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Biomedical subjects

M Gumbleton

Publications and source records attributed to M Gumbleton.

At least 37 records · Page 2Linked to original sources
PubMed1999

Transport of phosphatidylcholine in MDR3-negative epithelial cell lines via drug-induced MDR1 P-glycoprotein.

Human MDR1 P-glycoprotein (P-gp) is a membrane efflux pump for cytotoxics, whereas MDR3 P-gp is a phosphatidylcholine transporter. We have examined a role for MDR1 P-gp in phosphatidylcholine transport in MDR3-negative epithelial cells that have been induced to express the MDR1 P-gp by exposure to cytotoxics. The accumulation and retention of the fluorescently labelled phosphatidylcholine analogue, C12-NBD-PC, was studied in resistant, KBV1 and MCFadr, and sensitive, KB3-1 and MCF7, cells. Lower accumulation and decreased retention of C12-NBD-PC was evident in resistant cells, e.g., KBV1 accumulated 56%, and MCFadr accumulated 60%, of C12-NBD-PC levels in KB3-1 and MCF7, respectively. Treatment with the MDR1 P-gp inhibitor, verapamil, altered the kinetics of C12-NBD-PC in the resistant cells to more closely follow the pattern of C12-NBD-PC handling by sensitive cells. Comparison of C12-NBD-PC to that of the model MDR1 P-gp substrate, rhodamine-123, indicated phosphatidylcholine turnover kinetics by MDR1 P-gp to be relatively low. The transport by MDR1 P-gp of phosphatidylcholine from inner to outer membrane leaflet may regulate P-gp function and fulfill a role in the MDR1 multidrug-resistant phenotype.

4-Chloro-7-nitrobenzofurazan↗
PubMed1999

Temporal dependence of ectopeptidase expression in alveolar epithelial cell culture: implications for study of peptide absorption.

There is little data available regarding the extent of peptide metabolism encountered following inhalation to the lung. We have studied the activity of five ectopeptidases in primary rat alveolar epithelial cells, A549 cells and pulmonary macrophages. Peptidase activity was assayed in the plasma membrane fractions (PMF) of primary type II alveolar epithelial cells (ATII cells) after 2 days in culture and after 7 days in culture when they had formed monolayers of type I-like cells (ATI-like cells). Dipeptidyl peptidase IV (DPP) activity fell from 36.65 mU/mg protein to 16.29 mU/mg protein between day 2 and day 7 in culture, aminopeptidase N (AMN) activity increased from 16.16 mU/mg protein to 23.99 mU/mg protein, angiotensin-converting enzyme (ACE) activity was lost (4.29 mU/mg protein at day 2, not detected at day 7), and carboxypeptidase M (CPM) activity was acquired (not detected at day 2, 5.20 mU/mg protein at day 7). The profile of exopeptidase expression in A549 cells was similar to that of primary rat alveolar cells at day 7 in culture (DPP 24.24 mU/mg protein, AMN 47.74 mU/mg protein, CPM 4.28 mU/mg protein, ACE not detected). Macrophages expressed high levels of aminopeptidases (DPP 46.85 mU/mg protein, AMN 28.28 mU/mg protein) but carboxypeptidase activity was not detected. Low neutral endopeptidase 24.11 (NEP) activity was found in all cell types studied (0.96-2.41 mU/mg protein). The qualitative and quantitative changes in the peptidase activity of primary cultured rat alveolar cells between day 2 and day 7 in culture has implications for the use of alveolar cell monolayers as drug absorption models to investigate peptide absorption from the lung. Ectopeptidase activity in cultured alveolar cells can be used to infer the peptide-metabolising capacity of the surface of the alveolar epithelium. The aminopeptidase activity in particular, if representative of enzyme activity in vivo, would offer a significant metabolic barrier to systemic delivery of peptides via the lung.

Absorption↗
PubMed1999

Caveolin and its cellular and subcellular immunolocalisation in lung alveolar epithelium: implications for alveolar epithelial type I cell function.

Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.

Animals↗
PubMed1998

Synthesis, anti-human immunodeficiency virus activity and esterase lability of some novel carboxylic ester-modified phosphoramidate derivatives of stavudine (d4T).

