PubMed Health⌕ Search

Biomedical subjects

Irving W Wainer

Publications and source records attributed to Irving W Wainer.

At least 37 records · Page 2Linked to original sources
PubMed2004

Development and characterization of an open tubular column containing immobilized P-glycoprotein for rapid on-line screening for P-glycoprotein substrates.

Cellular membranes from a cell line expressing P-glycoprotein (Pgp(+)) and from a cell line that does not express Pgp (Pgp(-)) were immobilized on the surface of glass capillaries (25 cm x 100 microm i.d.) by non-covalent interactions using the avidin-biotin coupling system to create two open tubular columns, Pgp(+)-OT and Pgp(-)-OT. Frontal displacement chromatography on the Pgp(+)-OT demonstrated that the immobilized Pgp retained its ability to specifically bind the known Pgp substrates vinblastin and ketoconazole. The calculated affinities, expressed as K(d), for vinblastin and ketoconazole were 97 nM and 12.1 microM, which were comparable with previously reported K(d) values of 37 nM and 8.6 microM, respectively. The results confirm that the Pgp(+)-OT can be used to quantitatively estimate binding affinities for the Pgp. Frontal displacement chromatography on the Pgp(-)-OT demonstrated that the immobilized membranes retained the ability to bind some Pgp substrates, but that the binding was not due to specific binding to Pgp. A cohort of compounds containing high affinity Pgp substrates (vinblastin, prazosin) and moderate-low affinity Pgp substrates (doxorubicin, verapamil, ketoconazole) and a non-substrate (nicotine) were chromatographed on the Pgp(+)-OT and Pgp(-)-OT using fast frontal analysis and mass spectrometric detection. The results demonstrated that when the retention on the Pgp(+)-OT was corrected by subtraction of the retention on the Pgp(-)-OT, the test compounds could be accurately sorted into high, moderate-low and non-substrate categories. The data from the study indicates that a single 30-min parallel chromatographic experiment can be used to rank a compound based upon its relative affinity for the immobilized Pgp.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

The relationship between a urinary cachectic factor and weight loss in advanced cancer patients.

A 24K glycoprotein, the proteolysis-inducing factor (PIF), has been identified in mice and humans with cancer cachexia. Clinical cross-sectional studies found an association between the presence of PIF in urine and tumors of patients and weight loss. For the first time, we report results from a longitudinal study establishing the relationship between a urinary PIF pattern and persistent weight loss. Over time, cancer patients positive for the PIF pattern experienced weight loss, whereas those with a negative test gained weight.

Adult↗
PubMed2004

A population pharmacokinetic-metabolism model for individualizing ciprofloxacin therapy in ophthalmology.

The purpose of this study was to construct a population pharmacokinetic (PK) metabolism (MB) model to describe ciprofloxacin (C) concentrations in plasma and vitreous and aqueous humors in 26 patients. Ciprofloxacin was given as a 3-day oral prophylactic treatment to 26 patients before vitrectomy. Plasma, vitreous, and aqueous humor samples were collected from patients at different times on the day of surgery. Patients were phenotyped for CYP 1A2 activity using caffeine. Ciprofloxacin and caffeine concentrations were determined using validated HPLC assays. All concentrations of ciprofloxacin were simultaneously modeled using a four-compartment PK-MB model. Creatinine clearance and CYP 1A2 activity were modeled as surrogate markers of renal and hepatic clearances, respectively. Population PK was performed with IT2S, and simulations were performed with ADAPT-II. No eye infections were observed in any of the patients enrolled in the study, and there were only minimal effects on vitreous and aqueous concentrations after ocular drops were added to the oral treatments. The model that best described the concentrations of ciprofloxacin in serum and in aqueous and vitreous humor was a four-compartment PK linear model. Simulated AUCs of ciprofloxacin mean concentrations in the aqueous and vitreous humors were 17 +/- 9 and 10 +/- 8% of the systemic AUC, respectively. The terminal elimination half-life of the compound was (mean +/- SD) 5.0 +/- 2.8 hours. The apparent volume of distribution (Vss/F) was calculated to be 122.1 +/- 39.7 L. This PK-MB model may be very useful in optimizing treatments of various eye infections with ciprofloxacin. The results of this study suggest that giving ciprofloxacin orally for 2 days preceding surgery may prevent endophthalmitis from occurring, consequently abrogating the need for administering antibiotics via intraocular injections.

