Single-dose pharmacokinetics and lung function of nebulized niclosamide ethanolamine in sheep.

PURPOSE: Niclosamide is approved as an oral anthelminthic, but its low oral bioavailability hinders its medical use requiring high drug exposure outside the gastrointestinal tract. An optimized solution of niclosamide for nebulization and intranasal administration using the ethanolamine salt has been developed and tested in a Phase 1 trial. In this study we investigate the pulmonary exposure of niclosamide following administration via intravenous injection, oral administration or nebulization. METHODS: We characterized the plasma and pulmonary pharmacokinetics of three ascending doses of nebulized niclosamide in sheep, compare it to intravenous niclosamide for compartmental PK modelling, and to the human equivalent approved 2 g oral dose to investigate in the pulmonary exposure of different niclosamide delivery routes. Following a single-dose administration to five sheep, niclosamide concentrations were determined in plasma and epithelial lining fluid (ELF). Non-compartmental and compartmental modeling was used to characterize pharmacokinetic profiles. Lung function tests were performed in all dose groups. RESULTS: Administration of all niclosamide doses were well tolerated with no adverse changes in lung function tests. Plasma pharmacokinetics of nebulized niclosamide behaved dose-linear and was described by a 3-compartmental model estimating an absolute bioavailability of 86%. ELF peak concentration and area under the curve was 578 times and 71 times higher with nebulization of niclosamide relative to administration of oral niclosamide. CONCLUSIONS: Single local pulmonary administration of niclosamide via nebulization was well tolerated in sheep and resulted in substantially higher peak ELF concentration compared to the human equivalent oral 2 g dose.

Bulk Flow Optimisation of Amorphous Solid Dispersion Excipient Powders through Surface Modification.

Particulate amorphous solid dispersions (ASDs) have been recognised for their potential to enhance the performance of various solid dose forms, especially oral bioavailability and macromolecule stability. However, the inherent nature of spray-dried ASDs leads to their surface cohesion/adhesion, including hygroscopicity, which hinders their bulk flow and affects their utility and viability in terms of powder production, processing, and function. This study explores the effectiveness of L-leucine (L-leu) coprocessing in modifying the particle surface of ASD-forming materials. Various contrasting prototype coprocessed ASD excipients from both the food and pharmaceutical industries were examined for their effective coformulation with L-leu. The model/prototype materials included maltodextrin, polyvinylpyrrolidone (PVP K10 and K90), trehalose, gum arabic, and hydroxypropyl methylcellulose (HPMC E5LV and K100M). The spray-drying conditions were set such that the particle size difference was minimised, so that it did not play a substantial role in influencing powder cohesion. Scanning electron microscopy was used to evaluate the morphology of each formulation. A combination of previously reported morphological progression typical of L-leu surface modification and previously unreported physical characteristics was observed. The bulk characteristics of these powders were assessed using a powder rheometer to evaluate their flowability under confined and unconfined stresses, flow rate sensitivities, and compactability. The data showed a general improvement in maltodextrin, PVP K10, trehalose and gum arabic flowability measures as L-leu concentrations increased. In contrast, PVP K90 and HPMC formulations experienced unique challenges that provided insight into the mechanistic behaviour of L-leu. Therefore, this study recommends further investigations into the interplay between L-leu and the physico-chemical properties of coformulated excipients in future amorphous powder design. This also revealed the need to enhance bulk characterisation tools to unpack the multifactorial impact of L-leu surface modification.

LC-MSMS characterisations of scymnol and oxoscymnol biotransformations in incubation mixtures of rat liver microsomes.

