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Biomedical subjects

R Hargreaves

Publications and source records attributed to R Hargreaves.

At least 19 recordsLinked to original sources
PubMed2005

Segregation in aqueous methanol enhanced by cooling and compression.

Molecular segregation in methanol-water mixtures is studied across a wide concentration range as a function of temperature and pressure. Cluster distributions obtained from both neutron diffraction and molecular dynamics simulations point to significantly enhanced segregation as the mixtures are cooled or compressed. This evolution toward greater molecular heterogenity in the mixture accounts for the observed changes in the water-water radial distribution function and there are indications also of a change in the topology of the water clusters. The observed behavior is consistent with an approach to an upper critical solution point. Such a point would appear to be "hidden" below the freezing line, thereby precluding observation of the two-fluid region.

Journal Article↗
PubMed2004

Methanol-water solutions: a bi-percolating liquid mixture.

An extensive series of neutron diffraction experiments and molecular dynamics simulations has shown that mixtures of methanol and water exhibit extended structures in solution despite the components being fully miscible in all proportions. Of particular interest is a concentration region (methanol mole fraction between 0.27 and 0.54) where both methanol and water appear to form separate, percolating networks. This is the concentration range where many transport properties and thermodynamic excess functions reach extremal values. The observed concentration dependence of several of these material properties of the solution may therefore have a structural origin.

Journal Article↗
PubMed2003

Differential involvement of neurotransmitters through the time course of cisplatin-induced emesis as revealed by therapy with specific receptor antagonists.

Advances in antiemetic therapy for chemotherapy-induced emesis have resulted in improved protection against symptoms occurring within 24 h of chemotherapy. However, the vomiting which tends to occur beyond 24 h after chemotherapy (delayed-phase vomiting) is still relatively poorly controlled by the currently available drugs, suggesting that more than one mechanism may mediate these symptoms. The standard antiemetic regimen currently recommended for prevention of chemotherapy-induced emesis includes a serotonin (5-HT(3)) antagonist and a corticosteroid. The neurokinin-1 (NK(1)) antagonist aprepitant represents a new class of antiemetic currently in clinical development. Using data obtained in 2 Phase II clinical trials of aprepitant in patients receiving chemotherapy based on the highly emetogenic chemotherapeutic agent cisplatin, we compared the time course of antiemetic effect of aprepitant, a 5-HT(3) antagonist, or a combination of both. Over the entire observation period (up to 7 days post-cisplatin), patients who received the NK(1) antagonist had a superior prevention of emesis. However, in the first 24 h after cisplatin, emesis occurred in fewer patients who received the 5-HT(3) antagonist than in patients who did not receive this class of drug. Furthermore, the majority of treatment failures in patients who received the NK(1) antagonist occurred within the first 8-12 h of chemotherapy, whereas the treatment failures in patients who received a 5-HT(3) antagonist were more evenly distributed over time. Patients who received both drugs had superior control of symptoms compared with patients who received one or the other. The difference in the time course of emesis blockade observed with two different classes of receptor antagonists provides substantial evidence for involvement of separate pathophysiological mechanisms in chemotherapy-induced vomiting. Serotonin mediates the early vomiting process that occurs within 8-12 h following cisplatin-based chemotherapy, after which time substance P acting at NK(1) receptors becomes the dominant mediator of vomiting

Antiemetics↗
PubMed2001

Structure-based development of anticancer drugs: complexes of NAD(P)H:quinone oxidoreductase 1 with chemotherapeutic quinones.

BACKGROUND: NAD(P)H:quinone acceptor oxidoreductase (QR1) protects animal cells from the deleterious and carcinogenic effects of quinones and other electrophiles. Remarkably, the same enzyme activates cancer prodrugs that become cytotoxic only after two-electron reduction. QR1's ability to bioactivate quinones and its elevated expression in many human solid tumors makes this protein an excellent target for enzyme-directed drug development. Until now, structural analysis of the mode of binding of chemotherapeutic compounds to QR1 was based on model building using the structures of complexes with simple substrates; no structure of complexes of QR1 with chemotherapeutic prodrugs had been reported. RESULTS: Here we report the high-resolution crystal structures of complexes of QR1 with three chemotherapeutic prodrugs: RH1, a water-soluble homolog of dimethylaziridinylbenzoquinone; EO9, an aziridinylindolequinone; and ARH019, another aziridinylindolequinone. The structures, determined to resolutions of 2.0 A, 2.5 A, and 1.86 A, respectively, were refined to R values below 21% with excellent geometry. CONCLUSIONS: The structures show that compounds can bind to QR1 in more than one orientation. Surprisingly, the two aziridinylindolequinones bind to the enzyme in different orientations. The results presented here reveal two new factors that must be taken into account in the design of prodrugs targeted for activation by QR1: the enzyme binding site is highly plastic and changes to accommodate binding of different substrates, and homologous drugs with different substituents may bind to QR1 in different orientations. These structural insights provide important clues for the optimization of chemotherapeutic compounds that utilize this reductive bioactivation pathway.

