Predicting in vivo performance of fenofibrate amorphous solid dispersions using in vitro non-sink dissolution and dissolution permeation setup.

Amorphous solid dispersion (ASD) is emerging as a useful formulation strategy to increase the bioavailability of active pharmaceutical ingredients with poor solubility. In vitro dissolution testing under non-sink conditions has often been used to evaluate the ability of ASDs to generate and maintain supersaturation to predict the in vivo performance. However, such a single compartment dissolution setup can fail to predict the oral bioavailability, due to an interdependence between precipitation and permeation. Hence, the use of two compartment dissolution-permeation setups is emerging. In this study, three ASDs containing fenofibrate as model drug substance were developed using Soluplus®, and Hypromellose Acetate Succinate in two different grades (high and low), respectively. The aim was to compare the use of a small-scale in vitro non-sink dissolution setup and a small-scale in vitro dissolution-permeation setup to predict the in vivo oral exposure of the ASDs in rats. The maximum concentration (Cmax) and area under curve (AUC) obtained in the in vitro studies were used to predict the in vivo rank order of the formulations. The results showed that the two in vitro studies resulted in the same rank order based on both Cmax and AUC. Interestingly, Cmax resulted in a better in vitro/in vivo correlation than the in vitro AUC, and based on the in vitro Cmax, the in vivo rank order was predicted.

The GSTP1 Ile105 Val polymorphism modifies the metabolism of toluene di-isocyanate.

BACKGROUND: Toluene di-isocyanate (TDI) is widely used in the production of polyurethane foams and paints. As TDI causes respiratory disease in only a fraction of exposed workers, genetic factors may play a key role in disease susceptibility. Polymorphisms in TDI metabolising genes may affect elimination kinetics, resulting in differences in body retention, and in its turn differences in adverse effects. OBJECTIVES: To analyze how genotype modifies the associations between (i) TDI in air (2,4-TDI and 2,6-TDI) and its metabolites toluene diamine (TDA; 2,4-TDA and 2,6-TDA) in hydrolyzed urine; and (ii) 2,4-TDA and 2,6-TDA in hydrolyzed plasma and 2,4-TDA and 2,6-TDA in urine. METHODS: Workers exposed to TDI were analyzed for 2,4-TDI and 2,6-TDI in air (N=70), 2,4-TDA and 2,6-TDA in hydrolyzed urine (N=124) and in plasma (N=128), and genotype: CYP1A1*2A, CYP1A1*2B, GSTA1-52, GSTM1O, GSTM3B, GSTP1 I105V, GSTP1 A114V, GSTT1O, MPO-463, NAT1*3, *4, *10, *11, *14, *15, NAT2*5, *6, *7, and SULT1A1 R213H. RESULTS: GSTP1 105 strongly modified the relationship between 2,4-TDA in plasma and in urine: ValVal carriers had about twice as steep regression slope than IleIle carriers. A similar pattern was found for 2,6-TDA. CYP1A1*2A, GSTM1, GSTP1, GSTT1, and MPO possibly influenced the relationship between TDA in plasma and urine. CONCLUSION: Our results show, for the first time, genetic modification on the human TDI metabolism. The findings suggest that GSTP1 genotype should be considered when evaluating biomarkers of TDI exposure in urine and plasma. Moreover, the results support earlier findings of GSTP1 105 Val as protective against TDI-related asthma.

Eye and airway symptoms in low occupational exposure to toluene diisocyanate.

OBJECTIVES: Exposure to diisocyanates is a well known occupational hazard. The objective of this study was to determine the possibility of an association between low exposure to toluene diisocyanate (TDI) (airborne isocyanates and biomarkers of isocyanates in plasma and urine) and symptoms of the eyes and upper and lower airways. METHODS: Altogether 136 workers occupationally exposed to TDI and 118 unexposed employees were studied. A physician compiled thorough medical and occupational histories and registered symptoms, total and work-related, of the eyes, nose, and lower airways. The exposure was assessed with personal air measurements and with biomarkers of exposure in plasma and urine. The average exposure in the ambient air at the workplace of the exposed participants was below 1 ppb. RESULTS: Compared with the unexposed group, the exposed workers reported more total symptoms of the eyes and lower airways, as well as nose bleeding. A similar pattern, with even higher odds ratios, was observed for work-related symptoms. However, only eye symptoms proved to be significantly associated with the exposure, notably with all of the exposure measures. The risk was more pronounced for exposure to 2,4-TDI than for exposure to 2,6-TDI. CONCLUSIONS: Even very low exposure to TDI is related to negative health effects on exposed workers. Clear dose-response relationships were observed between three different measures of exposure and symptoms of the eyes.

