Application of a liquid chromatography-electrospray mass spectrometry (LC/MS) method to the biodistribution and excretion studies of novel 5'-chloro-2, 3-didehydroindolo (2', 3': 2, 3) betulinic acid (DRF-4012) in tumour-bearing mice.

Novel betulinic acid derivative 5'-chloro-2, 3-didehydroindolo [2', 3': 2, 3] betulinic acid (DRF-4012) is a new effective lupane type triterpenes with greater anticancer activity and efficacy than betulinic acid and currently under advanced preclinical investigation phase. In this study, a sensitive and rapid liquid chromatography-electrospray mass spectrometric (LC/MS) method has been developed for the determination of DRF-4012 in tumour-bearing mice plasma, urine, feces and tissues (liver, brain, lungs, heart, spleen, stomach, thigh muscle, kidneys, urinary bladder, small intestine and tumour). Biodistribution and excretion studies were performed for DRF-4012 nanoparticle (30 mg/kg body weight) after intravenous (i.v.) injection in tumour-bearing mice. DRF-4012 rapidly distributed throughout the body. After 0.5 h, tumour showed the second highest concentration, which was nearly half of the liver. After 4 and 24 h, the highest concentration of DRF-4012 was found in tumour indicating its retention in tumour site for a longer time. Excretion studies revealed that very low amount of unchanged DRF-4012 was observed in urine and primarily excreted through fecal route. This study may be useful to explain the manner in which DRF-4012 can inhibit tumour growth without apparent toxicity and preclinical/clinical evaluation of this potential antitumour agent.

Plasma polymerization of maleic anhydride: just what are the right deposition conditions?

Maleic anhydride plasma polymers enable amine-containing biomolecules and polymers to be covalently coupled to a surface from an aqueous solution without any intermediate chemistry. The challenge in developing these functionally active plasma polymers lies in determining the optimal deposition conditions for producing a stable, highly active film. Unlike many previous studies that explore highly varied pulsed and continuous wave (CW) deposition conditions, this paper focuses on the comparison of films deposited under the same low nominal power conditions (1 W) and compares a range of CW, millisecond, and microsecond pulsing parameters that can be used to produce this power condition. The use of attenuated total reflectance-Fourier transform infrared spectroscopy (ATR-FTIR) and X-ray photoelectron spectroscopy (XPS) has enabled the quantitative examination of the effects of processing parameters on the chemical functionality of the films. For the first time, the molecular specificity, surface sensitivity, and high mass resolution of time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) has been exploited to compare these films and multivariate analysis techniques used to explore the relationships between plasma processing parameters and surface chemistry. The results of the studies clearly demonstrate that a range of conditions can produce maleic anhydride films, with optimal functionality seen under microsecond pulsing regimes. Critically, the study demonstrates that the tight control and monitoring of the deposition parameters is critical if these films are to be manufactured with optimal functionality, stability, and minimum processing time.

Physical vs photolithographic patterning of plasma polymers: an investigation by ToF-SSIMS and multivariate analysis.

Physical and photolithographic techniques are commonly used to create chemical patterns for a range of technologies including cell culture studies, bioarrays and other biomedical applications. In this paper, we describe the fabrication of chemical micropatterns from commonly used plasma polymers. Atomic force microscopy (AFM) imaging, time-of-flight static secondary ion mass spectrometry (ToF-SSIMS) imaging, and multivariate analysis have been employed to visualize the chemical boundaries created by these patterning techniques and assess the spatial and chemical resolution of the patterns. ToF-SSIMS analysis demonstrated that well-defined chemical and spatial boundaries were obtained from photolithographic patterning, while the resolution of physical patterning via a transmission electron microscopy (TEM) grid varied depending on the properties of the plasma system including the substrate material. In general, physical masking allowed diffusion of the plasma species below the mask and bleeding of the surface chemistries. Multivariate analysis techniques including principal component analysis (PCA) and region of interest (ROI) assessment were used to investigate the ToF-SSIMS images of a range of different plasma polymer patterns. In the most challenging case, where two strongly reacting polymers, allylamine and acrylic acid were deposited, PCA confirmed the fabrication of micropatterns with defined spatial resolution. ROI analysis allowed for the identification of an interface between the two plasma polymers for patterns fabricated using the photolithographic technique which has been previously overlooked. This study clearly demonstrated the versatility of photolithographic patterning for the production of multichemistry plasma polymer arrays and highlighted the need for complementary characterization and analytical techniques during the fabrication plasma polymer micropatterns.

