Antibiotic pharmacokinetics in infected pleural effusions.

Pleural infection is usually treated with empirical broad-spectrum antibiotics, but limited data exist on their penetrance into the infected pleural space. We performed a pharmacokinetic study analysing the concentration of five intravenous antibiotics across 146 separate time points in 35 patients (amoxicillin, metronidazole, piperacillin-tazobactam, clindamycin and cotrimoxazole). All antibiotics tested, apart from co-trimoxazole, reach pleural fluid levels equivalent to levels within the blood and well above the relevant minimum inhibitory concentrations. The results demonstrate that concerns about the penetration of commonly used antibiotics, apart from co-trimoxazole, into the infected pleural space are unfounded.

Integrated and automated high-throughput purification of libraries on microscale.

We herein report the development of an automation platform for rapid purification and quantification of chemical libraries including reformatting of chemical matter to 10 mM DMSO stock solutions. This fully integrated workflow features tailored conditions for preparative reversed-phase (RP) HPLC-MS on microscale based on analytical data, online fraction QC and CAD-based quantification as well as automated reformatting to enable rapid purification of chemical libraries. This automated workflow is entirely solution-based, eliminating the need to weigh or handle solids. This increases process efficiency and creates a link between high-throughput synthesis and profiling of novel chemical matter with respect to biological and physicochemical properties in relevant assays.

Exploring the Pharmacokinetics of Phenoxymethylpenicillin (Penicillin-V) in Adults: A Healthy Volunteer Study.

This healthy volunteer study aimed to explore phenoxymethylpenicillin (penicillin-V) pharmacokinetics (PK) to support the planning of large dosing studies in adults. Volunteers were dosed with penicillin-V at steady state. Total and unbound penicillin-V serum concentrations were determined, and a base population PK model was fitted to the data.

Challenges in the bioanalysis of tetracyclines: Epimerisation and chelation with metals.

Tetracyclines (TCs) are important broad spectrum antibiotics which are active against gram-positive and gram-negative bacteria. TCs readily form epimers, especially under weakly acidic conditions. The epimers are reported to have different antibacterial and toxicological properties and pose a significant challenge for selective bioanalysis, being isobaric with the parent drug and possessing very similar physicochemical properties. During the development, validation and use of bioanalytical methods for minocycline in plasma, urine and renal dialysate there were two unexpected findings. The first was that the analyte and the internal standard, tetracycline, were found to be unexpectedly stable and resistant towards epimerisation in the presence of the deproteinising agent, trichloroacetic acid (TCA). The second was that keeping minocycline spiked dialysate in a freezer led to significant losses which were worse for low concentrations at lower storage temperatures. Investigations into the stability of tetracycline, minocycline, omadacycline and tigecycline in aqueous acidic solutions, under typical analytical conditions, revealed that TCA acts as a stabiliser with respect to both epimerisation and other degradation pathways for these TCs. This gives the rarely used TCA a significant advantage over the commonly used deproteinising agents such as acetonitrile when analysing TCs. Studies of the recoveries of tetracycline and tigecycline from frozen renal dialysis buffer demonstrated similar losses to those for minocycline. These were assigned to deposition of insoluble Mg2+ or Ca2+ complexes on freezing, as pre-storage treatment of the samples by incubation in EDTA coated tubes at room temperature prevented the losses. Minocycline was stable in renal dialysis buffer samples when frozen, for up to ca. 3 months, when this treatment was employed. The TCs were analysed using LC-MS/MS based methods developed in-house using the assay that was originally developed for minocycline in plasma, urine and dialysate as a template.

Development, validation and application of a novel HPLC-MS/MS method for the measurement of minocycline in human plasma and urine.

New treatments are urgently required to treat infections caused by multi-drug resistant Acinetobacter baumanni,. To address this need, a new formulation of Minocin®, (minocycline for injection) has been developed that allows for higher doses of minocycline to be administered. Phase 1 clinical trials were conducted in healthy volunteers to assess the safety and pharmacokinetics (PK) of this new formulation at higher doses. In order to generate PK data, novel, selective and simple HPLC-MS/MS based assays were developed and validated for the determination of minocycline (MC) in human plasma and urine. The respective working ranges were 0.05 to 30 mg/L and 0.1 to 30 mg/L. Removal of endogenous proteins with trichloroacetic acid was used as a simple means of extracting MC from the samples. An analogue, tetracycline was used as the internal standard (IS). Chromatographic separation, including that of MC from its 4-epimer (4-EMC), was achieved on a Waters XBridge BEH C18 column (50 x 4.6 mm ID, 5 µm) with gradient elution. The mobile phases comprised water containing 5 mM ammonium formate at a pH of 2.5, and methanol containing 5 mM ammonium formate. The internal standard (IS) was tetracycline, a structural analogue of minocycline. The methods were fully validated and met regulatory acceptance criteria for intra-run and inter-run accuracy and precision, carryover, dilution integrity and matrix effects. Mean extraction recoveries ranged between 64.3% and 84.6% for MC and 64.3% for the IS. There was no significant ion suppression or enhancement for MC or the IS. The validated assays were successfully applied to 1423 plasma and 689 urine samples from a Phase 1 clinical study. There was no evidence of instability, or significant interconversion between MC and 4-EMC, in stored clinical samples, spiked plasma and urine samples, or their extracts, under various test conditions.

