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Biomedical subjects

P Charlton

Publications and source records attributed to P Charlton.

At least 19 recordsLinked to original sources
PubMed2005

Upper limits on a stochastic background of gravitational waves.

The Laser Interferometer Gravitational-Wave Observatory has performed a third science run with much improved sensitivities of all three interferometers. We present an analysis of approximately 200 hours of data acquired during this run, used to search for a stochastic background of gravitational radiation. We place upper bounds on the energy density stored as gravitational radiation for three different spectral power laws. For the flat spectrum, our limit of omega0 < 8.4 x 10(-4) in the 69-156 Hz band is approximately 10(5) times lower than the previous result in this frequency range.

Journal Article↗
PubMed2005

Limits on gravitational-wave emission from selected pulsars using LIGO data.

We place direct upper limits on the amplitude of gravitational waves from 28 isolated radio pulsars by a coherent multidetector analysis of the data collected during the second science run of the LIGO interferometric detectors. These are the first direct upper limits for 26 of the 28 pulsars. We use coordinated radio observations for the first time to build radio-guided phase templates for the expected gravitational-wave signals. The unprecedented sensitivity of the detectors allows us to set strain upper limits as low as a few times 10(-24). These strain limits translate into limits on the equatorial ellipticities of the pulsars, which are smaller than 10(-5) for the four closest pulsars.

Journal Article↗
PubMed2001

Clinical protocol. A phase IIb, randomized, multicenter, double-blind study of the efficacy and safety of Trinam (EG004) in stenosis prevention at the graft-vein anastomosis site in dialysis patients.

Hemodialysis access complications remain a major cause of morbidity for patients with end-stage renal disease who are undergoing chronic hemodialysis. Vascular access complications occur in approximately 40% of patients with polytetrafluorethylene (PTFE) grafts within the first 6 months, primarily due to stenosis and thrombosis. Thrombosis at the site of vascular access increases the risk of infection and the need for hospitalization, and may lead to loss of potential new sites for vascular access. To a large extent, the failure of hemodialysis access is due to the rapid development of an intimal hyperplastic lesion in the region of anastomosis between the PTFE graft and the vein. The hospital costs related to hemodialysis access procedures are estimated to be around $1.3 billion per year and the total cost of hemodialysis complications to the US healthcare system is thought to be in excess of $2 billion per year. Ark Therapeutics Ltd. are developing a vascular endothelial growth factor D (VEGF-D) gene in an adenoviral vector which is delivered locally to the adventitial surface of a graft-vein anastomosis by means of a collagen collar device. The proposed indication for this product (Trinam) is the prevention of intimal hyperplasia at the graft-vein anastomosis site in patients who require vascular access to facilitate hemodialysis for end-stage renal disease. The rationale for Trinam to prevent intimal hyperplasia at the graft-vein anastomosis follows the discovery that VEGF has a 'vasculoprotective' action, resulting in inhibition of smooth muscle cell migration and proliferation. The fundamental mechanism for this vasculoprotective effect of VEGF, as distinct from its more widely appreciated 'angiogenic' role, is that VEGF acts on surface receptors on endothelial cells resulting in increased production of nitric oxide and prostacyclin. These entities diffuse into the media of the blood vessel wall and counter the tendency for intimal hyperplasia to develop. In an in vivo rabbit model of intimal thickening in carotid arteries, adventitial delivery of VEGF using a silastic collar as a gene delivery reservoir prevented smooth muscle cell proliferation without evidence of new blood vessel formation, indicating that the mechanism by which VEGF inhibited intimal hyperplasia did not involve angiogenesis. The objective of the proposed study is to assess the efficacy and safety of local delivery of Trinam when applied to the graft-vein anastomosis site in patients with end-stage renal disease who require vascular access for hemodialysis. At the time of surgical placement of a PTFE arm graft, patients will be randomized to either a single administration of Trinam or to 'no treatment' (i.e., control group). It is hypothesised that Trinam administration will result in less stenosis at the graft-vein anastomosis site (as measured by fistulography) compared with controls and therefore will reduce the need for interventions in dialysis patients. Approximately 210 patients will be enrolled from 10-15 centers and patients will be evaluated for efficacy and safety over 6 months. The total dose of Trinam will be 1 x 10(11) viral particles (replication-deficient adenoviral vector). This dose of Trinam was not associated with any significant toxicology findings in a preclinical study of pigs in which a PTFE loop-graft was anastomosed from the carotid artery to the internal jugular vein to mimic hemodialysis vascular access surgery.