Antineoplastic Agents↗
PubMed2000

Phosphorylated morpholine acetal human neurokinin-1 receptor antagonists as water-soluble prodrugs.

The regioselective dibenzylphosphorylation of 2 followed by catalytic reduction in the presence of N-methyl-D-glucamine afforded 2-(S)-(1-(R)-(3, 5-bis(trifluoromethyl)phenyl)ethoxy)-3-(S)-(4-fluoro)phenyl-4-(5-(2- phosphoryl-3-oxo-4H,-1,2,4-triazolo)methylmorpholine, bis(N-methyl-D-glucamine) salt, 11. Incubation of 11 in rat, dog, and human plasma and in human hepatic subcellular fractions in vitro indicated that conversion to 2 would be expected to occur in vivo most readily in humans during hepatic circulation. Conversion of 11 to 2 occurred rapidly in vivo in the rat and dog with the levels of 11 being undetectable within 5 min after 1 and 8 mg/kg doses iv in the rat and within 15 min after 0.5, 2, and 32 mg/kg doses iv in the dog. Compound 11 has a 10-fold lower affinity for the human NK-1 receptor as compared to 2, but it is functionally equivalent to 2 in preclinical models of NK-1-mediated inflammation in the guinea pig and cisplatin-induced emesis in the ferret, indicating that 11 acts as a prodrug of 2. Based in part on these data, 11 was identified as a novel, water-soluble prodrug of the clinical candidate 2 suitable for intravenous administration in humans.

Acetals↗
PubMed1999

Possible antimigraine mechanisms of action of the 5HT1F receptor agonist LY334370.

This study investigated whether the selective 5HT1F receptor agonist LY334370 has other possible antimigraine mechanisms in addition to the proposed inhibition of dural plasma extravasation. LY334370 (up to 10(-5) M) had no vasoconstrictor effects on human cerebral arteries in vitro. It had no effect (up to 10 mg kg-1, i.v.) on neurogenic vasodilation of dural blood vessels produced by electrical stimulation of the dura mater in anesthetized rats. Nor had it any effect (at 3 mg kg-1, i.v.) on the hyperalgesia produced by injection of carrageenan into the paw of conscious rats or on nociceptive reflex responses in the spinalized, decerebrate rabbit (up to 3 mg kg-1, i.v.), indicating that it has no general analgesic properties. However, it significantly inhibited activation of second-order neurons in the trigeminal nucleus caudalis produced by electrical stimulation of the dura mater in anesthetised rats at 3 mg kg-1, i.v. These results provide evidence to suggest that LY334370 has a central mechanism of action in blocking the transmission of nociceptive impulses within the trigeminal nucleus caudalis and that this may represent a mechanism through which it has its antimigraine effect.

Animals↗
PubMed1998

Structural optimization affording 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4- (3-oxo-1,2,4-triazol-5-yl)methylmorpholine, a potent, orally active, long-acting morpholine acetal human NK-1 receptor antagonist.

