Improved anticancer delivery of paclitaxel by albumin surface modification of PLGA nanoparticles

Background

Nanoparticles (NPs) play an important role in anticancer delivery systems. Surface modified NPs with hydrophilic polymers such as human serum albumin (HSA) have long half-life in the blood circulation system.

Methods

The method of modified nanoprecipitation was utilized for encapsulation of paclitaxel (PTX) in poly (lactic-co-glycolic acid) (PLGA). Para-maleimide benzoic hydrazide was conjugated to PLGA for the surface modifications of PLGA NPs, and then HSA was attached on the surface of prepared NPs by maleimide attachment to thiol groups (cysteines) of albumin. The application of HSA provides for the longer blood circulation of stealth NPs due to their escape from reticuloendothelial system (RES). Then the physicochemical properties of NPs like surface morphology, size, zeta potential, and in-vitro drug release were analyzed.

Results

The particle size of NPs ranged from 170 to 190 nm and increased about 20–30 nm after HSA conjugation. The zeta potential was about -6 mV and it decreased further after HSA conjugation. The HSA conjugation in prepared NPs was proved by Fourier transform infrared (FT-IR) spectroscopy, faster degradation of HSA in Differential scanning calorimetry (DSC) characterization, and other evidences such as the increasing in size and the decreasing in zeta potential. The PTX released in a biphasic mode for all colloidal suspensions. A sustained release profile for approximately 33 days was detected after a burst effect of the loaded drug. The in vitro cytotoxicity evaluation also indicated that the HSA NPs are more cytotoxic than plain NPs.

Conclusions

HSA decoration of PLGA NPs may be a suitable method for longer blood circulation of NPs.     

Determination of stress-induced degradation products of cetirizine dihydrochloride by a stability-indicating RP-HPLC method

Background

A new, simple and accurate stability-indicating reverse phase high performance liquid chromatography method was developed and validated during the early stage of drug development of an oral lyophilizate dosage form of cetirizine dihydrochloride.

Methods

For RP-HPLC analysis it was used an Eclipse XDB C8 column 150 mm × 4.6 mm, 5 μm (Agilent columns, Barcelona, Spain) as the stationary phase with a mobile phase consisted of a mixture of 0.2 M K₂HPO4 pH 7.00 and acetonitrile (65:35, v/v) at a flow rate of 1 mL min ⁻¹. Detection was performed at 230 nm using diode array detector. The method was validated in accordance with ICH guidelines with respect to linearity, accuracy, precision, specificity, limit of detection and quantification.

Results

The method results in excellent separation between the drug substance and its stress-induced degradation products. The peak purity factor is >950 for the drug substance after all types of stress, which confirms the complete separation of the drug substance peak from its stress induced degradation products.Regression analysis showed r² > 0.999 for cetirizine dihydrochloride in the concentration range of 650 μg mL ⁻¹ to 350 μg mL⁻¹ for drug substance assay and a r² > 0.999 in the concentration range of 0.25 μg mL⁻¹ to 5 μg mL⁻¹ for degradation products. The method presents a limit of detection of 0.056 μg mL ⁻¹ and a limit of quantification of 0.25 μg mL⁻¹. The obtained results for precision and accuracy for drug substance and degradation products are within the specifications established for the validation of the method.

Conclusions

The proposed stability-indicating method developed in the early phase of drug development proved to be a simple, sensitive, accurate, precise, reproducible and therefore useful for the following stages of the cetirizine dihydrochloride oral lyophilizate dosage form development.     

Thallium exists in opioid poisoned patients

Background

Thallium (Tl) is a toxic heavy metal that exists in nature. Tl poisoning (thallotoxicosis) may occur in opioid addicts. This study was designed to evaluate the frequency and level of urinary Tl in opioid abusers. In addition, clinical findings were evaluated.

Methods

A total of 150 subjects were examined. Cases with a history of at least 3 years of abuse were admitted in the Imam Reza Hospital as the case group; 50 non-opioid abusers from the target population were included as the control group. Twenty-four hour urinary qualitative and quantitative Tl analyses were performed on both groups.