Administration, Oral↗
PubMed2004

Effects of protein calorie malnutrition on the pharmacokinetics of ketamine in rats.

The effect of protein calorie malnutrition (PCM) on the pharmacokinetics of ketamine (KET) enantiomers has been investigated. Six control and six PCM rats were administered 85 mg/kg racemic KET by intramuscular injection, and plasma concentrations of (S)- and (R)-KET, norketamine (NKET), and 5,6-dehydronorketamine (DNK) were measured using enantioselective gas chromatography. Pharmacokinetic profiles were analyzed using standard noncompartmental and compartmental modeling methods. The volumes of distribution were similar between control and PCM rats for (S)- and (R)-KET. However, total clearance of both KET enantiomers was decreased, resulting in an increase in systemic exposure (p < 0.05). The KET absorption rate was also increased in PCM rats. A decrease in the clearance of both NKET enantiomers led to a significant increase in exposure in PCM rats (p < 0.005), and modeling results could not exclude the possibility that PCM induced an increase in the fraction of KET following the NKET pathway, which may further contribute to this increase in exposure. An increase in exposure to DNK enantiomers was also evident in PCM animals compared with controls [p < 0.005 (DNK1); N.S. (DNK2)], which was in concordance with the decrease in apparent clearance values. These results show that PCM significantly alters the pharmacokinetics of KET and several of its metabolites.

Animals↗
PubMed2003

Enantioselective interactions of dextromethorphan and levomethorphan with the alpha 3 beta 4-nicotinic acetylcholine receptor: comparison of chromatographic and functional data.

The enantioselectivity of the interaction of dextromethorphan (DM) and levomethorphan (LM) with an immobilized alpha 3 beta 4 subtype of the nicotinic acetylcholine receptor (nAChR) liquid chromatographic stationary phase has been compared to DM- and LM-induced non-competitive blockade of nicotine-stimulated 86Rb(+) efflux from cells expressing the alpha 3 beta 4-nAChR. The association rate constants (k(on)) and dissociation rate constants (k(off)) for the formation of the DM and LM complexes with the nAChR were determined using non-linear chromatographic techniques and the k(off) value for DM (1.01+/-0.01 s(-1)) was significantly lower than the k(off) for LM (1.55+/-0.002s(-1)) while the k(on) values did not significantly differ (23.66+/-0.61 and 18.61+/-0.36 microM(-1)s(-1), respectively). In thermodynamic studies using the van't Hoff approach, the enthalpy change (Delta H degrees) of the DM-nAChR complex was 330 calmol(-1) more stable than the LM-nAChR complex, while there was no significant difference in the entropy change (DeltaS degrees ). In the functional in vitro cell-based studies, there was no significant difference in the observed IC(50) values for DM (10.1+/-1.01 microM) and LM (10.9+/-1.08 microM), but the recovery from the DM-induced blockade was slower than the recovery from LM-induced blockade; after 7 min: 38.25+/-15.46% recovery from DM blockade, 63.30+/-16.08% from LM blockade; after 4h: 76.20+/-4.51% recovery from DM blockade and 93.12+/-8.76% from LM blockade. The enantioselective differences in the functional effects are consistent with the chromatographic and thermodynamic data and indicate that this difference is due to increased stability of the DM-nAChR complex. The results suggest that the chromatographic approach can be used to probe the interaction of non-competitive inhibitors (NCIs) with nAChRs and to predict relative duration of functional blockades.