Adenoviridae↗
PubMed2001

Dicationic bis(9-methylphenazine-1-carboxamides): relationships between biological activity and linker chain structure for a series of potent topoisomerase targeted anticancer drugs.

Bis(9-methylphenazine-1-carboxamides) joined by a variety of dicationic (CH(2))(n)()NR(CH(2))(m)NR(CH(2))(n) linkers of varying length (carboxamide N-N distances from 11.0 to 18.4 A) and rigidity were prepared by reaction of 9-methylphenazine-1-carboxylic acid imidazolide with the appropriate polyamines. The compounds were evaluated for growth inhibitory properties in P388 leukemia, Lewis lung carcinoma, and wild-type (JL(C)) and mutant (JL(A) and JL(D)) forms of human Jurkat leukemia with low levels of topoisomerase II (topo II). The compounds all had IC(50) ratios of <1 in the resistant Jurkat lines, consistent with topo II inhibition not being the primary mechanism of action. Analogues joined by an (CH(2))(2)NR(CH(2))(2)NR(CH(2))(2) linker were extremely potent cytotoxins, with selectivity toward the human cell lines, but absolute potencies declined sharply from R = H through R = Me to R = Pr and Bu. In contrast, (CH(2))(2)NR(CH(2))(3)NR(CH(2))(2) compounds showed reverse effects, with the R = Me analogue being more potent than the R = H one as well as being the most potent in the series [IC(50) in JL(C) cells 0.08 nM; superior to that for the clinical bis(naphthalimide) LU 79553]. Overall, the IC(50)s of analogues with linker chains (CH(2))(n)NH(CH(2))(m)NH(CH(2))(n) were inversely proportional to linker length. Constraining the rigidity of the linker chain by incorporating a piperazine ring did not decrease potency significantly. A representative compound bound tightly to DNA with high selectivity for GC sites, compatible with recent work suggesting that compounds of this type place their side chains in the major groove, making specific contacts with guanine bases. Representative compounds were susceptible to transport mediated resistance, being much less effective in cells that overexpressed P-glycoprotein. Overall the results suggest these compounds have a similar mode of action, mediated primarily by poisoning of topo I (possibly with some involvement of topo II). The bis(9-methylphenazine-1-carboxamides) show very high in vitro growth inhibitory potencies compared to their monomeric analogues. Two compounds showed in vivo activity in murine colon 38 syngeneic and HT29 human colon tumor xenograft models using intraperitoneal dosing.

Amides↗
PubMed2001

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576.

The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2000

Guanine specific binding at a DNA junction formed by d[CG(5-BrU)ACG](2) with a topoisomerase poison in the presence of Co(2+) ions.

The structure of the duplex d[CG(5-BrU)ACG](2) bound to 9-bromophenazine-4-carboxamide has been solved through MAD phasing at 2.0 A resolution. It shows an unexpected and previously unreported intercalation cavity stabilized by the drug and novel binding modes of Co(2+) ions at certain guanine N7 sites. For the intercalation cavity the terminal cytosine is rotated to pair with the guanine of a symmetry-related duplex to create a pseudo-Holliday junction geometry, with two such cavities linked through the minor groove interactions of the N2/N3 guanine sites at an angle of 40 degrees, creating a quadruplex-like structure. The mode of binding of the drug is shown to be disordered, with the major conformations showing the side chain bound to the N7 position of adjacent guanines. The other end of the duplex exhibits a terminal base fraying in the presence of Co(2+) ions linking symmetry-related guanines, causing the helices to intertwine through the minor groove. The stabilization of the structure by the intercalating drug shows that this class of compound may bind to DNA junctions as well as duplex DNA or to strand-nicked DNA ('hemi-intercalated'), as in the cleavable complex. This suggests a structural basis for the dual poisoning of topoisomerase I and II enzymes by this family of drugs.

