Microdosing of a Carbon-14 Labeled Protein in Healthy Volunteers Accurately Predicts Its Pharmacokinetics at Therapeutic Dosages.

Preclinical development of new biological entities (NBEs), such as human protein therapeutics, requires considerable expenditure of time and costs. Poor prediction of pharmacokinetics in humans further reduces net efficiency. In this study, we show for the first time that pharmacokinetic data of NBEs in humans can be successfully obtained early in the drug development process by the use of microdosing in a small group of healthy subjects combined with ultrasensitive accelerator mass spectrometry (AMS). After only minimal preclinical testing, we performed a first-in-human phase 0/phase 1 trial with a human recombinant therapeutic protein (RESCuing Alkaline Phosphatase, human recombinant placental alkaline phosphatase [hRESCAP]) to assess its safety and kinetics. Pharmacokinetic analysis showed dose linearity from microdose (53 µg) [(14) C]-hRESCAP to therapeutic doses (up to 5.3 mg) of the protein in healthy volunteers. This study demonstrates the value of a microdosing approach in a very small cohort for accelerating the clinical development of NBEs.

The determination of budesonide and fluticasone in human sputum samples collected from COPD patients using LC-MS/MS.

A bioanalytical method for the quantitative determination of budesonide and fluticasone in human sputum was developed. Sputolysin(®) Reagent was added to the sputum samples. After incubation (37°C; 60-70 min under shaking) and automated solid phase extraction the extracts were analysed using LC-MS/MS. Budesonide and fluticasone showed good linearity (r>0.99) over the range 0.1-100 nM in the first and second validation batch, and over the range 0.25-10,000 nM in the third and fourth validation batch. The lower limit of quantification (LLOQ) achieved was 5 nM for budesonide and fluticasone in 100 µL human sputum. Intra-run and inter-run RSD for four quality control levels (5-100 nM) were within 6.9% (budesonide) and 8.0% (fluticasone). The accuracy ranged from -11.4% to -1.6% (budesonide), and from -11.8% to 0.4% (fluticasone). The validated method was applied to clinical sputum samples from COPD patients.

Perfusion of immobilized isolated nerve terminals as a model for the regulation of transmitter release: release of different, endogenous transmitters, repeated stimulation, and high time resolution.

To study the release of neurotransmitters, i.e., the recruitment of transmitters for release and the regulation of the release process, isolated nerve terminals (synaptosomes) of the rat forebrain were immobilized in Sephadex gel inside a perfusion chamber. In this way, the following were achieved: (a) A very limited pressure stress was exerted on the synaptosomes, so that these remained viable for long periods (greater than 30 min) inside the chamber and did not elute from the chamber, which allowed long-term experiments with repeated stimulations; (b) estimation of the release of various endogenous transmitters, both in a Ca(2+)-dependent (exocytotic) and Ca(2+)-independent manner; (c) a step-like stimulation with depolarizing agents (rise time, 3-4 s) and a high time resolution (600-ms sampling); and (d) negligible reuptake of transmitter into the terminals or extracellular breakdown. It is concluded that this perfusion setup helps to provide new insights in the presynaptic stimulus-secretion coupling, co-transmission, and the exo-endocytosis cycle.

Hematological and clinicochemical profiles of healthy swine and swine with inflammatory processes.

The hematological and clinicochemical profiles of healthy swine and swine with inflammatory processes were investigated. Blood was collected at slaughter and postmortem examination was performed to select healthy swine and swine with pleuritis, pneumonia or abscesses. In healthy swine, the values of several variables revealed significant differences between gilts, barrows and boars. This was caused predominantly by the values obtained for boars. Inflammatory processes altered the values of most variables investigated, particularly for erythrocyte sedimentation rate, hemoglobin and hematocrit, for the activity of alkaline phosphatase, and for concentrations of iron, phosphate, albumin and fibrinogen in plasma. Compared with healthy swine, differences were largest for swine with metastatic abscesses and swine with both abscesses and other pathological lesions; differences were less pronounced in swine with solitary abscesses and were minor in swine with pneumonia and swine with pleuritis. Porcine hematological and clinicochemical profiles reflect the degree of inflammation.

Excretion of iodide in 24-h urine as determined by ion-pair reversed-phase liquid chromatography with electrochemical detection.

A simple method is presented for the routine analysis of iodide in urine. After a one-step sample clean-up, iodide was separated by ion-pair reversed-phase liquid chromatography and detected electrochemically with a silver electrode. The coefficient of variation of a single analysis of iodide in a pooled urine sample (530 nmol/l) was 7.6%. The detection limit, derived from a signal-to-noise ratio of 3, was 3 pmol, corresponding to 0.06 mumol/l. The recovery of iodide added to urine was 96 +/- 7%. The accuracy of the method was assessed by analysing ten different samples with neutron activation analysis. The data obtained with the two methods showed a high correlation (r = 0.991) and did not differ significantly. Excretion of iodide in samples of 24-h urine from a free-living population was shown to have a log-normal distribution and to be higher in men than in women. The iodide/creatinine ratio was independent of sex and increased with age.

Determination of free and total catecholamines and salsolinol in urine by ion-pair reversed-phase liquid chromatography with electrochemical detection after a one-step sample clean-up.

A simple method is presented for the routine determination of free and total catecholamines and salsolinol in urine. For the analysis of total catecholamines and salsolinol, the urine samples are first hydrolysed. The compounds are isolated by a one-step sample clean-up by cation-exchange chromatography and separated by ion-pair reversed-phase liquid chromatography and detected electrochemically. The method is suited for routine analysis. Pretreatment of ten samples for the high-performance liquid chromatographic analysis of free and total catecholamines and salsolinol is carried out in 4 h. A single analysis is completed in 30 min. The absolute losses of the compounds were less than 10%, except for epinephrine (20%). The detection limits were 0.9, 2.4, 4.8 and 6 nmol/l for norepinephrine, epinephrine, dopamine and salsolinol, respectively, in native urine. The values were twice as high for hydrolysed urine. The sample clean-up appears to be rather specific: apart from catecholamines and salsolinol, only those structurally related amines that contain a vicinal hydroxyl group are isolated.