Michael G. Bartlett

Chronic, intermittent exposure to chlorpyrifos in rats: Protracted effects on axonal transport, neurotrophin receptors, cholinergic markers, and information processing

Persistent behavioral abnormalities have been commonly associated with acute organophosphate (OP) pesticide poisoning; however, relatively little is known about the consequences of chronic OP exposures that are not associated with acute cholinergic symptoms. In this study, the behavioral and neurochemical effects of chronic, intermittent, and subthreshold exposures to the OP pesticide, chlorpyrifos (CPF), were investigated. Rats were injected with CPF s.c. (dose range, 2.5–18.0 mg/kg) every other day over the course of 30 days and then were given a 2-week CPF-free washout period. In behavioral experiments conducted during the washout period, dose-dependent decrements in a water-maze hidden platform task and a prepulse inhibition procedure were observed, without significant effects on open-field activity, Rotorod performance, grip strength, or a spontaneous novel object recognition task. After washout, levels of CPF and its metabolite 3,5,6-trichloro-2-pyridinol were minimal in plasma and brain; however, cholinesterase inhibition was still detectable. Furthermore, the 18.0 mg/kg dose of CPF was associated with (brain region-dependent) decreases in nerve growth factor receptors and cholinergic proteins including the vesicular acetylcholine transporter, the high-affinity choline transporter, and the α7-nicotinic acetylcholine receptor. These deficits were accompanied by decreases in anterograde and retrograde axonal transport measured in sciatic nerves ex vivo. Thus, low-level (intermittent) exposure to CPF has persistent effects on neurotrophin receptors and cholinergic proteins, possibly through inhibition of fast axonal transport. Such neurochemical changes may lead to deficits in information processing and cognitive function.

Determination of antimigraine compounds rizatriptan, zolmitriptan, naratriptan and sumatriptan in human serum by liquid chromatography/electrospray tandem mass spectrometry.

Development of a rapid, sensitive and selective method for the determination of antimigraine drugs from human serum is essential for understanding the pharmacokinetics of these drugs when administered concurrently. Solid phase extraction (SPE) using Oasis HLB was used to extract the drugs (sumatriptan, naratriptan, zolmitriptan and rizatriptan) and the internal standard bufotenine from serum. A method based on liquid chromatography/tandem mass spectrometry (LC/MS/MS) was developed and validated to simultaneously quantitate these antimigraine drugs from human serum. The precursor and major product ions of the analytes were monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. The base peak in all the analytes is formed by alpha cleavage associated with protonation of the secondary amine. Mechanisms for the formation of the collision-induced dissociation products of these antimigraine compounds are proposed. Linear calibration curves were generated from 1-100 ng/mL with all coefficients of determination greater than 0.99. The inter- and intraday precision (%RSD) were less than 9.3% and accuracy (%error) was less than 9.8% for all components. The limits of detection (LOD) for the method were 250 pg/mL for sumatriptan and 100 pg/mL for the remaining analytes based on a signal-to-noise ratio of 3.

Chromatographic methods for the determination of therapeutic oligonucleotides

Both DNA and RNA are being explored for their therapeutic potential against a wide range of diseases. As these new drugs emerge, new demands arise for the analysis and quantitation of these biomolecules. Pharmacokinetic and pharmacodynamic analysis requirements for drug approval place enormous challenges on the methods for analyzing these therapeutics. This review will focus on bioanalytical methods for DNA antisense and aptamers as well as small-interfering RNA (siRNA) therapeutics. Chromatography methods employing ultraviolet (UV), fluorescence and mass spectrometric (MS) detection along with matrix-assisted laser desorption/ionization (MALDI) will be covered. Sample preparation from biological matrices will be reviewed as well as metabolite analysis and identification. All of these techniques are important contributions toward oligonucleotide therapeutic development. They will also be important in microRNA (miRNA) biomarker discovery and RNomics in general, as more non-coding RNAs are inevitably discovered.