The bile alcohol 5ß-scymnol ([24R]-(+)-5ß-cholestan-3α,7α,12α,24,26,27-hexol) is a therapeutic nutraceutical derived from marine sources, however very little is known about its potential for biotransformation as a xenobiotic in higher vertebrates. In this study, biotransformation products of scymnol catalysed by liver microsomes isolated from normal and streptozotocin (STZ)-treated male Wistar rats were characterised by liquid chromatography-tandem mass spectroscopy (LC-MSMS). In order of increasing polarity relative to the reversed phase sorbent, structural assignments were made for four biotransformation products, namely 3-oxoscymnol (5ß-cholestan-3-one-7α,12α,24,26,27-pentol); 7-oxoscymnol (5ß-cholestan-7-one-3α,12α,24,26,27-pentol); 3ß-scymnol (5ß-cholestan-3ß,7α,12α,24,26,27-hexol) and 6ß-hydroxyscymnol (5ß-cholestan-3α,6ß,7α,12α,24,26,27-heptol). In addition, a total of eight biotransformation products were characterised from microsomal incubations of crude oxoscymnol compounds, namely 7ß-scymnol; 3,12-dioxoscymnol; 3,7-dioxoscymnol; 7,12-dioxoscymnol; 12-oxo-3ß-scymnol; 7-oxo-3ß-scymnol; 6ß-hydroxy-12-oxoscymnol and 6ß-hydroxy-7-oxoscymnol. Collectively, the results indicate hepatic enzyme-catalysed hydroxylation, dehydrogenation and epimerisation reactions on the steroid nucleus of scymnol, and provide an insight into biotransformation pathways for scymnol use as a therapeutic nutraceutical in higher vertebrates.

Comprehensive two-dimensional gas chromatography applied to illicit drug analysis.

Multidimensional gas chromatography (MDGC), and especially its latest incarnation--comprehensive two-dimensional gas chromatography (GC × GC)--have proved advantageous over and above classic one-dimensional gas chromatography (1D GC) in many areas of analysis by offering improved peak capacity, often enhanced sensitivity and, especially in the case of GC × GC, the unique feature of 'structured' chromatograms. This article reviews recent advances in MDGC and GC × GC in drug analysis with special focus on ecstasy, heroin and cocaine profiling. Although 1D GC is still the method of choice for drug profiling in most laboratories because of its simplicity and instrument availability, GC × GC is a tempting proposition for this purpose because of its ability to generate a higher net information content. Effluent refocusing due to the modulation (compression) process, combined with the separation on two 'orthogonal' columns, results in more components being well resolved and therefore being analytically and statistically useful to the profile. The spread of the components in the two-dimensional plots is strongly dependent on the extent of retention 'orthogonality' (i.e. the extent to which the two phases possess different or independent retention mechanisms towards sample constituents) between the two columns. The benefits of 'information-driven' drug profiling, where more points of reference are usually required for sample differentiation, are discussed. In addition, several limitations in application of MDGC in drug profiling, including data acquisition rate, column temperature limit, column phase orthogonality and chiral separation, are considered and discussed. Although the review focuses on the articles published in the last decade, a brief chronological preview of the profiling methods used throughout the last three decades is given.

Chemical signature of ecstasy volatiles by comprehensive two-dimensional gas chromatography.

A method for ecstasy volatiles 'signature' analysis based on two-dimensional gas chromatography separation and time-of-flight mass spectrometry detection (GC×GC-TOFMS) is presented. Organic impurity volatiles were extracted by head space solid phase microextraction (HS-SPME). The final column phase choice of the four different column combinations tested was a low-polarity 5% phenyl polysilphenylene-siloxane coupled with a polyethylene glycol phase, which best displayed the complex impurity profile. Second dimension ((2)D) retention time reproducibility was found to be about 1% RSD, and area reproducibility of SPME sampling was just over 5% RSD for compounds with S/N ratio of about 100. High similarity of TOFMS spectra of impurities was obtained against commercial MS libraries. 16 components from the two-dimensional profiles were selected for comparison of the 24 ecstasy tablets, most of which proved to be benzodioxole derived compounds. All tablets were correctly classified in eight groups according to their post-tabletting characteristics, when appropriate data pre-treatment was applied. Principal component analysis revealed clustering of samples according to the country of origin. Samples from Macedonia were elevated in N-formyl-MDMA and N-acetyl-MDMA while samples from Australia were elevated in 3,4-methylenedioxypropane and 3,4-methylenedioxyacetophenone. Furthermore, three components were found to be unique for one of the source countries. The additional separation of components on the (2)D column, increased response due to modulation, high acquisition rate with full mass spectra using TOFMS detection, and MS deconvolution extend the possibility of detecting additional markers and route-specific components, especially of low abundant, polar components.

