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Biomedical subjects

S E Unger

Publications and source records attributed to S E Unger.

At least 19 recordsLinked to original sources
PubMed2004

A novel incubation direct injection LC/MS/MS technique for in vitro drug metabolism screening studies involving the CYP 2D6 and the CYP 3A4 isozymes.

A direct injection LC/MS/MS method involving a novel incubation technique was developed for the inhibition screening of CYP 2D6 and CYP 3A4 isoenzymes using dextromethorphan and midazolam as probe substrates. Both assays were performed using an electrospray ionization source in the positive ion mode. Direct injection was possible by using a short C 18, LC column (2 mm x 20 mm) with large particle diameter packing (10 microm). Analytical characteristics of the direct injection technique were studied by examining matrix effects, which showed suppression of the ESI signal between 0.20 and 0.65 min. The retention times for analytes were adjusted to approximately 0.8 min (k'>3), resulting in no matrix effect. Column lifetime was evaluated and determined to be approximately 160 direct injections of the matrix. The precision and accuracy of the control samples for the quantitation of dextromethorphan was between -0.53 and -12.80, and 3.73 and 6.69% respectively. Unlike conventional incubation techniques, incubations were carried out in an autosampler equipped with a heating accessory. This novel incubation method, which involved no stirring of the incubation mixture, estimated the Cl(int in vitro) for dextromethorphan and midazolam in human liver microsomes to be 1.65+/-0.22 ml/(hmg) and 0.861 ml/(min mg) respectively. The autosampler tray maintained uniform temperature and was sensitive to changes in temperature between 33 and 41 degrees C. High-throughput screening was performed using known inhibitors of the CYP 2D6 isozyme, and the system was evaluated for its ability to differentiate between these inhibitors. The strong inhibitor quinidine resulted in a 25.6% increase in t(1/2), the medium potency inhibitor chlorpromazine resulted in an increase of 6.14% and the weak inhibitor primaquine had no significant effect on half-life. This technique involves no sample preparation, demonstrated run times of 2 min per injection and can be fully automated. The method should therefore prove to be a valuable tool in the drug discovery process.

Algorithms↗
PubMed2003

Simultaneous quantification of seven active metabolites of roxifiban in human plasma by LC/MS/MS in the presence of an interfering displacer at millimolar concentrations.

Roxifiban (DMP 754) is a glycoprotein (GP) IIb/IIIa antagonist. Following oral administration to humans, roxifiban is metabolized to its primary active zwitterionic form, XV459, and several minor, active, hydrolyzed and hydroxylated metabolites, namely, M1a (DPC-AD3508), M1b (DPC-AD6128), M2 (SW156), M3 (DPC-AG2185), M8a (DPC-AF5814), and M8b (DPC-AF5818). Quantification of these metabolites in humans was not workable with a previous analytical method due to ion suppression of at least four of the analytes by a competitive displacer, DMP 728. This compound, which is another GP IIb/IIIa antagonist with very high affinity for the platelet receptor, was added to harvested blood samples in millimolar quantity to liberate XV459 from the GP IIb/IIIa receptor. An automated ion exchange solid phase extraction (IX-SPE) procedure was developed to selectively extract the seven metabolites of roxifiban and its deuterated internal standard while specifically excluding DMP 728. Among the six hydroxylation metabolites, there were two pairs of epimeric diastereomers (M1a/M1b and M8a/M8b) and one pair of geometric isomers (M2/M3), corresponding to three critical chromatographic pairs that needed to be base-line resolved because of the lack of specificity of MS/MS detection for these isomers. A new LC/MS/MS assay was developed to simultaneously quantify the seven metabolites in human plasma. The assay method was validated under GLP conditions over the concentration range of 0.5 to 80 nM for each of the analytes and successfully applied to assaying approximately 500 plasma samples from clinical trials.

Amidines↗
PubMed2001

High-speed liquid chromatography/tandem mass spectrometry using a monolithic column for high-throughput bioanalysis.

