Efficient femtosecond optical parametric generation in group-velocity-matched MgO:PPLN at 10†MHz.

We report on efficient single-pass optical parametric generation (OPG) of broadband femtosecond pulses in the mid-infrared at 10 MHz by exploiting group-velocity-matched interaction in a 42-mm-long MgO:PPLN crystal. Using a microchip-started femtosecond amplified Mamyshev oscillator at 1064 nm as the pump, the OPG source can provide tunable femtosecond pulses across 1516-1566 nm in the signal and 3318-3568 nm in the idler, with slope efficiencies of ∼93% and ∼41%, respectively. For 650 mW of average input pump power, signal powers of up to 283 mW at 1524 nm are generated, with more than 200 mW over the entire tuning range. Idler average powers of up to 104 mW at 3450 nm, with more than 80 mW across the full range, are also obtained. For input pump pulses of ∼182 fs, the generated signal pulses have a duration of ∼460 fs at 1516 nm. The idler pulses have a typical bandwidth of ≥100 nm over the entire tuning range, and as wide as 181 nm at 3457 nm. The OPG source exhibits excellent passive power stability, better than 0.5% rms in the signal and 0.6% rms in the idler, over 1 h, both in Gaussian TEM00 spatial profile with M2 < 1.5.

Gut microbiota trajectory in pediatric patients undergoing hematopoietic SCT.

Acute GvHD (aGvHD) is the main complication of hematopoietic SCT (HSCT) during the treatment of hematological disorders. We carried out the first longitudinal study to follow the gut microbiota trajectory, from both the phylogenetic and functional points of view, in pediatric patients undergoing HSCT. Gut microbiota trajectories and short-chain fatty acid production profiles were followed starting from before HSCT and through the 3-4 months after transplant in children developing and not developing aGvHD. According to our findings, HSCT procedures temporarily cause a structural and functional disruption of the gut microbial ecosystem, describing a trajectory of recovery during the following 100 days. The onset of aGvHD is associated with specific gut microbiota signatures both along the course of gut microbiota reconstruction immediately after transplant and, most interestingly, prior to HSCT. Indeed, in pre-HSCT samples, non-aGvHD patients showed higher abundances of propionate-producing Bacteroidetes, highly adaptable microbiome mutualists that showed to persist during the HSCT-induced ecosystem disruption. Our data indicate that structure and temporal dynamics of the gut microbial ecosystem can be a relevant factor for the success of HSCT and opens the perspective to the manipulation of the pre-HSCT gut microbiota configuration to favor mutualistic persisters with immunomodulatory properties in the gut.

In vitro anti-angiogenic effects of Hemidesmus indicus in hypoxic and normoxic conditions.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for many purposes and poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. In the context of anticancer pharmacology, anti-angiogenic therapy has become an effective strategy for inhibiting new vessel formation and contrast tumor growth. These considerations are supported by the evidence that most tumors originate in hypoxic conditions and limitation of oxygen diffusion stimulates the formation of tumor abnormal microvasculature. Aim of this study was to evaluate the in vitro anti-angiogenic potential of Hemidesmus indicus (0.31-0.93 mg/mL) on human umbilical vein endothelial cells and delineate the main molecular mechanisms involved in its anti-angiogenic activity both in normoxia and hypoxia. MATERIALS AND METHODS: The decoction of Hemidesmus indicus was subjected to an extensive HPLC phytochemical characterization. Its in vitro anti-angiogenic potential was investigated in normoxia and hypoxia. Cell proliferation, apoptosis induction, and inhibition of endothelial cell migration and invasion were analyzed by flow cytometry. The endothelial tube formation assay was evaluated in matrix gel. The capillary tube branch points formed were counted using a Motic AE21 microscope and a VisiCam videocamera. The regulation of key factors of the neovascularization process such as VEGF, HIF-1α and VEGFR-2 was explored at mRNA and protein level by real time PCR and flow cytometry, respectively. RESULTS: Treatment with Hemidesmus resulted in a significant inhibition of cell proliferation and tube formation in both normoxia and hypoxia. Hemidesmus differently regulated multiple molecular targets related to angiogenesis according to oxygen availability. In normoxia, the inhibition of VEGF was the main responsible for its anti-angiogenic effect; the angiogenesis inhibition induced in hypoxia was regulated by a more complex mechanism involving firstly HIF-1α inhibition, and then VEGF and VEGFR-2 down-regulation. Additionally, the inhibition of endothelial cell migration and invasion by Hemidesmus was more pronounced in normoxia than in hypoxia, possibly due to the physiological enhanced induction of invasion characteristic of hypoxia. CONCLUSIONS: Our results indicate that Hemidesmus might represent a promising therapeutic strategy for diseases in which the inhibition of angiogenesis could be beneficial, such as cancer. The antiangiogenic activity of Hemidesmus is based on multiple interactions with critical steps in the angiogenic cascade. VEGF expression stimulated by HIF-1α as well as endothelial cell migration and differentiation represent important targets of Hemidesmus action and might contribute to its cancer therapeutic efficacy that is presently emerging and offer a scientific basis for its use in traditional medicine.