Metabolic Profiling of Systemic Lupus Erythematosus and Comparison with Primary Sjögren's Syndrome and Systemic Sclerosis.

Systemic lupus erythematosus (SLE) is a chronic inflammatory autoimmune disease which can affect most organ systems including skin, joints and the kidney. Clinically, SLE is a heterogeneous disease and shares features of several other rheumatic diseases, in particular primary Sjögrens syndrome (pSS) and systemic sclerosis (SSc), why it is difficult to diagnose The pathogenesis of SLE is not completely understood, partly due to the heterogeneity of the disease. This study demonstrates that metabolomics can be used as a tool for improved diagnosis of SLE compared to other similar autoimmune diseases. We observed differences in metabolic profiles with a classification specificity above 67% in the comparison of SLE with pSS, SSc and a matched group of healthy individuals. Selected metabolites were also significantly different between studied diseases. Biochemical pathway analysis was conducted to gain understanding of underlying pathways involved in the SLE pathogenesis. We found an increased oxidative activity in SLE, supported by increased xanthine oxidase activity and an increased turnover in the urea cycle. The most discriminatory metabolite observed was tryptophan, with decreased levels in SLE patients compared to control groups. Changes of tryptophan levels were related to changes in the activity of the aromatic amino acid decarboxylase (AADC) and/or to activation of the kynurenine pathway.

Applying dried blood spot sampling with LCMS quantification in the clinical development phase of tasquinimod.

BACKGROUND: Tasquinimod is an orally active anticancer drug in late clinical development. Here we describe the development and validation of a bioanalytical method based upon dried blood spot analysis in combination with LCMS/MS and stable isotope dilution. RESULTS & DISCUSSION: The present method was validated for accuracy, precision, linearity, selectivity, carry-over and ruggedness. Data elucidating stability of tasquinimod in dried blood spots and in blood at ambient temperature was investigated and found adequate. Furthermore, in a clinical study, incurred samples reanalysis was performed, and the correlation of blood concentration versus plasma concentrations of tasquinimod was investigated. CONCLUSION: The method described here is suitable for bioanalysis of tasquinimod in whole blood from humans in clinical studies.

Biological monitoring of exposure to toluene diisocyanate.

OBJECTIVES: Toluene diisocyanate (TDI) is used in the manufacture of polyurethane and is a potent inducer of diseases of the airways. In this study, 2,4- and 2,6-toluenediamine in hydrolyzed urine and plasma were evaluated as biomarkers of exposure to 2,4- and 2,6-TDI, respectively. METHODS: For 81 exposed workers from nine different plants, the personal 8-hour time-weighted-average exposure to TDI was monitored by a filter method with 1-(2-methoxyphenyl)piperazine. In parallel, urinary samples (U1) were collected during the last 4 hours of the workshift. On a different occasion, blood samples and additional urinary samples (U2) were collected from the exposed workers, and also from a reference group consisting of 121 unexposed workers. The biomarker levels were determined in urine and plasma by the use of alkaline hydrolysis. RESULTS: There were strong associations between the personal air and biomarker levels, with correlation coefficients in the range of 0.75-0.88 for the U1 samples and in the range of 0.50-0.78 for the plasma samples. By weighted linear regression, the relations were calculated between the air and biomarker levels. The slopes of the obtained regression curves ranged from 1.8 to 2.7 m3/1 for air-urine and from 2.2 to 2.9 m3/1 for air-plasma, and the intercepts were all close to the origin of the coordinates. Through the extrapolation of these regression curves, biological exposure limits were calculated. CONCLUSIONS: The biological monitoring methods and strategies presented in this report are useful for assessing exposure to TDI in practice.

Development and validation of a method using supported liquid extraction for the simultaneous determination of midazolam and 1'-hydroxy-midazolam in human plasma by liquid chromatography with tandem mass spectrometry detection.

The metabolic conversion of midazolam (MDZ) to its main metabolite 1'-hydroxy-midazolam (1-OH-MDZ) can be used as a probe drug for cytochrome P450 3A (CYP3A) activity. A sensitive method for the simultaneous determination of MDZ and its metabolite 1-OH-MDZ in human plasma using supported liquid extraction (SLE) in combination with liquid chromatography-tandem mass spectrometry (LC-MS/MS) detection was developed and validated. Plasma samples (100 µL) were diluted with 0.5M NH(3) (aq) containing deuterated internal standards. The samples were extracted with ethyl acetate on a 96-well SLE-plate. Separation was performed on a Symmetry Shield RP18 column using an acidic gradient running from 2% to 95% methanol in 3 min. Detection was performed using a triple quadrupole mass spectrometer running in positive electrospray selected reaction monitoring (SRM) mode. The validated dynamic range was 0.2-100 nmol/L for both analytes. In the concentration range 0.6-75 nmol/L the extraction recoveries were in the ranges 91.2-98.6% and 94.5-98.3% for MDZ and 1-OH-MDZ, respectively. Matrix effects were more pronounced for MDZ than for 1-OH-MDZ but the response was still 75.4% or higher compared to a reference. The overall repeatability was within 2.2-7.6% for both analytes, the overall reproducibility was within 3.1-10.2% for both analytes and the overall accuracy bias was within -1.1 to 7.5% for both analytes. The method was successfully applied to determine the plasma concentrations of MDZ and 1-OH-MDZ in 14 healthy volunteers up to 24h after administration of a single oral dose of 2mg MDZ. The SLE technology was found to be convenient and suitable for sample preparation, and the developed method was found to be rapid, selective and reproducible for the simultaneous determination of MDZ and 1-OH-MDZ in human plasma.