Determination of moxifloxacin in human plasma by liquid chromatography electrospray ionization tandem mass spectrometry

Moxifloxacin is an advanced-generation, 8-methoxy fluoroquinolone that is active against a broad spectrum of pathogens, including antibiotic resistant Streptococcus pneumoniae. Development of a rapid, sensitive and selective method for the determination of moxifloxacin in human plasma is essential for understanding the pharmacokinetics of the drug when administered orally or intravenously. Solid phase extraction (SPE) using Oasis(R) HLB was used to extract moxifloxacin and the internal standard lomefloxacin from plasma. A method based on liquid chromatography electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) was developed and validated to quantitate moxifloxacin in human plasma. The precursor and major product ion of the analyte was monitored on a triple quadrupole mass spectrometer with positive ion electrospray ionization (ESI) in the multiple reaction monitoring (MRM) mode. Mechanisms for the formation of collision-induced dissociation (CID) products of moxifloxacin are proposed. Linear calibration curves were generated from 1 to 1000 ng/ml with coefficients of determination greater than 0.999. The inter-day and intra-day precision (% CV) was less than 11.3% and accuracy (% error) was less than 10.0% for moxifloxacin. The limit of detection (LOD) for the method was 50 pg/ml based on a signal to noise ratio of 3.

Oral Haloperidol or Risperidone Treatment in Rats: Temporal Effects on Nerve Growth Factor Receptors, Cholinergic Neurons, and Memory Performance

First and second generation antipsychotics (FGAs and SGAs) ameliorate psychotic symptoms of schizophrenia, however, their chronic effects on information processing and memory function (i.e. key determinants of long term functional outcome) are largely unknown. In this rodent study the effects of different time periods (ranging from 2 weeks to 6 months) of oral treatment with the FGA, haloperidol (2.0 mg/kg/day), or the SGA, risperidone (2.5 mg/kg/day) on a water maze repeated acquisition procedure, the levels of nerve growth factor receptors, and two important cholinergic proteins, the vesicular acetylcholine transporter and the high affinity choline transporter were evaluated. The effects of the antipsychotics on a spontaneous novel object recognition procedure were also assessed during days 8-14 and 31-38 of treatment. Haloperidol (but not risperidone) was associated with impairments in water maze hidden platform trial performance at each of the time periods evaluated up to 45 days, but not when tested during days 83-90. In contrast, risperidone did not impair water maze task performance at the early time periods and it was actually associated with improved performance during the 83-90 day period. Both antipsychotics, however, were associated with significant water maze impairments during the 174-180 day period. Further, haloperidol was associated with decrements in short delay performance in the spontaneous novel object recognition task during both the 8-14 and 31-38 day periods of treatment, while risperidone was associated with short delay impairment during the 31-38 day time period. Both antipsychotics were also associated with time dependent alterations in the vesicular acetylcholine transporter, the high affinity choline transporter, as well as tyrosine kinase A, and p75 neurotrophin receptors in specific brain regions. These data from rats support the notion that while risperidone may hold some advantages over haloperidol, both antipsychotics can produce time-dependent alterations in neurotrophin receptors and cholinergic proteins as well as impairments in the performance of tasks designed to assess spatial learning and episodic memory.

Determination of acyclovir in maternal plasma, amniotic fluid, fetal and placental tissues by high-performance liquid chromatography.

Acyclovir [9-[(2-hydroxyethoxy)-methyl]-guanosine, Zovirax, ACV] is a synthetic purine nucleoside analog active against herpes simplex virus types 1 (HSV-1), 2 (HSV-2), and varicella zoster virus. Acyclovir has frequently been used in HSV-2 seropositive mothers to prevent prenatal transmission of herpes virus to their unborn children. A fast and reproducible HPLC method for the determination of the highly polar acyclvoir in maternal rat plasma, amniotic fluid, placental tissue, and fetal tissue has been developed and validated. Plasma and amniotic fluid samples were prepared by protein precipitation using 2 M perchloric acid and syringe filtering. Tissue samples were homogenized in distilled water, centrifuged, and extracted using a C(18) solid-phase extraction method prior to analysis. Baseline resolution was achieved for acyclovir and the internal standard gancyclovir, an anti-viral of similar structure to acyclovir, using an Agilent Eclipse XDB C(8) column (150 x 2.1 mm, 5 microm). The mobile phase used for the plasma and amniotic fluid was 10 mM acetate/citrate buffer-3.7 mM aqueous octanesulfonic acid (87.5:12.5, v/v) at a flow-rate of 0.2 ml/min. The mobile phase used for the tissue samples was 30 mM acetate/citrate buffer with 5 mM octanesulfonic acid-acetonitrile (99:1, v/v). Both aqueous mobile phase portions were pH adjusted to 3.08. All separations were done using an Agilent 1100 Series HPLC system with UV detection of 254 nm. The assay was validated for each matrix over a range of 0.25-100 microg/ml over 3 days using five replicates of three spiked concentrations. The relative standard deviation and percent error for each validation data set was <15% for middle and high quality control (QC) points and <20% for all low QC points. All calibration curves showed good linearity with an R(2)>0.99. The extraction efficiency for recovery of acyclovir from all matrices was >80%.

