A Quantitative LC-MS/MS Method for Distinguishing the Tau Protein Forms Phosphorylated and Nonphosphorylated at Serine-396.

Hyperphosphorylated tau protein is well-known to be involved in the formation of neurofibrillary tangles and the progression of age-related neurodegenerative diseases (tauopathies), including Alzheimer's Disease (AD). Tau protein phosphorylated at serine-396 (pS396-tau) is often linked to disease progression, and we therefore developed an analytical method to measure pS396-tau in cerebrospinal fluid (CSF) in humans and animal models of AD. In the S396-region, multiple phosphorylation sites are present, causing structural complexity and sensitivity challenges for conventional bottom-up mass spectrometry approaches. Here, we present an indirect LC-MS/MS method for quantification of pS396-tau. We take advantage of the reproducible miscleavage caused by S396 being preceded by a lysine (K395) and the proteolytic enzyme trypsin not cleaving when the following amino acid is phosphorylated. Therefore, treatment with trypsin discriminates between the forms of tau with and without phosphorylation at S396 and pS396-tau can be quantified as the difference between total S396-tau and nonphosphorylated S396-tau. To qualify the method, it was successfully applied for quantification of pS396-tau in human CSF from healthy controls and patients with Mild Cognitive Impairment and AD. In addition, the method was applied for rTg4510 mice where a clear dose dependent decrease in pS396-tau was observed in CSF following intravenous administration of a monoclonal antibody (Lu AF87908, hC10.2) targeting the tau epitope containing pS396. Finally, a formal validation of the method was conducted. In conclusion, this sensitive LC-MS/MS-based method for measurement of pS396-tau in CSF allows for quantitative translational biomarker applications for tauopathies including investigations of potential drug induced effects.

Inhibition of P2X7 receptors by Lu AF27139 diminishes colonic hypersensitivity and CNS prostanoid levels in a rat model of visceral pain.

Visceral pain is a prominent feature of various gastrointestinal diseases. The P2X7 receptor is expressed by multiple cell types including dorsal root ganglion satellite glial cells, macrophages, and spinal microglia, all of which have been implicated in nociceptive sensitization. We have used the selective and CNS penetrant P2X7 receptor antagonist Lu AF27139 to explore this receptor's role in distinct rat models of inflammatory and visceral hypersensitivity. Rats injected with CFA in the hindpaw displayed a marked reduction in hindpaw mechanical threshold, which was dose-dependently reversed by Lu AF27139 (3-30 mg/kg, p.o.). In rats injected with TNBS in the proximal colon, the colorectal distension threshold measured distally was significantly lower than sham treated rats at 7 days post-injection (P < 0.001), indicative of a marked central sensitization. Colonic hypersensitivity was also reversed by Lu AF27139 (10-100 mg/kg) and by the κ-opioid receptor agonist U-50,488H (3 mg/kg, s.c.). Moreover, both Lu AF27139 and U-50,488H prevented a TNBS-induced increase in spinal and brain levels of PGE2 and LTB4, as well as an increase in brain levels of PGF2α and TXB2. Lu AF27139 was well tolerated as revealed by a lack of significant effect on rotarod motor function and coordination at all doses tested up to 300 mg/kg. Thus, P2X7 receptor antagonism is efficacious in a rat model of visceral pain, via a mechanism which potentially involves attenuation of microglial function within spinal and/or supraspinal pain circuits, albeit a peripheral site of action cannot be excluded.

Screening novel CNS drug candidates for P-glycoprotein interactions using the cell line iP-gp: In vitro efflux ratios from iP-gp and MDCK-MDR1 monolayers compared to brain distribution data from mice.