Anticoagulant counter ion impact on bioanalytical LC-MS/MS assays: results from discussions and experiments within the European Bioanalysis Forum.

BACKGROUND: In regulated bioanalysis, the need for partial validation when changing the counter ion of the anticoagulant is currently being debated within the bioanalytical community. To date, industry and the health authorities have not yet reached a consensus on this issue. The aim of the present study was to evaluate the impact of a change in counter ion when using the same anticoagulant on LC-MS/MS assay performance for a broad array of new chemical entities, compiling data generated at companies within the European Bioanalysis Forum (EBF). RESULTS: In all, 15 EBF member companies provided experimental data on partial validation. In total, data from 42 LC-MS/MS assays were evaluated. The results show that a change in counter ion when using the same anticoagulant had no impact on assay performance. CONCLUSION: Based on these results and on conclusions from previous studies, the EBF recommends that in regulated bioanalysis, plasma samples containing different counter ions, but the same anticoagulant, should be regarded as equal matrices, thus removing any need for partial validation.

Diagnostic properties of metabolic perturbations in rheumatoid arthritis.

INTRODUCTION: The aim of this study was to assess the feasibility of diagnosing early rheumatoid arthritis (RA) by measuring selected metabolic biomarkers. METHODS: We compared the metabolic profile of patients with RA with that of healthy controls and patients with psoriatic arthritis (PsoA). The metabolites were measured using two different chromatography-mass spectrometry platforms, thereby giving a broad overview of serum metabolites. The metabolic profiles of patient and control groups were compared using multivariate statistical analysis. The findings were validated in a follow-up study of RA patients and healthy volunteers. RESULTS: RA patients were diagnosed with a sensitivity of 93% and a specificity of 70% in a validation study using detection of 52 metabolites. Patients with RA or PsoA could be distinguished with a sensitivity of 90% and a specificity of 94%. Glyceric acid, D-ribofuranose and hypoxanthine were increased in RA patients, whereas histidine, threonic acid, methionine, cholesterol, asparagine and threonine were all decreased compared with healthy controls. CONCLUSIONS: Metabolite profiling (metabolomics) is a potentially useful technique for diagnosing RA. The predictive value was without regard to the presence of antibodies against cyclic citrullinated peptides.

Incurred sample reproducibility: views and recommendations by the European Bioanalysis Forum.

Following intensive discussions, review, alignment of procedures and multiple surveys among their member companies, the European Bioanalysis Forum (EBF) is providing a recommendation on how to integrate incurred sample reproducibility (ISR) in the bioanalytical process. The recommendation aims to provide guidance throughout the lifecycle of a validated method, including the application of the method in study support. In its recommendation, the EBF considers both the internal discussions with EBF member companies, as well as the input provided in international meetings where ISR was discussed. The ultimate goal of the EBF recommendation is to ensure that bioanalytical methods can provide accurate and reproducible concentration data for pharmacokinetic and/or toxicokinetic evaluation, without any compromise, while safeguarding the optimal use of laboratory resources.

Determination of the immunomodulator laquinimod in human plasma by liquid chromatography/tandem mass spectrometry; development, validation and application of two methods in clinical pharmacokinetic profiling.