Results

Out of the 150 subjects, 128 (85 %) were negative for qualitative urinary Tl, followed by 5 % (trace), 7 % (1+), 2 % (2+), and 1 % (3+). Mean (standard error (SE), Min–Max) quantitative urinary Tl level was 14 μg/L (3.5 μg/L, 0–346 μg/L). Mean urinary Tl level in the case group was 21 μg/L (5 μg/L, 0–346 μg/L) and that in the controls was 1 μg/L (0.14 μg/L, 0–26 μg/L), which were significantly different (P = 0.001). The most frequent clinical findings were ataxia (86 %), sweating (81 %), and constipation (54 %). In all cases (n = 150), the mean (SE) value for cases with positive qualitative urinary Tl was 26.8 μg/L (0.9 μg/L) and that in the negative cases was 2.3 μg/L (0.2 μg/L), which were significantly different (P = 0.002).

Conclusions

This study showed that long-term opioid abuse may lead to Tl exposure. In opioid abusers with the clinical manifestation of thallotoxicosis, urinary Tl should be determined.     

Evaluation of in vitro toxicity of peptide (N-acetyl-Leu-Gly-Leu-COOH)-substituted-β-cyclodextrin derivative,a novel drug carrier,in PC-12 cells

Background

Cyclodextrins (CDs) have been shown to improve physicochemical and biopharmaceutical properties of drugs when low solubility and low safety limit their use in the pharmaceutical field. Recently, a new amphiphilic peptide-substituted-β-CD, hepta-(N-acetyl-Leu-Gly-Leu)-β-CD (hepta-(N-acetyl-LGL)-β-CD), is developed which exhibited good solubility, strong inclusion ability and an appropriate average molecular weight. However, there is limited information available about its toxic effects. This study was designed to evaluate cytotoxic effects of the hepta-(N-acetyl-LGL)-β-CD (50, 200, 400, and 800 μg/ml) on rat pheochromocytoma PC-12 cells.

Results

A significant reduction of cell viability with IC₅₀ values of 1115.0 μg/ml, 762.4 μg/ml, and 464.9 μg/ml at 6, 12, and 24 h post-treatment, respectively, as well as increased lipid peroxide levels and DNA damage were observed.

Conclusions

In conclusion, hepta-(N-acetyl-Leu-Gly-Leu)-β-CD exhibit significant toxic properties at high concentrations, probably through induction of oxidative stress and genotoxicity.     

Application of headspace solid phase microextraction for study of noncovalent interaction of borneol with human serum albumin

Aim:

To investigate noncovalent interactions between borneol and human serum albumin (HSA) under near-physiological conditions.

Methods:

A 65-μm polydimethylsiloxane (PDMS) fiber was selected for sampling. The extraction temperature was kept at 37 °C, and the extraction time was optimized at 10 min. Borneol solutions of different concentrations were equilibrated in 600 μmol/L HSA and 67 mmol/L phosphate buffer solution (pH 7.4, 37 °C) for 24 h prior to solid phase microextraction (SPME) using headspace mode. The binding properties were obtained based on the calculation of extracted borneol amount using gas chromatography (GC) determination.

Results:

The headspace SPME extraction method avoided disturbance from the HSA binding matrix. The recovery showed good linearity for the borneol concentrations over the range of 0.4–16.3 μmol/L with a regression coefficient (R²) of 0.9998. The limit of detection and lower limit of quantitation were determined to be 0.01 μmol/L and 0.4 μmol/L, respectively. The binding constant and the percentage binding rate were estimated to be 2.4×10³(mol/L)^-1 and 59.5%, respectively.

Conclusion:

Headspace SPME coupled to GC is a simple, sensitive and rapid method for the study of borneol binding to HSA. The method may be applied in the determination of other protein binding properties in human plasma.     

Doxorubicin-conjugated PLA-PEG-Folate based polymeric micelle for tumor-targeted delivery: Synthesis and in vitro evaluation

Background

Selective delivery of anticancer agents to target areas in the body is desirable to minimize the side effects while maximizing the therapeutic efficacy. Anthracycline antibiotics such as doxorubicin (DOX) are widely used for treatment of a wide variety of solid tumors.This study evaluated the potential of a polymeric micellar formulation of doxorubicin as a nanocarrier system for targeted therapy of a folate-receptor positive human ovarian cancer cell in line.