Drug efflux by P-glycoprotein (P-gp, ABCB1) is considered as a major obstacle for brain drug delivery for small molecules. P-gp-expressing cell monolayers are used for screening of new drug candidates during early states of drug development. It is, however, uncertain how well the in vitro studies can predict the in vivo P-gp mediated efflux at the blood-brain barrier (BBB). We previously developed a novel cell line of porcine origin, the iP-gp cell line, with high transepithelial resistance and functional expression of human P-gp. The aim of the present study was to evaluate the applicability of the cell line for screening of P-gp interactions of novel drug candidates. For this purpose, bidirectional fluxes of 14 drug candidates were measured in iP-gp cells and in MDCK-MDR1 cells, and compared with pharmacokinetic data obtained in male C57BL/6 mice. The iP-gp cells formed extremely tight monolayers (>15 000 Ω∙cm2) as compared to the MDCK- MDR1 cells (>250 Ω∙cm2) and displayed lower Papp,a-b values. The efflux ratios obtained with iP-gp and MDCK-MDR1 monolayers correlated with Kp,uu,brain values from the in vivo studies, where compounds with the lowest Kp,uu,brain generally displayed the highest efflux ratios. 12 of the tested compounds displayed a poor BBB penetration in mice as judged by Kp,uu less than 1. Of these compounds, nine compounds were categorized as P-gp substrates in the iP-gp screening, whereas analysis of data estimated in MDCK-MDR1 cells indicated four compounds as potential substrates. The results suggest that the iP-gp cell model may be a sensitive and useful screening tool for drug screening purposes to identify possible substrates of human P-glycoprotein.

In vitro and in vivo characterization of Lu AA41178: A novel, brain penetrant, pan-selective Kv7 potassium channel opener with efficacy in preclinical models of epileptic seizures and psychiatric disorders.

Activation of the voltage-gated Kv7 channels holds therapeutic promise in several neurological and psychiatric disorders, including epilepsy, schizophrenia, and depression. Here, we present a pharmacological characterization of Lu AA41178, a novel, pan-selective Kv7.2-7.5 opener, using both in vitro assays and a broad range of in vivo assays with relevance to epilepsy, schizophrenia, and depression. Electrophysiological characterization in Xenopus oocytes expressing human Kv7.2-Kv7.5 confirmed Lu AA41178 as a pan-selective opener of Kv7 channels by significantly left-shifting the activation threshold. Additionally, Lu AA41178 was tested in vitro for off-target effects, demonstrating a clean Kv7-selective profile, with no impact on common cardiac ion channels, and no potentiating activity on GABAA channels. Lu AA41178 was evaluated across preclinical in vivo assays with relevance to neurological and psychiatric disorders. In the maximum electroshock seizure threshold test and PTZ seizure threshold test, Lu AA41178 significantly increased the seizure thresholds in mice, demonstrating anticonvulsant efficacy. Lu AA41178 demonstrated antipsychotic-like activity by reducing amphetamine-induced hyperlocomotion in mice as well as lowering conditioned avoidance responses in rats. In the mouse forced swim test, a model with antidepressant predictivity, Lu AA41178 significantly reduced immobility. Additionally, behavioral effects typically observed with Kv7 openers was also characterized. In vivo assays were accompanied by plasma and brain exposures, revealing minimum effective plasma levels <1000 ng/ml. Lu AA41178, a potent opener of neuronal Kv7 channels demonstrate efficacy in assays of epilepsy, schizophrenia and depression and might serve as a valuable tool for exploring the role of Kv7 channels in both neurological and psychiatric disorders.

Investigating surrogate cerebrospinal fluid matrix compositions for use in quantitative LC-MS analysis of therapeutic antibodies in the cerebrospinal fluid.