We report the design, synthesis and antiviral evaluation of a series of lipophilic, masked phosphoramidate derivatives of the anti-human immunodeficiency virus (HIV) nucleoside analogue d4T, designed to act as membrane-soluble prodrug forms for the free nucleotide. In particular, we report a series of 12 novel compounds with systematic variation in the structure of the carboxylate ester function. In order to rationalize the changes in antiviral action with variation of this moiety we applied our recently developed 31P NMR-based assay for carboxyesterase lability to this series. However, no clear positive correlation emerged, indicating that, at least within this series, factors other than simple esterase lability may be the major determinants of antiviral potency.

3T3 Cells↗
PubMed1994

Pharmacokinetic-pharmacodynamic (PK-PD) modelling in non-steady-state studies and arterio-venous drug concentration differences.

In conducting a non-steady-state pharmacokinetic (PK)-pharmacodynamic (PD) study there is potential for the observed effect (E) vs time, and venous plasma drug concentration (C) vs time, profiles to display temporal displacement with respect to each other. This is most frequently observed when there exists a distributional nonequilibrium across the effect organ giving rise to hysteresis, i.e. observed C preceding E in the time domain, with the resulting potential for a counterclockwise loop to be generated in the observed E vs C plot (when data are connected in time-order). Such temporal displacement does not afford direct prediction of the steady-state E vs C PD relationship. When an arterio-venous (A-V) difference exists across the tissues of the blood sampling compartment (i.e. the arm), and this arises solely from an elimination process then drug concentration in the respective peripheral arterial plasma and venous plasma compartments will be in equilibrium at all times during a non-steady-state PK experiment. If there are no other sources of temporal displacement in the relationship between E and C then the observed E vs C plot will be a direct predictor of the steady-state E vs C PD relationship. In contrast when the A-V difference is of a distributional nature then proteresis, i.e. observed E preceding C in the time domain, will arise with the potential for the generation of a clockwise loop in the observed E vs C relationship. Simulated error-incorporated E vs time, and C vs time, data was analysed by semi-parametric implementation of an effect-compartment link-model that affords accurate steady-state E vs C PD predictions (without the requirement of sampling arterial blood) from data that incorporates the concurrent presence of: (i) distributional nonequilibrium across the effect organ, and (ii) distributional A-V non-equilibrium. Accurate steady-state E vs C PD predictions were achieved irrespective of the comparative magnitudes of the two nonequilibria, i.e. whether the rate of equilibration across the effect organ was faster than, or slower than, the rate of equilibration across the arm (resulting in a clockwise or counterclockwise loop in the observed E vs C plot, respectively), or indeed if one or other of the nonequilibria is essentially absent. When the rate of equilibration across the effect organ is slower than the rate of A-V equilibration (i.e. counterclockwise loop generated in the observed E vs C plot) then the need to model for the underlying A-V nonequilibrium is redundant, i.e. accurate steady-state E vs C PD predictions can be achieved with implementation (strictly incorrectly) of a more simple link parameterised solely to model for distributional nonequilibrium across the effect organ.(ABSTRACT TRUNCATED AT 400 WORDS)

Computer Simulation↗
PubMed1993

Simultaneous pharmacodynamic modeling of the non-steady-state effects of three oral doses of 1,3-glyceryl dinitrate upon blood pressure in healthy volunteers.

The organic nitrate 1,3-glyceryl dinitrate (1,3-GDN) is one of the primary dinitrate metabolites of the antianginal agent nitroglycerin (GTN). Investigational New Drug Approval was sought to administer oral solution doses of 1,3-GDN to a small number (n = 3) of healthy volunteers; each subject receiving three doses at 1.2, 2.4, and 3.6 mg. With volunteers confined to a semirecumbent posture for the duration of each treatment (4-hr period postdose), diastolic blood pressure (DBP) was recorded and plasma samples collected for 1,3-GDN concentration analysis. Appreciable concentration-related decreases in DBP were observed, with maximal decreases from predose baseline values approximating 11 to 25 mm Hg. For each subject parametric pharmacodynamic modeling was performed with simultaneous analysis utilizing the DBP vs. time data from all three doses; an inhibitory Emax pharmacodynamic model was adopted. The temporal relationship between plasma 1,3-GDN concentrations and DBP displayed rapid equilibration. For subjects 1, 2 and 3, respectively, Emax was predicted as 12.9, 23.4, and 29.7 mm Hg, representing 21.5, 31.6, and 39.5% decreases in DBP from predose baseline values; plasma concentrations at half Emax (C50) were 2.75, 2.43, and 5.93 micrograms/L. Utilizing pharmacokinetic-pharmacodynamic modeling, 1,3-GDN plasma concentrations appear to relate to a systemic "effect measure" that is mechanistically representative of the therapeutic actions of organic nitrates as peripheral vasodilators. The establishment of a GDN plasma concentration-effect relationship together with the relatively high plasma levels of GDN achieved following GTN dosing supports the hypothesis that the GDNs contribute significantly to the hemodynamic effect observed with GTN.