Organic depth profiling of a binary system: the compositional effect on secondary ion yield and a model for charge transfer during secondary ion emission.

In recent years, it has been demonstrated that cluster ion beams may be used to sputter some materials, particularly organic materials, without the significant accumulation of damage. It is therefore possible to use cluster ion beam sputtering in conjunction with a surface analytical technique, such as SIMS, to obtain depth profiles and three-dimensional images of the distribution of organic species in the near-surface region. For SIMS organic depth profiling to be useful as an analytical tool, it is important that it is able to measure physically meaningful quantities, such as the local concentration of a species within a blend. In this paper, we investigate a model system of a miscible binary mixture of codeine and poly(lactide). We show that there is a strong surface enrichment of poly(lactide), which provides a reference signal and permits the direct comparison of different samples in terms of secondary ion yield behavior. We demonstrate that it is possible to relate secondary ion intensities to local concentrations for a binary system and that there is a direct correspondence between the yield enhancement of one component and the yield suppression of the other. The dependence of secondary ion yield on composition is described using a model of the kinetically limited transfer of charge between secondary ions and secondary neutrals. Application of the model to pure materials under the assumption that only highly fragmented secondary ions are initially produced and interact with unfragmented secondary neutrals leads to the prediction that high molecular mass quasi-molecular ions have intensities proportional to the square of the total secondary ion yield. This relationship has been independently observed in other work (Seah, M. P. Surf. Interface Anal. 2007, 39, 634.).

Oral bioavailability enhancement of exemestane from self-microemulsifying drug delivery system (SMEDDS).

Limited aqueous solubility of exemestane leads to high variability in absorption after oral administration. To improve the solubility and bioavailability of exemestane, the self-microemulsifying drug delivery system (SMEDDS) was developed. SMEDDS comprises of isotropic mixture of natural or synthetic oil, surfactant, and cosurfactant, which, upon dilution with aqueous media, spontaneously form fine o/w microemulsion with less than 100 nm in droplet size. Solubility of exemestane were determined in various vehicles. Ternary phase diagrams were plotted to identify the efficient self-emulsification region. Dilution studies, droplet size, and zeta potential of the formulations were investigated. The release of exemestane from SMEDDS capsules was studied using USP dissolution apparatus in different dissolution media and compared the release of exemestane from a conventional tablet. Oral pharmacokinetic study was performed in female Wistar rats (n = 8) at the dose of 30 mg kg(-1). The absorption of exemestane from SMEDDS form resulted in about 2.9-fold increase in bioavailability compared with the suspension. Our studies illustrated the potential use of SMEDDS for the delivery of hydrophobic compounds, such as exemestane by the oral route.

Intraoperative aberrometry versus preoperative biometry for intraocular lens power selection in short eyes.

