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D A Smith

Publications and source records attributed to D A Smith.

At least 55 records · Page 3Linked to original sources
PubMed2001

Identification of the cytochrome P450 enzymes involved in the N-demethylation of sildenafil.

AIMS: To characterize the cytochrome P450 (CYP) enzymes responsible for the N-demethylation of sildenafil to its main metabolite, UK-103 320, to investigate the potential inhibitory effects of sildenafil on CYP enzymes and to evaluate the potential of selected drugs to affect sildenafil metabolism. METHODS: The metabolic pathways of sildenafil N-demethylation were studied using human liver microsomes, as well as microsomes expressing individual human CYP enzymes. Further studies to identify the individual enzymes were performed at 2.5 and 250 microM sildenafil, and employed a combination of chemical inhibition, correlation analysis, and metabolism by expressed recombinant CYP enzymes. In addition, the effect of sildenafil on the activity of the six major drug metabolizing enzymes was investigated. RESULTS: Sildenafil conversion was found to be mediated by at least two CYP enzymes, for which the mean kinetic parameters were Km1 = 6(+/-3 microM), Km2 = 81(+/-45 microM), Vmax1 = 22(+/-9 pmol) and Vmax2 = 138(+/-77 pmol) UK-103 320 formed min(-1) mg(-1). At 250 microM sildenafil, N-demethylation was primarily mediated through the low-affinity, high-Km enzyme (approximately 83%), whilst at 2.5 microM there was a greater role for the high-affinity, low-Km enzyme (approximately 61%). Ketoconazole strongly inhibited metabolism at both sildenafil concentrations and was the only significant inhibitor at 250 microM sildenafil. At the lower sildenafil concentration, sulphaphenazole and quinidine also inhibited formation of UK-103 320. Overall, 75% or more of the N-demethylation of sildenafil at any concentration is probably attributable to CYP3A4. These results were supported by experiments using expressed human CYP enzymes, in which only CYP3A4 and CYP2C9 exhibited substantial sildenafil N-demethylase activity (respective Km values of 221 microM and 27 microM). Sildenafil metabolism was inhibited by potent CYP3A4 inhibitors which are used clinically, but was found to be only a weak inhibitor of drug metabolizing enzymes itself, the strongest inhibition occurring against CYP2C9 (Ki = 80 microM). CONCLUSIONS: Evidence is provided for CYP3A4 and to a lesser extent CYP2C9-mediated metabolism of sildenafil. There is the possibility that elevated plasma concentrations of sildenafil could occur with coadministration of known inhibitors of CYP2C9 or CYP3A4. Since peak plasma concentrations of clinical doses of sildenafil are only 200 ng ml(-1) ( approximately 0.4 microM) it is very unlikely that sildenafil will significantly alter the plasma concentration of other compounds metabolized by cytochrome P450 enzymes.

Aryl Hydrocarbon Hydroxylases↗
PubMed2001

Characterization of auxotrophic mutants of Mycobacterium tuberculosis and their potential as vaccine candidates.

Auxotrophic mutants of Mycobacterium tuberculosis have been proposed as new vaccine candidates. We have analyzed the virulence and vaccine potential of M. tuberculosis strains containing defined mutations in genes involved in methionine (metB), proline (proC), or tryptophan (trpD) amino acid biosynthesis. The metB mutant was a prototrophic strain, whereas the proC and trpD mutants were auxotrophic for proline and tryptophan, respectively. Following infection of murine bone marrow-derived macrophages, H37Rv and the metB mutant strain survived intracellularly for over 10 days, whereas over 90% of proC and trpD mutants were killed during this time. In SCID mice, both H37Rv and the metB mutant were highly virulent, with mouse median survival times (MST) of 28.5 and 42 days, respectively. The proC mutant was significantly attenuated (MST, 130 days), whereas the trpD mutant was essentially avirulent in an immunocompromised host. Following infection of immunocompetent DBA mice with H37Rv, mice survived for a median of 83.5 days and the metB mutant now showed a clear reduction in virulence, with two of five infected mice surviving for 360 days. Both proC and trpD mutants were avirulent (MST of >360 days). In vaccination studies, prior infection with either the proC or trpD mutant gave protection equivalent (proC mutant) to or better (trpD mutant) than BCG against challenge with M. tuberculosis H37Rv. In summary, proC and trpD genes are essential for the virulence of M. tuberculosis, and mutants with disruptions in either of these genes show strong potential as vaccine candidates.