Microneedle biosensors for real-time, minimally invasive drug monitoring of phenoxymethylpenicillin: a first-in-human evaluation in healthy volunteers.

BACKGROUND: Enhanced methods of drug monitoring are required to support the individualisation of antibiotic dosing. We report the first-in-human evaluation of real-time phenoxymethylpenicillin monitoring using a minimally invasive microneedle-based ß-lactam biosensor in healthy volunteers. METHODS: This first-in-human, proof-of-concept study was done at the National Institute of Health Research/Wellcome Trust Imperial Clinical Research Facility (Imperial College London, London, UK). The study was approved by London-Harrow Regional Ethics Committee. Volunteers were identified through emails sent to a healthy volunteer database from the Imperial College Clinical Research Facility. Volunteers, who had to be older than 18 years, were excluded if they had evidence of active infection, allergies to penicillin, were at high risk of skin infection, or presented with anaemia during screening. Participants wore a solid microneedle ß-lactam biosensor for up to 6 h while being dosed at steady state with oral phenoxymethylpenicillin (five 500 mg doses every 6 h). On arrival at the study centre, two microneedle sensors were applied to the participant's forearm. Blood samples (via cannula, at -30, 0, 10, 20, 30, 45, 60, 90, 120, 150, 180, 210, 240 min) and extracellular fluid (ECF; via microdialysis, every 15 min) pharmacokinetic (PK) samples were taken during one dosing interval. Phenoxymethylpenicillin concentration data obtained from the microneedles were calibrated using locally estimated scatter plot smoothing and compared with free-blood and microdialysis (gold standard) data. Phenoxymethylpenicillin PK for each method was evaluated using non-compartmental analysis. Area under the concentration-time curve (AUC), maximum concentration, and time to maximum concentration were compared. Bias and limits of agreement were investigated with Bland-Altman plots. Microneedle biosensor limits of detection were estimated. The study was registered with ClinicalTrials.gov, number NCT03847610. FINDINGS: Ten healthy volunteers participated in the study. Mean age was 42 years (SD 14). Seven (70%) were men. Microdialysis and microneedle results were similar for phenoxymethylpenicillin ECF maximum concentration (0·74 mg/L vs 0·64 mg/L; 95% CI -0·24 to 0·44; p=0·53), time to maximum concentration (1·18 h vs 1·10 h; -0·52 to 0·67; p=0·79), and AUC (1·54 mg × h/L vs 1·67 mg × h/L; -1·10 to 0·85; p=0·79). In total, 440 time points were compared with mean difference between measurements -0·16 mg/L (95% CI -1·30 to 0·82). Mean phenoxymethylpenicillin AUCs for free serum and microneedle PK were similar (1·77 mg × h/L [SD 0·59] vs 1·67 mg × h/L [1·00]; -0·77 to 0·97; p=0·81). Median coefficient of variation between sensors within individuals was 7% (IQR 4-17). Limit of detection for the microneedles was estimated at 0·17 mg/L. INTERPRETATION: This study is proof-of-concept of real-time, microneedle sensing of penicillin in vivo. Future work will explore microneedle use in patient populations, their role in data generation to inform dosing recommendations, and their incorporation into closed-loop control systems for automated drug delivery. FUNDING: National Institute for Health Research Imperial Biomedical Research Centre, Mérieux Foundation.

Development, validation and application of a novel HPLC-MS/MS method for the quantification of atorvastatin, bisoprolol and clopidogrel in a large cardiovascular patient cohort.