With the ever-increasing workload from a variety of in vitro and in vivo screening procedures, new analytical methodologies to perform bioanalysis in an accurate and high-throughput manner are in great demand. In this work, monolithic columns were used instead of conventional particulate HPLC columns to perform chromatographic separations. Because the pressure drop on a monolithic column was considerably lower than that on a particulate column, a high flow rate (6 mL/min) was used for a 4.6 x 50 mm monolithic column with a total backpressure of about 61 bar measured using acetonitrile/water (50:50). The capability of using a regular column length at high flow rates, combined with the extremely small dependency of separation efficiency on linear flow velocity, allowed for the generation of sufficient chromatographic resolving power in a significantly reduced runtime. As demonstrated in this work, a plasma extract of a mixture of tempazepam, tamoxifen, fenfluramine, and alprozolam were baseline separated within a total analysis time of one minute. An average peak width at half maximum of approximately one second was noted using a generic broad gradient. It was also found that the separation efficiency and signal/noise (S/N) ratios for this separation remained almost constant at flow rates of 1, 3, and 6 mL/min, respectively. The ruggedness of the separation was evaluated by injecting 600 plasma extracts containing the replicates of a standard curve of the above mixture during an overnight run. The chromatographic retention time, separation quality, peak response and sensitivity were highly reproducible throughout the run. This high-speed liquid chromatography/tandem mass spectrometry (LC/MS/MS) system has been used routinely in the authors' laboratory to support drug discovery programs.

Acetonitriles↗
PubMed2001

Multiple-sprayer tandem mass spectrometry with parallel high flow extraction and parallel separation for high-throughput quantitation in biological fluids.

In this work, an on-line four-column high-flow parallel extraction device was set up and interfaced to a four-column parallel separation system with a four-sprayer tandem mass spectrometric detector. This system allowed for the direct and simultaneous sampling, extraction, separation, and detection of four samples in biological fluids, resulting in significantly increased throughput. The performance of this system was evaluated using rat plasma samples spiked with methotrexate as a test compound. The results showed that the relative standard deviation for responses across the four parallel channels was less than 10% for a concentration range from 10 to 2500 nM, resulting in comparable sensitivity (slope of curve) for the analyte on all channels. A ruggedness test was performed using a mixed set of new and used extraction columns and the relative standard deviation in analyte response was found to be below 15%. The inter-channel crosstalk was measured to be about 0.1%. A comparison between the data generated on this system and on a conventional system for a rat pharmacokinetic study showed no more than 20% difference in measured concentrations. This parallel system is a viable tool for high-throughput bioanalysis in a drug discovery environment.

Animals↗
PubMed2000

Application of directly coupled LC-NMR-MS to the structural elucidation of metabolites of the HIV-1 reverse-transcriptase inhibitor BW935U83.

The human in vivo metabolism of the HIV-1 reverse transcriptase inhibitor 5-chloro-1-(2',3'-dideoxy-3'-fluoro-erythro-pentofuranosyl)uracil (BW935U83) was studied using 19F NMR spectroscopy, directly coupled LC-NMR and LC-NMR-MS. The number and relative proportions of the drug metabolites were obtained from 19F NMR spectra of whole human urine. The novel use of the continuous-flow 19F detected LC-NMR experiment yielded chromatographic retention times and 19F chemical shifts for the parent drug, the glucuronide conjugate of the parent and an early eluting polar metabolite. The parent drug and its glucuronide conjugate were easily characterised by directly coupled 1H LC-NMR spectroscopy and two-dimensional TOCSY experiments. The identification of the second metabolite was achieved using 19F NMR and directly coupled 1H LC-NMR-MS which furnished the molecular weight, and through the use of MS-MS techniques, information on the fragment ions. This species was identified as 3-fluoro-ribolactone.

Anti-HIV Agents↗
PubMed2000

Direct plasma sample injection in multiple-component LC-MS-MS assays for high-throughput pharmacokinetic screening.