Cations, Divalent↗
PubMed2000

Polymerization of plasminogen activator inhibitor-1.

The activity of the serine proteinase inhibitor (serpin) plasminogen activator inhibitor-1 (PAI-1) is controlled by the intramolecular incorporation of the reactive loop into beta-sheet A with the generation of an inactive latent species. Other members of the serpin superfamily can be pathologically inactivated by intermolecular linkage between the reactive loop of one molecule and beta-sheet A of a second to form chains of polymers associated with diverse diseases. It has long been believed that PAI-1 is unique among active serpins in that it does not form polymers. We show here that recombinant native and latent PAI-1 spontaneously form polymers in vitro at low pH although with distinctly different electrophoretic patterns of polymerization. The polymers of both the native and latent species differ from the typical loop-A-sheet polymers of other serpins in that they readily dissociate back to their original monomeric form. The findings with PAI-1 are compatible with different mechanisms of linkage, each involving beta-strand addition of the reactive loop to s7A in native PAI-1 and to s1C in latent PAI-1. Glycosylated native and latent PAI-1 can also form polymers under similar conditions, which may be of in vivo importance in the low pH environment of the platelet.

Amino Acid Sequence↗
PubMed2000

Drug binding sites on P-glycoprotein are altered by ATP binding prior to nucleotide hydrolysis.

P-glycoprotein (P-gp) confers multiple drug resistance on cancer cells by acting as a plasma membrane localized ATP-dependent drug efflux pump. Currently, there is little information on the nature of the communication between the energy-providing nucleotide binding domains (NBDs) and the drug binding sites of P-gp to generate transport of substrate. Many substrates and modulators cause alterations in ATP hydrolysis, but what effect do the various stages of the catalytic cycle have on drug interaction with P-gp? Vanadate trapping of Mg.ADP caused a reversible decrease in the binding capacity of the transported substrate [(3)H]-vinblastine and the nontransported modulator [(3)H]XR9576 to P-gp in CH(r)B30 cell membranes. The non-hydrolyzable nucleotide analogue ATP-gamma-S also caused a reduction in the binding capacity of [(3)H]-vinblastine but not for the modulator [(3)H]XR9576. This indicates that signaling to the NBDs following binding of a nontransported modulator is different to that transmitted upon interaction of a transported substrate. Second, it appears that the binding of nucleotide, rather than its hydrolysis, causes the initial conformational shift in the drug-binding site during a transport cycle.

4-Chloro-7-nitrobenzofurazan↗
PubMed2000

An investigation into the formation of N- [2-(dimethylamino)ethyl]acridine-4-carboxamide (DACA) and 6-[2-(dimethylamino)ethylamino]- 3-hydroxy-7H-indeno[2, 1-C]quinolin-7-one dihydrochloride (TAS-103) stabilised DNA topoisomerase I and II cleavable complexes in human leukaemia cells.

The antitumour agents DACA (XR5000; N-[2-(dimethylamino)ethyl]acridine-4-carboxamide) and TAS-103 (6-[2-(dimethylamino)ethylamino]-3-hydroxy-7H-indeno[2, 1-c]quinolin-7-one dihydrochloride) have been shown to inhibit two essential nuclear enzymes in vitro, DNA topoisomerase I and DNA topoisomerase (topo) II. To examine whether DACA or TAS-103 stabilise topo I, topo IIalpha, and topo IIbeta cleavable complexes in human leukaemia CCRF-CEM cells, the TARDIS assay (trapped in agarose DNA immunostaining) was used. This assay can reveal drug-stabilised topo-DNA complexes formed in situ in individual cells. The results showed that both DACA and TAS-103 can stabilise topo IIalpha cleavable complexes in these cells. Topo IIbeta cleavable complexes were also formed, but only at high concentrations of DACA and TAS-103. The effect on topo I was less clear, with TAS-103 showing only low levels of cleavable complex formation and DACA having no detectable effect under these assay conditions. This is in contrast to the purified enzyme cleavable complex assay, where both DACA and TAS-103 poisoned topo I. Although both DACA and TAS-103 show a preference for topo IIalpha in whole cells using the TARDIS assay, the formation of low levels of topo I or topo IIbeta cleavable complexes may still play a role in cell death.

