extracellular heat shock protein (eHSP70), to intense exercise and to investigate the relationship between salivary eHSP70 and salivary immunoglobulin A (SIgA) levels in response to exercise. Sixteen healthy sedentary young males (means ± SD 23.8 ± 1.5 years, 172.2 ± 6.4 cm, 68.3 ± 7.4 kg) performed 59 min of cycling exercise at 75 % VO2_max. Saliva and whole blood samples were collected before (Pre), immediately after (Post), and at 1, 2, 3, and 4 h after completion of the exercise (1, 2, 3, and 4 h). The salivary eHSP70 and SIgA levels were measured by enzyme-linked imunosorbent assay (ELISA), and the secretion rates were computed by multiplying the concentration by the saliva flow rate. White blood cells were analyzed using an automated cell counter with a direct-current detection system. The salivary eHSP70 secretion rates were 1.11 ± 0.86, 1.51 ± 1.47, 1.57 ± 1.32, 2.21 ± 2.04, 3.36 ± 2.72, and 6.89 ± 4.02 ng · min⁻¹ at Pre, Post, and 1, 2, 3, and 4 h, respectively. The salivary eHSP70 secretion rate was significantly higher at 4 h than that at Pre, Post, 1, and 3 h (p < 0.05). The SIgA secretion rates were 26.9 ± 12.6, 20.3 ± 10.4, 19.6 ± 11.0, 21.8 ± 12.8, 21.5 ± 11.9, and 21.9 ± 11.7 μg · min⁻¹ at Pre, Post, 1, 2, 3, and 4 h, respectively. The salivary SIgA secretion rate was significantly lower between 1 and 4 h than that at Pre (p < 0.05). There was a positive correlation between salivary eHSP70 and SIgA in both concentration and secretion rates before exercise (p < 0.05). The absolute number of white blood cells significantly increased after exercise, with a maximum at 2 h (p < 0.05). The neutrophil/lymphocyte ratio was significantly increased from 1 to 4 h when compared with that in the Pre samples (p < 0.05). The present study revealed that salivary eHSP70 significantly increased at 4 h after the 59 min of intense exercise in sedentary male subjects. Exercise stress can induce elevated salivary eHSP70 level and upregulate oral immune function partially.     

Zinc deficiency or excess within the physiological range increases genome instability and cytotoxicity, respectively, in human oral keratinocyte cells

Zinc (Zn) is an essential component of Zn-finger proteins and acts as a cofactor for enzymes required for cellular metabolism and in the maintenance of DNA integrity. The study investigated the genotoxic and cytotoxic effects of Zn deficiency or excess in a primary human oral keratinocyte cell line and determined the optimal concentration of two Zn compounds (Zn Sulphate (ZnSO₄) and Zn Carnosine (ZnC)) to minimise DNA damage. Zn-deficient medium (0 μM) was produced using Chelex treatment, and the two Zn compounds ZnSO₄ and ZnC were tested at concentrations of 0.0, 0.4, 4.0, 16.0, 32.0 and 100.0 μM. Cell viability was decreased in Zn-depleted cells (0 μM) as well as at 32 μM and 100 μM for both Zn compounds (P < 0.0001) as measured via the MTT assay. DNA strand breaks, as measured by the comet assay, were found to be increased in Zn-depleted cells compared with the other treatment groups (P < 0.05). The Cytokinesis Block Micronucleus Cytome assay showed a significant increase in the frequency of both apoptotic and necrotic cells under Zn-deficient conditions (P < 0.05). Furthermore, elevated frequencies of micronuclei (MNi), nucleoplasmic bridges (NPBs) and nuclear buds (NBuds) were observed at 0 and 0.4 μM Zn, whereas these biomarkers were minimised for both Zn compounds at 4 and 16 μM Zn (P < 0.05), suggesting these concentrations are optimal to maintain genome stability. Expression of PARP, p53 and OGG1 measured by western blotting was increased in Zn-depleted cells indicating that DNA repair mechanisms are activated. These results suggest that maintaining Zn concentrations within the range of 4–16 μM is essential for DNA damage prevention in cultured human oral keratinocytes.     

Plasma levels of alpha-1-antichymotrypsin are elevated in patients with chronic heart failure,but are of limited prognostic value

Background

There is increasing interest in utilising novel markers of cardiovascular disease risk in patients with chronic heart failure (HF). Recently, it was shown that alpha-1-antichymotrypsin (ACT), an acute-phase protein and major inhibitor of cathpesin G, plays a role in the pathophysiology of HF and may serve as a marker for myocardial distress.