PURPOSE: To compare the accuracy of preoperative biometry-based formulas to intraoperative aberrometry (IA) with respect to predicting refractive outcomes after cataract surgery in short eyes. SETTING: Private practice and community-based ambulatory surgery center. DESIGN: Retrospective consecutive case series. METHODS: Eyes with an axial length (AL) shorter than 22.1 mm underwent cataract extraction and intraocular lens (IOL) implantation. The predicted residual refractive error was calculated preoperatively using Hoffer Q, Holladay 2, Haigis, Barrett Universal II, and Hill-RBF formulas and intraoperatively using IA. The postoperative spherical equivalent (SE) was compared with the predicted SE to evaluate the accuracy of each aforementioned method. RESULTS: Fifty-one eyes from 38 patients met criteria to be included in the analysis. Without optimizing the formulas specifically for short eyes, the mean numerical errors (MNEs) associated with Hoffer Q, Holladay 2, Haigis, Barrett Universal II, Hill-RBF, and IA were -0.08 (95% confidence interval [CI], -0.30 to 0.13), -0.14 (95% CI, -0.35 to 0.07), +0.26 (95% CI, 0.05 to 0.47), +0.11 (95% CI, -0.10 to 0.32), +0.07 (95% CI, -0.14 to 0.28), and +0.00 (95% CI, -0.21 to 0.21), respectively (P < .001). The proportion of eyes within ±0.5 diopter (D) of the predicted SE were 49.0%, 43.1%, 52.9%, 52.9%, 60.8%, and 58.8%, respectively (P = .06). The prediction outcomes from IA were statistically better than Haigis, but not other formulas. When formula and IA predictions differed by 0.5 D or more, IA's ability to recommend a more emmetropic outcome was no better than chance (50%). CONCLUSIONS: Intraoperative aberrometry is not significantly different from the best preoperative biometry-based methods available for IOL power selection in short eyes.

Non-fouling microfluidic chip produced by radio frequency tetraglyme plasma deposition.

This Technical Note presents the direct surface modification of a glass/PTFE hybrid microfluidic chip, via radio frequency glow discharge plasma polymerisation of tetraethlylene glycol dimethylether (tetraglyme), to produce hydrophilic, non-fouling, PEO-like surfaces. We use several techniques including X-ray photoelectron spectroscopy (XPS), direct enzyme-linked immunosorbent assays (ELISA) and immunofluorescent imaging to investigate the channel coatings. Our results indicate the successful deposition of a PEO-like coating onto microchannel surfaces that has both solution and shelf stability (>3 months) and is capable of preventing fibrinogen adsorption to the microchannel surfaces.

Concurrent determination of topotecan and model permeability markers (atenolol, antipyrine, propranolol and furosemide) by reversed phase liquid chromatography: utility in Caco-2 intestinal absorption studies.

A simple, sensitive, specific and high-resolution reversed-phase liquid chromatographic method utilizing ultraviolet detection has been developed and validated for simultaneous determination of topotecan and four intestinal permeability markers (atenolol, antipyrine, propranolol and furosemide) as suggested by US-FDA. Chromatography was carried out on C-18 column with mobile phase comprising water (pH 3.0) and acetonitrile gradient pumped at a flow rate of 1 ml min(-1). The validation parameters included specificity, accuracy, precision, sensitivity and stability studies. Topotecan, an anti-cancer drug widely used in metastatic carcinoma, is a P-glycoprotein substrate having oral bioavailability of 30% with large inter-patient variability. The present method was successfully applied for demonstrating P-gp mediated transport of topotecan and its inhibition using verapamil in Caco-2 cell monolayer. The method can be used in identification of novel P-gp inhibitors for topotecan and estimating the contribution of P-gp in affecting oral bioavailability of topotecan. The other applications of method include its use in validation of Caco-2 monolayer assay for getting biowaiver based on Biopharmaceutic Classification System and its extrapolation to in situ and/or in vivo studies.

Evaluation of in vitro Anti-psoriatic Activity of a Novel Polyherbal Formulation by Multiparametric Analysis.