The simultaneous dosing of numerous compounds followed by multiple-component analysis using LC-MS-MS (the N-in-1 approach) has significantly improved the throughput of the drug-screening process. However, plasma samples still need to be extracted before LC-MS-MS analysis, which frequently limits the throughput of the assay. In this work, a high-throughput on-line extraction technique has been developed for multiple-component LC-MS-MS assays using a high-flow column-switching technique. In N-in-1 LC-MS-MS assays, high sensitivity is required since the dose level is generally reduced to minimize drug-drug interactions. In addition, good chromatographic separation is essential to minimize interference and suppression effects. The direct plasma sample injection method developed in this work has successfully met the two requirements for multiple-component LC-MS-MS assays in high-throughput pharmacokinetic screening. Plasma samples containing a large number of potential drug candidates were directly injected onto an extraction column operated under a flow rate sufficiently high to exhibit a turbulent-flow profile. The extracted analytes were then eluted onto an analytical column via column switching for LC-MS-MS analysis. The use of turbulent flow resulted in a faster and more rugged extraction with reduced carryover compared with results obtained under laminar-flow conditions. Meanwhile, the use of a column-switching method maintained the chromatographic resolving power and high sensitivity of the LC-MS-MS assay. Separation efficiency, dynamic range, accuracy, and precision comparable with those of solid-phase extraction have been achieved with the turbulent-flow column-switching technique. As a result, this technique has been successfully and routinely used for high-throughput pharmacokinetic screening.

Animals↗
PubMed1998

Pharmaceutical development and specification of stereoisomers.

The pharmaceutical development of chiral drugs requires the activities of many different research and development groups. Guidelines which help to coordinate the activities of these groups and assist in the successful development of compounds with either single or multiple chiral centers are outlined and discussed.

Clinical Trials as Topic↗
PubMed1997

NMR and HPLC-NMR spectroscopic studies of futile deacetylation in paracetamol metabolites in rat and man.

HPLC-NMR spectroscopy has been used to investigate the level of deacetylation followed by reacetylation (futile deacetylation) of metabolites of paracetamol detected in human and rat urine. This has been achieved through the synthesis and administration of paracetamol isotopically labeled at the acetyl group with C2H3, 13CH3 and 13CO-13CH3. Using paracetamol-C2H3 it had been shown that in the rat the sulphate metabolite present in the urine shows 10-13% futile deacetylation depending on the dose, whereas for paracetamol-13CO-13CH3 the corresponding value was about 8%. After solid phase extraction, it was also possible to determine the level of futile deacetylation in the glucuronide metabolite using directly-coupled HPLC-NMR. This approach was facilitated by the use of acetonitrile-d3 as an HPLC eluent and the HPLC-NMR analyses showed that the level of futile deacetylation in the sulphate and glucuronide metabolites were equal at about 9%. The glucuronide of paracetamol-C2H3 was the predominant metabolite in man and following separation using HPLC-NMR, the level of futile deacetylation was shown to be 1% for the glucuronide and 2% for the sulphate, these values being equal within experimental error. This work demonstrates the utility of NMR and HPLC-NMR spectroscopy for isotope exchange studies.

Acetaminophen↗
PubMed1996

Combined HPLC, NMR spectroscopy, and ion-trap mass spectrometry with application to the detection and characterization of xenobiotic and endogenous metabolites in human urine.

The direct coupling of HPLC with NMR spectroscopy has been extended by splitting the HPLC eluent after conventional UV detection and sending part to a NMR spectrometer and part to an ion-trap mass spectrometer in a "triplehyphenated" HPLC-NMR-MS system. Combined UV, 1H NMR, and positive-ion electrospray MS detection was achieved in the continuous-flow mode using whole human urine from a subject dosed with acetaminophen. By means of HPLC-NMR-MS, the structural information available from the complementary spectroscopic techniques provided rapid confirmation of the identity of the acetaminophen glucuronide and sulfate metabolites, together with a number of endogenous metabolites. In particular, the HPLC-NMR-MS approach allowed the unequivocal identification of phenylacetylglutamine in human urine, an endogenous metabolite not previously observed in 1H NMR spectra of urine because of extensive overlap with resonances from other metabolites. The analytical advantages and complementarity of NMR and MS techniques in direct hyphenation with HPLC are discussed. The new technique of HPLC-NMR-MS will provide the scope for more comprehensive and fully automated analysis of biofluids and other complex mixtures than was previously available from single hyphenation of these instruments.

Acetaminophen↗
PubMed1994

Detection of malignancy-associated metabolites in the sera of cancer patients by electron capture gas chromatography.

