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1.
Brain Res Bull ; 52(1): 51-9, 2000 May 01.
Article in English | MEDLINE | ID: mdl-10779703

ABSTRACT

The mechanism underlying the pathology of Huntington's disease (HD) is unknown, although there is substantial evidence supporting a role for excitotoxicity. The discovery of abnormal aggregations of protein in the brains of patients with HD, as well as in the brains of transgenic mice modeling this disease, has led to the suggestion that these "inclusions" have a pathogenic role. However, the relationship between inclusion formation and the progressive neurodegeneration in HD remains unclear. Here, we used mice transgenic for the first exon of the HD gene and an expanded CAG repeat (R6/2 line) to examine the role of neuronal intranuclear inclusions in kainic acid (KA) excitotoxicity. Unexpectedly, we found that the toxicity of KA was markedly attenuated in R6/2 mice compared with wild-type mice. In particular, the number and severity of KA-induced seizures in R6/2 mice was significantly reduced. When seizures occurred in 3-week-old R6/2 mice, we found lesions in the CA3 region of the hippocampus. However, neuronal intranuclear inclusions were not induced by KA in 3-week-old mice. Further, in older mice (9 weeks), the pre-existence of inclusions in CA3 neurons did not increase the vulnerability of neurons to KA, since no lesions were seen in 9-week R6/2 mouse brain. Our results suggest that an increased susceptibility to excitotoxic stimuli does not underlie the early phase of the neurological phenotype in R6/2 mice, although a role in later stages is not excluded by our findings. The significance of these findings is discussed in the context of the R6/2 mouse as a model for HD.


Subject(s)
Huntington Disease/genetics , Kainic Acid/poisoning , Mutation/physiology , Animals , Brain/ultrastructure , Cell Nucleus/ultrastructure , Drug Resistance/physiology , Hindlimb/drug effects , Hindlimb/physiopathology , Hippocampus/drug effects , Hippocampus/pathology , Humans , Inclusion Bodies/ultrastructure , Mice , Mice, Transgenic/genetics , Neurons/ultrastructure , Pyramidal Cells/drug effects , Pyramidal Cells/ultrastructure , Seizures/chemically induced , Seizures/physiopathology , Survival Analysis , Time Factors
2.
J Chromatogr B Biomed Sci Appl ; 709(2): 243-54, 1998 May 29.
Article in English | MEDLINE | ID: mdl-9657221

ABSTRACT

Short narrow analytical HPLC columns have been used successfully with high linear flow-rates and combined with mass spectrometric detection to produce a generic approach to quantitative bioanalysis. The approach has been used to validate several assays in the low ng/ml region and an example is given in this paper. When combined with a simple solid-phase extraction process the need for complicated, time consuming method development has been removed for the majority of pharmaceutical compounds. The approach takes advantage of not only the extra selectivity of the MS-MS detector but the excellent resolution and peak shape produced by gradient elution.


Subject(s)
Chromatography, High Pressure Liquid/methods , Mass Spectrometry/methods , Pharmaceutical Preparations/analysis , Acetanilides/analysis , Animals , Blood Chemical Analysis/methods , Chromatography, High Pressure Liquid/instrumentation , Cresols/analysis , Ketones/analysis , Pharmaceutical Preparations/metabolism , Rats , Reproducibility of Results , Sensitivity and Specificity , Spectrophotometry, Ultraviolet , Theophylline/analysis , Uracil/analysis
3.
Rapid Commun Mass Spectrom ; 12(5): 217-24, 1998.
Article in English | MEDLINE | ID: mdl-9519475

ABSTRACT

A liquid chromatography tandem mass spectrometry (LC/MS/MS) method has been developed for the fast routine analysis of selected CYP450 probe substrate metabolites in microsomal incubations, with no sample pretreatment. This has allowed fast and simple assessment of the potential effects which drug candidates may or may not have on the metabolism of specific CYP450 probe substrates, providing information which can then be used to rationalize in vivo interaction studies required in the clinic. This methodology takes advantage of fast gradient chromatography as a generic means of sample separation and analysis. It provides high throughput analysis compared to conventional gradient HPLC, with no significant loss in chromatographic performance.


