An analytical approach for on-site analysis of breath samples for Δ9-tetrahydrocannabinol (THC).

Increased acceptance of cannabis containing the psychoactive component, Δ9-tetrahydrocannabinol (THC), raises concerns about the potential for impaired drivers and increased highway accidents. In contrast to the "breathalyzer" test, which is generally accepted for determining the alcohol level in a driver, there is no currently accepted roadside test for THC in a motorist. There is a need for an easily collectible biological sample from a potentially impaired driver coupled with an accurate on-site test to measure the presence and quantity of THC in a driver. A novel breath collection device is described, which includes three separate sample collectors for collecting identical A, B, and C breath samples from a subject. A simple one-step ethanol extraction of the "A" breath collector sample can be analyzed by UHPLC/selected ion monitoring (SIM) liquid chromatography/mass spectrometry (LC/MS) to provide qualitative and quantitative determination of THC in breath sample in less than 4 min for samples collected up to 6 h after smoking a cannabis cigarette. SIM LC/MS bioanalyses employed d3-THC as the stable isotope internal standard fortified in negative control breath samples for quantitation including replicates of six calibrator standards and three quality control (QC) samples. Subsequent confirmation of the same breath sample in the B collectors was then confirmed by a reference lab by LC/MS/MS analysis. Fit-for-purpose bioanalytical validation consistent with pharmaceutical regulated bioanalyses produced pharmacokinetic (PK) curves for the two volunteer cannabis smokers. These results produced PK curves, which showed a rapid increase of THC in the breath of the subjects in the first hour followed by reduced THC levels in the later time points. A simpler single-point calibration curve procedure with calibrators and QC prepared in ethanol provided similar results. Limitations to this approach include the higher cost and operator skill sets for the instrumentation employed and the inability to actually determine driver impairment.

Conserved valproic-acid-induced lipid droplet formation in Dictyostelium and human hepatocytes identifies structurally active compounds.

Lipid droplet formation and subsequent steatosis (the abnormal retention of lipids within a cell) has been reported to contribute to hepatotoxicity and is an adverse effect of many pharmacological agents including the antiepileptic drug valproic acid (VPA). In this study, we have developed a simple model system (Dictyostelium discoideum) to investigate the effects of VPA and related compounds in lipid droplet formation. In mammalian hepatocytes, VPA increases lipid droplet accumulation over a 24-hour period, giving rise to liver cell damage, and we show a similar effect in Dictyostelium following 30 minutes of VPA treatment. Using (3)H-labelled polyunsaturated (arachidonic) or saturated (palmitic) fatty acids, we shown that VPA treatment of Dictyostelium gives rise to an increased accumulation of both types of fatty acids in phosphatidylcholine, phosphatidylethanolamine and non-polar lipids in this time period, with a similar trend observed in human hepatocytes (Huh7 cells) labelled with [(3)H]arachidonic acid. In addition, pharmacological inhibition of ß-oxidation in Dictyostelium phenocopies fatty acid accumulation, in agreement with data reported in mammalian systems. Using Dictyostelium, we then screened a range of VPA-related compounds to identify those with high and low lipid-accumulation potential, and validated these activities for effects on lipid droplet formation by using human hepatocytes. Structure-activity relationships for these VPA-related compounds suggest that lipid accumulation is independent of VPA-catalysed teratogenicity and inositol depletion. These results suggest that Dictyostelium could provide both a novel model system for the analysis of lipid droplet formation in human hepatocytes and a rapid method for identifying VPA-related compounds that show liver toxicology.

Liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel profiling tool for drug distribution and metabolism analysis: the terfenadine example.