A reliable test that detects malignancy and indicates response to therapy is needed. Frequency-pulsed electron-capture gas-liquid chromatography (FPEC-GLC), a selective analytical technique that is sensitive to 15 fmol quantities of metabolites, was used to analyse derivatised acidic chloroform extracts of sera from patients with biopsy-proven cancer, non-malignant infectious and non-infectious disease, and healthy controls. Two peaks designated P1 and P10, not found in serum from healthy controls (n = 7) or patients with non-malignant disease (n = 85), were detected in biopsy-proven samples (n = 52) from cancer patients. P1 and P10 were later shown by chemical and mass spectral studies to be carboxylic acids. When one or both of these peaks were detected in the sera of non-treated patients they were always associated with malignancy. In patients responding to therapy, a reduction or disappearance of these peaks was observed. Further, it was noted that P10 persisted or increased in sera of patients with progressive cancer not responding to therapy. We conclude that this test has potential in diagnosis and for following the response of the disease to therapy.

Adenocarcinoma↗
PubMed1991

Dihydropyrimidine calcium channel blockers. 3. 3-Carbamoyl-4-aryl-1,2,3,4-tetrahydro-6-methyl-5-pyrimidinecarboxylic acid esters as orally effective antihypertensive agents.

In order to explain the potent antihypertensive activity of the modestly active (IC50 = 3.2 microM) dihydropyrimidine calcium channel blocker 5, we carried out drug metabolism studies in the rat and found 5 is metabolized to compounds 6-10. Two of the metabolites, 6 (IC50 = 16 nM) and 7 (IC50 = 12 nM), were found to be responsible for the antihypertensive activity of compound 5. Potential metabolism of 6 into 7 in vivo precluded our interest in pursuing compounds related to 6. Structure-activity studies aimed at identifying additional aryl-substituted analogues of 7 led to 17g,j,p with comparable potential in vivo, though these compounds were less potent than 7 in vitro. To investigate the effects of absolute stereochemistry on potency, we resolved 7 via diastereomeric ureas 19a,b, prepared from 18 by treatment with (R)-alpha-methylbenzylamine. Our results demonstrate that the active R-(-)-enantiomer 20a of 7 is both more potent and longer acting than nifedipine (1) as an antihypertensive agent in the SHR. The in vivo potency and duration of 20a is comparable to the long-acting dihydropyridine amlodipine. The superior oral antihypertensive activity of 20a compared to that of previously described carbamates 2 (R2 = COOEt) could be explained by its improved oral bioavailability, possibly resulting from increased stability of the urea functionality.

Administration, Oral↗
PubMed1990

Metabolism of methylenedioxymethamphetamine: formation of dihydroxymethamphetamine and a quinone identified as its glutathione adduct.

The in vitro conversion of (+)-3,4-methylenedioxymethamphetamine and (-)-3,4-methylenedioxymethamphetamine to the corresponding catecholamine, 3,4-dihydroxymethamphetamine (N-methyl-alpha-methyldopamine), by rat liver microsomes was examined. Metabolite formation was monitored after short-term incubations using high-performance liquid chromatography-electrochemical detection to determine concentrations of the catecholamine. The formation of N-methyl-alpha-methyldopamine exhibited enantioselectivity and levels were significantly higher after incubation of the (+)-isomer. The reaction appears to be cytochrome P-450 dependent as it was sensitive to SKF 525A and carbon monoxide. The catecholamine was unstable and was metabolized rapidly to a compound capable of forming an adduct with glutathione (GSH) and other thiol compounds. This second oxidation did not appear to be cytochrome P-450-dependent but required NADPH and microsomal protein. Catecholamine oxidation was inhibited by superoxide dismutase and by reducing agents. The same catecholamine oxidation product, characterized as the GSH adduct, could be generated by a xanthine-xanthine oxidase mixture and by tyrosinase. Mass spectral data showed that it was a 1:1 amine GSH adduct. These results indicate that MDMA is oxidized by cytochrome P-450 to the catechol and the catecholamine oxidized by superoxide to a quinone to which GSH or other thiol functions add. The formation of this quinone and its thiol adducts may account for some of the irreversible actions of this compound on serotonergic neurons.

