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Biomedical subjects

G E Padbury

Publications and source records attributed to G E Padbury.

10 recordsLinked to original sources
PubMed1999

Equilibrium distribution of HIV antiviral drugs into human peripheral blood mononuclear cells (PBMC) is controlled by free drug concentration in the extracellular medium.

Effect of protein binding on the equilibrium distribution of selected HIV antiviral drugs into isolated human peripheral blood mononuclear cells (PBMC, mainly lymphocytes) was investigated. Human PBMC from a single healthy human donor were isolated, purified, and cryopreserved. Uptake of non-peptide HIV-1 protease inhibitors PNU-96988 and PNU-103017 by these cells in vitro was evaluated as a function of increasing concentration of human serum in the cell incubation media. Both PNU-96988 and PNU-103017 were extensively bound to serum proteins. Uptake/efflux kinetics were very rapid such that accumulation by the cells was thermodynamically, not kinetically, controlled. Accumulation by human PBMCs in vitro was directly proportional to the free and not the total drug concentration in the media. For comparative purposes, the serum protein binding effect on the distribution of two HIV reverse transcriptase (RT) inhibitors, delavirdine (RESCRIPTOR) and zidovudine (AZT), was also evaluated. Like the HIV-1 protease inhibitors, delavirdine was found to be extensively associated with serum proteins and its accumulation by human PBMCs in vitro to be proportional to the free and not total drug concentration. In contrast, AZT was not bound to serum proteins to any significant extent. The uptake of this drug by human PBMCs in vitro was independent of serum concentration. However, the intrinsic cellular accumulation of PNU-96988, PNU-103017 and delavirdine were all greater than AZT. Thus, the extent to which drugs uptake by cells is affected by serum appears proportional to the binding affinity of the serum proteins for the drug.

Adult↗
PubMed1999

Increased lipophilicity and subsequent cell partitioning decrease passive transcellular diffusion of novel, highly lipophilic antioxidants.

Oxidative stress is considered a cause or propagator of acute and chronic disorders of the central nervous system. Novel 2, 4-diamino-pyrrolo[2,3-d]pyrimidines are potent inhibitors of iron-dependent lipid peroxidation, are cytoprotective in cell culture models of oxidative injury, and are neuroprotective in brain injury and ischemia models. The selection of lead candidates from this series required that they reach target cells deep within brain tissue in efficacious amounts after oral dosing. A homologous series of 26 highly lipophilic pyrrolopyrimidines was examined using cultured cell monolayers to understand the structure-permeability relationship and to use this information to predict brain penetration and residence time. Pyrrolopyrimidines were shown to be a more permeable structural class of membrane-interactive antioxidants where transepithelial permeability was inversely related to lipophilicity or to cell partitioning. Pyrrole substitutions influence cell partitioning where bulky hydrophobic groups increased partitioning and decreased permeability and smaller hydrophobic groups and more hydrophilic groups, especially those capable of weak hydrogen bonding, decreased partitioning, and increased permeability. Transmonolayer diffusion for these membrane-interactive antioxidants was limited mostly by desorption from the receiver-side membrane into the buffer. Thus, in this case, these in vitro cell monolayer models do not adequately mimic the in vivo situation by underestimating in vivo bioavailability of highly lipophilic compounds unless acceptors, such as serum proteins, are added to the receiving buffer.

Animals↗
PubMed1998

Species-dependent enantioselective pharmacokinetics of PNU-103017, a pyrone HIV protease inhibitor.