Liquid extraction surface analysis mass spectrometry (LESA-MS) is a novel surface profiling technique that combines micro-liquid extraction from a solid surface with nano-electrospray mass spectrometry. One potential application is the examination of the distribution of drugs and their metabolites by analyzing ex vivo tissue sections, an area where quantitative whole body autoradiography (QWBA) is traditionally employed. However, QWBA relies on the use of radiolabeled drugs and is limited to total radioactivity measured whereas LESA-MS can provide drug- and metabolite-specific distribution information. Here, we evaluate LESA-MS, examining the distribution and biotransformation of unlabeled terfenadine in mice and compare our findings to QWBA, whole tissue LC/MS/MS and MALDI-MSI. The spatial resolution of LESA-MS can be optimized to ca. 1 mm on tissues such as brain, liver and kidney, also enabling drug profiling within a single organ. LESA-MS can readily identify the biotransformation of terfenadine to its major, active metabolite fexofenadine. Relative quantification can confirm the rapid absorption of terfendine after oral dosage, its extensive first pass metabolism and the distribution of both compounds into systemic tissues such as muscle, spleen and kidney. The elimination appears to be consistent with biliary excretion and only trace levels of fexofenadine could be confirmed in brain. We found LESA-MS to be more informative in terms of drug distribution than a comparable MALDI-MS imaging study, likely due to its favorable overall sensitivity due to the larger surface area sampled. LESA-MS appears to be a useful new profiling tool for examining the distribution of drugs and their metabolites in tissue sections.

Liquid extraction surface analysis (LESA) of food surfaces employing chip-based nano-electrospray mass spectrometry.

An automated surface-sampling technique called liquid extraction surface analysis (LESA), coupled with infusion nano-electrospray high-resolution mass spectrometry and tandem mass spectrometry (MS/MS), is described and applied to the qualitative determination of surface chemical residues resulting from the artificial spraying of selected fresh fruits and vegetables with representative pesticides. Each of the targeted pesticides was readily detected with both high-resolution and full-scan collision-induced dissociation (CID) mass spectra. In the case of simazine and sevin, a mass resolution of 100,000 was insufficient to distinguish the isobaric protonated molecules for these compounds. When the surface of a spinach leaf was analyzed by LESA, trace levels of diazinon were readily detected on the spinach purchased directly from a supermarket before they were sprayed with the five-pesticide mixture. A 30 s rinse under hot running tap water appeared to quantitatively remove all remaining residues of this pesticide. Diazinon was readily detected by LESA analysis on the skin of the artificially sprayed spinach. Finally, incurred pyrimethanil at a level of 169 ppb in a batch slurry of homogenized apples was analyzed by LESA and this pesticide was readily detected by both high-resolution mass spectrometry and full-scan CID mass spectrometry, thus showing that pesticides may also be detected in whole fruit homogenized samples. This report shows that representative pesticides on fruit and vegetable surfaces present at levels 20-fold below generally allowed EPA tolerance levels are readily detected and confirmed by the title technologies making LESA-MS as interesting screening method for food safety purposes.

The embryonic stem cell test as tool to assess structure-dependent teratogenicity: the case of valproic acid.

Teratogenicity can be predicted in vitro using the embryonic stem cell test (EST). The EST, which is based on the morphometric measurement of cardiomyocyte differentiation and cytotoxicity parameters, represents a scientifically validated method for the detection and classification of chemicals according to their teratogenic potency. Furthermore, an abbreviated protocol applying flow cytometry of intracellular marker proteins to determine differentiation into the cardiomyocyte lineage is available. Although valproic acid (VPA) is in worldwide clinical use as antiepileptic drug, it exhibits two severe side effects, i.e., teratogenicity and hepatotoxicity. These limitations have led to extensive research into derivatives of VPA. Here we chose VPA as model compound to test the applicability domain and to further evaluate the reliability of the EST. To this end, we study six closely related congeners of VPA and demonstrate that both the standard and the molecular flow cytometry-based EST are well suited to indicate differences in the teratogenic potency among VPA analogs that differ only in chirality or side chain length. Our data show that identical results can be obtained by using the standard EST or a shortened protocol based on flow cytometry of intracellular marker proteins. Both in vitro protocols enable to reliably determine differentiation of murine stem cells toward the cardiomyocyte lineage and to assess its chemical-mediated inhibition.