Results

DOX-conjugated targeting and non-targeting micelles prepared by the dialysis method were about 188 and 182 nm in diameter, respectively and their critical micelle concentration was 9.55 μg/ml. The DOX-conjugated micelles exhibited a potent cytotoxicity against SKOV3 human ovarian cancer cells. Moreover, the targeting micelles showed higher cytotoxicity than that of non-targeting ones (IC₅₀ = 4.65 μg/ml vs 13.51 μg/ml).

Conclusion

The prepared micelle is expected to increase the efficacy of DOX against cancer cells and reduce its side effects.     

Enhancement of cellular uptake and cytotoxicity of curcumin-loaded PLGA nanoparticles by conjugation with anti-P-glycoprotein in drug resistance cancer cells

Aim:

To compare the anti-cancer activity and cellular uptake of curcumin (Cur) delivered by targeted and non-targeted drug delivery systems in multidrug-resistant cervical cancer cells.

Methods:

Cur was entrapped into poly (DL-lactide-co-glycolide) (PLGA) nanoparticles (Cur-NPs) in the presence of modified-pluronic F127 stabilizer using nano-precipitation technique. On the surface of Cur-NPs, the carboxy-terminal of modified pluronic F127 was conjugated to the amino-terminal of anti-P-glycoprotein (P-gp) (Cur-NPs-APgp). The physical properties of the Cur-NPs, including particle size, zeta potential, particle morphology and Cur release kinetics, were investigated. Cellular uptake and specificity of the Cur-NPs and Cur-NPs-APgp were detected in cervical cancer cell lines KB-V1 (higher expression of P-gp) and KB-3-1 (lower expression of P-gp) using fluorescence microscope and flow cytometry, respectively. Cytotoxicity of the Cur-NPs and Cur-NPs-APgp was determined using MTT assay.

Results:

The particle size of Cur-NPs and Cur-NPs-APgp was 127 and 132 nm, respectively. The entrapment efficiency and actual loading of Cur-NPs-APgp (60% and 5 μg Cur/mg NP) were lower than those of Cur-NPs (99% and 7 μg Cur/mg NP). The specific binding of Cur-NPs-APgp to KB-V1 cells was significantly higher than that to KB-3-1 cells. Cellular uptake of Cur-NPs-APgp into KB-V1 cells was higher, as compared to KB-3-1 cells. However, the cellular uptake of Cur-NPs and Cur-NPs-IgG did not differ between the two types of cells. Besides, the cytotoxicity of Cur-NPs-APgp in KB-V1 cells was higher than those of Cur and Cur-NPs.

Conclusion:

The results demonstrate that Cur-NPs-APgp targeted to P-gp on the cell surface membrane of KB-V1 cells, thus enhancing the cellular uptake and cytotoxicity of Cur.     

Non-genomic vasorelaxant effects of 17β-estradiol and progesterone in rat aorta are mediated by L-type Ca2+ current inhibition

Aim:

The sex hormones 17β-estradiol (βES) and progesterone (PRG) induce rapid non-genomic vasodilator effects which could be protective for the cardiovascular system. The purpose of this study was to analyze the mechanisms underlying their vasodilator effect in rat aortic smooth muscle preparations.

Methods:

Endothelium-denuded aorta artery rings were prepared from male Wistar rats and incubated in an organ bath. The contractions of the preparation were recorded through isometric transducers. The effects of the hormones on K⁺ current and L-type Ca²⁺ current (LTCC) were analyzed by using the whole cell voltage-clamp technique in A7r5 cells.

Results:

Both βES and PRG (1–100 μmol/L) concentration-dependently relaxed the endothelium-denuded aortic rings contracted by (–)-Bay K8644 (0.1 μmol/L) or by KCl (60 mmol/L). The IC₅₀ values of the two hormones were not statistically different. The K_V channel blocker 4-aminopyridine (2 mmol/L), BK_Ca channel blocker tetraethylammonium (1mmol/L) and K_ATP channel blocker glibenclamide (10 μmol/L) did not significantly modify the relaxant effect of the hormones. On the other hand, the blockage of the intracellular βES and PRG receptors with estradiol receptor antagonists ICI 182,780 (1 μmol/L) and PRG receptor antagonist mifepristone (30 μmol/L), respectively, did not significantly modify the relaxant action of the hormones. In A7r5 cells, both the hormones (1–100 μmol/L) rapidly and reversibly inhibited the basal and BAY-stimulated LTCC. However, these hormones had no effect on the basal K⁺ current.