Hemidesmus indicus induces apoptosis as well as differentiation in a human promyelocytic leukemic cell line.

ETHNOPHARMACOLOGICAL RELEVANCE: The decoction of the roots of Hemidesmus indicus is widely used in the Indian traditional medicine for the treatment of blood diseases, dyspepsia, loss of taste, dyspnea, cough, poison, menorrhagia, fever, and diarrhea. Poly-herbal preparations containing Hemidesmus are often used by traditional medical practitioners for the treatment of cancer. The aim of this study was to investigate the cytodifferentiative, cytostatic and cytotoxic potential of a decoction of Hemidesmus indicus's roots (0.31-3 mg/mL) on a human promyelocytic leukemia cell line (HL-60). MATERIALS AND METHODS: The decoction of Hemidesmus indicus was characterized by HPLC to quantify its main phytomarkers. Induction of apoptosis, cell-cycle analysis, levels of specific membrane differentiation markers were evaluated by flow cytometry. The analysis of cell differentiation by nitroblue tetrazolium (NBT) reducing activity, adherence to the plastic substrate, α-napthyl acetate esterase activity and morphological analysis was performed through light microscopy (LM) and transmission electron microscopy (TEM). RESULTS: Starting from the concentration of 0.31 mg/ml, Hemidesmus indicus induced cytotoxicity and altered cell-cycle progression, through a block in the G0/G1 phase. The decoction caused differentiation of HL-60 cells as shown by NBT reducing activity, adherence to the plastic substrate, α-naphtyl acetate esterase activity, and increasing expression of CD14 and CD15. The morphological analysis by LM and TEM clearly showed the presence of granulocytes and macrophages after Hemidesmus indicus treatment. CONCLUSIONS: The cytodifferentiating, cytotoxic and cytostatic activities of Hemidesmus indicus offers a scientific basis for its use in traditional medicine. Its potent antileukemic activity provides a pre-clinical evidence for its traditional use in anticancer pharmacology. Further experiments are worthwhile to determine the in vivo anticancer potential of this plant decoction and its components.

Volumetry: an alternative to assess therapy response for malignant pleural mesothelioma?

The purpose of our study was to assess robustness of volumetric measurement of malignant pleural mesothelioma (MPM) before and after chemotherapy to modified RECIST (response evaluation criteria in solid tumours) criteria. 30 patients with digitally available chest computed tomography (CT) scans before and after three cycles of chemotherapy were included. Three readers independently assessed tumour response using two different methods: 1) the modified RECIST criteria; and 2) the tumour volumetric approach using dedicated software (Myrian; Intrasense, Paris, France). Inter-rater reliability of unidimensional and volumetric measurements was assessed using intraclass correlation. Tumour response classification for modified RECIST was compared to the volumetric approach applying unidimensional RECIST volumetric equivalent criteria. The determination of unidimensional tumour measurement (RECIST) revealed a low inter-rater reliability (0.55) and a low interobserver agreement for tumour response classification (general κ 0.33). Only 14 patients were classified equally. A high inter-rater reliability (0.99) and interobserver agreement (general κ 0.9) were found for absolute tumour volumes (volumetric measurements). 27 cases were classified equally. The number of cases classified as "stable disease" was higher for the volumetric approach using tumour-equivalent criteria compared to modified RECIST. Volumetric measurement of MPM on CT using Myrian software is a reliable, reproducible and sensitive method to measure tumour volume and, thus, therapy response after induction chemotherapy.

Micellar complexes of all-trans retinoic acid with polyvinylalcohol-nicotinoyl esters as new parenteral formulations in neuroblastoma.