Matrix vapor deposition/recrystallization and dedicated spray preparation for high-resolution scanning microprobe matrix-assisted laser desorption/ionization imaging mass spectrometry (SMALDI-MS) of tissue and single cells.

Matrix preparation techniques such as air spraying or vapor deposition were investigated with respect to lateral migration, integration of analyte into matrix crystals and achievable lateral resolution for the purpose of high-resolution biological imaging. The accessible mass range was found to be beyond 5000 u with sufficient analytical sensitivity. Gas-assisted spraying methods (using oxygen-free gases) provide a good compromise between crystal integration of analyte and analyte migration within the sample. Controlling preparational parameters with this method, however, is difficult. Separation of the preparation procedure into two steps, instead, leads to an improved control of migration and incorporation. The first step is a dry vapor deposition of matrix onto the investigated sample. In a second step, incorporation of analyte into the matrix crystal is enhanced by a controlled recrystallization of matrix in a saturated water atmosphere. With this latter method an effective analytical resolution of 2 microm in the x and y direction was achieved for scanning microprobe matrix-assisted laser desorption/ionization imaging mass spectrometry (SMALDI-MS). Cultured A-498 cells of human renal carcinoma were successfully investigated by high-resolution MALDI imaging using the new preparation techniques.

A novel chromatographic method allows on-line reanalysis of the proteome.

Liquid chromatography combined with electrospray ionization is widely used for direct analysis of polar and labile molecules by LCMS. The on-line coupling in LCMS is a major strength but also causes a principal limitation that each eluting analyte has to be analyzed immediately and is not available for detailed interrogation after the LCMS run. Here we developed a new chromatographic strategy, which removes this limitation. After column separation the flow is split, one portion is analyzed directly, and the other is diverted to a capture capillary. After the direct LCMS run, the flow is switched, and the portion stored in the capillary is analyzed ("replay run"). We describe a setup consisting of an analytical column, a splitting valve, and a focusing column, which performs at full sensitivity and undiminished chromatographic resolution. We demonstrate three principal advantages of this system: nearly continuous MS utilization, duplicate analysis without requirement for additional sample, and targeting of important but undersampled features in the replay run.

Cellular expression and crystal structure of the murine cytomegalovirus major histocompatibility complex class I-like glycoprotein, m153.

Mouse cytomegalovirus (MCMV), a beta-herpesvirus that establishes latent and persistent infections in mice, is a valuable model for studying complex virus-host interactions. MCMV encodes the m145 family of putative immunoevasins with predicted major histocompatibility complex, class I (MHC-I) structure. Functions attributed to some family members include down-regulation of host MHC-I (m152) and NKG2D ligands (m145, m152, and m155) and interaction with inhibitory or activating NK receptors (m157). We present the cellular, biochemical, and structural characterization of m153, which is a heavily glycosylated homodimer, that does not require beta2m or peptide and is expressed at the surface of MCMV-infected cells. Its 2.4-A crystal structure confirms that this compact molecule preserves an MHC-I-like fold and reveals a novel mode of dimerization, confirmed by site-directed mutagenesis, and a distinctive disulfide-stabilized extended N terminus. The structure provides a useful framework for comparative analysis of the divergent members of the m145 family.

Novel valproic acid derivatives with potent differentiation-inducing activity in myeloid leukemia cells.

The anti-epileptic drug valproic acid harbors anti-tumoral activity in solid and leukemic tumor cell models and is currently evaluated in clinical trials. However, the plasma trough concentrations obtained in patients by common anti-epileptic dose regimens are below concentrations required for exerting anti-tumor effects in vitro. Here, we describe the identification of three novel valproic acid derivatives with superior differentiation-inducing and anti-proliferative activities in K562 bcr/abl-positive chronic myeloid leukemia cells and HL60 promyelocytic leukemia cells at achievable therapeutic VPA concentrations. These compounds reveal potent inhibition of histone deacetylase activity, induction of p21Cip/Waf expression as well as low toxicity on CD34+ bone marrow cells.