As quantitative analysis of biotherapeutics in cerebrospinal fluid (CSF) with LC-MS becomes increasingly widespread, there is a need for method developments towards higher sensitivity. By using artificial CSF (aCSF) in the development phase, the consumption of costly and sparsely available CSF can be limited. The aCSF compositions tested here were made from various dilutions of bovine serum albumin (BSA) or rat plasma to mimic the total protein concentration found in CSF. Focusing on monoclonal antibodies, the aCSF was spiked with human immunoglobulin (hIgG) and prepared with the bottom-up analysis technique using LC-MS. Assuming that the composition of the aCSF would affect the digest, the response from aCSF matrices was compared with CSF from rat, monkey, and dog in terms of estimated sample concentration and matrix effects. The samples were spiked with hIgG in the range of 10 to 1000 ng/mL and volumes of 10 µL were transferred to sample preparation. The results indicate that BSA dilutions from 300 to 2000 µg/mL and rat plasma dilutions of 0.5-2% provide the most accurate concentration estimates when compared with rat CSF. 1000 µg/mL BSA did not produce significantly different concentration estimates for 500 ng/mL samples when compared with CSF from rat, monkey, and dog, and can therefore be used as aCSF for several different species.

Lack of Exposure in a First-in-Man Study Due to Aldehyde Oxidase Metabolism: Investigated by Use of 14C-microdose, Humanized Mice, Monkey Pharmacokinetics, and In Vitro Methods.

Inclusion of a microdose of 14C-labeled drug in the first-in-man study of new investigational drugs and subsequent analysis by accelerator mass spectrometry has become an integrated part of drug development at Lundbeck. It has been found to be highly informative with regard to investigations of the routes and rates of excretion of the drug and the human metabolite profiles according to metabolites in safety testing guidance and also when additional metabolism-related issues needed to be addressed. In the first-in-man study with the NCE Lu AF09535, contrary to anticipated, surprisingly low exposure was observed when measuring the parent compound using conventional bioanalysis. Parallel accelerator mass spectrometry analysis revealed that the low exposure was almost exclusively attributable to extensive metabolism. The metabolism observed in humans was mediated via a human specific metabolic pathway, whereas an equivalent extent of metabolism was not observed in preclinical species. In vitro, incubation studies in human liver cytosol revealed involvement of aldehyde oxidase (AO) in the biotransformation of Lu AF09535. In vivo, substantially lower plasma exposure of Lu AF09535 was observed in chimeric mice with humanized livers compared with control animals. In addition, Lu AF09535 exhibited very low oral bioavailability in monkeys despite relatively low clearance after intravenous administration in contrast to the pharmacokinetics in rats and dogs, both showing low clearance and high bioavailability. The in vitro and in vivo methods applied were proved useful for identifying and evaluating AO-dependent metabolism. Different strategies to integrate these methods for prediction of in vivo human clearance of AO substrates were evaluated.

Multi-component analysis of tetracyclines, sulfonamides and tylosin in swine manure by liquid chromatography-tandem mass spectrometry.

A multi-component method focussing on thorough sample preparation has been developed for simultaneous analysis of swine manure for three classes of antibiotic-tetracyclines, sulfonamides, and tylosin. Liquid manure was initially freeze-dried and homogenised by pulverization before extraction by pressurised liquid extraction. The extraction was performed at 75 degrees C and 2,500 psig in three steps using two cycles with 0.2 mol L(-1) citric acid buffer (pH 4.7) and one cycle with a mixture of 80% methanol with 0.2 mol L(-1) citric acid (pH 3). After liquid-liquid extraction with heptane to remove lipids, the pH of the manure was adjusted to 3 with formic acid and the sample was vacuum-filtered through 0.6 mum glass-fibre filters. Finally the samples were pre-concentrated by tandem SPE (SAX-HLB). Recoveries were determined for manure samples spiked at three concentrations (50-5,000 microg kg(-1) dry matter); quantification was achieved by matrix-matched calibration. Recoveries were >70% except for oxytetracycline (42-54%), sulfadiazine (59-73%), and tylosin (9-35%) and did not vary with concentration or from day-to-day. Limits of quantification (LOQ) for all compounds, determined as a signal-to-noise ratio of 10, were in the range 10-100 microg kg(-1) dry matter. The suitability of the method was assessed by analysis of swine manure samples from six different pig-production sites, e.g. finishing pigs, sows, or mixed production. Residues of antibiotics were detected in all samples. The largest amounts were found for tetracyclines (up to 30 mg kg(-1) dry matter for the sum of CTC and ECTC). Sulfonamides were detected at concentrations up to 2 mg kg(-1) dry matter (SDZ); tylosin was not detected in any samples.