All-trans-retinoic acid (ATRA) is now included in many antitumor therapeutic schemes for the treatment of acute promyelocytic leukemia, Kaposi's sarcoma, head and neck squamous cell carcinoma, ovarian carcinoma, bladder cancer, and neuroblastoma. Unfortunately, its poor aqueous solubility hampers its parenteral formulation, whereas oral administration of ATRA is associated with progressively diminishing drug levels in plasma, which is related to induction of retinoic acid-binding proteins and increased drug catabolism by cytochrome P450-mediated reactions. An ATRA formulation, obtained by complexation of the drug into polymeric micelles, might be suitable for parenteral administration overcoming these unwanted effects. To this purpose, amphiphilic polymers were prepared by polyvinylalcohol (PVA) partial esterification with nicotinoyl moieties and their functional properties evaluated with regard to ATRA complexation. The physicochemical characteristics of the polymers and the complexes were analyzed by 1H-NMR, Dynamic Light Scattering (DLS), Capillary Electophoresis (CE), and were correlated with the complex ability to improve the drug solubilization and release the free drug in an aqueous environment. Subsequently, the best complex, providing the highest ATRA solubilization and release, was evaluated in vitro to test its cytotoxicity towards neuroblastoma cell lines. The PVA substitution degree calculated from 1H-NMR was found to be 5.0%, 8.2%, 15.3% (nicotinoyl moiety:PVA monomer molar ratio), while capillary electrophoresis analysis on the complexes revealed that the drug loadings were 0.95%, 1.20%, 4.76% (ATRA:polymer w:w) for PVA substitution degrees of 5.0%, 8.2%, and 15.3%, respectively. Complexation strongly increased ATRA aqueous solubility, which reached 1.20 +/- 0.25 mg/mL. The DLS measurements of the polymers and the complexes in aqueous solutions revealed mean sizes always below 400 nm, low polydispersity (min 0.202 +/- 0.013, max 0.450 +/- 0.032), and size almost unaffected by concentration. Drug fractional release did not exceed 8% after 48 h. The cytotoxicity studies against neuroblastoma cell lines outlined a significant growth inhibition effect of complexed ATRA with respect to free ATRA. These data suggest that the systems analyzed may be suitable carriers for parenteral administration of ATRA and other hydrophobic antitumor drugs, where the carriers are required to improve drug aqueous solubility and delay drug release almost after their accumulation in solid tumors.

Oxalate consumption by lactobacilli: evaluation of oxalyl-CoA decarboxylase and formyl-CoA transferase activity in Lactobacillus acidophilus.

AIMS: This study was undertaken to evaluate the oxalate-degrading activity in several Lactobacillus species widely used in probiotic dairy and pharmaceutical preparations. Functional characterization of oxalyl-CoA decarboxylase and formyl-CoA transferase in Lactobacillus acidophilus was performed in order to assess the possible contribution of Lactobacillus in regulating the intestinal oxalate homeostasis. METHODS AND RESULTS: In order to determine the oxalate-degrading ability in 60 Lactobacillus strains belonging to 12 species, a screening was carried out by using an enzymatic assay. A high variability in the oxalate-degrading capacity was found in the different species. Strains of Lact. acidophilus and Lactobacillus gasseri showed the highest oxalate-degrading activity. Oxalyl-CoA decarboxylase and formyl-CoA transferase genes from Lact. acidophilus LA14 were cloned and sequenced. The activity of the recombinant enzymes was assessed by capillary electrophoresis. CONCLUSIONS: Strains of Lactobacillus with a high oxalate-degrading activity were identified. The function and significance of Lact. acidophilus LA14 oxalyl-CoA decarboxylase and formyl-CoA transferase in oxalate catabolism were demonstrated. These results suggest the potential use of Lactobacillus strains for the degradation of oxalate in the human gut. SIGNIFICANCE AND IMPACT OF THE STUDY: Identification of probiotic strains with oxalate-degrading activity can offer the opportunity to provide this capacity to individuals suffering from an increased body burden of oxalate and oxalate-associated disorders.

Fenretinide-polyvinylalcohol conjugates: new systems allowing fenretinide intravenous administration.