Conclusion:

The vasorelaxant effects of βES and PRG are due to the inhibition of LTCC. The K⁺ channels are not involved in the effects.     

Serum Concentration of Lignocaine After Pertubation: An Observational Study

Objective

The objective of this study was to report the serum concentration of lignocaine after pertubation in patients with endometriosis.

Design

Prospective observational study.

Setting

The study was carried out at a gynaecological outpatient unit in Stockholm, Sweden.

Population

Eligible patients had endometriosis with a dysmenorrhoic pain score of >50 mm on a visual analogue scale, and patent fallopian tubes.

Methods

Patients with endometriosis (n = 25) were included in the study. The patients received pre-ovulatory pertubations with lignocaine hydrochloride 10 mg (n = 16) or ringer acetate (placebo, n = 9). The procedure comprised passing the study solution through the uterus and the fallopian tubes via an intra-cervical balloon catheter. Serum samples were collected at 0, 5, 15 and 30 min after pertubation.

Main Outcome Measures

The serum samples were analysed for the concentration of lignocaine with an LCMS-SIM method.

Results

Low levels of lignocaine were detected in the serum samples following pertubation of 10 mg lignocaine hydrochloride. The highest observed concentration was seen after 30 min (mean 0.050 μg/ml), with an individual maximum of 0.124 μg/ml. Maximum concentration (C_max) and time to C_max (T_max) could not be calculated, since the highest values were observed in the 30-min samples, which was the last sample obtained. Lignocaine was not detected after pertubation with placebo.

Conclusions

The serum levels of lignocaine following pertubation of 10 mg lignocaine hydrochloride are detectable but low. Lignocaine pertubated through the fallopian tubes reaches the peritoneal cavity and diffuses through the peritoneum into the blood circulation. Pertubation with lignocaine is safe and has no lignocaine-related adverse events.     

Binary Solvents Dispersive Liquid—Liquid Microextraction (BS-DLLME) Method for Determination of Tramadol in Urine Using High-Performance Liquid Chromatography

Background

Tramadol is an opioid, synthetic analog of codeine and has been used for the treatment of acute or chronic pain may be abused. In this work, a developed Dispersive liquid liquid microextraction (DLLME) as binary solvents-based dispersive liquid-liquid microextraction (BS-DLLME) combined with high performance liquid chromatography (HPLC) with fluorescence detection (FD) was employed for determination of tramadol in the urine samples. This procedure involves the use of an appropriate mixture of binary extraction solvents (70 μL CHCl3 and 30 μL ethyl acetate) and disperser solvent (600 μL acetone) for the formation of cloudy solution in 5 ml urine sample comprising tramadol and NaCl (7.5%, w/v). After centrifuging, the small droplets of extraction solvents were precipitated. In the final step, the HPLC with fluorescence detection was used for determination of tramadol in the precipitated phase.

Results

Various factors on the efficiency of the proposed procedure were investigated and optimized. The detection limit (S/N = 3) and quantification limit (S/N = 10) were found 0.2 and 0.9 μg/L, respectively. The relative standard deviations (RSD) for the extraction of 30 μg L of tramadol was found 4.1% (n = 6). The relative recoveries of tramadol from urine samples at spiking levels of 10, 30 and 60 μg/L were in the range of 95.6 – 99.6%.

Conclusions

Compared with other methods, this method provides good figures of merit such as good repeatability, high extraction efficiency, short analysis time, simple procedure and can be used as microextraction technique for routine analysis in clinical laboratories.     

Preparation,characterization and optimization of sildenafil citrate loaded PLGA nanoparticles by statistical factorial design

Background and the aim of the study

The objective of the present study was to formulate and optimize nanoparticles (NPs) of sildenafil-loaded poly (lactic-co-glycolic acid) (PLGA) by double emulsion solvent evaporation (DESE) method. The relationship between design factors and experimental data was evaluated using response surface methodology.