Teratogenic effects mediated by inhibition of histone deacetylases: evidence from quantitative structure activity relationships of 20 valproic acid derivatives.

The widely used antiepileptic drug valproic acid (VPA), which is also used in migraine prophylaxis and the treatment of bipolar disorders, is also under trial as an anticancer agent. Despite its wide range of therapeutic applications, VPA also has two severe side effects: acute liver toxicity and teratogenicity. The mechanism of action for all these properties is unknown to date, but recently, it was shown that VPA is able to inhibit the enzyme class of histone deacetylases (HDACs), proteins with a fundamental impact on gene expression and therefore possible molecular targets of VPA-induced signaling cascades. The purpose of this study was to determine if teratogenic side effects of VPA could be linked to its HDAC inhibition ability by studying a large set of structurally diverse derivatives based on the VPA core structure. We demonstrate that only VPA derivatives with a teratogenic potential in mice are able to induce a hyperacetylation in core histone H4 in teratocarcinoma F9 cells. We also demonstrate that this marker of functional HDAC inhibition occurs almost immediately (15 min) after exposure of F9 cells to VPA, whereas no influence on the HDAC protein levels (HDAC 2 and HDAC 3) could be detected even after 24 h of treatment. Further measurement of the IC50(HDAC) values of VPA derivatives in a human HDAC enzyme test system revealed an activity range from 10 to 10 000 microM; in some derivatives, HDAC inhibition ability was 40 times that of VPA. We also show a quantitative correlation between the IC50(HDAC) and the teratogenic potential of VPA derivatives, which clearly points toward HDACs as the formerly described teratogenic receptors of VPA-induced neural tube defects (NTDs).

S-2-pentyl-4-pentynoic hydroxamic acid and its metabolite s-2-pentyl-4-pentynoic acid in the NMRI-exencephaly-mouse model: pharmacokinetic profiles, teratogenic effects, and histone deacetylase inhibition abilities of further valproic acid hydroxamates and amides.

Structure-activity relationship studies of valproic acid (VPA) derivatives have revealed a quantitative correlation between histone deacetylase (HDAC) inhibition and induction of neural tube defects (NTDs) in the NMRI-exencephaly-mouse model, but this correlation has been, so far, limited to congeners with a carboxylic acid function. Whereas the classical HDAC inhibitor trichostatin A is active only as a hydroxamate but not as a carboxylic acid, we found that neither VPA amides nor hydroxamates inhibit HDACs, but can cause NTDs; e.g., 2-pentyl-4-pentynoic hydroxamic acid with its S-enantiomer being the potent teratogen. We therefore investigated the hypothesis that hydroxamic acid derivatives of VPA might be metabolized in vivo and may possibly be pro-teratogenic, as had been shown for valpromide but not valproic hydroxamic acid. We developed two stereoselective quantification methods based on chiral derivatization of VPA hydroxamates with (1R,2S,5R)-(-)-menthylchloroformate and carboxylic acid derivatives with (S)-(-)-1-naphthylethylamine, followed by gas chromatography-nitrogen phosphor detector analysis of biological samples. We then determined the pharmacokinetic profiles of S-2-pentyl-4-pentynoic hydroxamic acid and of S-2-pentyl-4-pentynoic acid in mice. S-2-Pentyl-4-pentynoic hydroxamic acid was found to be extensively metabolized to the corresponding carboxylic acid without affecting the stereochemistry at position C2. Furthermore, the metabolite S-2-pentyl-4-pentynoic acid was found to be very stable in vivo, with an extended half-life of 4.2 h compared with that of VPA, 1.4 h. Comparison of the individual HDAC inhibition abilities of additional VPA amides and hydroxamates, as measured by cellular and enzymatic assays, led us to the conclusion that both classes of VPA derivatives can be pro-teratogenic.

Increased replication of human cytomegalovirus in retinal pigment epithelial cells by valproic acid depends on histone deacetylase inhibition.