N-(4-hydroxyphenyl)retinamide (fenretinide, 4-HPR) has been shown to be active toward many tumors without appreciable side effects. However its in vitro activity does not match a correspondent efficacy in vivo. The main reason is that the drug's hydrophobicity hinders its bioavailability in the body fluids. Even if the drug is previously dissolved in organic solvents, such as ethanol or DMSO, the subsequent dilution in body fluids trigger its precipitation in fine aggregates characterized by very low dissolution efficiency, never reaching amounts suitable for therapeutic response. To date no intravenous formulation of 4-HPR exists on the market. The 4-HPR linkage to a hydrophilic polymer by a covalent bond easily hydrolyzable in aqueous environment is expected to increase the drug's aqueous solubility, providing the free drug after hydrolysis of the covalent bond. This may be a useful tool for the preparation of aqueous intravenous formulations of 4-HPR. For this purpose, we linked 4-HPR to polyvinylalcohol (PVA) by a carbonate bond at different drug/hydroxy vinyl monomer molar ratios. We demonstrated that conjugation increased 4-HPR aqueous solubility and strongly inhibited neuroblastoma cell proliferation. In addition, in an in vivo neuroblastoma metastatic model, we obtained a significant antitumor effect as a consequence of the improved drug bioavailability.

Analysis of Amaryllidaceae alkaloids from Narcissus by GC-MS and capillary electrophoresis.

Amaryllidaceae are known as ornamental plants, furthermore some species of this family contain galanthamine, an acetylcholinesterase inhibitor approved for the treatment of Alzheimer's disease, and other alkaloids with interesting pharmacological activity. In the present work, the quali- and quantitative analysis of Amaryllidaceae-type alkaloids in the bulbs of Narcissus species is presented using different analytical approaches. Extracts of Narcissus pseudonarcissus cv. Carlton and Narcissus jonquilla Quail, were first examined by GC-MS using a Rtx-5 MS (programmed temperature) and the major alkaloids were identified. Together with galanthamine, high contents of haemanthamine, were found. Galanthamine was reliably quantified by GC-MS, whereas haemanthamine partly decomposed under the GC conditions, thus alternative analytical methods were investigated. Firstly, reversed-phase HPLC-ESI-MS was applied to identify and isolate at semipreparative levels haemanthamine. The compound was fully characterized by MS/MS and (1)H NMR and then used as a reference substance. The quantitation of both galanthamine and haemanthamine was then accomplished by capillary electrophoresis with spectrophotometric detection. A non-aqueous (NACE) approach was selected in order to use a running buffer fully compatible with samples in organic solvent. In particular, a mixture methanol-acetonitrile (75:25, v/v) containing ammonium acetate (90 mM) was used as a background electrolyte. The same analytical sample was subjected to GC-MS and NACE analysis; the different selectivity displayed by these techniques allowed different separation profiles that can be useful in phytochemical characterization of the extracts. The GC-MS and NACE methods were validated and applied to the quantitation of galanthamine (GC-MS and NACE) and haemanthamine (NACE) in bulbs of N. jonquilla.

Methotrexate determination in pharmaceuticals by enantioselective HPLC.

A simple, sensitive and selective high performance liquid chromatographic method with UV detection for the chiral separation of racemic methotrexate (rac-Mtx) and enantiomeric purity of L-methotrexate in pharmaceutical formulations was developed and validated. The chiral separation was optimized studying both the nature of the stationary phase by using Chirobiotic T, Chiracel OJ and human serum albumin columns and the effect of the mobile phase composition. The best results in terms of enantioresolution and enantioselectivity were achieved with a polar organic mobile phase on Chirobiotic T stationary phase. Essential steps in method validation such as precision, accuracy, suitability and stability were studied according to ICH guidelines. At wavelength 303 nm, the limit of detection (S/N=3) was found to be 0.9 microg/ml for rac-Mtx. The separation of D-Mtx at 0.2% (w/w) level (as limit of quantitation) from the main drug L-Mtx was successfully obtained with 1.72 enantioresolution value. Enantiomeric purity of L-Mtx was determined in pharmaceutical formulations (tablets and injections) with inter- and intra-days relative standard deviation < or = 1.6%. Under the validated stereoselective HPLC conditions for methotrexate, folic acid was also analysed.

HPLC-DAD and LC-ESI-MS analysis of doxycycline and related impurities in doxipan mix, a medicated premix for incorporation in medicated feedstuff.