Animals↗
PubMed2001

Production of matrix metalloproteinases in response to mycobacterial infection.

Matrix metalloproteinases (MMPs) constitute a large family of enzymes with specificity for the various proteins of the extracellular matrix which are implicated in tissue remodeling processes and chronic inflammatory conditions. To investigate the role of MMPs in immunity to mycobacterial infections, we incubated murine peritoneal macrophages with viable Mycobacterium bovis BCG or Mycobacterium tuberculosis H37Rv and assayed MMP activity in the supernatants by zymography. Resting macrophages secreted only small amounts of MMP-9 (gelatinase B), but secretion increased dramatically in a dose-dependent manner in response to either BCG or M. tuberculosis in vitro. Incubation with mycobacteria also induced increased MMP-2 (gelatinase A) activity. Neutralization of tumor necrosis alpha (TNF-alpha), and to a lesser extent interleukin 18 (IL-18), substantially reduced MMP production in response to mycobacteria. Exogenous addition of TNF-alpha or IL-18 induced macrophages to express MMPs, even in the absence of bacteria. The immunoregulatory cytokines gamma interferon (IFN-gamma), IL-4, and IL-10 all suppressed BCG-induced MMP production, but through different mechanisms. IFN-gamma treatment increased macrophage secretion of TNF-alpha but still reduced their MMP activity. Conversely, IL-4 and IL-10 seemed to act by reducing the amount of TNF-alpha available to the macrophages. Finally, infection of BALB/c or severe combined immunodeficiency (SCID) mice with either BCG or M. tuberculosis induced substantial increases in MMP-9 activity in infected tissues. In conclusion, we show that mycobacterial infection induces MMP-9 activity both in vitro and in vivo and that this is regulated by TNF-alpha, IL-18, and IFN-gamma. These findings indicate a possible contribution of MMPs to tissue remodeling processes that occur in mycobacterial infections.

Animals↗
PubMed2001

Corneal lipid deposition in Cuban tree frogs (Osteopilus septentrionalis) and its relationship to serum lipids: an experimental study.

To evaluate the association between corneal lipid infiltration (corneal arcus) and dietary cholesterol in Cuban tree frogs (Osteopilus septentrionalis), 47 wild-caught frogs were fed diets of either regular or high-cholesterol crickets containing 0.7% and 1.7% cholesterol dry matter, respectively. Serum total cholesterol and triglycerides were measured when the frogs were euthanized after 17 mo. In a subsample of frogs, serum lipoproteins were characterized using high-performance liquid chromatography. The first case of corneal lipid deposition occurred in a female frog after 13 mo on the high-cholesterol diet. In the subsequent 4 mo, 5/11 males and 11/35 females developed the disease. Four of these affected frogs were females on the regular diet. Frogs with corneal lipid deposition had elevated serum total cholesterol (27.3 +/- 19.8 mmol/L) and low-density lipoproteins (LDL, 17.8 +/- 18.9 mmol/L) compared with unaffected captive frogs (16.5 +/- 20.4 and 9.0 +/- 7.6 mmol/L, respectively). Corneal lipid deposition was more prevalent in frogs on the high-cholesterol diet, and this group had higher serum total cholesterol (34.1 +/- 15.2 mmol/L in females, 22.8 +/- 14.8 mmol/L in males) than did frogs on the diet of regular crickets (12.3 +/- 8.7 mmol/L in females, 10.4 +/- 3.1 mmol/L in males). Captive frogs on both diets had higher serum total cholesterol than did wild frogs (3.1 +/- 2.1 mmo/L in females, 5.3 +/- 2.6 mmo/L in males). This additional serum cholesterol was primarily carried on very low-density lipoproteins (VLDL) and LDL rather than high-density lipoproteins (HDL), as indicated by the significantly higher ratio of VLDL cholesterol and LDL cholesterol over HDL cholesterol in captive frogs compared with wild frogs. Elevation in this ratio was significantly higher in captive females than in captive males. There was no evidence that increased serum cholesterol in captive females was directly related to the process of vitellogenesis.

