Biological conversion of a water-soluble prodrug of cyclosporine A.

UNIL088 is a water-soluble prodrug of cyclosporine A (CsA) designed for topical ocular delivery. The pro-moiety is grafted via an ester function to CsA and the solubilizing group is a phosphate ion. The aim of this study was to elucidate the conversion mechanisms by which UNIL088 generates CsA. UNIL088 was incubated in rabbit tears at physiological temperature to study its enzymatic and chemical conversion, respectively. Metabolites and intermediates were identified using a quadrupole-time of flight (QqTOF) mass spectrometer, which allowed biotransformation pathways to be deduced. Conversion is activated by the chemical or enzymatic hydrolysis of the terminal ester function of the pro-moiety, leading to the phospho-serine-sarcosine-cyclosporine A that spontaneously converts into CsA. In addition to the main biotransformation pathway, a secondary reaction involved hydrolysis of the phosphate ester group of the pro-moiety, probably by phosphatases present in tears.

Optimized release of dexamethasone and gentamicin from a soluble ocular insert for the treatment of external ophthalmic infections.

In the case of external ophthalmic infections, repeated instillations of antibiotics are required to reach therapeutic level, above the minimal inhibitory concentration (MIC). An additional administration of a corticosteroid is often needed, in order to limit the precorneal damages caused by the infection. However, repeated administration of a corticosteroid can increase intraocular pressure and thus lead to glaucoma. To overcome the disadvantages of separated and repeated instillations of two products and to avoid the side effects of dexamethasone, a soluble insert containing gentamicin sulfate and dexamethasone phosphate was developed. The new system ensures the concomitant release of the two drugs during the first 10 h of treatment, followed by an adequate concentration of gentamicin sulfate, above the MIC of 4.0 microgram ml-1, during 50 h, due to a combination of gentamicin sulfate with cellulose acetate phthalate, which reduces the solubility of gentamicin.

Use of large particles support for fast analysis of methadone and its primary metabolite in human plasma by liquid chromatography-mass spectrometry.

A bioanalytical method was developed for the quantitation of methadone (MTD) and its primary metabolite, (EDDP) in plasma. The extraction step was performed within a capillary column packed with large particles (35x0.3 mm I.D.; d(p) 30 micrometer) at high flow-rate conditions (450 microliter/min). The separation was performed on a microbore analytical column (55x2 mm I.D.; d(p) 3 micrometer) coupled to a mass spectrometer (MS). This procedure was based on a column-switching unit. Analytes of interest were retained on the precolumn by hydrophobic interactions and backflushed from the precolumn to the analytical column. The detection was carried out with a MS single quadrupole equipped with an electrospray interface. The total analysis time was 6 min. The limits of quantification were evaluated at 10 and 25 ng/ml for MTD and EDDP, respectively. At this level, good accuracies were obtained for both analytes with repeatability values less than 18%.

A poly(ortho ester) designed for combined ocular delivery of dexamethasone sodium phosphate and 5-fluorouracil: subconjunctival tolerance and in vitro release.

A viscous hydrophobic poly(ortho ester) (POE) has been developed as a biocompatible, biodegradable sustained release system for selected cases of glaucoma filtering surgery. Dexamethasone and 5-fluorouracil (5-FU) are frequently administered together post-operatively, for their anti-fibroblastic and anti-inflammatory properties, respectively. A combined sustained release of both drugs could be advantageously used. Drug release kinetics were studied using specially designed thermostated cells. Subconjunctival tolerance was evaluated on New Zealand albino rabbits by clinical evaluation. Due to its basicity, the addition of dexamethasone sodium phosphate (DEX-P) stabilized the polymer and prolonged 5-FU in vitro release from 2 to 4 days. Both therapeutic agents were released concomitantly, according to a linear profile. The presence of 5-FU only slightly affected the overall subconjunctival tolerance of POE in rabbits, whereas the addition of DEX-P markedly improved POE tolerance by reducing the hyperemia of the conjunctiva to a minimal grade.

Concomitant and controlled release of dexamethasone and 5-fluorouracil from poly(ortho ester).

A viscous bioerodible and hydrophobic poly(ortho ester) has been developed as a biocompatible, sustained drug release system for an ophthalmic application in intraocular proliferative disorders. The combination of wound healing modulators such as 5-fluorouracil and dexamethasone is a major advantage since these drugs act at different stages of these diseases. Since 5-fluorouracil is an acidic, water-soluble compound and dexamethasone exists in three chemical forms, i.e. the water-insoluble base, the highly hydrophobic acetate ester or the basic phosphate salt, it was of interest to investigate whether the physicochemical properties of the drugs have an influence on their release rates, and whether a concomitant and sustained release of both 5-fluorouracil and dexamethasone could be achieved. It has been found that lipophilicity and acidobasicity play a major role in controlling drug release rates and polymer degradation. The combination of 5-fluorouracil and dexamethasone phosphate allows a sustained and concomitant release of both drugs, due to the basic characteristics of the corticosteroid which stabilize the polymer. This system appears to be promising for concomitant and controlled drug delivery aimed at the pharmacological treatment of intraocular proliferative disorders.