HPLC-DAD and LC-ESI-MS methods have been developed for the analysis of doxycycline (DOX), including the identification of the related impurities metacycline (MTC) and 6-epidoxycycline (EDOX) and its determination in a medicated premix. The chromatographic separations have been performed on Luna C18 stationary phase and on Synergi (4 microm) Polar-RP 80A, using both acidic (pH 2.5) and basic (pH 8.0) mobile phases. The Synergi Polar-RP column, in combination with a mobile phase of oxalic acid (0.02 M; pH 2.5)-acetonitrile 82:18 (v/v), allowed the complete separation of MTC, EDOX and DOX. The same separation was also obtained using Luna C18 stationary phase with a pH 8 mobile phase. Application of a LC-ESI-MS system and MS/MS analysis, using both positive and negative polarity, allowed the peak identity to be confirmed. A method based on Luna C18 column and UV detection at 346 nm was validated for the determination of DOX in a medicated premix for incorporation in medicated feedstuff.

Photostability studies on nicardipine-cyclodextrin complexes by capillary electrophoresis.

Nicardipine (NC)-cyclodextrin solid systems were prepared in equimolar ratios and their photostability in aqueous solution under exposure to UV(A)-UV(B) radiations was evaluated. The photodegradation process was monitored by a capillary electrophoresis (CE) method able to provide the enantioresolution of the rac-nicardipine. Enantioresolution was achieved using the mixture 3.0% sulfate-beta-cyclodextrin (SbetaCD) and 2.0% heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin (TMbetaCD) as chiral selector in 20mM triethanolammonium phosphate solution (pH 3.0). The photostability studies were carried out on inclusion complexes of rac-nicardipine with alpha-cyclodextrin (alphaCD), beta-cyclodextrin (betaCD), gamma-cyclodextrin (gammaCD), hydroxypropyl-alpha-cyclodextrin (HPalphaCD), hydroxypropyl-beta-cyclodextrin (HPbetaCD), hydroxypropyl-gamma-cyclodextrin (HPgammaCD), (2-hydroxyethyl)-beta-cyclodextrin (HEbetaCD) and methyl-beta-cyclodextrin (MbetaCD). A photoprotective effect was observed by betaCD, HPalphaCD, HEbetaCD, whereas gammaCD, MbetaCD, HPbetaCD and HPgammaCD did not affect the nicardipine photostability. Conversely, alphaCD was found to favour the drug photodegradation. Evidences for CDs-mediated stereoselective photodegradation of rac-nicardipine were observed only for the beta-CD complex. In this case, two distinct photodegradation profiles, with two different kinetic constants (k), were observed for the nicardipine enantiomers.

Drug affinity to immobilized target bio-polymers by high-performance liquid chromatography and capillary electrophoresis.

This review addresses the use of high-performance liquid chromatography (HPLC) and capillary electrophoresis (CE) as affinity separation methods to characterise drugs or potential drugs-bio-polymer interactions. Targets for the development of new drugs such as enzymes (IMERs), receptors, and membrane proteins were immobilized on solid supports. After the insertion in the HPLC system, these immobilized bio-polymers were used for the determination of binding constants of specific ligands, substrates and inhibitors of pharmaceutical interest, by frontal analyses and zonal elution methods. The most used bio-polymer immobilization techniques and methods for assessing the amount of active immobilized protein are reported. Examples of increased stability of immobilized enzymes with reduced amount of used protein were shown and the advantages in terms of recovery for reuse, reproducibility and on-line high-throughput screening for potential ligands are evidenced. Dealing with the acquisition of relevant pharmacokinetic data, examples concerning human serum albumin binding studies are reviewed. In particular, papers are reported in which the serum carrier has been studied to monitor the enantioselective binding of chiral drugs and the mutual interaction between co-administered drugs by CE and HPLC. Finally CE, as merging techniques with very promising and interesting application of microscale analysis of drugs' binding parameters to immobilized bio-polymers is examined.

Determination of the dissociation constants (pKa) of basic acetylcholinesterase inhibitors by reversed-phase liquid chromatography.