Method

A Box-Behnken design was made considering the mass ratio of drug to polymer (D/P), the volumetric proportion of the water to oil phase (W/O) and the concentration of polyvinyl alcohol (PVA) as the independent agents. PLGA-NPs were successfully prepared and the size (nm), entrapment efficiency (EE), drug loading (DL) and cumulative release of drug from NPs post 1 and 8 hrs were assessed as the responses.

Results

The NPs were prepared in a spherical shape and the sizes range of 240 to 316 nm. The polydispersity index of size was lower than 0.5 and the EE (%) and DL (%) varied between 14-62% and 2-6%, respectively. The optimized formulation with a desirability factor of 0.9 was selected and characterized. This formulation demonstrated the particle size of 270 nm, EE of 55%, DL of 3.9% and cumulative drug release of 79% after 12 hrs. In vitro release studies showed a burst release at the initial stage followed by a sustained release of sildenafil from NPs up to 12 hrs. The release kinetic of the optimized formulation was fitted to Higuchi model.

Conclusions

Sildenafil citrate NPs with small particle size, lipophilic feature, high entrapment efficiency and good loading capacity is produced by this method. Characterization of optimum formulation, provided by an evaluation of experimental data, showed no significant difference between calculated and measured data.     

Compatibility and Stability of Morphine Sulphate and Naloxone Hydrochloride in 0.9% Sodium Chloride for Injection

Background

Naloxone may be administered in conjunction with morphine to reduce the risk of opioid-induced pruritis. Combining these drugs for coadministration may be beneficial, but little is known about their physical compatibility and stability in combined solutions.

Objective:

To describe the physical compatibility and stability of morphine sulphate and naloxone hydrochloride (at various concentrations) in IV admixtures.

Methods:

The physical compatibility and stability of admixtures of morphine 1000 μg/mL and naloxone 4 μg/mL, 12.5 μg/mL, and 25 μg/mL in 0.9% sodium chloride were studied. For each concentration of naloxone, one bag was stored at room temperature (22°C) for 72 h and one bag was stored under refrigeration (4°C) for 30 days. For all preparations, physical characteristics, including pH, colour, and formation of precipitate, were evaluated. The samples were also analyzed by a stability-indicating high-performance liquid chromatographic method. Stability was defined as the retention of at least 90% of the initial concentration.

Results:

No notable changes in pH or colour and no macroprecipitation were observed in any of the preparations after storage at 22°C for up to 72 h or at 4°C for up to 30 days. All preparations maintained more than 90% of the initial concentrations of morphine and naloxone at the end of the respective study periods. The calculated lower limit of the 95% confidence interval also indicated that 90% or more of the initial concentration remained at the end of each study period.

Conclusion:

Admixtures of morphine sulphate and naloxone hydrochloride were stable for 72 h at room temperature and for 30 days with refrigeration.     

Dehydroandrographolide enhances innate immunity of intestinal tract through up-regulation the expression of hBD-2

Background

Dehydroandrographolide (DA) is one of major active components in the well-known oriental herbal medicine Andrographis paniculata (Burm.f) Nees which belongs to the Acanthaceae family. DA is used for the treatment of infections in China. However, DA has not been found to significantly inhibit bacterial and viral growth directly. The current study investigates the effect of DA on the expression of human β –defensin-2 (hBD-2) in human intestinal epithelial cells and the possible signaling pathways.

Methods

Human intestinal epithelial HCT-116 cells were incubated with 1–100 μM DA for 2–24 h. RT–PCR and Western blot were used to assess the expression of hBD-2. The specific inhibitors were used and the levels of phosphorylation of signaling molecules were detected for dissecting the signaling pathways leading to the induction of hBD-2.

Results

MTT assay showed there was no obvious cytotoxicity for HCT-116 cells by 1–100 μM DA treatment. RT-PCR and Western blot assays showed that DA (1–100 μM) could up-regulate the expression of hBD-2, and the effect lasted longer than 24 h. By using SB203580 and SB202190 (inhibitors of p38), the enhancement of hBD-2 expression were significantly attenuated. However, inhibitor of ERK and inhibitor of JNK could not block the effect of DA. Furthermore, Western blot found activation of p38 but not ERK and JNK in DA-treated HCT-116 cells.

Conclusion

The results suggested that DA enhanced innate immunity of intestinal tract by up-regulating the expression of hBD-2 through the p38 MAPK pathways.     