Acridines↗
PubMed2000

Communication between multiple drug binding sites on P-glycoprotein.

P-glycoprotein, a member of the ATP-binding cassette transporter family, is able to confer resistance on tumors against a large number of functionally and chemically distinct cytotoxic compounds. Several recent investigations suggest that P-glycoprotein contains multiple drug binding sites rather than a single site of broad substrate specificity. In the present study, radioligand-binding techniques were used to directly characterize drug interaction sites on P-glycoprotein and how these multiple sites interact. The drugs used were classified as either 1) substrates, which are known to be transported by P-glycoprotein (e.g., vinblastine) or 2) modulators, which alter P-glycoprotein function but are not themselves transported by the protein (e.g., XR9576). Drug interactions with P-glycoprotein were either competitive, at a common site, or noncompetitive, and therefore at distinct sites. Based on these data, we can assign a minimum of four drug binding sites on P-glycoprotein. These sites fall into two categories: transport, at which translocation of drug across the membrane can occur, and regulatory sites, which modify P-glycoprotein function. Intriguingly, however, some modulators interact with P-glycoprotein at a transport site rather than a regulatory site. The pharmacological data also demonstrate that both transport and regulatory sites are able to switch between high- and low-affinity conformations. The multiple sites on P-glycoprotein display complex allosteric interactions through which interaction of drug at one site switches other sites between high- or low-affinity conformations. The data are discussed in terms of a model for the mechanism of transport by P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

In vivo efficacy of XR9051, a potent modulator of P-glycoprotein mediated multidrug resistance.

Overexpression of P-glycoprotein (P-gp) is a potential cause of multidrug resistance (MDR) in tumours. We have previously reported that XR9051 (N-(4-(2-(6,7-dimethoxy-1,2,3,4-tetrahydro-2-isoquinolyl)ethyl)phe nyl)-3-((3Z,6Z)-6-benzylidene-1-methyl-2,5-dioxo-3-pipera zinylidene)methylbenzamide) is a potent and specific inhibitor of P-gp, which reverses drug resistance in several murine and human MDR cell lines. In this study we have evaluated the in vivo efficacy of this novel modulator in a panel of murine and human tumour models and examined its pharmacokinetic profile. Efficacy studies in mice bearing MDR syngeneic tumours (P388/DX Johnson, MC26) or human tumour xenografts (A2780AD, CH1/DOXr, H69/LX) demonstrated that co-administration of XR9051 significantly potentiated the anti-tumour activity of a range of cytotoxic drugs. This modulatory activity was observed following parenteral and oral co-administration of XR9051. In addition, the combination schedules were well-tolerated. Following intravenous administration in mice, XR9051 is rapidly distributed and accumulates in tumours and other tissues. In addition, the compound is well-absorbed after oral administration. These data suggest that XR9051 has the potential for reversing clinical MDR mediated by P-glycoprotien.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

The molecular interaction of the high affinity reversal agent XR9576 with P-glycoprotein.

1 The kinetics and nature of equilibrium binding were used to characterize the molecular interaction of the anthranilic acid derivative [3H]-XR9576 with the multidrug resistance P-glycoprotein (P-gp). XR9576 displayed specific high-affinity binding to P-gp (Bmax = 275 pmol mg-1, Kd = 5.1 nM). The transport substrates [3H]-vinblastine and [3H]-paclitaxel displayed 4 fold and 20 fold lower affinity respectively for P-gp. The duration of action of XR9576 with P-gp was increased in comparison to that of vinblastine which displayed a slower rate of association and a faster dissociation rate. 2 The relative affinities of several modulators and transport substrates to interact with P-gp were determined from displacement drug equilibrium binding assays. Vinblastine and paclitaxel could only fractionally displace [3H]-XR9576 binding, displaying Ki values significantly different from their measured Kd values. This suggests a non-competitive interaction between XR9576 and the P-gp substrates vinblastine and paclitaxel. 3 XR9576 was shown to be a potent modulator of P-gp mediated [3H]-vinblastine and [3H]-paclitaxel transport as it increased the steady-state accumulation of these cytotoxics in CHrB30 cells to levels observed in non-P-gp-expressing AuxB1 cells (EC50 = 487+/-50 nM). This inhibition of drug transport is not mediated through competition for transport since [3H]-XR9576 accumulation was not influenced by P-gp expression or function. 4 These results demonstrate that the P-gp modulator XR9576 exhibits greater selectivity, duration of inhibition and potency of interaction with this transporter than any other reported modulators. Several lines of evidence suggest that XR9576 inhibits P-gp function by binding at a site which is distinct from the site of interaction of transport substrates. The two sites may be classified as serving modulatory or transport functions.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1999