Cardiovascular disease is a leading cause of morbidity, mortality, and healthcare expenditure worldwide. Importantly, there is interindividual variation in response to cardiovascular medications, leading to variable efficacy and adverse events. Therefore a rapid, selective, sensitive and reproducible multi-analyte HPLC-MS/MS assay for the quantification in human plasma of atorvastatin, its major metabolites 2-hydroxyatorvastatin, atorvastatin lactone and 2-hydroxyatorvastatin lactone, plus bisoprolol and clopidogrel-carboxylic acid has been developed, fully validated, and applied to a large patient study. Fifty microliter plasma samples were extracted with a simple protein precipitation procedure involving acetonitrile with acetic acid (0.1%, v/v). Chromatographic separation was via a 2.7 µm Halo C18 (50 × 2.1 mm ID, 90 Å) column and gradient elution at a flow rate of 500 µL/min consisting of a mobile phase of water (A) and acetonitrile (B), each containing 0.1% formic acid (v/v), over a 6.0 min run time. The six analytes and their corresponding six deuterated internal standards underwent positive ion electrospray ionisation and were detected with multiple reaction monitoring. The developed method was fully validated with acceptable selectivity, carryover, dilution integrity, and within-run and between-run accuracy and precision. Mean extraction recovery for the analytes was 92.7-108.5%, and internal standard-normalised matrix effects had acceptable precision (coefficients of variation 2.2-12.3%). Moreover, all analytes were stable under the tested conditions. Atorvastatin lactone to acid interconversion was assessed and recommendations for its minimisation are made. The validated assay was successfully applied to analyse 1279 samples from 1024 patients recruited to a cardiovascular secondary prevention prospective study.

Human OATP1B1 (SLCO1B1) transports sulfated bile acids and bile salts with particular efficiency.

Human OATP1B1 is highly expressed at the basolateral membrane of the hepatocyte. It plays an important role in the sodium-independent transport of bile acids and bile salts and contributes to the systemic clearance of many drugs. In this study, the interaction of at least one representative of all major chemical classes of bile acids and bile salts, which include the bile acid chenodeoxycholate (CDC), monovalent (amidated) bile salts glycochenodeoxycholate (GCDC), taurochenodeoxycholate (TCDC) and taurocholate (TC), a sulfated bile acid 3-sulfo-chenodeoxycholate (3S-CDC) and a divalent (amidated and sulfated) bile salt 3-sulfo-glycolithocholate (3S-GLC) were tested with OATP1B1 overexpressed in HEK293 cells. All bile acid derivatives except for CDC showed an efficient transport by OATP1B1. 3S-GLC gave the lowest KM (0.708 ±â€¯0.125 µM) and 3S-CDC showed the highest Vmax value (158 ±â€¯87.3 pmol/mg protein/min). The ranking of Clint values (3S-GLC > 3S-CDC > TCDC > GCDC > TC) also showed a preference for sulfated derivatives. In summary, human OATP1B1 transports sulfate esters of bile acids and bile salts more efficiently than monovalent bile salts.

The utility of HepG2 cells to identify direct mitochondrial dysfunction in the absence of cell death.

Drug-induced mitochondrial dysfunction has been hypothesized to be an important determining factor in the onset of drug-induced liver injury. It is essential to develop robust screens with which to identify drug-induced mitochondrial toxicity and to dissect its role in hepatotoxicity. In this study we have characterised a mechanistically refined HepG2 model, using a panel of selected hepatotoxicants and non-hepatotoxicants. We have demonstrated that acute metabolic modification, via glucose-deprivation over a 4 h period immediately prior to compound addition, is sufficient to allow the identification of drugs which induce mitochondrial dysfunction, in the absence of cell death over a short exposure (2-8 h) using a plate-based screen to measure cellular ATP content and cytotoxicity. These effects were verified by measuring changes in cellular respiration, via oxygen consumption and extracellular acidification rates. Overall, these studies demonstrate the utility of HepG2 cells for the identification of mitochondrial toxins which act directly on the electron transport chain and that the dual assessment of ATP content alongside cytotoxicity provides an enhanced mechanistic understanding of the causes of toxicity.

Automated approach for the rapid identification of purification conditions using a unified, walk-up high performance liquid chromatography/supercritical fluid chromatography/mass spectrometry screening system.

Integration of supercritical fluid/mass spectrometry (SFC/MS) and reversed phase liquid (HPLC/MS) chromatographic screening techniques into a single chromatographic system and utilized in "walk up" mode, enabled us to produce an orthogonal data set for selecting purification conditions for medicinal chemistry compounds. To streamline the overall workflow, we also demonstrate the use of automated batch data processing of individual data files to identify suitable separation conditions without user intervention. We have addressed the chromatographic challenges that hinder the identification of the intended target and thus the selection of ideal purification conditions. For instance, multiple component-of-interest (COI) peaks, co-elution of impurities with the COI, and chromatographic suitability factors such as retention times and peak shapes are all important considerations when selecting appropriate methods for purification and, therefore, are bottlenecks to an automated approach. Since SFC and HPLC data were collected in parallel from separate instruments in our workflow, the time required for the separation scientist to analyze acquired data from both systems was a time-limiting factor. To reduce data processing time and accelerate or "FastTrack" samples to purification, two unique and automated solutions were introduced. We describe the implementation of an integrated, multi-column, walk-up HPLC/SFC/MS system, and the implementation of an intelligent, automated method selection application which uses advanced data evaluation criteria to selectively score and identify the most practical separation conditions for SFC/MS and HPLC/MS methodologies.