Animals↗
PubMed2001

Hemispheric specialization in emotion: attention, arousal, and EEG activation in unilateral temporal lobe epilepsy.

To investigate the neural processing of emotion in right and left temporal lobe epilepsy (TLE), we monitored EEG during neutral and emotionally positive and negative auditory stimulation. Left TLE patients displayed increased right lateralization and a negative emotional stimulus rating bias, while right TLE patients did not. This EEG arousal and negative affect may reflect deactivated inhibition of the right hemisphere, symptomatically expressed as arousal and anxiety. Failure to increase lateralization to emotional stimuli in right TLE may reflect compromised emotional attention/arousal centers in the right hemisphere. Results support a two-dimensional theory of the neural processing of emotion.

Acoustic Stimulation↗
PubMed2001

Absorption, distribution, metabolism, and excretion considerations in selection of orally active indole-containing endothelin antagonist.

A series of potent indole-containing endothelin antagonists were evaluated in rat pharmacokinetic studies as part of a rational drug design program. Early compounds in this series were found to show poor gastrointestinal absorption, limiting their utility as oral agents. Structural modifications and pharmacokinetic studies indicated that reducing the overall H-bonding potential, through a reduction in the number of H-bond donors and acceptors, could increase absorption of the molecules. There was a correlation between calculated H-bonding capacity and rate of permeability across Caco-2 monolayers for this series of compounds. Caco-2 permeability was also shown to be indicative of the estimated extent of absorption in rats. Balancing the requirements of absorption and systemic clearance lead to the selection of an alcohol-containing compound, compound 7a (single enantiomer of compound 7) that was moderately absorbed after oral administration and converted to an active acid metabolite, which itself was of low intrinsic clearance. Species differences were observed between the absorption of compound 7a in rat and dog and also in the extent of conversion to the acid metabolite. Absorption was estimated at 30% in rat and 100% in dog. Approximately 30% of the absorbed drug was converted to systemically available acid metabolite in rat, compared with only 3% in dog.

Administration, Oral↗
PubMed2001

The registered nurse first assistant classroom that isn't really a classroom.

Faculty and students in the 21st century need to be skilled in the use of computer technology. In this registered nurse first assistant (RNFA) program, distance learning modalities are used to link students and faculty from different locales, expanding the accessibility of RNFA education. Independent study is used to stimulate critical thinking and skill acquisition in a safe, user friendly environment. The strategies described are simple yet powerful and can be similarly developed by faculty in other RNFA programs without the need for large budgetary expenditures or sophisticated technology.

Clinical Competence↗
PubMed2000

Protein folding: pulling back the frontiers.

In the past few years, it has become possible to measure the forces required to mechanically unfold single protein molecules. Recently, the mechanical properties of heteropolyproteins have been studied, shedding new light on the mechanical design of modular proteins such as titin.

Connectin↗
PubMed2000

Partially unfolded species populated during equilibrium denaturation of the beta-sheet protein Y74W apo-pseudoazurin.