2C-B: a new psychoactive phenylethylamine recently discovered in Ecstasy tablets sold on the Swiss black market.

This study sought to identify, by means of several analytical methods (GC-MS, HPLC-DAD, CE-DAD, FTIR, and NMR), 4-bromo-2,5-dimethoxyphenethylamine (2C-B), which was found in two sets of tablets obtained from the Swiss black market. Unequivocal identification of 2C-B was only achieved by a combination of mass spectrometric and NMR analysis. Quantitation of 2C-B was performed by HPLC-DAD and CE-DAD. The amounts of 2C-B found in the tablets (3-8 mg) were in the range of the minimum quantity required to induce the effects characteristic of this drug.

Quantitation of polar analytes using column-switching: application to oxycodone and three metabolites in human plasma.

We present herein a sensitive and selective assay for the determination of oxycodone and its main metabolites, oxymorphone, noroxycodone and noroxymorphone in human plasma, using column-switching and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Sample preparation comprised protein precipitation with perchloric acid. After neutralization, the supernatant was injected without any evaporation step onto a polymeric, pH-resistant cartridge (HySphere Resin GP 10-12 microm) for sample clean-up (Prospekt II). The latter operation was achieved by using alkaline conditions to ensure retention of analytes and methanol for matrix interference removal. More than two hundred plasma samples could be analyzed with a single cartridge. Analytes were desorbed in the backflush mode and were separated on a conventional reversed phase column (XTerra MS 4.6 x 50 mm, 3.5 microm), using an acidic mobile phase (i.e. containing 0.1% of formic acid). Mass spectrometric detection was achieved with a 4000 Q TRAP equipped with an atmospheric pressure chemical ionization (APCI) source, in positive ionization mode, operated in the selected reaction monitoring mode (SRM). Starting from a plasma volume of 250 microl, quantification ranges were 25-10,000 pg/ml for OXM and NOXM and 50-10,000 pg/ml for OXC and NOXC. Accuracy was found to be within 98% and 108% and precision better than 7%. Replicate determination of incurred or study samples ensured the method to be reproducible and usable for clinical studies.

Determination of dexamethasone in tears by capillary electrophoresis.

A selective capillary zone electrophoresis (CZE) microassay was developed for the simultaneous determination of dexamethasone phosphate and its major metabolite, dexamethasone, in tears. The calibration was carried out in the biological matrix with indoprofen as an internal standard which allowed the separation of dexamethasone phosphate and dexamethasone from the tear constituents. The limits of detection and quantification of the assay were 0.5 and 2.0 microg ml(-1), respectively. This quantification method is essential for the in vivo determination of dexamethasone concentration-time profiles in tears after application of the antiinflammatory drug.

Selectivity manipulation using nonaqueous capillary electrophoresis. Application to tropane alkaloids and amphetamine derivatives.

Non-aqueous capillary electrophoresis was investigated for its potential in the analysis of pharmaceutical compounds, namely tropane alkaloids and amphetamine derivatives. The separation of these drugs was compared in aqueous and organic media such as methanol and/or acetonitrile. Selectivity, migration times and efficiency were critically affected by the composition of the methanol/acetonitrile mixture, as well as by the nature and the concentration of the electrolyte. In particular, the migration orders of two positional isomers, littorine and hyoscyamine, were inverted in the presence of trifluoroacetic acid in the nonaqueous medium. The same behavior was observed for amphetamine-methamphetamine and for two methylenedioxyamphetamine derivatives.

Binding constant dependency of amphetamines with various commercial methylated beta-cyclodextrins.

Cyclodextrins play an important role in enantioselective separations. They represent the major class of chiral selectors used by capillary electrophoresis. Unfortunately, the purity of commercial cyclodextrins is often not well characterized, and similar selectors sold by various suppliers may show totally different enantioselectivities. In this study, the composition of several commercial methylated-beta-cyclodextrins is evaluated by means of previously developed analytical methods. Then, different calculation methodologies, such as graphical determinations, as well as nonlinear or linear regression approaches, are evaluated in order to calculate the binding constants of inclusion complexes formed by some amphetamine derivatives with methylated-beta-cyclodextrins. The nonlinear curve-fitting methodology proves to be the most suitable for these determinations. Comparisons are made between the different selectors and several hypotheses are proposed concerning the formation of the inclusion complex. Finally, enantiomeric resolutions are evaluated for these selectors and conclusions drawn about the knowledge of selector composition.