BACKGROUND: We have developed a novel aqueous polyherbal formulation (SIRB-001) consisting of 3 herbs; Rheum palmatum L., Lonicera Japonica and Rehmannia glutinosa Libosch in the ratio 1:1:3. SIRB-001 has demonstrated efficacious effects in psoriasis patients. OBJECTIVE: This study was aimed at scientifically evaluating the in vitro antipsoriatic activity of SIRB-001. METHOD: The in vitro anti-psoriatic properties of SIRB-001 were assessed in human keratinocyte cell line; HaCaT. Anti-proliferative effect was studied using MTT assay. Apoptosis was examined by flow cytometry and colorimetric methods. Inflammatory markers and VEGF were determined by ELISA. IL-17/IL-23 secretion was assessed in immune cells. Signaling markers (kinases) by enzymatic assay and Topoisomerase-II activity by Kinetoplast DNA Cleavage assay was tested. RESULTS: SIRB-001 significantly inhibited (p<0.01) proliferation of HaCaT cells and induced apoptosis. Significant (p<0.01) downregulation of pro-inflammatory markers (TNF- α, IFN-γ, IL-6, NO, sPLA2) and VEGF was observed. IL-17/IL-23 secretion was significantly (p<0.01) alleviated in immune cells (RAW264.7 and THP-1). Inhibition of signaling markers (AKT1, FLT3, MAPK1, PRKCA, MAP2K) was observed. SIRB-001 demonstrated inhibition of Topoisomerase-II activity. High Performance Liquid Chromatography (HPLC) analysis of SIRB-001 was carried out using standard marker compounds chlorogenic acid (tR=13.98min), Acteoside (tR=24.22 min) and Rhein (tR=53.76 min). CONCLUSION: The in vitro results substantiate the anti-psoriatic effect of SIRB-001 in patients. SIRB-001 exerted anti-psoriatic effects at cellular level via multiple arms (antiproliferative, pro-apoptotic, anti-inflammatory, anti-angiogenic). This study provides insight into mechanism of action of SIRB-001 and highlights its promising potential for development as a herbal therapeutic agent for psoriasis, emphasizing the need of further pharmacological evaluation and toxicological studies.

Antidiabetic potential of polyherbal formulation DB14201: Preclinical development, safety and efficacy studies.

ETHNOPHARMACOLOGICAL RELEVANCE: The poly-herbal formulation DB14201 is a new combination of ayurvedic ingredients for treatment of diabetes. The aim of present study was to investigate safety and in vivo efficacy of DB14201 extract. Further this work was aimed to develop, characterize and standardize DB14201 extract and develop it as a botanical drug. MATERIALS AND METHODS: The polyherbal extract was standardized using four chemical markers. The LC-MS/MS method was developed for identification and quantification of mangiferin, berberine, kaempferol and curcumin. The extract was standardized for heavy metal content, aflotoxins, and microbial tests. The mechanism of action of DB14201 extract was explored through glucose uptake by adipocytes, TNF-α production and free fatty acid release, in vitro, was studied using murine adipocytes (3T3-L1). The effect of extract on insulin release was evaluated using murine pancreatic beta cell (ß TC-6). The safety and in vivo efficacy of extract was studied using suitable animal model. Hematology and blood biochemistry parameters were also assessed. RESULTS: In vitro studies of DB14201 in murine adipocytes and murine pancreatic beta cells demonstrated the plausible mechanism of action of DB14201 could be through increase in glucose uptake and by stimulation of insulin release by RIN-5f cells. The microbial load, heavy metals were found to be within the AYUSH permissible limits and aflotoxins were absent. Preclinical efficacy studies in animal models proved the anti-diabetic potential of the extract. The preclinical acute dose toxicity study and 90-days repeated dose toxicity study of DB14201 extract in wistar rats by oral route indicated that the extract is safe up to 1000mg/kg dose. Hematology and blood biochemistry parameters were within the normal range. CONCLUSIONS: The data presented herein demonstrated anti-diabetic potential of developed DB14201 extract and this study will serve as the benchmark for the further research on this polyherbal formulation.

Thin head and single use microkeratomes reduce epithelial defects during LASIK.

PURPOSE: To evaluate the effect of microkeratome design and head dimension (flap thickness) on the rate of epithelial defects. METHODS: A retrospective review of 4000 LASIK procedures performed between May 2000 and December 2003 was conducted. Intraoperative epithelial defects were identified in 326 cases. The microkeratome design (reusable vs disposable) and head dimensions (100, 130, and 150 micron) were recorded along with age, sex, central ultrasonic pachymetry, keratometry, preoperative refraction (sphere, cylinder, and axis), and Schirmer testing. The chi-square and Student t test were used in data analysis. RESULTS: A total of 326 (8.2%) procedures had an associated epithelial defect. With the reusable microkeratome, heads that produced thinner flaps were associated with a lower incidence of epithelial defects (P<.05 for all three groups, 100, 130 and 150 micron head). When compared to a reusable head of the same dimension and to the entire population of reusable heads, the disposable 130 head exhibited a significantly lower rate of epithelial defects (P<.0001). CONCLUSIONS: The results of this study support that both microkeratome design and head dimension (and resulting corneal flap thickness) play a role in the formation of epithelial defects. These are surgeon selectable factors, unrelated to technique or patient risk factors, which can be used to reduce the rate of epithelial defects and associated complications.