Persistence of testosterone and 17beta-estradiol in soils receiving swine manure or municipal biosolids.

Natural and synthetic steroidal hormones can be carried to agricultural soil through fertilization with municipal biosolids, livestock manure, or poultry manure. The persistence and pathways of dissipation of [4-(14)C]-testosterone and of [4-(14)C]-17beta-estradiol in organic-amended soils were investigated using laboratory microcosms. Testosterone dissipation was investigated over a range of amendment concentrations, temperatures, and soil types. Under all conditions the parent compound and transformation products were dissipated within a few days. Addition of swine manure slurry to soil hastened the transformation of testosterone and 17beta-estradiol to the corresponding less hormonally active ketones, 4-androstene-3,17-dione and estrone. Two other testosterone transformation products, 5alpha-androstan-3,17-dione and 1,4-androstadiene-3,17-dione, were also detected. Experiments with sterilized soil and sterilized swine manure slurry suggested that the transformation of (14)C-labeled hormonal parent compounds was mainly caused by microorganisms in manure slurry, while mineralization of the hormones to (14)CO(2) required viable soil microorganisms. Organic amendments transiently inhibited the mineralization of [4-(14)C]-testosterone, perhaps by inhibiting soil microorganisms, or by enhancing sorption and reducing the bioavailability of testosterone or transformation products. Overall, organic amendments influenced the pathways and kinetics of testosterone and estradiol dissipation, but did not increase their persistence.

Dissipation and effects of chlortetracycline and tylosin in two agricultural soils: a field-scale study in southern Denmark.

Presently, there is a basic lack of information concerning the accumulation of antibacterial agent residues in agricultural soils. In this field study, performed in southern Denmark, we assess the dissipation of chlortetracycline (CTC), and tylosin A (TYL A) as a function of time. Field soils were classified as a sandy loam soil (field A) and a sandy soil (field B) and each field was sampled on six occasions during the 155-d experimental period from May to October 2000 for chemical analysis and counts of colony-forming units (CFU) detecting the level of aerobic bacteria surviving antibiotic exposure. Colony-forming units and TYL A were detected throughout the entire sampling period, with respective starting soil concentrations of 30 and 50 microg kg(-1) soil declining to 1 and 5 microg kg(-1) soil, on day 155. Compound half-lives (95% confidence limits in parentheses) were estimated for both fields and T1/2 for CTC was 25 d (20-34) and 34 d (28-42) in fields A and B, respectively, and T1/2 for TYL A was 67 d (54-86) and 49 d (40-64) in fields A and B, respectively. No significant difference was determined between compound half-lives on the two fields. The level of aerobic antibiotic-resistant bacteria in the soil over time and soil fauna community was assessed in relation to application of manure containing antibacterial agents to the agricultural fields. The level of both CTC- and TYL-resistant bacteria was affected in the soil by amendment of manure, but declined during the study to the same level as observed at the beginning.

Simultaneous extraction of tetracycline, macrolide and sulfonamide antibiotics from agricultural soils using pressurised liquid extraction, followed by solid-phase extraction and liquid chromatography-tandem mass spectrometry.