Anti-cancer effect of Cordyceps militaris in human colorectal carcinoma RKO cells via cell cycle arrest and mitochondrial apoptosis

Background

Cordyceps militaris has been used as a traditional medicine in Asian countries for a long time. Different types of Cordyceps extract were reported to have various pharmacological activities including an anti-cancer effect. We investigated the inhibitory effect of Cordyceps militaris ethanol extract on a human colorectal cancer-derived cell line, RKO.

Methods

RKO cells were treated with various concentrations of nucleosides-enriched ethanol extract of Cordyceps militaris for 48 h and cytotoxicity was measured using a CCK-8 assay. Then, xenograft Balb/c nude mice were injected with RKO cells and subsequently orally administered with ethanol extract of Cordyceps militaris every day for 3 weeks to examine the inhibitory effect on tumor growth. Lastly, the effect of Cordyceps militaris on cell cycle as well as apoptosis was measured using flow cytometry. Also, the expression of p53, caspase 9, cleaved caspase-3, cleaved PARP, Bim, Bax, Bak, and Bad were detected using western blot assay.

Results

RKO cells were highly susceptible to the ethanol extract of Cordyceps militaris (CME) and the growth of RKO cells-derived tumor was significantly delayed by the treatment of Cordyceps militaris. Cordyceps militaris induced cell cycle arrest in G2/M phase (untreated; 20.5 %, CME 100 μg/ml; 61.67 %, CME 300 μg/ml; 66.33 %) and increased early apoptosis (untreated; 1.01 %, CME 100 μg/ml; 8.48 %, CME 300 μg/ml; 18.07 %). The expression of p53, cleaved caspase 9, cleaved caspase-3, cleaved PARP, Bim, Bak, and Bad were upregulated by the treatment of Cordyceps militaris.

Conclusion

Ethanol extract of Cordyceps militaris was highly cytotoxic to human colorectal carcinoma RKO cells and inhibited the growth of tumor in xenograft model. The anti-tumor effect of Cordyceps militaris was associated with an induction of cell cycle arrest and mitochondrial-mediated apoptosis.     

Biological activity and microscopic characterization of Lythrum salicaria L

Background

There are several plants have been used worldwide in the folk medicine with high incidence for treatment of human disorders, of which Lythrum salicaria belongs to the Lythraceae family has traditionally reputation for some medicinal usage and recently many biological and pharmacological activity of the plant have been studied.

Methods

In this study, microscopic characterizations of the aerial parts of the plant were determined. Moreover, the plant extract (aqueous methanol 80%) was subjected to an anti-diabetic activity test (in a rat model of streptozocin induced diabetes), anti-Helicobacter pylori (using disc diffusion method) along with antioxidant activity against DPPH (stable free radical) tests. Besides, total flavonoids, phenols, tannins, as well as polysaccharides contents have been assessed using spectroscopic methods.

Results

The microscopic properties of the plant fragments revealed anomocytic stomata, conical shape trichomes, and abundant spherical pollen grains as a characteristic pattern for the aerial parts of the plant. The extract of the plant at concentration of 15 g/kg showed mild lowering activity on blood glucose level to 12.6% and 7.3% after 2 and 3 h of administration. Additionally, clinically isolated H. pylori strain was inhibited with the plant extract at concentration of 500 mg/mL (zone of inhibition: 17 ± 0.08 mm). Moreover, IC₅₀ values for DPPH inhibition of the plant extract, vitamin E, BHA were examined as 13.5, 14.2, and 7.8 μg/mL, respectively. Total flavonoids, phenols, tannin, and polysaccharides contents of the extract were successfully evaluated as 5.8 ± 0.4 μg QE/mg EXT, 331 ± 3.7 μg GAE/mg EXT, 340 ± 2.3 μg TAE/mg EXT, 21 ± 0.2 μg GE/mg EXT, respectively.

Conclusions

The results suggested that L. salicaria has low anti-diabetic and anti-Helicobacter pylori effects, but high antioxidant activity, just the same as positive standard (vitamin E), which might be attributed to the high content of phenolic compounds in the extract.     

Use of Propranolol Blockade to Explore the Pharmacology of GSK961081, a Bi-Functional Bronchodilator,in Healthy Volunteers: Results from Two Randomized Trials

Purpose

The objective of this study was to explore the pharmacology of GSK961081, a bi-functional bronchodilator, in healthy volunteers.