PURPOSE: Human cytomegalovirus (HCMV) replication depends on different cellular pathways, including histone acetylation and extracellular-signal regulated kinases 1 and 2 (Erk 1/2). In the present study, the influence of therapeutic valproic acid (VPA) concentrations was investigated on HCMV replication in retinal pigment epithelial (RPE) cells. METHODS: HCMV antigen expression and replication were detected by immunostaining, real-time RT-PCR, and determination of virus titers. Histone acetylation and Erk 1/2 phosphorylation were detected by Western blot. RESULTS: Pretreatment with VPA < or =1 mM enhanced HCMV antigen expression and replication by up to ninefold. In addition to histone deacetylase (HDAC) inhibition, VPA stimulated Erk 1/2 phosphorylation in RPE cells. Investigation of six VPA derivatives revealed that S-2-pentyl-4-pentynoic acid was the only derivative that induced histone hyperacetylation, indicating HDAC inhibition, in the observed concentrations < or =1 mM and that increased HCMV antigen expression. Other derivatives did not enhance HCMV replication in the tested concentrations, although some were found to induce Erk 1/2 phosphorylation. The mitogen-activated protein kinase kinase (MEK) inhibitor PD98059 inhibited VPA-induced Erk 1/2 phosphorylation but did not affect VPA-induced increased HCMV replication. In addition, the structurally nonrelated HDACI trichostatin A enhanced HCMV replication but did not affect Erk 1/2 phosphorylation in RPE cells. CONCLUSIONS: The data demonstrate that VPA stimulates HCMV replication by HDAC inhibition independent of Erk 1/2 phosphorylation in therapeutic concentrations in RPE cells. Therefore, patients at risk of HCMV retinitis who are treated with VPA or other HDAC inhibitors should be carefully monitored.

Increased human cytomegalovirus replication in fibroblasts after treatment with therapeutical plasma concentrations of valproic acid.

Valproic acid (2-propylpentanoic acid, VPA), an effective inhibitor of histone deacetylases (HDAC) is used for the treatment of epilepsia. In this study, structure-activity relationships for the action of structurally modified VPA derivatives on human cytomegalovirus (HCMV) replication and HDAC inhibition were defined. Pretreatment of human foreskin fibroblasts with VPA (0.125-1mM) caused a concentration-dependent increase of HCMV immediate early and antigen late antigen expression. Structure-activity relationships of VPA derivatives for HCMV stimulation were compared to those for teratogenic action and those for HDAC inhibition. Side chain elongation and introduction of a triple bond in 4-position of the other chain caused teratogenicity, stimulated HCMV replication, and increased HDAC inhibition, as demonstrated by enhanced levels of acetylated histones. Teratogenic VPA derivatives with a branched chain in 3-position as well as a non-teratogenic anticonvulsive active VPA derivative did not stimulate HCMV or accumulation of acetylated histones. This demonstrates a strict correlation between inhibition of HDAC and increased HCMV replication.

Liposomal daunorubicin (DaunoXome) in multiple myeloma: a modified VAD regimen using short-term infusion.

Liposomal daunorubicin replacing conventional anthracyclines may reduce toxicity and enhance efficacy of chemotherapy. In this phase I study, we evaluated liposomal daunorubicin (DaunoXome) in combination with vincristine and dexamethasone for toxicity, pharmacokinetics and potential efficacy in patients with multiple myeloma. The main objective was to determine the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of liposomal daunorubicin combined with vincristine and dexamethasone (VLDD). Additionally, pharmacokinetics were determined at higher dose levels. Seventeen multiple myeloma patients were enrolled in this trial; 76% of the patients had relapsed or refractory multiple myeloma. Successive cohorts received liposomal daunorubicin at doses of 40, 60, 80 and 100 mg/m2 on day 1 in combination with vincristine 1.4 mg/m2 (day 1) and oral dexamethasone (40 mg, days 1-4). DLT occurred at 100 mg/m2. Liposomal daunorubicin at 80 mg/m2 was well tolerated in this protocol and should be used for further phase II studies with the VLDD regimen. In this phase I trial, 64% of the patients achieved a partial remission or a minor response.