Hydroxysteroid dehydrogenase transformations of 5ß-scymnol and identification of oxoscymnol transformation products by liquid chromatography-tandem mass spectroscopy.

A new and sensitive high performance liquid chromatography (HPLC) separation procedure coupled with tandem mass spectroscopy (MS and MS(2)) detection was developed to identify for the first time the oxidation products of 5ß-scymnol [(24R)-(+)-5ß-cholestan-3α,7α,12α,24,26,27-hexol] catalysed by bacterial hydroxysteroid dehydrogenase (HSD) reactions in vitro. The authentic scymnol (MW 468) standard yielded a protonated molecular ion [M+H](+) at m/z 469 Da, and higher mass adduct ions attributed to [M+NH(4)](+) (m/z 486), [M+H+CH(3)OH](+) (m/z 501) and [M+H+CH(3)COOH](+) (m/z 530). (24R)-(+)-5ß-Cholestan-3-one-7α,12α,24,26,27-pentol (3-oxoscymnol, m/z 467 Da, relative retention time (RRT)=0.89) was identified as the principle molecular species of scymnol in the reaction with 3α-HSD pure enzyme. [S](0.5) for the reaction of 3α-HSD with scymnol as substrate was 0.7292 mM. (24R)-(+)-5ß-cholestan-7-one-3α,12α,24,26,27-pentol (7-oxoscymnol, m/z 467 Da, RRT=0.79) and (24R)-(+)-5ß-cholestan-12-one-3α,7α,24,26,27-pentol (12-oxoscymnol, m/z 467 Da, RRT=0.81) were similarly identified as principle molecular species in the respective 7α-HSD and 12α-HSD reactions. Polarity of the oxoscymnol species was established as 7-oxoscymnol>12-oxoscymnol>3-oxoscymnol>scymnol (in order from most polar to least polar). Confirmation that 5ß-scymnol is an oxidative substrate for steroid-metabolising enzymes was made possible by the use of sophisticated liquid chromatography-mass spectrometry (LC-MS) techniques that will likely provide the basis for further exploration of scymnol as a therapeutic compound.

Prophylactic and therapeutic effects of Mytilus edulis fatty acids on adjuvant-induced arthritis in male Wistar rats.

Lipid-rich fractions from the flesh tissue of Mytilus edulis were obtained by solvent extraction and chromatographic separation, and tested for anti-inflammatory (AI) activity in vitro and in vivo. Inhibition of leukotriene production by isolated human neutrophils in response to calcium ionophore stimulation in the presence of exogenous arachidonic acid substrate was demonstrated for the hydrolysed triglyceride fraction of the crude lipid extract. This fraction was subsequently tested for in vivo AI activity using the mycobacterial adjuvant-induced polyarthritis rat model. The hydrolysed triglyceride fraction showed significant AI activity when dosed therapeutically (10 mg/kg BW/day, p.o., for 6 days from the onset of arthritis), decreasing body weight loss by 55% and hind paw swelling by 65% compared to the arthritic control. The (non-hydrolysed) crude lipid extract was effective when dosed prophylactically (30 mg/kg BW/day, p.o., for 16 days starting on day -2 of arthritigen inoculation). Structural analysis by GC and GC-MS revealed in the extracts an abundance of EPA (20:5n-3) and DHA (22:6n-3) (37% of total fatty acids), along with a small quantity of a rare anti-inflammatory n-3 analogue of arachidonic acid, namely 7, 11, 14, 17-eicosatetraenoic acid (20:4n-3).

Phytochemical analysis and biological screening of leaf and twig extracts from Kunzea ericoides.

Kunzea ericoides is a member of the Myrtle group of tea trees. Leaf and twig material of K. ericoides was extracted with different solvents to afford terpene (including the essential oil), flavonoid and lipid classes (but no alkaloid class), which were subsequently screened for antibacterial, antitumour, cytotoxic, antioxidant and antiinflammatory activity. Differences were observed in the biological activity for the chemical classes tested, and in general, the leaf extracts were comparatively more bioactive than the twig extracts. The leaf lipid extract was the most bioactive fraction, exhibiting antibacterial, antitumour and antiinflammatory activity. Thin layer chromatography and gas chromatography-mass spectroscopy analysis of each extract revealed previously identified phytochemicals that may be responsible for the observed bioactivities.