Methods

Two randomized, double-blind, placebo-controlled studies were conducted. Following optimization of the propranolol dosing regimen (study 1), we conducted a five-period crossover study (study 2) in which subjects received the following treatments: dry powder inhaler (DPI) GSK961081 400 µg + oral placebo, DPI GSK961081 1,200 µg + oral placebo, DPI GSK961081 400 µg + oral propranolol 80 mg, DPI GSK961081 1,200 µg + oral propranolol 80 mg and DPI and oral placebo. GSK961081 (or inhaled placebo) was dosed at 0 h. Propranolol (or oral placebo) was dosed at −8, −2, 4, 10, and 16 h. The primary endpoint for both studies was bronchodilation, measured by specific airway conductance (sGaw), which was assessed at 0, 1, 4, 7, 12, 22, and 24 h in study 2. Tolerability and pharmacokinetics were secondary endpoints.

Results

Studies 1 and 2 enrolled 18 and 23 subjects, respectively. In study 2, bronchodilation was seen for 24 h following GSK961081 400 and 1,200 μg. In the presence of β₂ blockade, GSK961081 1,200 μg demonstrated bronchodilation in the first 4 h after dosing (treatment difference from placebo at 1 h: 1.206; 90 % confidence interval [CI] 1.126–1.292; and at 4 h: 1.124; 90 % CI 1.078–1.173) but not at 7 h onwards. In the presence of β₂ blockade, GSK961081 400 μg demonstrated bronchodilation in the first 1 h after dosing (treatment difference from placebo: 1.193; 90 % CI 1.117–1.274), but not at 4 h onwards. Adverse events were reported for 21 (study 1) and 15 subjects (study 2); none were serious, and there were no deaths.

Conclusion

The duration of bronchodilation as a result of receiving the muscarinic antagonist component alone was shorter than that from the muscarinic antagonist β₂ agonist combination. Removing the β₂ agonist component may underestimate the contribution of the muscarinic antagonist component to the bronchodilation of the combination.     

Enhancement of cytotoxicity of antimicrobial peptide magainin II in tumor cells by bombesin-targeted delivery

Aim:

To investigate whether the conjugation of magainin II (MG2), an antimicrobial peptides (AMPs), to the tumor-homing peptide bombesin could enhance its cytotoxicity in tumor cells.

Methods:

A magainin II-bombesin conjugate (MG2B) was constructed by attaching magainin II (MG2) to bombesin at its N-terminus. The peptides were synthesized using Fmoc-chemistry. The in vitro cytotoxicity of the peptide in cancer cells was quantitatively determined using the CCK-8 cell counting kit. Moreover, the in vivo antitumor effect of the peptide was determined in tumor xenograft models.

Results:

The IC₅₀ of MG2B for cancer cells (10–15 μmol/L) was at least 10 times lower than the IC₅₀ of unconjugated MG2 (125 μmol/L). Moreover, the binding affinity of MG2B for cancer cells was higher than that of unconjugated MG2. In contrast, conjugation to a bombesin analog lacking the receptor-binding domain failed to increase the cytotoxicity of MG2, suggesting that bombesin conjugation enhances the cytotoxicity of MG2 in cancer cells through improved binding. Indeed, MG2B selectively induced cell death in cancer cells in vitro with the IC₅₀ ranging from 10 to 15 μmol/L, which was about 6–10 times lower than the IC₅₀ for normal cells. MG2B (20 mg/kg per day, intratumorally injected for 5 d) also exhibited antitumor effects in mice bearing MCF-7 tumor grafts. The mean weights of tumor grafts in MG2B- and PBS-treated mice were 0.21±0.05 g and 0.59±0.12 g, respectively.

Conclusion:

The results suggest that conjugation of AMPs to bombesin might be an alternative approach for targeted cancer therapy.     

Adenosine deaminase activity modulation by some street drug: molecular docking simulation and experimental investigation

Background

Adenosine deaminase (ADA) is an enzyme that plays important roles in proliferation, maturation, function and development of the immune system. ADA activity may be altered by variety of substances including synthetic or natural products. Morphine, cocaine and their analogs exert immune suppressive activities by decreasing immune system function. The purpose of this study is to confirm that this possible effect may be modulated by interaction of these substances with ADA activity by experimental and computational method.