Objective

To assess whether ACT is independently associated with long-term mortality in chronic HF patients.

Methods

ACT plasma levels were categorised into quartiles. Survival times were analysed using Kaplan-Meier curves and Cox proportional hazards regression, without and with correction for clinically relevant risk factors, including sex, age, duration of HF, kidney function (MDRD), ischaemic HF aetiology and NT-proBNP.

Results

Twenty healthy individuals and 224 patients (mean age 71 years, 72 % male, median HF duration 1.6 years) with chronic HF were included. In total, 159 (71 %) patients died. The median survival time was 5.3 (95 % CI 4.5–6.1) years. ACT was significantly elevated in patients (median 433 μg/ml, IQR 279–680) in comparison with controls (median 214 μg/ml, IQR 166–271; p < 0.001). Cox regression analysis demonstrated that ACT was not independently related to long-term mortality in chronic HF patients (crude HR = 1.03, 95 % CI 0.75–1.41, p = 0.871; adjusted HR = 1.12, 95 % CI 0.78–1.60, p = 0.552), which was confirmed by Kaplan-Meier curves.

Conclusion

ACT levels are elevated in chronic HF patients, but no independent association with long-term mortality can be established.     

In vitro propagation and withaferin A production in Withania ashwagandha,a rare medicinal plant of India

Withania ashwagandha, belonging to the family Solanaceae, is an important medicinal herb of India with restricted geographic distribution. It is a rich source of withaferin A (WA) and other bioactive withanolides. In the present study a rapid in vitro mass propagation protocol of W. ashwagandha was developed from nodal explants. Nodal explants were cultured on MS medium supplemented with various concentrations and combinations of plant growth regulators (PGRs). The highest number of regenerated shoots per ex-plant (33 ± 2.7) and highest WA (13.4 ± 1.15 mg/g of DW) production was obtained on MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). In vitro raised shoots were further rooted on half-strength MS medium containing 2.0 μM Indole-3-butyric acid (IBA) and analyzed for WA production. The rooted plantlets when transferred to poly bags in the greenhouse showed 90 % survival frequency. Levels of WA were higher in the in vitro and ex vitro derived shoot and root tissues as compared to field grown mother plants. In an attempt to further maximize WA production, shoot cultures were further grown in liquid MS medium supplemented with 5.0 μM 6-benzyladenine (BA) and 1.0 μM Kinetin (Kn). Root cultures were grown on half strength MS liquid medium fortified with 2.0 μM of IBA. WA production in the liquid cultures was significantly higher compared to the static composition of the same media. This protocol, first of its kind in this plant, can be successfully employed for conservation, proliferation and large-scale production of WA. The regenerated plants can also be used in traditional medicine as an alternative to naturally collected plants.     

Comparative Evaluation of Porous Versus Nonporous Mucoadhesive Films as Buccal Delivery System of Glibenclamide

The present research work focused on the comparative assessment of porous versus nonporous films in order to develop a suitable buccoadhesive device for the delivery of glibenclamide. Both films were prepared by solvent casting technique using the 3² full factorial design, developing nine formulations (F1–F9). The films were evaluated for ex vivo mucoadhesive force, ex vivo mucoadhesion time, in vitro drug release (using a modified flow-through drug release apparatus), and ex vivo drug permeation. The mucoadhesive force, mucoadhesion time, swelling index, and tensile strength were observed to be directly proportional to the content of HPMC K4M. The optimized porous film (F4) showed an in vitro drug release of 84.47 ± 0.98%, ex vivo mucoadhesive force of 0.24 ± 0.04 N, and ex vivo mucoadhesion time of 539.11 ± 3.05 min, while the nonporous film (NF4) with the same polymer composition showed a release of 62.66 ± 0.87%, mucoadhesive force of 0.20 ± 0.05 N, and mucoadhesive time of 510 ± 2.00 min. The porous film showed significant differences for drug release and mucoadhesion time (p < 0.05) versus the nonporous film. The mechanism of drug release was observed to follow non-Fickian diffusion (0.1 < n < 0.5) for both porous and nonporous films. Ex vivo permeation studies through chicken buccal mucosa indicated improved drug permeation in porous films versus nonporous films. The present investigation established porous films to be a cost-effective buccoadhesive delivery system of glibenclamide.KEY WORDS: buccoadhesive drug delivery, glibenclamide, in vitro release and ex vivo permeation, porous film