PNU-103017, 4-Cyano-N-(3-(cyclopropyl(5,6,7,8,9,10-hexahydro-4-hydroxy- 2-oxo-2H-cycloocta(b) pyran-3-yl)methyl)phenyl)-benzenesulfonamide, is a selective HIV aspartyl protease inhibitor under evaluation as a potential oral treatment of Acquired Immunodeficiency Diseases. PNU-103017 is a racemic mixture of two enantiomers, designated PNU-103264 (R-) and PNU-103265 (S-). Stereoselective pharmacokinetics of the two enantiomers of PNU-103017 were observed in the dog, rat, and human after single and multiple dose administration of the racemate and were apparently species-dependent. Mean enantiomeric ratios of plasma concentrations (R-/S-) at each time point were greater than 1 in the dog, ranging from 1.22 to 3.06, but less than 1 in the rat and in the human, ranging from 0.44 to 0.80 and 0.23 to 0.73, respectively. A trend towards increased or decreased (farther from 1:1, R-/S-) enantiomeric ratio of plasma concentrations with time after each administration was also observed. The enantiomeric ratio remained unchanged after multiple dose administration in the rat, dog, and human although enzyme induction and increased plasma clearance were observed for both enantiomers.

Animals↗
PubMed1997

Contribution of serum protein association to discrepancy between the in vivo and in vitro UDS results for 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843).

U-89843 has been shown to undergo biotransformation, both in vitro and in vivo, to form U-97924 as a major primary metabolite. U-89843 was found to be positive in an in vitro UDS mutagenesis screen conducted with primary rat hepatocytes in serum-free media. In contrast to in vitro results, no evidence of genetic toxicity of U-89843 was observed in rats in the in vivo/in vitro version of the UDS test with single oral doses up to 1400 mg/kg. The negative results may be related to more robust in vivo detoxification mechanisms or relatively lower exposure to reactive metabolites formed by bioactivation of U-89843 as compared to that observed in the serum-free in vitro hepatocyte test system. Further studies showed rat serum suppressed the in vitro metabolism of U-89843 as well as the formation of the corresponding hydroxylated metabolite, U-97924, the putative precursor of proposed reactive electrophilic metabolite. The measured in vivo systemic clearance of U-89843 (0.53 l/h/kg) in rats was about 1000-fold slower than the in vitro intrinsic clearance (606 l/h/kg) estimated by measuring the formation of U-97924 in rat liver microsomal incubations. Since U-89843 is extensively associated with serum proteins a poor extraction ratio into the liver may account for the slower biotransformation of U-89843 in vivo as compared to that exhibited in in vitro serum-free hepatocyte incubations. Addition of bovine serum albumin (1-40 mg/ml) to the in vitro UDS assay medium decreased the UDS mean net grains per nucleus response of U-89843. These results suggest that the effect of serum protein should be considered when comparing serum-free in vitro UDS and in vivo UDS results for highly serum protein bound compounds.

Animals↗
PubMed1997

Pyrrolopyrimidines: novel brain-penetrating antioxidants with neuroprotective activity in brain injury and ischemia models.

A novel group of antioxidant compounds, the pyrrolopyrimidines, has been discovered recently. Many of these possess significantly improved oral bioavailability (56-70% in rats), increased efficacy and potency in protecting cultured neurons against iron-induced lipid peroxidative injury and as much as a 5-fold increase in brain uptake compared with the 21-aminosteroid antioxidant compound, tirilazad mesylate (U-74006F), described earlier. They appear to quench lipid peroxidation reactions by electron-donating and/or radical-trapping mechanisms. Several compounds in the series, such as U-101033E and U-104067F, demonstrate greater ability than tirilazad to protect the hippocampal CA1 region in the gerbil transient (5-min) forebrain ischemia model. Delaying treatment until 4 hr after the ischemic insult still results in significant CA1 neuronal protection. U-101033E is still effective in salvaging a portion of the CA1 neuronal population when the ischemic duration is extended to 10 min. In addition, U-101033E has been found to be protective in the context of focal cerebral ischemia, reducing infarct size in the mouse permanent middle cerebral artery occlusion model, in contrast to tirilazad which is minimally effective. These results suggest that antioxidant compounds with improved brain parenchymal penetration are better able to limit certain types of ischemic brain damage than those which are localized in the cerebral microvasculature. However, the activity of U-101033E in improving early post-traumatic recovery in mice subjected to severe concussive head injury is similar to that of tirilazad. Last, the oral bioavailability of many pyrrolopyrimidines suggests that they may be useful for certain chronic neurodegenerative disorders in which lipid peroxidation plays a role.