Chromatography, Liquid↗
PubMed2003

Quantitative determination of the enantiomers of methadone and its metabolite (EDDP) in human saliva by enantioselective liquid chromatography with mass spectrometric detection.

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection (LC-MS) has been developed and validated for the simultaneous determination of saliva concentrations of (R)- and (S)-methadone (Met) and (R)- and (S)-2-ethylidene-1,5-dimethyl-3,3-diphenyl-pyrrolidine (EDDP, a primary metabolite of Met). Saliva specimens were collected using Salivette devices (Sarsedt), and centrifuged; collected saliva was then spiked with deuterated internal standards, D3-Met and D3-EDDP, and directly injected into the LC-MS. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase (CSP) based upon immobilized alpha(1)-acid glycoprotein (AGP) using a mobile phase composed of acetonitrile: ammonium acetate buffer (10mM, pH 7.0) in a ratio of 18:82 (v/v), a flow rate of 0.9 ml/min and a temperature of 25 degrees C. Under these conditions, enantioselective separations were observed for methadone (alpha=1.30) and EDDP (alpha=1.17) within 15 min. Met, EDDP, D3-Met and D3-EDDP were detected using selected ion monitoring at m/z 310.20, 278.20, 313.20 and 281.20, respectively. Linear relationships between peak height ratio and drug-enantiomer concentrations were obtained for methadone in the range of 5.0-600.0 ng/ml, and for EDDP from 0.5 to 15.0 ng/ml per enantiomer with correlation coefficients better than 0.9994, where lower limit of quantification (LLOQ) for Met was 5 ng/ml and for EDDP 0.5 ng/ml. Acceptable intra- and inter-day precision of the method (CVs<4.0%) and accuracy (CVs<4.0%) were obtained. These findings demonstrate the accuracy and precision of the method used to successfully analyze saliva obtained from patients enrolled in a methadone-maintenance program.

Chromatography, Liquid↗
PubMed2003

Syntheses of immobilized G protein-coupled receptor chromatographic stationary phases: characterization of immobilized mu and kappa opioid receptors.

Opioid receptors are members of the superfamily of G protein-coupled receptors (GPCRs). Liquid chromatographic stationary phases containing either the human mu or kappa opioid receptor subtypes have been developed to study the binding between the opioid receptors and their ligands. The receptors were obtained from Chinese hamster ovary cells stably transfected with human mu or kappa receptor subtypes. The receptors were isolated through partial solubilization with sodium cholate detergent, and the partially purified receptor complex was immobilized in the phospholipid analogue monolayer of an immobilized artificial membrane liquid chromatographic stationary phase. The resulting phase was packed into a glass column (1.8 x 0.5 (i.d.) cm) and used in the on-line chromatographic determination of drug/ligand binding affinities to the immobilized opioid receptors. Preliminary on-line binding studies employing frontal chromatographic techniques were conducted with the known mu antagonist (naloxone) and a kappa agonist (U69593). The calculated dissociation constants (Kd) were 110 nM for naloxone and 84 nM for U69593. The results indicate that the immobilized receptors retained their ability to specifically bind ligands and were active for 1200 column volumes applied over two weeks. Zonal chromatographic experiments were also conducted, and competitive displacements of the marker ligands were observed. The results suggest that the immobilized opioid receptor stationary phases can be used to qualitatively assess the binding affinities of compounds to the immobilized receptors and represent the first example of the use of immobilized GPCRs in a chromatographic system.

Animals↗
PubMed2003

Determination of the enantiomers of ketamine and norketamine in human plasma by enantioselective liquid chromatography-mass spectrometry.