Influence of age and bladder dysfunction on the contractile properties of isolated human detrusor smooth muscle.

OBJECTIVE: To test the hypothesis that the in vitro contractile properties of human detrusor smooth muscle are dependent on the age, gender and lower urinary tract pathology of the patient. MATERIALS AND METHODS: Contractions were elicited in isolated human detrusor smooth muscle preparations by nerve-mediated electrical field stimulation, agonist application (carbachol, α,ß-methylene ATP and high-K solutions) or direct muscle electrical stimulation. Biopsies (n = 227) were obtained from four groups of patients with: stable bladders (control), bladder outlet obstruction (BOO), idiopathic (IDO), or neurogenic (NDO) detrusor overactivity. RESULTS: The magnitude of nerve-mediated contractions declined as a function of patients' age in each of the BOO, IDO and NDO groups but not in the control group. Contractions elicited by direct muscle activation (10 µM carbachol or electrical stimulation with 20 ms pulses in the presence of 1 µM tetrodotoxin) did not vary with patient age. Carbachol contractions were significantly smaller in samples from NDO bladders. Atropine resistance was more prevalent in the pathology groups compared with the control group and was greatest in the IDO group. There was no influence of age in the prevalence or magnitude of atropine-resistant contractions in any group. Muscle excitability to direct electrical stimulation was similar in all groups. CONCLUSIONS: In the human bladder there is no evidence for a decline of detrusor smooth muscle contractility or excitability as a function of age, nor any gender difference or presence of pathology. In the pathology groups there was evidence for a decline of functional innervation with age.

Utilization of DBS within drug discovery: development of a serial microsampling pharmacokinetic study in mice.

BACKGROUND: Dried blood spots (DBS) are rapidly gaining a foothold in the pharmaceutical industry. However, applications in exploratory drug discovery are limited mainly owing to method development time. The development of a generic DBS assay is presented with its application in serial microsampling from mice. RESULTS: A generic 'fit-for-purpose' assay was developed on FTA(®) Elute, which allowed 90% of compounds tested to reach sensitivity levels of 1 ng/ml. A ten-time point serial mouse pharmacokinetic study was conducted using 20-µl microsamples and DBS. Application of generic 'fit-for-purpose' approach did not compromise data delivery or quality. CONCLUSIONS: Serial microsampling and DBS in exploratory mouse pharmacokinetic has been shown to provide superior data quality when compared with traditional plasma-based composite studies.

Determination of a potent urokinase-type plasminogen activator, UK-356,202, in plasma at pg/mL levels using column-switching HPLC and fluorescence detection.

A rapid, sensitive and selective method using column-switching HPLC with fluorescence detection has been developed for the determination of UK-356,202, a potent urokinase-type plasminogen activator, in human plasma. A structural isomer of UK-356,202 is used as an internal standard. The lower limit of quantification is 20 pg/mL and the method is linear over a 100-fold concentration range. UK-356,202 is extracted from plasma simply through the removal of proteins by precipitation with acetonitrile. The HPLC system comprises three columns and the cycle time is 9.5 min per sample. The eluate from the extraction column is heart-cut onto a trace enrichment cartridge which is then back-flushed onto a narrow-bore Supelco ABZ+ Plus analytical column. The method has been used to analyze many thousands of samples from clinical and toxicological studies support. Its ruggedness is demonstrated by the use of a single extraction column for the analysis of over 1200 clinical samples.

Late onset dopa-responsive dystonia with tremor, gait freezing and behavioural disturbance and a normal dopamine transporter scan.

A 79-year-old woman presented with dystonic posturing of the right leg while walking and an action tremor of her hands, both of which were levodopa responsive. She subsequently developed gait freezing. However, there was neither generalised bradykinesia nor rigidity. Structural imaging showed no significant changes, and a dopamine transporter scan was normal. She subsequently required rapidly escalating doses of levodopa in order to achieve symptom control, raising concerns over the possible development of a dopamine dysregulation syndrome. Issues raised included the difficulties of managing patients with a rare diagnosis and the role of dopaminergic medication with the potential for abuse.