Apo-pseudoazurin is a single domain cupredoxin. We have engineered a mutant in which a unique tryptophan replaces the tyrosine residue found in the tyrosine corner of this Greek key protein, a region that has been proposed to have an important role in folding. Equilibrium denaturation of Y74W apo-pseudoazurin demonstrated multistate unfolding in urea (pH 7.0, 0.5 M Na(2)SO(4) at 15 degrees C), in which one or more partially folded species are populated in 4. 3 M urea. Using a variety of biophysical techniques, we show that these species, on average, have lost a substantial portion of the native secondary structure, lack fixed tertiary packing involving tryptophan and tyrosine residues, are less compact than the native state as determined by fluorescence lifetimes and time-resolved anisotropy, but retain significant residual structure involving the trytophan residue. Peptides ranging in length from 11 to 30 residues encompassing this region, however, did not contain detectable nonrandom structure, suggesting that long-range interactions are important for stabilizing the equilibrium partially unfolded species in the intact protein. On the basis of these results, we suggest that the equilibrium denaturation of Y74W apo-pseudoazurin generates one or more partially unfolded species that are globally collapsed and retain elements of the native structure involving the newly introduced tryptophan residue. We speculate on the role of such intermediates in the generation of the complex Greek key fold.

Amino Acid Sequence↗
PubMed2000

Selecting the perioperative patient focused model.

The Project Team on a Perioperative Model was charged with finding or creating a theoretical model for perioperative nursing. Team members evaluated 15 existing theories and decided to use the conceptual model developed by AORN's Data Elements Coordinating Committee. The Perioperative patient Focused Model is logical, and the concepts and principles are supported in practice environments.

Decision Making↗
PubMed2000

Induction and drug development.

Enzyme induction is an undesirable drug interaction, effecting the efficacy of co-administered drugs, rather than safety. The number of clinically used drugs which induce P-450 enzymes is, in fact, quite limited. However, in certain disease areas (AIDS, epilepsy) many of the drugs used, whether for primary or secondary indications, have the potential for enzyme induction. Induction is often seen pre-clinically, due to the elevated dose levels used, but this potential rarely transfers to the clinical situation. Some screening of induction potential can be conducted by the use of human hepatocytes, but supply and variable response limits their use. Robust and routine screens for enzyme induction are being developed based on nuclear receptors. However, whilst genuine structure activity relationships may emerge, with these new technologies, none is evident from the clinical information other than in general structures are diverse but most are lipophilic as defined by a positive calculated LogP value. A critical factor in P-450 induction in the clinic, based on the known drugs, is the question of dose size. The major inducible form of P-450 in man is CYP3A4. The drugs that induce CYP3A4 are given in high doses. In contrast to these amounts, many clinically used drugs, which are non-inducers are effective at doses up two orders of magnitude lower. As a first rule for drug discovery/development programmes it seems prudent to obey the "Golden Rules" of drug design: "Ensure moderate daily dose size by having chosen a viable mechanism and then increase potency against the target whilst optimising pharmacokinetics". This approach is exemplified by the antidiabetic compound troglitzone, a clinical CYP3A4 inducer which has a clinical dose of 200-600 mg and rosiglitazone a more potent analogue dose (2-12 mg) which is devoid of CYP3A4 induction in the clinic.

Cytochrome P-450 CYP3A↗
PubMed2000

The role of P-glycoprotein in determining the oral absorption and clearance of the NK2 antagonist, UK-224,671.

UK-224,671 has been shown to exhibit low oral bioavailability in vivo due to poor absorption from the GI tract. The purpose of this study was to investigate the underlying reason for this observation. In Caco-2 cell flux experiments, the absorptive (A to B) flux of UK-224,671 was low, consistent with poor in vivo absorption. However, flux in the B to A direction was significantly greater, suggesting that UK-224,671 can permeate the membrane of the gut wall cell. Such a Caco-2 cell flux is indicative of transporter mediated efflux, possibly by P-glycoprotein. In P-glycoprotein knockout mice, the oral bioavailability of UK-224,671 was 22%, representing a significant increase over the P-glycoprotein expressing wild type mice (<2%). However, in the knockout mice absorption was still incomplete, suggesting that both P-glycoprotein mediated efflux and poor membrane permeation combine to limit the oral absorption of UK-224,671 in wild type mice. Lack of P-glycoprotein expression had no effect on the clearance of UK-224,671 in mice, which suggests that uptake from the blood into the excretory cell is mediated by a transporter other than P-glycoprotein. Bile duct cannulated rat experiments show that approximately 20% of the clearance of UK-224,671 occurs by direct secretion across the gut wall into the faeces. This clearance pathway requires UK-224,671 to cross both the basolateral and apical membranes of the gut wall cell. P-glycoprotein is likely to be involved in the passage of the compound across the apical membrane as has been observed for other P-glycoprotein substrates.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Protein folding mechanisms: new methods and emerging ideas.