A sensitive enantioselective liquid chromatographic assay with mass spectrometric detection has been developed and validated for the simultaneous determination of plasma concentrations of (R)- and (S)-ketamine, and (R)- and (S)-norketamine. The compounds were extracted from human plasma using solid-phase extraction and then directly injected into the LC-MS system for detection and quantification. Enantioselective separations were achieved on a liquid chromatographic chiral stationary phase based upon immobilized alpha(1)-acid glycoprotein (the Chiral AGP column). The separations were achieved using a mobile phase composed of 2-propanol-ammonium acetate buffer (10 mM, pH 7.6) (6:94, v/v), a flow-rate of 0.5 ml/min and a temperature of 25 degrees C. Under these conditions, the analysis time was 20 min. Detection of the ketamine, norketamine and bromoketamine (internal standard) enantiomers was achieved using selected ion monitoring at m/z 238.1, 224.1 and 284.0, respectively. Extracted calibration curves were linear from 1 to 125 ng/ml per enantiomer for each analyte with correlation coefficients better than 0.9993 and intra- and inter-day RSDs of less than 8.0%. The method was applied to samples from a clinical study of ketamine in pain management.

Calibration↗
PubMed2003

Development and characterization of an immobilized enzyme reactor based on glyceraldehyde-3-phosphate dehydrogenase for on-line enzymatic studies.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) has been extensively studied as a target for new drugs to be used in the treatment of various parasitic diseases. The standard approach to the determination of GAPDH activity utilizes solubilized free enzyme and is limited by the enzyme's low stability. In the current study the stability of GAPDH was significantly increased through the covalent immobilization of the enzyme on a wide-pore silica support containing glutaraldehyde (Glut-P). The optimal conditions for the immobilization were: 100 mg Glut-P stationary phase, approximately 150 microg of enzyme dissolved in pyrophosphate buffer (15 mM, pH 8.5). The mixture was gently agitated for 6 h at 4 degrees C. Under these conditions 91.3% of protein was immobilized on 100 mg of Glut-P support with retention of 2.97% of the initial enzymatic activity. The activity of the immobilized GAPDH was stable for over 30 days. The GAPDH-Glut-P stationary phase was packed into a glass column to produce a GAPDH immobilized enzyme reactor (GAPDH-IMER). The activity and kinetic parameters of the GAPDH-IMER were investigated and the results demonstrated that the enzyme retained its activity and sensitivity to the competitive inhibitor agaric acid.

Citric Acid↗
PubMed2003

Immobilized nicotinic receptor stationary phases: going with the flow in high-throughput screening and pharmacological studies.

The nicotinic acetylcholine receptor (nAChR) subtypes alpha3beta4-nAChR and alpha4beta2-nAChR have been immobilized and the resulting stationary phases used to determine binding affinities. The alpha3beta4-nAChR column was coupled to a C(18) column and a mixture of 18 compounds was sorted into ligands and non-ligands for the alpha3beta4-nAChR. The results demonstrate that the nAChR stationary phases can be used for on-line high-throughput screening (HTS).

Animals↗
PubMed2003

Verapamil metabolites: potential P-glycoprotein-mediated multidrug resistance reversal agents.

Multidrug resistance in cancer chemotherapy frequently correlates with overexpression of the P-glycoprotein drug transporter. Attempts to reverse P-glycoprotein-mediated multidrug resistance with racemic verapamil or its less toxic (R)-enantiomer have been complicated by cardiotoxicity. The objective of this study was to investigate the effects of the major verapamil metabolite, norverapamil, as well as the PR-22 and D-620 metabolites, on P-glycoprotein-mediated drug transport. We measured the basolateral-to-apical fluxes of the P-glycoprotein substrates digoxin and vinblastine in the presence and absence of verapamil, (R)-norverapamil, (S)-norverapamil, racemic norverapamil, PR-22, or D-620 across confluent monolayers of Madin-Darby canine kidney (MDCK) cells that express P-glycoprotein on their apical membranes. Verapamil and norverapamil nonstereospecifically inhibited the renal tubular secretion of digoxin and vinblastine similarly in a dose-dependent manner. However, there was no decrease in the cellular accumulation of digoxin and vinblastine, suggesting that neither verapamil nor norverapamil prevent the substrates from entering the MDCK cells. Furthermore, the norverapamil metabolite P-22 also inhibited the secretion of these P-glycoprotein substrates. Our results suggest that the verapamil metabolites norverapamil and PR-22, which are less cardiotoxic than the parent compound, have comparable inhibitory abilities to verapamil (norverapamil greater than PR-22) and may be useful in reversing resistance to P-glycoprotein substrates.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Displacement and nonlinear chromatographic techniques in the investigation of interaction of noncompetitive inhibitors with an immobilized alpha3beta4 nicotinic acetylcholine receptor liquid chromatographic stationary phase.