Subject(s)
Cytochrome P-450 Enzyme System/metabolism , Pharmaceutical Preparations/analysis , Pharmaceutical Preparations/metabolism , Chromatography, Liquid , Humans , In Vitro Techniques , Mass Spectrometry , Microsomes/enzymology , Pharmaceutical Preparations/chemistry , Spectrophotometry, Ultraviolet
4.
J Chromatogr A ; 828(1-2): 199-207, 1998 Dec 18.
Article in English | MEDLINE | ID: mdl-9916306

ABSTRACT

The use of ultra-high flow-rate chromatography coupled to mass spectrometry offers great potential for the rapid, on-line analysis of pharmaceutical compounds in plasma as it permits high throughput direct analysis of plasma samples without any time-consuming sample preparation such as solid-phase extraction. The coupling of mass spectrometry with high-performance liquid chromatography often results in enhanced selectivity and sensitivity compared to, for example, ultraviolet absorbance detection. This can remove the need for complete resolution of the analyte from endogenous materials in the matrix. The use of large particle size stationary phases, and therefore, the ability to use large porosity column end frits, coupled with the added selectivity and sensitivity of the mass spectrometer allows an on-line analysis approach to be used for the direct analysis of pharmaceuticals in biological matrices with extremely high throughput. This paper presents an overview of the manner in which we have optimised this technique for the analysis of plasma samples, in terms of gradient profile, system configuration and optimal injection volume for maximum throughput and robustness. The nature of the mobile phase flow is also discussed.


Subject(s)
Chromatography, Liquid/methods , Isoquinolines/blood , Humans , Mass Spectrometry , Reference Standards , Reproducibility of Results
5.
Br J Clin Pharmacol ; 43(6): 579-87, 1997 Jun.
Article in English | MEDLINE | ID: mdl-9205817

ABSTRACT

AIMS: Two open studies in healthy volunteers were conducted to determine the absolute bioavailability and metabolic disposition of zolmitriptan (311C90), a novel 5HT1D agonist for the acute treatment of migraine. METHODS: After an initial test i.v. infusion, bioavailability was assessed by comparison of AUC after an i.v. infusion (3.5 mg) and an oral tablet (10 mg), in six men and six women using a randomised, crossover design. Disposition was studied by administration of a 25 mg capsule, labelled with 100 microCi [14C]-zolmitriptan, to five men and one woman on a single occasion. RESULTS: Zolmitriptan was well tolerated by both i.v. and oral routes. Adverse events were mostly mild, consistent with earlier studies and characteristic of this class of drug. Reports were similar in nature and number after both oral and i.v. dosing. Mean +/- s.d. oral bioavailability was 0.49 +/- 0.24 (0.38 +/- 0.16 in men and 0.60 +/- 0.28 in women). After oral dosing, Cmax and AUC values in women were approximately double those in men. Relative to zolmitriptan concentrations, metabolite concentrations were higher after oral dosing than after i.v., and higher in men compared with women. Half-life was significantly longer after oral dosing (mean 22%, 95% CI 6-35%). Mean +/- s.d. values for CL, V2 and t1/2,z after i.v. dosing (all subjects) were 8.7 +/- 1.7 ml min-1 kg-1, 122 +/- 321 and 2.30 +/- 0.59 h respectively. Following administration of 25 mg [14C]-zolmitriptan, 91.5% of the dose was recovered in 7 days, 64.4 +/- 6.5% in urine and 27.1 +/- 6.0% in faeces. Less than 10% was recovered unchanged in urine, with 31.1 +/- 6.4% recovered as the inactive indole acetic acid metabolite. Most of the faecal material was unchanged zolmitriptan, representing unabsorbed drug. Plasma concentrations of [14C] were slightly higher than those of the summed concentrations of known analytes zolmitriptan, the active N-desmethyl metabolite (183C91), the inactive N-oxide (1652W92) and indole acetic acid (2161W92) metabolites, which accounted for 86% of total plasma radioactivity. No other significant metabolites were detected in plasma. Some minor additional metabolites were detected in urine, none of which contributed more than 5% of the dose. CONCLUSIONS: The data suggest that zolmitriptan undergoes first-pass metabolism and this is more extensive in men than in women. Zolmitriptan has suitable bioavailability for an acute oral migraine treatment and there are no significant unidentified metabolites in man.