Measuring Compositions in Organic Depth Profiling: Results from a VAMAS Interlaboratory Study.

We report the results of a VAMAS (Versailles Project on Advanced Materials and Standards) interlaboratory study on the measurement of composition in organic depth profiling. Layered samples with known binary compositions of Irganox 1010 and either Irganox 1098 or Fmoc-pentafluoro-l-phenylalanine in each layer were manufactured in a single batch and distributed to more than 20 participating laboratories. The samples were analyzed using argon cluster ion sputtering and either X-ray photoelectron spectroscopy (XPS) or time-of-flight secondary ion mass spectrometry (ToF-SIMS) to generate depth profiles. Participants were asked to estimate the volume fractions in two of the layers and were provided with the compositions of all other layers. Participants using XPS provided volume fractions within 0.03 of the nominal values. Participants using ToF-SIMS either made no attempt, or used various methods that gave results ranging in error from 0.02 to over 0.10 in volume fraction, the latter representing a 50% relative error for a nominal volume fraction of 0.2. Error was predominantly caused by inadequacy in the ability to compensate for primary ion intensity variations and the matrix effect in SIMS. Matrix effects in these materials appear to be more pronounced as the number of atoms in both the primary analytical ion and the secondary ion increase. Using the participants' data we show that organic SIMS matrix effects can be measured and are remarkably consistent between instruments. We provide recommendations for identifying and compensating for matrix effects. Finally, we demonstrate, using a simple normalization method, that virtually all ToF-SIMS participants could have obtained estimates of volume fraction that were at least as accurate and consistent as XPS.

Effect of P-glycoprotein inhibitor, verapamil, on oral bioavailability and pharmacokinetics of irinotecan in rats.

The objective of present investigation was to study the effect of verapamil on the pharmacokinetics of irinotecan in order to evaluate the role of P-glycoprotein (P-gp) in irinotecan disposition. An in vitro study using Caco-2 intestinal cell monolayer was first carried out to determine the effect of verapamil on the function of intestinal P-gp. Verapamil (25mg/kg) was administered orally 2h before irinotecan oral (80 mg/kg) or intravenous (20mg/kg) dosing in female Wistar rats. Plasma and biliary samples were collected at specified time points from control and treated animals to determine irinotecan and its metabolite, SN-38 concentrations. Bi-directional transport and inhibition studies in Caco-2 cells indicated irinotecan to be a P-gp substrate and the function of intestinal P-gp was significantly inhibited in presence of verapamil. After oral irinotecan dosing, the mean area under the plasma concentration-time curve (AUC) was found to be 14.03+/-2.18 microgh/ml which was increased significantly, i.e. 61.71+/-15.0 microgh/ml when verapamil was co-administered (P<0.05). Similarly, the mean maximum plasma concentration of irinotecan increased from 2.93+/-0.37 microg/ml (without verapamil) to 10.75+/-1.0 microg/ml (with verapamil) (P<0.05). There was approximately 4-5-folds increase in apparent bioavailability. On the other hand, the intravenous irinotecan administration with verapamil resulted in small but statistically significant effect on AUC (10.76+/-2.0 to 23.3+/-3.8 microgh/ml; P<0.05) and systemic clearance (1206.4+/-159.7 to 713.5+/-78.2 ml/(hkg)). In addition, SN-38 showed significant change in oral pharmacokinetic parameters and minor changes in intravenous pharmacokinetic profile. Biliary excretion curves of both irinotecan and SN-38 were lowered by verapamil. The mean percent of irinotecan excreted into bile over 5h following intravenous and oral administration was found to be 8% and 1%, respectively, which was further reduced to half when treated with verapamil. These results are quite stimulating for further development of a clinically useful oral formulation of irinotecan based on P-gp inhibition.