Administration, Oral↗
PubMed1993

Interpretation and utilization of effect and concentration data collected in an in vivo pharmacokinetic and in vitro pharmacodynamic study.

The effect (E) of some drugs may be monitored in vitro using the plasma drug concentration (C) following the in vivo dosing of drug. When using a specific analytical assay, counterclockwise hysteresis in the E vs C relationship can be explained only by the presence of an agonistic metabolite (MA); the extent of hysteresis will depend upon the pharmacokinetics and relative "potency" of C and MA. If a nonspecific assay is used, plots of E vs C may actually relate to E vs total agonist (C + MA), and unusual hysteresis may be observed, e.g., clockwise hysteresis when C is more "potent" than MA. Here, we simulate data for three models of relative C and MA pharmacokinetics. The E vs C and E vs MA data are simulated for both linear and noncompetitive agonist Emax models. When C and MA are equally potent, hysteresis will not be observed in a plot of E vs C + MA. However, when C and MA are of differing "potencies," hysteresis will be observed (the direction of hysteresis is dependent on the relative potency of C and MA). By appropriately "weighting" a respective agonist (C or MA), hysteresis will "collapse" and the relative potencies of C and MA can be estimated.

Models, Theoretical↗
PubMed1992

Improved gas chromatographic assay for the simultaneous determination of nitroglycerin and its mono- and dinitrate metabolites.

A sensitive, specific capillary gas chromatographic-electron-capture detection method for the simultaneous determination of nitroglycerin (GTN), 1,2- and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN, respectively) and 1- and 2-glyceryl mononitrate (1-GMN and 2-GMN, respectively) is reported. The minimum quantifiable concentration for GTN, GDNs and GMNs is 0.4 ng/ml in plasma, with extraction recoveries for GMNs greater than 76% and for GTN and the GDNs greater than 95%. Over the full range of quantifiable concentrations the inter-run assay precision and accuracy were less than 13 and 11%, respectively, for all five nitrates. Similar intra-run assay precision and accuracy values were found. The method was employed in the preliminary in vitro examination of GTN, GDN and GMN kinetics in human blood. Following addition of GTN to human blood, the ratio of 1,2-GDN to 1.3-GDN maximum concentrations (Cmax) was ca. 7:1, reflecting preferential denitration of the GTN molecule at the primary positions, while the Cmax ratio for 2-GMN to 1-GMN in this system was ca. 6:1, representing a highly selective if not specific primary denitration of the 1,2-GDN molecule. Following the intravenous administration of 1,2-GDN to five healthy male volunteers, 2-GMN/1-GMN Cmax ratios averaged 8.8:1, representing a highly selective but not specific formation of 2-GMN from the 1,2-GDN molecule. The assay will find utility in in vitro studies attempting to address the molecular pharmacology of GTN and its metabolites, and in in vivo clinical pharmacology studies attempting to address the relationship between pharmacokinetics and pharmacodynamics of GTN and its metabolites.

Chromatography, Gas↗
PubMed1992

Comparison of vasodilatory responses to nitroglycerin and its dinitrate metabolites in human veins.