Subject(s)
Oxazoles/pharmacokinetics , Oxazolidinones , Serotonin Receptor Agonists/pharmacokinetics , Absorption , Administration, Oral , Adult , Analysis of Variance , Area Under Curve , Biological Availability , Chromatography, High Pressure Liquid , Chromatography, Liquid , Cross-Over Studies , Feces/chemistry , Female , Half-Life , Humans , Indoleacetic Acids/metabolism , Infusions, Intravenous , Male , Mass Spectrometry , Middle Aged , Migraine Disorders/drug therapy , Oxazoles/administration & dosage , Oxazoles/blood , Oxazoles/chemistry , Oxazoles/urine , Serotonin Receptor Agonists/administration & dosage , Serotonin Receptor Agonists/blood , Serotonin Receptor Agonists/chemistry , Serotonin Receptor Agonists/therapeutic use , Serotonin Receptor Agonists/urine , Sex Factors , Tryptamines
6.
Rapid Commun Mass Spectrom ; 11(18): 1953-8, 1997.
Article in English | MEDLINE | ID: mdl-9450350

ABSTRACT

The use of turbulent flow chromatography coupled to mass spectrometry (turbulent flow LC/MS) shows great potential for the rapid, direct analysis of pharmaceutical compounds in plasma and serum. The use of turbulent flow LC/MS has removed the need for any time-consuming sample preparation such as solid phase extraction, and allowed a total sample analysis time of approximately 2.5 min to be achieved. The coupling of a mass spectrometer with HPLC often not only results in greater sensitivity, but also the added specificity of the mass spectrometer reduces the need for complete resolution of the analyte from endogenous material in the matrix. This allows an on-line analysis approach to be used for the analysis of pharmaceuticals in biological matrices. Turbulent flow chromatography is achieved by the use of high flow rates and large particle size stationary phases. When coupled with mass spectrometric detection, the technique allows the direct analysis of plasma or serum samples with very rapid chromatography and, therefore, extremely high throughout. This work demonstrates the suitability of this technique for the validated analysis of biological samples for a novel isoquinoline pharmaceutical and offers some ideas on the future continued development, optimization and application of turbulent flow liquid chromatography.


Subject(s)
Pharmaceutical Preparations/analysis , Plasma/chemistry , Calibration , Chromatography, High Pressure Liquid , Gas Chromatography-Mass Spectrometry , Humans , Isoquinolines/blood , Quality Control , Reference Standards
7.
Br J Clin Pharmacol ; 35(4): 419-25, 1993 Apr.
Article in English | MEDLINE | ID: mdl-8485022

ABSTRACT

1. Tucaresol (589C80; 4[2-formyl-3-hydroxyphenoxymethyl] benzoic acid) interacts stoichiometrically with haemoglobin to increase oxygen affinity. By decreasing the proportion of insoluble deoxy sickle haemoglobin at capillary oxygen concentrations, tucaresol may be of therapeutic benefit in sickle cell anaemia. 2. In this study, which involved the first administration to man, the pharmacokinetics and pharmacodynamics of tucaresol were studied in healthy male volunteers following oral doses of 200-3600 mg. 3. Peak drug concentrations in plasma and erythrocytes were linearly related to dose; mean (s.d.) values were 95.8 (26.1) and 1035 (67) micrograms ml-1, respectively, at the highest dose. Median tmax in plasma was 6.5 h and in erythrocytes 24.5 h, when approximately 60% of the administered dose was in the target tissue. Plasma drug concentrations fell biexponentially with commencement of the apparent terminal elimination phase at approximately 24 h. The terminal elimination half-life from plasma increased with dose (r = 0.77; P < 0.0001) from 133-190 h at 400 mg to a mean (s.d.) of 289 (30) h at 3600 mg. Erythrocyte drug concentrations declined mono-exponentially with a half-life that was always shorter than the apparent terminal half-life in plasma: overall mean (95% CI) of t1/2 erythrocyte/t1/2 plasma ratio was 0.57 (0.53, 0.61). The erythrocyte AUC/plasma AUC ratio increased with dose (r = 0.67; P < 0.001). 4. The proportion of haemoglobin modified to a form with high oxygen affinity (%MOD) increased in a dose-related manner above doses of 800 mg reaching 19-26% after the 3600 mg dose.(ABSTRACT TRUNCATED AT 250 WORDS)