Laquinimod (ABR-215062) is a synthetic compound currently undergoing clinical development for oral treatment of multiple sclerosis. The present paper describes the development, validation and clinical application of two rapid and sensitive methods for the determination of ABR-215062 in human plasma. Both methods use liquid chromatography/tandem mass spectrometry (LC/MS/MS) with electrospray ionization in positive mode and a stable isotope (13C6)-labeled ABR-215062 as internal standard for calibration. The selected reaction monitoring was based on the transitions m/z 357.1 --> 236.1 for ABR-215062, and either m/z 363.2 --> 236.1 or 365.2 --> 238.1 for 13C6-ABR-215062. Method 1, aimed and validated for low level determinations (0.4-100 nmol/L) of ABR-215062, utilizes solid-phase extraction followed by isocratic elution. Method 2, aimed and validated for wide range determinations (0.75-15000 nmol/L) of ABR-215062, utilizes protein precipitation followed by fast gradient elution. The methods were validated with respect to selectivity, lower limit of quantification (LLOQ), dynamic range, precision, accuracy, extraction recovery and ruggedness. Furthermore, the stability of low level ABR-215062 in plasma was investigated. The methods were also applied in clinical trials. The LLOQs were 0.4 and 0.75 nmol/L for methods 1 and 2, respectively. The intra- and inter-day precision for the methods were 1.6-3.5% and 2.1-5.7%. The extraction recoveries were 90-97% for both methods. ABR-215062 was stable in plasma for at least 3 months when stored at -20 degrees C. In conclusion, our developed methods were found to be selective, sensitive, robust and suitable for applications in clinical pharmacokinetic profiling.

Upper reference limits for biomarkers of exposure to aromatic diisocyanates.

OBJECTIVES: The objectives of this study were to determine the levels of 2,4-toluenediamine, 2,6-toluenediamine, 1,5-naphthtalenediamine and 4,4'-methylenediphenyldianiline in hydrolyzed urine and plasma for occupationally unexposed workers and to calculate upper reference limits (URLs). These analytes are biomarkers of exposure to 2,4-toluene diisocyanate and 2,6-toluene diisocyanate (2,4-TDI and 2,6-TDI), 1,5-naphtalene diisocyanate (NDI) and 4,4'-methylenediphenyl diisocyanate (MDI), respectively. METHODS: The biomarker levels were determined in urinary and plasma samples obtained from 121 occupationally unexposed workers. Based on these biomarkers levels and the biomarker levels in an occupationally exposed group of workers, URLs were calculated. The method used for these calculations was based on the receiver operator characteristic (ROC) curve method technique, and the URLs were set at the optimum of sum of sensitivity and specificity. RESULTS: The URLs for the different diisocyanates were calculated to be in the range of 0.1-0.5 microg/L. Occupationally unexposed workers had detectable biomarker levels of the diisocyanates investigated. Especially abundant was the biomarkers of MDI which were found in 97% of both urinary and plasma samples. For the other biomarkers, 0-15% of the unexposed workers had detectable levels. The detected levels were mostly close to the limit of detection (LOD), but urinary levels of biomarkers of MDI up to 60 times the LOD were found. The sensitivities and specificities for classification of the workers as occupationally exposed or not, were in the range of 88-100% and 97-100%, respectively. CONCLUSIONS: The URLs were calculated that may be applicable when screening for occupational exposure. A worker with a biomarker level above the URL will be classified as occupationally exposed. Biomarkers of aromatic diisocyanates, especially biomarkers of MDI, were present among occupationally unexposed workers, but the source and nature of the exposure is unknown.

Development, validation and characterization of an analytical method for the quantification of hydrolysable urinary metabolites and plasma protein adducts of 2,4- and 2,6-toluene diisocyanate, 1,5-naphthalene diisocyanate and 4,4'-methylenediphenyl diisocyanate.

Occupational exposure to diisocyanates within the plastic industry causes irritation and disorders in the airway. The aim of this study was to develop, validate and characterize a method for the determination of 2,4-toluenediamine (2,4-TDA), 2,6-toluenediamine (2,6-TDA), 1,5-diaminonaphthalene (1,5-NDA) and 4,4'-methylenedianiline (4,4'-MDA) in hydrolysed urine and plasma, and to study the correlation between the plasma and urinary levels of these potential biomarkers of 2,4-toluene diisocyanate (2,4-TDI), 2,6-toluene diisocyanate (2,6-TDI), 1,5-naphthalene diisocyanate (1,5-NDI) and 4,4'-methylenediphenyl diisocyanate (4,4'-MDI), respectively. Samples were hydrolysed with 0.3 M NaOH at 100 degrees C for 24 h. The diamines were extracted, derivatized with pentafluoropropionic acid anhydride, and quantified by selected ion monitoring on gas chromatography-mass spectrometry. The repeatability and reproducibility of the method were 7-18% and 7-19%, respectively. Dialysis experiments showed that the metabolites of 2,4-TDI, 2,6-TDI, 1,5-NDI and 4,4'-MDI in plasma were exclusively protein adducts. No free diamines were found in urine, indicating that all diisocyanate-related metabolites were in a conjugated form. For each diisocyanate-related biomarker, there were strongly significant correlations (p<0.001) between individual levels of metabolites in plasma and urine, with Spearman's rank correlation coefficient (rs) values of 0.74-0.90. The methods presented here will be valuable for the development of biological monitoring methods for diisocyanates.