Independent of the route of nitroglycerin administration, substantial amounts of 1,2-glyceryl dinitrate (1,2-GDN) and 1,3-glyceryl dinitrate (1,3-GDN) metabolites accumulate in humans. Thus far their pharmacologic activity in comparison to nitroglycerin in humans is unknown. To compare the venodilatory potency of nitroglycerin and of 1,2-GDN and 1,3-GDN in vivo, cumulative dose-response curves were established in nine healthy volunteers by use of the dorsal hand vein compliance technique. Separated by a washout period, two of the three venodilators were infused in randomized order after preconstriction with phenylephrine. Values for maximum vasodilation were similar for all compounds: nitroglycerin, 109%; 1,2-GDN, 100%; and 1,3-GDN, 106%. The respective values for the dose rate exerting 50% of maximum vasodilation were 5.1, 43, and 60 ng/min, indicating that the dinitrates were about 10 times less potent than nitroglycerin (p < 0.001) but not significantly different from each other. The findings support the hypothesis that activity of nitroglycerin metabolites is related to the number of nitrate groups in the molecule and are in agreement with lower dinitrate potencies found in animal experiments.

Adult↗
PubMed1991

Pharmacokinetic studies of the nitroglycerin metabolites, 1,2- and 1,3- glyceryl dinitrates, in the rat.

1,2- and 1,3-glyceryl dinitrates (1,2-GDN and 1,3-GDN) are the primary metabolites of glyceryl trinitrate, a commonly used anti-anginal agent. The goal of this study was to examine the pharmacokinetic properties of these metabolites in rats. Sprague-Dawley rats were infused intravenously with 0.25 or 2.0 micrograms min-1 of either 1,2- or 1,3-GDN for 70 min, during which steady state blood concentrations were achieved. Post-infusion blood samples were collected for 30 min. 1,2-GDN was found to possess slightly higher clearance (32.3 vs 20.8 ml min-1 kg-1) and volume of distribution (695 vs 454 ml kg-1) than 1,3-GDN; however, the two metabolites exhibited similar mean residence times (22.0 vs 21.8 min). Upon an 8-fold increase in the infusion rate, the pharmacokinetic parameters were not significantly altered for either 1,2- or 1,3-GDN. When each GDN was co-infused with an 8-fold higher dose of the other GDN, there were also no significant changes in the parameters.

Animals↗
PubMed1991

Percutaneous penetration kinetics of nitroglycerin and its dinitrate metabolites across hairless mouse skin in vitro.

The percutaneous penetration kinetics of the antianginal, nitroglycerin (GTN), and its primary metabolites, 1,2- and 1,3-glyceryl dinitrate (1,2- and 1,3-GDN), were evaluated in vitro, using full-thickness hairless mouse skin. GTN and the 1,2- and 1,3-GDNs were applied (a) in aqueous solution as pH 7.4 phosphate-buffered saline (PBS) and (b) incorporated into lipophilic ointment formulations. The cutaneous transformation of GTN to its dinitrate metabolites was detected, but no interconversion between 1,2-GDN and 1,3-GDN was observed. Following application of the nitrates in PBS solution, all three compounds exhibited steady-state transport kinetics. The steady-state flux of GTN (8.9 +/- 1.5 nmol cm-2 hr-1) was significantly greater (P less than 0.05) than those of 1,2-GDN (0.81 +/- 0.54 nmol cm-2 hr-1) and 1,3-GDN (0.72 +/- 0.20 nmol cm-2 hr-1). The corresponding permeability coefficient (rho) for GTN (20 +/- 3 x 10(-3) cm hr-1) was significantly larger than the corresponding values for 1,2-GDN (1.4 +/- 0.9 x 10(-3) cm hr-1) and 1,3-GDN (1.2 +/- 0.4 x 10(-3) cm hr-1), which were statistically indistinguishable (P greater than 0.05). Further analysis of the transport data showed that the differences between GTN and the GDNs could be explained by the relative stratum corneum/water partition coefficient (Ks) values of the compounds. The apparent partition parameters, defined as kappa = Ks.h [where h is the diffusion path length through stratum corneum (SC)] were 19.8 +/- 2.5 x 10(-2) cm for GTN and 1.91 +/- 1.07 x 10(-2) and 1.81 +/- 0.91 x 10(-2) cm for 1,2- and 1,3-GDN, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Cutaneous↗
PubMed1990

Differential effects of anesthetic regimens on gentamicin pharmacokinetics in the rat: a comparison with chronically catheterized conscious animals.