Subject(s)
Antisickling Agents/pharmacology , Benzaldehydes/pharmacology , Benzoates/pharmacology , Administration, Oral , Adult , Antisickling Agents/adverse effects , Antisickling Agents/pharmacokinetics , Benzaldehydes/adverse effects , Benzaldehydes/pharmacokinetics , Benzoates/adverse effects , Benzoates/pharmacokinetics , Blood Cell Count/drug effects , Blood Pressure/drug effects , Heart Rate/drug effects , Hemoglobins/drug effects , Hemoglobins/metabolism , Humans , Male
8.
J Pharm Sci ; 78(10): 874-6, 1989 Oct.
Article in English | MEDLINE | ID: mdl-2600797

ABSTRACT

A method for the determination of 2-cyano-1-methyl-3-[4-(4-methyl-6-oxo-1,4,5,6-tetrahydropyridazin- 3- yl)phenyl]guanidine (SK&F94836, 1) in plasma is presented. The method involves liquid-solid extraction of the drug by C18 cassettes, with subsequent elution by an AASP LC module for determination by HPLC with UV detection. The assay is rapid, precise, accurate, and specific. The between-day CV values over the concentration range 50-500 ng/mL are 4% or less; this rises to 8% at 25 ng/mL. The corresponding between-day bias is less than 1% over the same range of concentrations. The limit of quantification is 25 ng/mL and the assay can be used for measuring 1 in samples from preclinical studies following either oral or intravenous administration of the compound.


Subject(s)
Guanidines/blood , Pyridazines/blood , Administration, Oral , Animals , Chromatography, High Pressure Liquid , Dogs , Guanidines/administration & dosage , Infusions, Intravenous , Pyridazines/administration & dosage , Spectrophotometry, Ultraviolet
9.
J Pharm Biomed Anal ; 6(1): 75-85, 1988.
Article in English | MEDLINE | ID: mdl-16867442

ABSTRACT

A selective and specific assay for SK&F 94120 [5-(4-acetamidophenyl)pyrazin-2(1H)-one] and its four metabolites in plasma has been developed. The method incorporates a single liquid-solid extraction step using C18 Analytichem Automated Sample Processor (AASP) cassettes. This is followed by successive elutions of the solid phase with two mobile phases of increasing acetonitrile content, by an AASP liquid chromatography module. A mobile phase containing 10% acetonitrile elutes a glucuronide metabolite from the cartridge which is then chromatographed and quantified intact. A second mobile phase, containing 20% acetonitrile, is then used to elute the unchanged drug and the three other metabolites from the same cartridge. The assay shows good accuracy and precision (less than 10% for all analytes) and is able to determine SK&F 94120 and its metabolites in plasma at concentrations between 0.05 and 1.00 mg l(-1).

10.
J Chromatogr ; 353: 371-8, 1986 Feb 26.
Article in English | MEDLINE | ID: mdl-3700521

ABSTRACT

A selective and specific assay for 5-(4-acetamidophenyl)pyrazin-2(1H)-one (SK&F 94120), a novel inotropic agent, has been developed. The method incorporates a liquid-solid extraction step with a C18 Analytichem automated sample processor (AASP) cassette, which consists of ten miniature extraction columns. The cassette is then loaded into the AASP auto injector, ready for automated liquid chromatography with UV detection. The AASP consists of a high-pressure sealing chamber which encapsulates each column. The high-performance liquid chromatographic mobile phase is directed through the chamber, and the analytes are eluted onto the analytical column for subsequent separation and measurement. The assay is sufficiently accurate and precise to determine SK&F 94120 at concentrations as low as 0.5 mg/l. The mean coefficient of variation for the concentration range 0.5-10.0 mg/l was 2% with a bias of +/- 1%. The assay has been used for pharmacokinetic and bioavailability studies in several species, including rat, dog, and cynomolgus monkey.


Subject(s)
Cardiotonic Agents/analysis , Pyrazines/analysis , Animals , Autoanalysis , Cardiotonic Agents/blood , Chromatography, High Pressure Liquid/methods , Dogs , Drug Stability , Pyrazines/blood , Spectrophotometry, Ultraviolet
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