During the past year, advances in our understanding of folding mechanisms have been made through detailed experimental and theoretical studies of a number of proteins. The development of new methods has allowed the earliest events in folding to be probed and the measurement of folding at the level of individual molecules is now possible, opening the door to exciting new experiments.

Animals↗
PubMed2000

Hepatocellular response to chemical stress in CD-1 mice: induction of early genes and gamma-glutamylcysteine synthetase.

Exposure of cells to toxic chemical species can result in reduced glutathione (GSH) depletion, generation of free radicals, and/or binding to critical cell determinants. Chemical stress is usually followed by a concerted cellular response aimed at restoring homeostasis, although the precise initial stimulus for the response is unclear. We have focused on one component of this stress response, the up-regulation of gamma-glutamylcysteine synthetase (gamma-GCS) and the preceding molecular events involved in its regulation in an in vivo mouse model. Male CD-1 mice received buthionine sulphoximine (BSO; 7.2 mmol/kg), diethyl maleate (DEM; 4.2 mmol/kg), paracetamol (APAP; 3.5 and 1.0 mmol/kg), or carbon tetrachloride (CCl(4); 1.0 and 0.2 mmol/kg). Biochemical (serum transaminase and hepatic GSH levels) and molecular (c-jun and c-fos messenger RNA [mRNA] levels and activator protein 1 [AP-1] DNA binding activity) parameters were measured, as well as the consequent effects on gamma-GCS levels and activity. All compounds produced GSH depletion, but only the higher doses of APAP and CCl(4) caused liver damage. DEM, APAP, and CCl(4) increased c-jun and c-fos mRNA levels, together with an increase in AP-1 binding; BSO failed to induce AP-1 despite an increase in c-fos. Interestingly, the effects on gamma-GCS varied markedly according to the compound: BSO and DEM increased gamma-GCS enzyme activity, although only DEM, but not BSO, resulted in an increase in gamma-GCS(h) mRNA and protein. In contrast, APAP and CCl(4) both increased gamma-GCS(h) mRNA and protein; however, there was a marked dose-dependent decrease in gamma-GCS activity. These data indicate that the effect of chemical stress on the liver is compound specific and is not merely dependent on depletion of GSH.

Acetaminophen↗
PubMed2000

An unusual renal tumour.

Primary renal lymphoma is a rare disease. The basis for diagnosis of this entity has been described and its existence is widely accepted. Localized deposition of amyloid is also a rare phenomenon. A case of primary lymphoma with localized amyloid deposition is reported here.

Aged↗
PubMed2000

Pharmacokinetics and metabolism of a sulphamide NK2 antagonist in rat, dog and human.

1. UK-224,671 is a sulphamide-containing NK2 antagonist with moderate lipophilicity and basicity. 2. The physicochemical properties of UK-224,671 are reflected in its pharmacokinetics following intravenous (i.v.) administration. The compound partitioned extensively into red blood cells in all species examined and the blood clearance was moderate to low with respect to liver blood flow and distribution into tissues was extensive. 3. UK-224,671 exhibited species differences in oral bioavailability. In dog, the compound exhibited moderate bioavailability (55%), whereas in rat and man oral bioavailability was < 10%. 4. In rat and dog, the major excreted form after i.v. administration was unchanged UK-224,671 in both urine and faeces. In addition, of three metabolites observed, the most abundant was the N-descyclopropylmethyl (UK-280,045). 5. The profile of radioactivity in rat following oral administration of [14C]-UK-224,671 was not consistent with a 10% absorbed compound with 40% of the dose present as metabolites. This suggests that the low bioavailability of UK-224,671 in rat is due to a combination of moderate intestinal permeability and extensive first-pass metabolism by the gut and does not result from poor gastrointestinal absorption per se.

Administration, Oral↗