Comprehensive two-dimensional gas chromatography--quadrupole mass spectrometric analysis of drugs.

The use of comprehensive two-dimensional gas chromatography coupled to quadrupole mass spectrometry (GC x GC-qMS) for drug screening is investigated with 77 underivatised drug standards in methanolic solution. The GC x GC-qMS setup involved a reduced mass scan range of 42-235 u and minimum quadrupole sampling time to achieve quadrupole scanning frequency of 19.36 Hz. Only 26% of the drugs investigated gave fair-to-acceptable library matches with full mass scan range commercial libraries. The creation of a new "truncated" library based on the mass spectra of the drug standards in the applied mass scan range of 42-235 u extends the feasibility of the currently adopted GC x GC-qMS approach to higher molecular weight compounds and is investigated with blank blood spiked with drug standards. With the new library, 75% of the drugs yielded matches of at least 90%. The time-of-flight mass spectrometer (TOFMS) is expected to address the limitations of the present GC x GC-qMS setup and a brief comparison between GC x GC-qMS and GC x GC-TOFMS is also provided in this study.

Protopine alkaloids in horse urine.

Protopine was extracted from Fumaria officinalis and purified by column chromatography. Urine samples were collected from horses and a human volunteer that had been administered either F. officinalis or protopine free base. Plant and urine samples were acetylated and analysed by GCMS after solid-phase extraction (SPE). The urinary metabolites of protopine were identified as 4,6,7,13-tetrahydro-9,10-dihydroxy-5-methyl-benzo[e]-l,3-benzodioxolo [4,5-1][2] benzazecin-12(5H)-one, 4,6,7,13-tetrahydro-10-hydroxy-9-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-1][2] benzazecin-12(5H)-one and 4,6,7,13-tetrahydro-9-hydroxy-10-methoxy-5-methyl-benzo[e]-1,3-benzodioxolo[4,5-l][2] benzazecin-12(5H)-one, chelianthifoline, isochelianthifoline and 2-O-desmethylchelianthifoline. The metabolic formation of the tetrahydroprotoberberines by closure of the bridge across N5 and C13 is rate limited and protopine-like metabolites accumulate only when the route is overloaded. Metabolism was qualitatively similar in the horse and human.

3-Methoxytyramine as an indicator of dopaminergic manipulation in the equine athlete.

The influence of sampling variables on the concentration of the dopamine metabolites 3-methoxytyramine (3MT), dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA) was examined in equine urine. A logarithmic transformation of the data for all horses gave distribution which approximated the normal distributions for each metabolite. The mean urinary concentration of 3 MT in horses was 214 ng/mL and the application of a threshold with a probability of 1 in 10,000 gave an actionable level of 4 microg/mL. Environmental variables were not forensically significant in determining the population distribution. HVA was not found to be a reliable indicator of dopamine or levodopa administration.

Comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (GC x GC-TOFMS) for drug screening and confirmation.

Comprehensive two-dimensional gas chromatography (GC x GC) is applied to analysis of drug standard mixtures containing 78 drugs of interest in forensic samples. For this study, underivatised drugs were employed. While several of the drugs were not detected at the low concentrations employed in the samples, most could be satisfactorily assigned their first and second dimension retentions in the GC x GC retention plane. For this study, time-of-flight mass spectrometry (TOFMS) detection was used. The enhanced separation possible in GC x GC is demonstrated, and typical linearity and apparatus precision are shown for tramadol, diazepam, olanzapine and desipramine using selected qualifier ions. Mass spectral library search quality for the detection of drugs in a selection of authentic forensic cases, along with retention position in the 2D retention plane, is used to support positive identification of the presence of the drugs. The analysis of 'difficult' drugs paracetamol and phenytoin is shown to produce anomalous chromatographic peak shape in the 2D plane, whereas most drugs gave acceptable peak shapes. The GC x GC technique was applied to screening drugs in forensic samples, with either flame ionisation (FID) or TOFMS detection, and compared favourably with conventional single column GC-MS analysis when tested for diazepam in an authentic forensic study.