3,4-Methylenedioxyamphetamine↗
PubMed1987

Xylocandin: a new complex of antifungal peptides. II. Structural studies and chemical modifications.

Xylocandins A1, A2, B1, B2, C1, C2, D1 and D2 are new antifungal peptides isolated from Pseudomonas cepacia ATCC 39277. The molecular weights of the xylocandins were determined by fast atom bombardment mass spectrometry (A1 m/z 1,215; A2 1,199; B1 1,229; B2 1,213; C1 1,097; C2 1,081; D1 1,083; D2 1,067). Each xylocandin is a cyclic peptide containing glycine, serine, asparagine (1-3 residues), beta-hydroxytyrosine, and an unusual amino acid with the formula C18H37NO5. Additionally A1, A2, D1 and D2 contain 2,4-diaminobutyric acid; A1, B1, C1 and D1 contain erythro-beta-hydroxyasparagine; and A1, A2, B1 and B2 contain xylose. For each xylocandin pair, an erythro-beta-hydroxyasparagine residue in the first component of the pair is thus replaced by an asparagine in the second component, accounting for the 16 dalton mass difference for each pair. Chemical modification of A1 and A2 at the diaminobutyric acid and beta-hydroxytyrosine residues was used to probe structural requirements for activity.

Anti-Bacterial Agents↗
PubMedDate not supplied

Transformation of dopamine and alpha-methyldopamine by NG108-15 cells: formation of thiol adducts.

The catecholamines, alpha-methyldopamine (alpha-MeDA) and dopamine (DA), have been implicated in 3,4-(methylenedioxy)amphetamine (MDA) toxicity. The toxicity and metabolic fate of alpha-MeDA, a metabolite of MDA, and DA, a neurotransmitter released by MDA administration, were examined in NG108-15 cells. Both catechols were found to accumulate intracellularly into NG108-15 cells. alpha-MeDA was about 4 times more toxic than DA in the cells. The depletion of glutathione (GSH) by buthionine sulfoximine (BSO) resulted in a drastic increase (10 times) in the alpha-MeDA mediated toxicity while the toxicity of DA was enhanced by 2 times. DA was largely metabolized to dihydroxyphenylacetic acid (DOPAC) and, to a smaller extent, formed an adduct with GSH. alpha-MeDA was primarily metabolized to a GSH adduct. alpha-MeDA was also metabolized to a product which was identified as the cysteinyl adduct. These adducts were identified by HPLC coelution with authentic standards. The GSH and cysteinyl adducts are presumably formed through conjugation of the thiols with intermediary quinone oxidation products of DA and alpha-MeDA. Previous studies indicate that alpha-MeDA is significantly more toxic than DA, especially under conditions of GSH depletion. The results of this study suggest that alpha-MeDA toxicity may occur through cytoplasmic accumulation and oxidation to a reactive quinone species followed by reaction with vital thiol functions or generation of reactive oxygen species. Cytoplasmic DA levels, on the other hand, appear to be significantly lower due to MAO metabolism and vesicular storage, and therefore, DA appears less likely to form conjugates with thiol groups or participate in possible redox cycling.

3,4-Methylenedioxyamphetamine↗
PubMedDate not supplied

Degradation of peptide drugs by immobilized digestive proteases.

An in vitro assay using immobilized digestive enzymes and LC/MS has been developed to measure the susceptibility of orally administered peptide drugs to hydrolysis by the digestive system. Six different bioactive peptides and peptide drugs were digested using an established in vitro system incorporating four sets of immobilized digestive enzymes, which simulated gastric digestion in the stomach, digestion by pancreatic enzymes in the intestinal lumen, and digestion by mucosal peptidases at the intestinal brush border. Hydrolysis products were rapidly identified using LC/MS and coincided with those expected based on the substrate specificities of the immobilized enzymes. The LC/MS system consisted of a reversed phase HPLC connected to a mass spectrometer through a continuous-flow fast atom bombardment ionization system. Data acquired on the susceptibility of peptide drugs to hydrolysis by digestive enzymes could be used during the formulation of orally administered drugs to protect them from degradation prior to absorption in the gut. Also, this assay could be used to quickly identify those compounds in a structurally related series of new drugs that are most resistant to digestive proteases.

Amino Acid Sequence↗