A liquid chromatographic column containing immobilized alpha3beta4 nicotinic acetylcholine receptors (alpha3beta4-nAChRs) has been used to determine the equilibrium association constants (Ka), desorption rate constants (kd), and adsorption rate constants (ka) for the noncompetitive inhibitors: mecamylamine, ketamine, bupropion, and dextromethorphan. Displacement chromatography, with mecamylamine as the displacer, was used to verify that the four compounds bound to the same site on the immobilized alpha3beta4-nAChRs. Nonlinear chromatographic techniques were then utilized to calculate the Ka, ka, and kd values associated with the formation of the noncompetitive inhibitor-alpha3beta4-nAChR complexes. The ka values determined in this study ranged from 19.7 to 10.5 microM(-1) sec(-1), with a relative order of mecamylamine > dextromethorphan > or = ketamine > bupropion. The kd values determined in this study indicated that dextromethorphan-induced inhibition should produce a longer recovery time than the other three NCIs. This was consistent with results from a previous in vitro study. The data from this study indicate that the immobilized alpha3beta4-nAChR column and nonlinear chromatography can be used in the study of NCIs at the alpha3beta4-nAChR.

Chromatography, Liquid↗
PubMed2002

The effect of grapefruit juice and seville orange juice on the pharmacokinetics of dextromethorphan: the role of gut CYP3A and P-glycoprotein.

The objective of this study was to determine the effects of grapefruit juice and seville orange juice on dextromethorphan (DM) pharmacokinetics. Eleven healthy volunteers were studied over a 3-week period consisting of 5 study days each separated by a three-day washout. All subjects refrained from drinking caffeine containing beverages (coffee, soda, etc.) 8 h before orally taking DM (30 mg) with 200 ml water, 200 ml grapefruit juice, 200 ml water, 200 ml seville orange juice, and 200 ml water on Study Days 1 to 5. Aliquots of urine samples were assayed and analysed for DM, and the DM metabolites dextrorphan, 3-methoxymorphinan and 3-hydroxymorphinan using a validated HPLC method employing a phenyl column and a fluorescence detection. Results suggests that DM could provide some useful information on P-glycoprotein or related membrane efflux protein activity in the human gastro-intestinal tract. Bioavailability (F) of DM increased significantly with grapefruit and seville orange juice, but only returned to half the baseline value after three days of washout. This confirms that grapefruit and seville orange juice are long-lasting and perhaps irreversible inhibitors of gut CYP3A/P-glycoprotein. Grapefruit and seville orange juice appeared to have the same overall effect on DM pharmacokinetics. In addition, this paper presents a novel method of phenotyping for CYP2D6, CYP3A and P-glycoprotein using DM as a probe.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Multidimensional on-line screening for ligands to the alpha3beta4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase.