The veterinary antibacterial agents chlortetracycline (CTC), oxytetracycline (OTC), sulfadiazine (SDZ), erythromycin (ERY) and tylosin (TYL A, B, C and D) were extracted from soil using pressurized liquid extraction (PLE). Citric acid (pH 4.7) and methanol was used as extraction buffer, followed by tandem-solid-phase extraction (SPE) clean-up (SAX + HLB) for all compounds. For quantification two slightly different methods were employed using LC-MS-MS with MRM detection. The soil extraction method was validated using a loamy sand soil and a sandy soil, representing two typical Danish agricultural soils. Recoveries were 50-80% for the tetracyclines (CTC and OTC) and sulfadiazine (SDZ) and 60-100% for the macrolides (TYL and ERY). Limits of detection for the soil extraction method (LOD(soil)) were 0.6-5.6 microg kg(-1) soil for CTC and OTC, 0.9-2.9 microg kg(-1) soil for SDZ and 2.4-5.5 microg kg(-1) soil for TYL A and ERY. Furthermore, the method was applied to field samples taken from two agricultural fields fertilised with liquid manure containing CTC and TYL A. These results showed a decline in the content of antibacterial agents throughout the sampling period of 155 days from 10 to 15 microg CTC kg(-1) soil and 20-55 microg TYL A kg(-1) soil to below or near the LOD(soil) listed above. Finally, the method was applied to barley grains harvested from the fields. None of the antibacterial agents were measured in grain samples, but recoveries for spiked grain samples were similar to soil recoveries.

Influence of soil moisture on linear alkylbenzene sulfonate-induced toxicity in ammonia-oxidizing bacteria.

Moisture affects bioavailability and fate of pollutants in soil, but very little is known about moisture-induced effects on pollutant toxicity. We here report on a modifying effect of moisture on degradation of linear alkylbenzene sulfonates (LASs) and on their toxicity towards ammonia-oxidizing bacteria (AOB) in agricultural soil. In soil spiked with two LAS levels (250 or 1,000 mg/kg) and incubated at four different moisture levels (9-100% of water-holding capacity), degradation was strongly affected by both soil moisture and initial LAS concentration, resulting in degradation half-lives ranging from 13 to more than 160 d. Toxicity towards AOB assessed by a novel Nitrosomonas europaea luxAB-reporter assay was correlated to total LAS concentration, indicating that LAS remained bioavailable over time without accumulation of toxic intermediates. Toxicity towards indigenous AOB increased with increasing soil moisture. The results indicate that dry soil conditions inhibit LAS degradation and provide protection against toxicity within the indigenous AOB, thus allowing for a rapid recovery of this population when LAS degradation is resumed and completed after rewetting. We propose that the protection of microbial populations against toxicity in dry soil may be a general phenomenon caused primarily by limited diffusion and thus a low bioavailability of the toxicant.

Degradation and mobility of linear alkylbenzene sulfonate and nonylphenol in sludge-amended soil.

Degradation and mobility of the surfactants linear alkylbenzene sulfonate (LAS) and nonylphenol (NP) were investigated in a lysimeter study using a sandy loam soil and 45-cm soil columns. Anaerobically digested sewage sludge was incorporated in the top-15-cm soil layer to an initial content of 38 mg LAS and 0.56 mg NP kg(-1) dry wt., respectively. Spring barley (Hordeum vulgare L.) was sown onto the columns. The lysimeters were placed outdoors and therefore received natural precipitation, but were also irrigated to a total amount of water equivalent to 700 mm of precipitation. Leachate and soil samples from three soil layers were collected continuously during a growth period of 110 d. Leachate samples and soil extracts were concentrated by solid-phase extraction (SPE) and analyzed using high performance liquid chromatography (HPLC) with fluorescence detection. The concentrations in the top-15-cm soil layer declined to 25 and 45% of the initial contents for LAS and NP, respectively, within the first 10 d of the study. At the end of the study, less than 1% LAS was left, while the NP content was below the detection limit. Assuming first-order degradation kinetics, half-lives of 20 and 37 d were estimated for LAS and NP, respectively. The surfactants were not measured in leachate samples in concentrations above the analytical detection limits of 4.0 and 0.5 microg L(-1) for LAS and NP, respectively. In addition, neither LAS nor NP were measured in concentrations above the detection limits of 150 and 50 microg kg(-1) dry wt., respectively, in soil layers below the 15 cm of sludge incorporation, indicating negligible downward transport of the surfactants in the lysimeters.