Animals↗
PubMed1996

Bioactivation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) to reactive intermediates that bind covalently to macromolecules and produce genotoxicity.

U-89843 is a novel pyrrolo[2,3-d]pyrimidine antioxidant with prophylactic activity in animal models of lung inflammation. During preclinical safety evaluation, U-89843 was found to give a positive response in the in vitro unscheduled DNA synthesis (UDS) assay, an assay which measures DNA repair following chemically-induced DNA damage in metabolically competent rat hepatocytes. Incubation of [14C]U-89843 with liver microsomes resulted in covalent binding of radioactive material to macromolecules by a process that was NADPH-dependent. U-89843 has been shown to undergo C-6 methylhydroxylation to give U-97924, in rat both in vivo and in vitro, in a reaction catalyzed by cytochrome P450 2C11. Synthetical U-97924 is chemically reactive and undergoes dimerization in aqueous solution. The dimerization of U-97924 was significantly inhibited by addition of nucleophiles such as methanol, glutathione, and N-acetylcysteine. Characterization of the corresponding methanol, glutathione, and N-acetylcysteine adducts of U-97924 supported the hypothesis of a reaction pathway involving reactive iminium species formed via dehydration of U-97924. The metabolism-dependent irreversible covalent binding of radioactive material to liver microsomal protein and DNA also is dramatically reduced in the presence of reduced glutathione (GSH). A trifluoromethyl analog of U-89843 was prepared in an effort to block the corresponding metabolic hydroxylation pathway. This new compound (U-107634) was found to be negative in the in vitro UDS assay, and its metabolic susceptibility toward hydroxylation at the C-6 methyl group was eliminated. These observations suggest that the positive in vitro UDS results of U-89843 are mediated by the bioactivation of U-89843, leading to reactive electrophilic intermediates derived from the (hydroxymethyl)pyrrole metabolite U-97924.

Animals↗
PubMed1996

In vitro and in vivo biotransformation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-D]pyrimidine (U-89843) in the rat.

The biotransformation of 6,7-dimethyl-2,4-di-1-pyrrolidinyl-7H-pyrrolo[2,3-d]pyrimidine (U-89843) has been studied in rat both in vitro and in vivo. Major metabolites observed by HPLC analysis of rat plasma, liver cytosol, and microsomal incubations were characterized by UV, LC/MS, and comparison with synthetic standards. The structures of the metabolites were shown to be the C-6 hydroxymethyl (U-97924), C-6 formyl (U-97865), and C-6 carboxyl analogs of U-89843. In the male rat, formation of U-97924 is mediated by cytochrome P4502C11. Kinetic analysis of U-97924 formation indicated that it was a high-affinity/high-capacity process (KM = 4.2 +/- 0.5 microM; Vmax = 21.2 +/- 0.8 nmol/mg/min). Formation of U-97865 via enzymatic oxidation from the primary metabolite U-97924 was catalyzed by both the microsomal subcellular fraction in a NADPH-dependent process (presumably via cytochrome P450) and in cytosol by NAD(+)-dependent alcohol dehydrogenase. Upon incubation with cytosolic fractions, U-97865 was found to undergo NAD(+)-dependent oxidation, mediated by aldehyde dehydrogenase, to the corresponding carboxylic acid. Although significant levels of U-89843, U-97924, and U-97865 were observed in vivo in rat plasma, only a minor amount of the carboxylic acid together with larger amounts of unidentified polar metabolites were excreted in urine and feces.

Administration, Oral↗
PubMed1994

Fully automated high-performance liquid chromatographic method for the determination of carzelesin (U-80,244) and metabolites (U-76,073 and U-76,074) in human plasma.