On-line capillary electrophoresis-electrospray mass spectrometry for the stereoselective analysis of drugs and metabolites.

The on-line combination of partial-filling capillary electrophoresis and electrospray ionization mass spectrometry was demonstrated for the enantioseparation of pharmaceutical drugs and metabolites, namely amphetamines, methadone, venlafaxine and selected tropane alkaloids. The partial-filling technique proved to be a suitable and efficient approach to avoid mass spectrometry (MS) source contamination, as well as signal suppression due to nonvolatile additives. To achieve chiral separation, various chiral selectors were applied, including neutral and particularly negatively charged cyclodextrins. Because of the countercurrent contribution, charged cyclodextrins were found more suitable for the on-line MS detection of separated enantiomers. Hyphenation of capillary electrophoresis (CE) with mass spectrometry was found appropriate for the stereoselective analysis of methadone in real serum samples. Moreover, the use of MS in the selected ion monitoring mode resulted in a very high selectivity, as well as improved sensitivity compared to UV detection. Finally, with atropine as a model compound, the quantitative performances of the method were evaluated and showed high sensitivity, as well as good repeatability in terms of migration time and peak area ratio.

[Ecstasy--the status in French-speaking Switzerland. Composition of seized drugs, analysis of biological specimens and short review of its pharmacological action and toxicity]. / Ecstasy--la situation en Suisse romande. Composition des saisies, analyse des échantillons biologiques et brève revue de son action pharmacologique et de sa toxicité.

Methylenedioxy-N-methylamphetamine (MDMA, "Ecstasy") and other related phenylethylamines are nowadays used extensively in Western Switzerland at dance clubs and raves. There is a widely held belief among teenagers and misusers that ecstasy is safe. In the last years however, an increasing number of reports of MDMA-related deaths has been reported. Acute clinical toxicity problems following MDMA ingestion include hyperthermia, convulsions and arrhythmias. There is also growing concern that these phenylethylamines are neurotoxic and cause long-term damage to serotonineric nerve terminals in animal brains. Qualitative analyses by GC-MS of street samples of ecstasy showed that only a part of them contain MDMA or related phenylethylamines (MDA, MDEA, MBDB and 2C-B). Most of them were mixed with caffeine and an excipient (sugars or polyols [e.g. mannitol]). Amphetamine cut with caffeine and other drugs (e.g. testosterone), stimulants (e.g. pseudoephedrine) and other drugs unrelated to stimulants and phenylethylamines (e.g. LSD, chloroquine, vasodilators) were also detected. Quantitative determinations performed by HPLC-DAD or EC-DAD reveal huge fluctuations in the amount of active substance(s) per tablet. MDMA and related compounds display unique psychoactive properties, acting as a stimulant and inducing feelings of empathy. The effects of MDMA intake are very likely the results of the large release of serotonin (5-HT) in the synaptic cleft, of the inhibition of the re-uptake inactivation of 5-HT and of the inhibition of a key-enzyme involved in the biosynthesis of 5-HT. Forensic investigations performed at our institute showed significant blood levels of MDMA, MDEA and MDA in samples drawn from people suspected of driving under the influence of psychoactive drugs. Up to now, no death could be attributed to MDMA intoxication only because our analyses always revealed the additional presence of toxic amounts of other psychoactive drugs (e.g. opiates, cocaine). Our study shows that because of the variable composition of ecstasy tablets, unpredictable types and amounts of drugs may be taken by MDMA misusers. Moreover, there is considerable concern that traffic accidents may be caused by MDMA-abusers. MDMA intake could result in severe intoxication and even death, especially when combined with other types of drugs.

Central composite design in the chiral analysis of amphetamines by capillary electrophoresis.

We have studied a central composite design for the chiral separation of amphetamines using capillary electrophoresis. Five variables, i.e., buffer concentration, pH, chiral selector concentration, temperature, and applied voltage, were investigated. Enantiomeric resolutions as well as analysis time and generated power were established as responses for each experiment. A model of each response was obtained by multiple regression of a quadratic-degree mathematical expression. From the models, we determined the most favorable conditions for the chiral separation by optimizing the resolution and setting the other responses at threshold values. Results were compared with a previous study in which a systematic investigation of the operating parameters was carried out. In order to visualize the robustness of the method, response surfaces were drawn for the significant variables. We have concluded that experimental designs offer a rapid means of optimizing several variables and provide an efficient test for the robustness of the analytical method.