High performance liquid chromatography method for the pharmacokinetic study of bicalutamide SMEDDS and suspension formulations after oral administration to rats.

Bicalutamide is a non-steroidal antiandrogen and is an oral medication that is used for treating prostate cancer. To evaluate the bioavailability of bicalutamide from bicalutamide self-microemulsifying drug delivery systems (SMEDDS) and bicalutamide suspension formulations, a sensitive, specific reversed-phase high performance liquid chromatographic (HPLC) method using ultraviolet detection was developed and validated for the analysis of bicalutamide (BCT) in rat blood plasma. Letrozole (LZ) was used as the internal standard. The chromatographic separation was achieved on C18 column at 35 degrees C, with a mobile phase consisting of water: acetonitrile (adjusted to pH 3.0 with 20% o-phosphoric acid) (60:40), at a flow rate of 1.0 mL min(-1). Bicalutamide and letrozole were well separated with retention times of 10.9+/-0.2 and 5.7+/-0.2 min, respectively. The method was successfully used to determine pharmacokinetics of bicalutamide, following oral administration of bicalutamide suspension and bicalutamide SMEDDS to wistar rats. Significant difference was observed in main pharmacokinetic parameters of tmax, Cmax and AUC(0 --> infinity) between SMEDDS and suspension, and a two fold increase in the relative bioavailability of bicalutamide was observed with the SMEDDS compared with suspension formulation. It was concluded that the absorption of bicalutamide from SMEDDS was enhanced.

Quantitative XPS depth profiling of codeine loaded poly(l-lactic acid) films using a coronene ion sputter source.

The controlled release of active pharmaceutical ingredients from polymers over prolonged periods of time is vital for the function of drug eluting stents and other drug loaded delivery devices. Characterisation of the drug distribution in polymers allows the in vitro and in vivo performance to be rationalised. We present the first X-ray photoelectron spectroscopy (XPS) depth profiling study of such a drug eluting stent system for which we employ a novel coronene ion sputter source. The rationale for this is to ascertain quantitative atomic concentration data through the thickness of flat films containing codeine and poly(l-lactic acid) (PLA) as a model of a drug loaded polymer device. A range of films of thickness of up to 96 nm are spun cast from chloroform onto Piranha cleaned silicon wafers. Ellipsometry of the films is undertaken prior to depth profiling to determine the total film thickness and provide a measure of the relative loading of drug within the PLA matrix through spectroscopic analysis. Progressive XPS analysis of the bottom of the sputter crater with sputter time indicated codeine to be depleted from the surface and segregated to the bulk of the polymer films by comparison with a uniform distribution calculated from the bulk loading. This serves to illustrate that surface depletion of drug occurs, which poses important implications for drug loaded polymer delivery systems.

Multisensory enhancement of localization under conditions of induced myopia.

Enhanced behavioral performance mediated by multisensory stimuli has been shown using a variety of measures, including response times, orientation behaviors, and even simple stimulus detection. However, there has been little evidence for a multisensory-mediated improvement in stimulus localization. We suggest that this lack of effect may be a result of the high acuity of the visual system. To examine whether normal visual acuity may be masking any potential multisensory benefit for stimulus localization, we examined the ability of human subjects to localize visual, auditory and combined visual-auditory targets under conditions of normal and degraded vision. Under conditions of normal vision, localization precision (i.e., variability) was equivalent for visual and multisensory targets, and was significantly worse for auditory targets. In contrast, under conditions of induced myopia, visual localization performance was degraded by an average of 25%, while auditory localization performance was unaffected. However, during induced myopia, multisensory (i.e., visual-auditory) localization performance was significantly improved relative to visual performance. These results show a multisensory-mediated enhancement in human localization ability, and illustrate the cross-modal benefits that can be obtained when spatial information in one sense is compromised or ambiguous.