Carzelesin (U80,244, I) is a cyclopropylpyrroloindole prodrug analog. The compound exerts its cytotoxic activity after conversion, via U-76,073 (II) to U-76,074 (III) by binding to the DNA minor groove in a sequence selective fashion. In pre-clinical investigations the drug displayed a broad spectrum activity against human xenografts in mice. To enable pharmacokinetic monitoring during the phase I clinical trials, we have developed a selective and sensitive assay for the parent compound and its two metabolites. Sample pre-treatment has been automated by using the ASPEC system and involves solid-phase extraction of the diluted plasma sample (1:3 in 20% v/v acetic acid) on an SPE-C18 precolumn followed by two consecutive washings with water and acetonitrile. The compounds are eluted with 600 microliters of dimethylacetamide and 500 microliters is injected on a Spherisorb-CN column. The sample is chromatographed using a linear gradient from 24% to 60% (v/v) acetonitrile in 20 mM phosphate buffer (pH 6.5). The eluate fraction containing the three compounds is heart-cutted in a 2-ml sample loop, switched onto a Spherisorb-ODS column and separation is accomplished using a mobile phase of acetonitrile-20 mM phosphate buffer pH 6.5 (64:36, v/v). UV detection is used with absorbance monitored at 360 nm. This highly selective method offers a lower limit of detection of less than 1 ng/ml for each of the compounds using 1000 microliters of plasma and enables the quantification of the analytes with an acceptable precision and accuracy over a range of 1 to 50 ng/ml. The assay has been implemented in a phase I clinical trial.

Antineoplastic Agents↗
PubMed1992

Cytotoxicity and antitumor activity of carzelesin, a prodrug cyclopropylpyrroloindole analogue.

The cyclopropylpyrroloindole analogues are DNA minor-groove binders containing a cyclopropyl group, which mediates N3-adenine covalent adduct formation in a sequence-selective fashion. Carzelesin (U-80244) is a cyclopropylpyrroloindole prodrug containing a relatively nonreactive chloromethyl precursor to the cyclopropyl function. Activation of carzelesin requires two steps, (a) hydrolysis of a phenylurethane substituent to form U-76073, followed by (b) ring closure to form the cyclopropyl-containing DNA-reactive U-76074. The formation of the DNA-reactive U-76074, via U-76073, from carzelesin was shown to proceed very slowly in phosphate-buffered saline (t1/2 greater than 24 h) but to occur rapidly in plasma from mouse, rat, dog, and human (initial t1/2 values ranging from 18 min for mouse to 52 min for rat) and in cell culture medium (t1/2 approximately 40 min). Although carzelesin was less potent in terms of in vitro cytotoxicity and in vivo optimal dosage and showed low affinity for binding to DNA, it was therapeutically more efficacious against mouse L1210 leukemia than was U-76074 or adozelesin (U-73975), another cyclopropylpyrroloindole analogue which is currently in phase I clinical trials. Carzelesin also proved to be more efficacious than U-76074 or adozelesin against mouse pancreatic ductal 02 adenocarcinoma, a system reported to be resistant to every agent tested. Carzelesin was highly effective against this tumor and produced 97% tumor growth inhibition. In addition, i.v. administered carzelesin showed significant activity (National Cancer Institute criteria) against i.v. or s.c. implanted Lewis lung carcinoma, i.p. or s.c. implanted B16 melanoma, s.c. implanted colon 38 carcinoma, and five s.c. implanted human tumor xenografts, including clear cell Caki-1 carcinoma, colon CX-1 adenocarcinoma, lung LX-1 tumor, ovarian 2780 carcinoma, and prostatic DU-145 carcinoma. Carzelesin treatment produced 100% complete remissions (no palpable tumor mass at the termination of the experiment) in mice bearing early-stage human ovarian 2780. Pharmacologically, carzelesin proved to be relatively schedule and route independent and was highly active against i.p. implanted L1210 leukemia, regardless of whether the analogue was given i.v., i.p., s.c., or p.o. These results, collectively, suggest that carzelesin is absorbed and distributed well. Both carzelesin and adozelesin caused marked tumor shrinkage in mice bearing human lung LX-1 or advanced-stage human ovarian 2780 carcinoma; however, tumor regrowth occurred shortly after the treatment with adozelesin was stopped. Little or no apparent tumor regrowth occurred after treatment with carzelesin.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenocarcinoma↗