Determination of degradation products of sumatriptan succinate using LC-MS and LC-MS-MS.

Acid, base, heat, oxidation and UV irradiation stress methods were applied to study the stability of the bulk drug form of sumatriptan succinate. Liquid chromatography coupled with mass spectrometry (LC-MS and LC-MS-MS) was used to analyze the degraded samples and tentative structural identifications were assigned based upon known reactivity of the drug, molecular weight measurements and MS-MS fragmentation patterns. Sumatriptan succinate was found to be stable to exposure of acid, base, oxidation and UV irradiation at ambient conditions, but was found to degrade under acidic, basic and oxidative conditions when heated to 90 degrees C.

Collision‐induced dissociation mass spectra of cocaine, and its metabolites and pyrolysis products

As the first step in the development of a liquid chromatographic/tandem mass spectrometric method for the quantitation of cocaine, its metabolites and its pyrolytic degradation products from biological matrices, the collision-induced dissociation of the protonated and selected deuterium-labeled species was studied. The fragment ions generated from the CID mass spectra were assigned and fragmentation mechanisms were proposed.

ELECTROSPRAY COLLISION-INDUCED DISSOCIATION MASS SPECTRA OF POSITIVELY CHARGED OLIGONUCLEOTIDES

Positive ion collision-induced dissociation (CID) mass spectra of oligonucleotides provide qualitatively similar fragmentation patterns to CID mass spectra generated from negative ion precursors. The major sequence ion series w and a-base are still abundant enough to be useful for the determination of the sequence of an oligonucleotide. The application of sequencing to both positively and negatively charged precursor ions up to the 20-mer level are shown. The positive ion electrospray charge-state distributions are found to be sensitive to the base composition of the oligonucleotide. In the case of homopolymers containing the bases adenine, guanine and cytodine the average charge state is found to be similar. However, the average charge state of a homopolymer containing the base thymine is significantly lower. In addition, the CID mass spectra of this oligonucleotide results in the formation of x-2H and z-2H type ions exclusively. These differences are thought to be caused due to the phosphate backbone being the site of protonation rather than the base.

Targeting melanoma growth and viability reveals dualistic functionality of the phosphonothionate analogue of carba cyclic phosphatidic acid

BackgroundAlthough the incidence of melanoma in the U.S. is rising faster than any other cancer, the FDA-approved chemotherapies lack efficacy for advanced disease, which results in poor overall survival. Lysophosphatidic acid (LPA), autotaxin (ATX), the enzyme that produces LPA, and the LPA receptors represent an emerging group of therapeutic targets in cancer, although it is not known which of these is most effective.ResultsHerein we demonstrate that thio-ccPA 18:1, a stabilized phosphonothionate analogue of carba cyclic phosphatidic acid, ATX inhibitor and LPA1/3 receptor antagonist, induced a marked reduction in the viability of B16F10 metastatic melanoma cells compared with PBS-treated control by 80-100%. Exogenous LPA 18:1 or D-sn-1-O-oleoyl-2-O-methylglyceryl-3-phosphothioate did not reverse the effect of thio-ccPA 18:1. The reduction in viability mediated by thio-ccPA 18:1 was also observed in A375 and MeWo melanoma cell lines, suggesting that the effects are generalizable. Interestingly, siRNA to LPA3 (siLPA3) but not other LPA receptors recapitulated the effects of thio-ccPA 18:1 on viability, suggesting that inhibition of the LPA3 receptor is an important dualistic function of the compound. In addition, siLPA3 reduced proliferation, plasma membrane integrity and altered morphology of A375 cells. Another experimental compound designed to antagonize the LPA1/3 receptors significantly reduced viability in MeWo cells, which predominantly express the LPA3 receptor.ConclusionsThus the ability of thio-ccPA 18:1 to inhibit the LPA3 receptor and ATX are key to its molecular mechanism, particularly in melanoma cells that predominantly express the LPA3 receptor. These observations necessitate further exploration and exploitation of these targets in melanoma.