An RP-HPLC study for the pKa determination of a series of basic compounds related to caproctamine, a dibenzylaminediamide reversible inhibitor of acetylcholinesterase, is reported. The 2-substituted analogues, bearing substituents with different electronegativity, were analysed by RP-HPLC by using C18 C4 stationary phases with a mobile phase consisting of mixture of acetonitrile and triethylamine phosphate buffer (pH range comprised between 4 and 10). Typical sigmoidal curves were obtained, showing the dependence of the capacity factors upon pH. In general, the retention of the investigated basic analytes increased with increasing of the pH. The inflection point of the pH sigmoidal dependence was used for the dissociation constant determination at a fixed acetonitrile percentage. When plotting pKa vs. percent of acetonitrile in the mobile phase for two representative compounds, linear regression were obtained: the y intercept gave the aqueous pKa(w). The pKa estimation by HPLC method was found to be useful to underline the difference of benzylamine basicity produced by the ortho aromatic substituents. The variation of pKa values (6.15-7.80) within the series of compounds was correlated with the electronic properties of the ortho-substituents through the Hammett sigma parameter, whereas the ability of substituents to accept H-bond was found to play a role in determining the conformational behavior of the molecules.

Prostaglandin E(1)-based vasoactive cocktails in erectile dysfunction: how environmental conditions affect PGE(1) efficacy.

INTRODUCTION: The chemical stability of prostaglandin E(1) (PGE(1)) in physiological solution has been studied in different environmental conditions. However, very little data exist regarding the PGE(1) stability and the consequent breakdown products in PGE(1)-based vasoactive cocktails under different environmental conditions. OBJECTIVE: To evaluate the loss of the therapeutic efficacy of PGE(1) either alone or in combination with other vasoactive substances under different storage conditions. MATERIALS AND METHODS: Utilizing high performance liquid chromatography the PGE(1) content was evaluated alone and in association with papaverine and papaverine plus phentolamine at temperatures of 2-8 and at 20 degrees C, and after 15, 30, 60, 90 and 120 days of storage using multivariate statistical analysis of variance. RESULTS: We found that the time of storage significantly affects PGE(1) activity. Furthermore, both the storage temperature and cocktail composition had a significant effect on PGE(1) stability. The chromatographic studies did not disclose the presence of the principal degradation products of PGE(1) (PGA(1), PGB(1)). The presence of papaverine and temperature of 20 degrees C have the greatest effect on the degradation of PGE(1 )during the first 30 days of storage. DISCUSSION: Temperature and time are prevalent factors determining the slow and progressive deterioration curve of PGE(1) after 30 days of storage. None of the environmental conditions evaluated was so drastic to determine the presence of PGA(1) and PGB(1). CONCLUSION: For clinical use, one should note that PGE(1) maintains 50-80% of its efficacy for about 1 month even if stored at room temperature (20 degrees C) and/or combined with papaverine.

Stereoselective binding of 2-(4-biphenylyl)-3-substituted-3-hydroxypropionic acids on an immobilised human serum albumin chiral stationary phase.

A series of 2-(4-biphenylyl)-3,3'-hydroxy-substituted phenyl propionic acid, with anti-inflammatory properties, bearing two chiral centres, were studied by HPLC upon HSA-CSP (human serum albumin-based chiral stationary phase). The compounds were analysed in their stereoisomeric erythro and threo forms. The study involved the enantioselective analysis on HSA-CSP, the determination of the racemate lipophilicity (log k'(w)), a QSRR (quantitative structure-retention relationship) analysis and CD study for the assessment of the absolute configuration of the most retained enantiomer. Lipophilicity was found to be an important factor affecting the affinity of the compounds for the HSA stationary phase, but electronic properties seemed to play a role. The position of the substituent of the phenyl group on carbon 3 was found important to modulate stereoselective interaction, the highest value of enantioselectivities being found for the erythro ortho-substituted phenyl derivatives. The previously proposed two steps mechanism of enantiodiscrimination for cyclohexylphenyl substituted derivatives was confirmed for this series of derivatives bearing the biphenylyl moiety.

Use of an immobilised human serum albumin HPLC column as a probe of drug-protein interactions: the reversible binding of valproate.

The reversible binding of valproate to human serum albumin determines a decrease of the binding of ligands that selectively bind to site I, site II, and bilirubin binding site. The binding inhibition was followed by displacement chromatography methodology using increasing concentrations of the competitor, i.e. valproate, in the mobile phase. Significant binding inhibition was observed for drugs binding at site I and site II. The greater displacement was observed for the more retained enantiomer of benzodiazepines and profens. A reduction of the affinity was observed also in the case of phenol red, this compound being selected as representative of bilirubin binding site. Difference circular dichroism spectroscopy was also used to characterise the binding of valproate to human serum albumin. This antiepilectic drug was proved to affect the binding at site I, II, and bilirubin binding site. The data have physiological relevance because significant inhibition of the binding resulted at clinic concentrations of valproate.