The intravenous disposition of gentamicin was compared in the conscious chronically catheterized rat with that in rats anesthetized using five injectable laboratory anesthetics. Gentamicin plasma clearance in the conscious rat was significantly higher than in animals anesthetized with urethane, fentanyl/fluanisone/midazolam, pentobarbitone, or ketamine/midazolam but similar to that in rats anesthetized with alphaxolone/alphadolone. Urethane anaesthesia resulted in a significantly lower gentamicin clearance than in all other groups. Gentamicin clearance in rats anesthetized with alphaxolone/alphadolone was significantly higher than in rats anesthetized with either fentanyl/fluanisone/midazolam or urethane. No significant differences in the volume of distribution of gentamicin were observed between any of the groups studied, either anesthetized or conscious. Carboxyinulin blood clearance in the conscious group was significantly higher than that with urethane, fentanyl/fluanisone/midazolam, pentobarbitone, or ketamine/midazolam but not significantly different from alphaxolone/alphadolone-anesthetized animals. The differences in carboxyinulin clearance were noted to be proportional to the differences in gentamicin clearance (r2 = 0.98). These results demonstrate that the choice of anesthetic used in laboratory pharmacokinetic studies is important. Gentamicin clearance was higher in conscious than anesthetized rats, and it may be prudent to use chronically catheterized animals in pharmacokinetic studies.

Anesthetics↗
PubMed1990

Differential influence of laboratory anaesthetic regimens upon renal and hepatosplanchnic haemodynamics in the rat.

Renal blood flow in rats anaesthetized with the combination alphaxolone/alphadolone (3.90 mL min-1 (g tissue)-1) was significantly (P less than 0.05) greater than in rats anaesthetized with ketamine midazolam (3.24 mL min-1 (g tissue)-1, pentobarbitone (3.19 mL min-1 (g tissue)-1), fentanyl/fluanisone midazolam (2.84 mL min-1 (g tissue)-1) or urethane (1.99 mL min-1 (g tissue)-1). Renal blood flow in the urethane anaesthetized rats was significantly (P less than 0.05) lower than in animals anaesthetized with the other anaesthetic regimens, and is consistent with literature reports of a depressive effect of urethane anaesthesia upon xenobiotic renal clearance in the rat. Hepatosplanchnic blood flow was highest in the alphaxolone/alphadolone anaesthetized animals (71.7 mL min-1 kg-1), with the urethane anaesthetized animals demonstrating a significantly (P less than 0.05) lower (33.4 mL min-1 kg-1) blood flow. The fentanyl fluanisone/midazolam (65.4 mL min-1 kg-1), pentobarbitone (61.1 mL min-1 kg-1), and ketamine/midazolam (51.4 mL min-1 kg-1) regimens resulted in hepatosplanchnic blood flows of intermediate magnitude. The observed marked differential effects of the anaesthetic regimens upon renal and hepatosplanchnic blood flows may dramatically influence drug disposition in the experimental animal, and be of significance to laboratory pharmacokinetic studies in which anaesthesia is used.

Anesthetics↗
PubMed1988

In vitro assessment of cephaloridine nephrotoxicity: comparison of renal cortical slice and renal tubule fragment techniques.

Renal cortical slices and a suspension of renal tubule fragments were prepared from male Wistar rats that had received a single s.c. dose of either cephaloridine (100 mg/kg) or normal saline (1 ml/kg) 48 h previously. The comparative sensitivity of these tissue preparations as in vitro models to assess nephrotoxin-induced changes in renal function was investigated by measuring the ability of the preparations to undertake the active accumulation of [3H]para-aminohippuric acid and to undertake gluconeogenesis from sodium pyruvate. Through the use of the cortical slice technique, [3H]para-aminohippuric acid tissue accumulation and glucose production in the cephaloridine-treated group were not significantly different from control. In contrast, using the tubule fragment technique, significant (p less than 0.05) reductions in the accumulation of [3H]para-aminohippuric acid and in the production of glucose via gluconeogenesis, between cephaloridine and normal saline control treatments were observed. Control values in the tubule fragment technique, for para-aminohippuric acid transport and glucose production via gluconeogenesis, were observed to be much greater than control values obtained from the cortical slice technique. It is suggested that the tubule fragment technique may be a more valuable in vitro preparation to assess the effects of potential nephrotoxins on tubule transport function than the still widely used cortical slice technique. The use of specific metabolic substrates, such as acetate, that will stimulate cellular metabolism and substrate transport, will also enhance the value of the technique.

Aminohippuric Acids↗