Analgesia after intracranial surgery: a double-blind, prospective comparison of codeine and tramadol.

We have compared codeine and tramadol in a prospective, double-blind study of postoperative analgesia in 75 patients after elective intracranial surgery. Twenty-five patients received codeine 60 mg, tramadol 50 mg or tramadol 75 mg i.m. Patients receiving codeine had significantly lower pain scores over the first 48 h after operation (P < 0.0001). Although there was no difference in visual analogue scale (VAS) scores between the three groups at 24 h, the codeine group had significantly lower scores at 48 h (P < 0.0001). The tramadol 75 mg group had significantly higher scores for both sedation and nausea and vomiting (P < 0.0001 for both scores). We conclude that codeine 60 mg i.m. provided better postoperative analgesia than tramadol after craniotomy and that tramadol 75 mg should be avoided because of its side effects of increased sedation and nausea and vomiting.

Adolescent↗
PubMed1999

Comparison of low-dose epidural with combined spinal-epidural analgesia for labour.

We have performed a randomized comparison of two low-dose epidural regimens for analgesia in labour, differing only in the manner in which initial analgesia was established. In the epidural (EPI) group, 484 women received a loading dose of 20 ml of 0.1% bupivacaine with fentanyl 2 micrograms ml-1. In the combined spinal-epidural (CSE) group, 524 women received a spinal injection of plain bupivacaine 2.5 mg with fentanyl 25 micrograms. In both groups, these initial doses were followed by 0.1% bupivacaine with fentanyl 2 micrograms ml-1 infused at a rate of 12 ml h-1, with 20-ml top-ups for breakthrough pain. The groups were compared for midwife assessment of analgesic efficacy, delivery mode, patient assessments of first stage analgesia, second stage analgesia, overall analgesia, motor block and complications. Midwives, who were not blinded to the treatment groups, assessed 61.6% of CSE as providing 'excellent' analgesia compared with 56.4% of epidurals (P = 0.02). Patients assessed overall analgesia as 'excellent' in 74.8% of CSE compared with 71.7% of epidurals (P = 0.14). Other comparisons between groups revealed no differences. These findings may have been affected by an uneven distribution of multiparous women between the groups (25% in the EPI group and 34.2% in the CSE group; P = 0.002). However, subgroup analysis of primiparous and multiparous women did not alter the results.

Adolescent↗
PubMed1999

Preliminary clinical experience of a contracture correction device.

Joint contractures which do not respond to conventional physiotherapy can be difficult to treat. Serial plastering has been used effectively but is expensive, inconvenient to the patient and does not permit daily hygiene or clinical inspection. A mechanical device has been developed consisting a hinged orthosis which spans the affected joint to which is attached a gas strut to provide a corrective moment about the anatomical joint. Such an arrangement enables prescribed corrective moments to be applied accurately following clinical assessment using routine physiotherapy techniques. The inherently low spring rate of a gas strut ensures that the specified corrective torque is maintained as correction occurs. Initial treatment experience under the control of the developers had generated wider interest in the system. A geographically distant independent orthotic supply centre was trained in the techniques of application. They treated nine elbow and three knee joints in patients who had not responded to physiotherapy treatment. All of the patients experienced improvement. The average for elbow joints was a reduction in the contracture of 25.6 degrees with a corrective moment of 6.8 Nm over a period of 3.9 weeks. For the knee joints the averages were a reduction in contracture of 10.7 degrees with a corrective moment of 12.7 Nm over a period of 4 weeks. The results confirmed the practicality of transferring the system to independent clinical centres and provide evidence to support funding for a formal prospective clinical trial of the treatment approach.

Adult↗