The alpha3beta4 subtype of the neuronal nicotinic acetylcholine receptor (nAChR) subtype was immobilized on a liquid chromatographic support and the resulting column used for the rapid and direct on-line screening for nAChR ligands. A multidimensional chromatographic system was developed consisting of the immobilized receptor column (NR column) connected via a switching valve to a C(18) column that was, in turn, connected to a single quadrupole mass spectrometer. A mixture of 18 compounds, containing alpha3beta4 nAChR (7) and compounds that are not alpha3beta4 nAChR ligands (11), was injected onto the NR column. The mobile phase consisted of ammonium acetate (10 mM, pH 7.4)-methanol (95:5, v/v) and the flow-rate was 0.2 ml/min. For the first 8 min the eluent was directed to waste. At t=8 min, the switching valve was rotated and the NR column connected to the C(18) column. The eluent from the NR column was directed to the C(18) column for 12 min. At t=20 min, the switching valve was rotated and the NR column was disconnected from the C(18) column. The compounds trapped on the C(18) column were separated and eluted onto the mass spectrometer using a mobile phase of ammonium acetate (10 mM, pH 7.4)-methanol (40:60, v/v) at a flow-rate of 1.0 ml/min. Detection was accomplished using total ion monitoring. The multidimensional system correctly isolated six of the seven alpha3beta4 nAChR ligands and only one of the 11 non-ligands was found with the alpha3beta4 nAChR ligands. The results indicate that the multidimensional liquid chromatographic system can be used for the on-line screening of chemical mixtures for alpha3beta4 nAChR ligands.

Chromatography, Liquid↗
PubMed2002

Biosynthesis in an on-line immobilized-enzyme reactor containing phenylethanolamine N-methyltransferase in single-enzyme and coupled-enzyme formats.

An immobilized-enzyme reactor (IMER) based upon phenylethanolamnie N-methyltransferase (PNMT) has been developed. The activity of the PNMT-IMER and its applicability for on-line N-methylation of normetanephrine was investigated. The reactor was connected through a switching valve to a cyano (CN) and ODS stationary phase connected in series. The substrate was injected onto the PNMT-IMER and the unreacted substrate and product were eluted and transported via a switching valve onto the analytical columns. The results from the PNMT-IMER/CN-ODS chromatographic system demonstrate that the enzyme retained its catalytic activity. Known substrates and inhibitors for PNMT were examined and the chromatographic system was utilized to carry out both quantitative and qualitative determinations. The PNMT-IMER/ CN-ODS system proves to be useful in basic biochemical studies, an ideal for the high throughput screening of substances for PNMT substrate-inhibitor properties. The PNMT-IMER was then coupled in series using switching-valve technology with a previously developed dopamine beta-hydroxylase immobilized-enzyme reactor and used to carry out the on-line two-step synthesis of epinephrine from dopamine.

Animals↗
PubMed2002

Immobilized receptor- and transporter-based liquid chromatographic phases for on-line pharmacological and biochemical studies: a mini-review.

This review addresses the synthesis and characterization of two different types of receptor-based liquid chromatographic supports, one based upon a trans-membrane ligand gated ion channel receptor (the nicotinic acetylcholine receptor) and the other a soluble nuclear receptor (the estrogen receptor). In addition, studies with the P-glycoprotein transporter are also reported. The nicotinic receptor was immobilized via hydrophobic insertion into the interstitial spaces of an immobilized artificial membrane (IAM) stationary phase. the estrogen receptor was tethered to a hydrophilic stationary phase and the membranes containing the Pgp transporter were coated on the surface of the IAM stationary phase. The stationary phases were characterized using known ligands and substrates for the respective non-immobilized proteins. The results from zonal and frontal chromatographic experiments demonstrated that the stationary phases could be used to determine binding affinities (expressed as dissociation constants, Kd,'s) and to resolve mixtures of ligands according to their relative affinities. In addition. competitive ligand binding studies on the P-glycoprotein-based stationary phase have established that this phase can be used to identify and characterize competitive displacement and allosteric interactions. These studies demonstrate that immobilized-receptor phases can be used for on-line pharmacological studies and as rapid screens for the isolation and identification of lead drug candidates from complex biological or chemical mixtures.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2002

Plasma protein binding study of oxybutynin by high-performance frontal analysis.

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.

Blood Proteins↗