Evidences of cyclodextrin-mediated enantioselective photodegradation of rac-nicardipine by capillary electrophoresis.

Capillary electrophoresis (CE) was applied to photostability studies on rac-nicardipine, a dihydropyridine chiral drug. CE methods were developed able to provide the enantioresolution of drug and its separation from the photodegradation products. Enantioresolution was achieved using 5% sulfated-beta-cyclodextrin (S-beta-CD) as chiral selector in 20 mM triethanolammonium phosphate solution (pH 3). The photostability studies were carried out on inclusion complexes of rac-nicardipine with beta-cyclodextrin (beta-CD) and (2-hydroxypropyl)-beta-cyclodextrin (HP-beta-CD) in aqueous solutions (pH 7.4 and 5). The CE analysis of the solutions exposed to UV-A and UV-B radiations showed a photoprotective effect by beta-CD; conversely, HP-beta-CD proved to favor the drug photodegradation. Moreover, evidences for CDs-mediated stereoselective photodegradation of rac-nicardipine were obtained. In fact, two distinct photodegradation profiles were observed for the nicardipine enantiomers in the presence of the CDs. The photodegradation was found to follow an apparent first-order kinetics and two different kinetic constants (k) were obtained for the two enantiomers. After exposure to UV-A and UV-B radiations, the solutions contained residual nicardipine with a significant change in the enantiomeric ratio; this effect was depending on the CD used for the inclusion complexation.

Determination of 5-aminosalicylic acid related impurities by micellar electrokinetic chromatography with an ion-pair reagent.

A micellar electrokinetic chromatographic (MEKC) method was developed for the quantification of mesalazine or 5-aminosalicylic acid (5-ASA) and its major impurities 3-aminosalicylic acid, salicylic acid, sulfanilic acid and 4-aminophenol. The optimisation of the experimental conditions was carried out considering some important requirements: resolution, reproducibility, detection limits of 0.1% (m/m) or less, short total analysis time. Preliminary investigations employing sodium dodecyl sulfate (SDS) as surfactant did not lead to the necessary resolution of the studied compounds; the addition of tetrabutylammonium bromide (TBAB) to the SDS micellar system resulted in the complete separation of all the compounds. The effects on the separation by several parameters such as TBAB concentration, SDS concentration, background electrolyte pH and concentration, were evaluated. Using a fused-silica capillary (8.5 cm effective length) complete analysis was obtained in a very short time. Under the optimised final conditions [120 mM 3-(cyclohexylamino)-2-hydroxy-1-propanesulfonic acid buffer, pH 10.20, 65 mM SDS in the presence of 55 mM TBAB and 5% methanol] the method was validated for specificity, precision, linearity, limits of detection and quantitation, and robustness: the 5-ASA related impurities can be quantified at least at the 0.1% (m/m) level.

Determination of inhibitors' potency (IC50) by a direct high-performance liquid chromatographic method on an immobilised acetylcholinesterase column.

An immobilised acetylcholinesterase (AChE) stationary phase was prepared by using an in situ AChE immobilisation procedure. A stainless steel column packed with epoxide silica was connected to the HPLC system and the enzyme solution at pH 5.8 was recycled through the column at a flow-rate of 0.5 ml/min for 24 h. The activity of the immobilised AChE was determined by injecting the substrate acetylthiocholine, using as mobile phase 0.1 M phosphate buffer (pH 7.4) containing Ellman's reagent [5,5'-dithio-bis(2-nitrobenzoic acid)] and measuring the area of the obtained peak with UV detection at 412 nm. The effect of AChE inhibitors tacrine, edrophonium and donepezil were evaluated by the simultaneous injection of each inhibitor with the substrate. The resulting decrease in the AChE activity, as expressed by the decrease of the peak area detected at 412 nm, was related to the concentration and potency of the solutes. The obtained IC50 values were compared with those derived by the conventional spectrophotometric method. This immobilized enzyme reactor, included in a chromatographic system, can be used for the rapid screening for new inhibitors allowing for the on-line determination of a compound's inhibitory potency. The advantages over the conventional methods are the increased enzyme stability and system automation which allows a large number of compounds to be analysed continuously.