Methods

The structural changes in ADA have been studied in presence of cocaine, ethylmorphine, homatropine, morphine and thebaine by determination of ADA hydrolytic activity, circular dichroism and fluorescence spectroscopy in different concentrations. Docking study was performed to evaluate interaction method of test compound with ADA active site using AutoDock4 software.

Results

According to in-vitro studies all compounds inhibited ADA with different potencies, however thebaine activated it at concentration below 50 μM, ethylmorphine inhibited ADA at 35 μM. Moreover, fluorescence spectra patterns were differed from compounds based on structural resemblance which were very considerable for cocaine and homatropine.

Conclusion

The results of this study confirms that opioids and some other stimulant drugs such as cocaine can alter immune function in illegal drug abusers. These findings may lead other investigators to develop a new class of ADA activators or inhibitors in the near future.     

Novel microtubule-targeted agent 6-chloro-4-(methoxyphenyl) coumarin induces G2-M arrest and apoptosis in HeLa cells

Aim:

To identify a novel coumarin analogue with the highest anticancer activity and to further investigate its anticancer mechanisms.

Methods:

The viability of cancer cells was investigated using the MTT assay. The cell cycle progression was evaluated using both flow cytometric and Western blotting analysis. Microtubule depolymerization was observed with immunocytochemistry in vivo and a tubulin depolymerization assay in vitro. Apoptosis was demonstrated using Annexin V/Propidium Iodide (PI) double-staining and sub-G₁ analysis.

Results:

Among 36 analogues of coumarin, 6-chloro-4-(methoxyphenyl) coumarin showed the best anticancer activity (IC₅₀ value about 200 nmol/L) in HCT116 cells. The compound had a broad spectrum of anticancer activity against 9 cancer cell lines derived from colon cancer, breast cancer, liver cancer, cervical cancer, leukemia, epidermoid cancer with IC₅₀ value of 75 nmol/L–1.57 μmol/L but with low cytotocitity against WI-38 human lung fibroblasts (IC₅₀ value of 12.128 μmol/L). The compound (0.04–10 μmol/L) induced G₂-M phase arrest in HeLa cells in a dose-dependent manner, which was reversible after the compound was removed. The compound (10–300 μmol/L) induced the depolymerization of purified porcine tubulin in vitro. Finally, the compound (0.04–2.5 μmol/L) induced apoptosis of HeLa cells in dose- and time-dependent manners.

Conclusion:

6-Chloro-4-(methoxyphenyl) coumarin is a novel microtubule-targeting agent that induces G₂–M arrest and apoptosis in HeLa cells.     

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit in vitro

Aim:

To examine the effects of anisomycin on glioma cells and the related mechanisms in vitro.

Methods:

The U251 and U87 human glioblastoma cell lines were tested. The growth of the cells was analyzed using a CCK-8 cell viability assay. Apoptosis was detected using a flow cytometry assay. The expression of proteins and phosphorylated kinases was detected using Western blotting.

Results:

Treatment of U251 and U87 cells with anisomycin (0.01–8 μmol/L) inhibited the cell growth in time- and concentration-dependent manners (the IC₅₀ values at 48 h were 0.233±0.021 and 0.192±0.018 μmol/L, respectively). Anisomycin (4 μmol/L) caused 21.5%±2.2% and 25.3%±3.1% of apoptosis proportion, respectively, in U251 and U87 cells. In the two cell lines, anisomycin (4 μmol/L) activated p38 MAPK and JNK, and inactivated ERK1/2. However, neither the p38 MAPK inhibitor SB203580 (10 μmol/L) nor the JNK inhibitor SP600125 (10 μmol/L) prevented anisomycin-induced cell death. On the other hand, anisomycin (4 μmol/L) reduced the level of PP2A/C subunit (catalytic subunit) in a time-dependent manner in the two cell lines. Treatment of the two cell lines with the PP2A inhibitor okadaic acid (100 nmol/L) caused marked cell death.

Conclusion:

Anisomycin induces glioma cell death via down-regulation of PP2A catalytic subunit. The regulation of PP2A/C exression by anisomycin provides a clue to further study on its role in glioma therapy.