Structural modifications requiring novel synthetic chemistry were made to the morpholine acetal human neurokinin-1 (hNK-1) receptor antagonist 4, and this resulted in the discovery of 2-(R)-(1-(R)-3, 5-bis(trifluoromethyl)phenylethoxy)-3-(S)-(4-fluoro)phenyl-4-(3-ox o-1 ,2,4-triazol-5-yl)methyl morpholine (17). This modified compound is a potent, long-acting hNK-1 receptor antagonist as evidenced by its ability to displace [125I]Substance P from hNK-1 receptors stably expressed in CHO cells (IC50 = 0.09 +/- 0.06 nM) and by the measurement of the rates of association (k1 = 2.8 +/- 1.1 x 10(8) M-1 min-1) and dissociation (k-1 = 0.0054 +/- 0.003 min-1) of 17 from hNK-1 expressed in Sf9 membranes which yields Kd = 19 +/- 12 pM and a t1/2 for receptor occupancy equal to 154 +/- 75 min. Inflammation in the guinea pig induced by a resiniferatoxin challenge (with NK-1 receptor activation mediating the subsequent increase in vascular permeability) is inhibited in a dose-dependent manner by the oral preadmininstration of 17 (IC50 (1 h) = 0.008 mg/kg; IC90 (24 h) = 1.8 mg/kg), indicating that this compound has good oral bioavailbility and peripheral duration of action. Central hNK-1 receptor stimulation is also inhibited by the systemic preadministration of 17 as shown by its ability to block an NK-1 agonist-induced foot tapping response in gerbils (IC50 (4 h) = 0.04 +/- 0.006 mg/kg; IC50 (24 h) = 0.33 +/- 0.017 mg/kg) and by its antiemetic actions in the ferret against cisplatin challenge. The activity of 17 at extended time points in these preclinical animal models sets it apart from earlier morpholine antagonists (such as 4), and the piperidine antagonists 2 and 3 and could prove to be an advantage in the treatment of chronic disorders related to the actions of Substance P. In part on the basis of these data, 17 has been identified as a potential clinical candidate for the treatment of peripheral pain, migraine, chemotherapy-induced emesis, and various psychiatric disorders.

Acetals↗
PubMed1997

Effect of plasma protein binding on in vivo activity and brain penetration of glycine/NMDA receptor antagonists.

A major issue in designing drugs as antagonists at the glycine site of the NMDA receptor has been to achieve good in vivo activity. A series of 4-hydroxyquinolone glycine antagonists was found to be active in the DBA/2 mouse anticonvulsant assay, but improvements in in vitro affinity were not mirrored by corresponding increases in anticonvulsant activity. Here we show that binding of the compounds to plasma protein limits their brain penetration. Relative binding to the major plasma protein, albumin, was measured in two different ways: by a radioligand binding experiment or using an HPLC assay, for a wide structural range of glycine/NMDA site ligands. These measures of plasma protein binding correlate well (r = 0.84), and the HPLC assay has been used extensively to quantify plasma protein binding. For the 4-hydroxyquinolone series, binding to plasma protein correlates (r = 0.92) with log P (octanol/pH 7.4 buffer) over a range of log P values from 0 to 5. The anticonvulsant activity increases with in vitro affinity, but the slope of a plot of pED50 versus pIC50 is low (0.40); taking plasma protein binding into account in this plot increases the slope to 0.60. This shows that binding to albumin in plasma reduces the amount of compound free to diffuse across the blood-brain barrier. Further evidence comes from three other experiments: (a) Direct measurements of brain/blood ratios for three compounds (2, 16, 26) show the ratio decreases with increasing log R. (b) Warfarin, which competes for albumin binding sites dose-dependently, decreased the ED50 of 26 for protection against seizures induced by NMDLA. (c) Direct measurements of brain penetration using an in situ brain perfusion model in rat to measure the amount of drug crossing the blood-brain barrier showed that compounds 2, 26, and 32 penetrate the brain well in the absence of plasma protein, but this is greatly reduced when the drug is delivered in plasma. In the 4-hydroxyquinolones glycine site binding affinity increases with lipophilicity of the 3-substituent up to a maximum at a log P around 3, then does not improve further. When combined with increasing protein binding, this gives a parabolic relationship between predicted in vivo activity and log P, with a maximum log P value of 2.39. Finally, the plasma protein binding studies have been extended to other series of glycine site antagonists, and its is shown that for a given log P these have similar protein binding to the 4-hydroxyquinolones, except for compounds that are not acidic. The results have implications for the design of novel glycine site antagonists, and it is suggested that it is necessary to either keep log P low or pKa high to obtain good central nervous system activity.

Animals↗
PubMed1997

In the absence of the invariant chain, HLA-DR molecules display a distinct array of peptides which is influenced by the presence or absence of HLA-DM.

The independent influences of invariant chain (Ii) and HLA-DM molecules on the array of naturally processed peptides displayed by HLA-DR molecules were studied using transfected cell lines. The absence of Ii led to an altered set of HLA-DR-bound peptides as judged by the discriminating responses of alloreactive T cell clones. While most T cell clones raised against DR+Ii+DM+ peripheral blood mononuclear cells (PBMC) failed to respond to DR+Ii-DM- cells, T cell clones raised against DR+Ii-DM- transfectants were not stimulated by DR+Ii+DM+ cells. Furthermore, coexpression of HLA-DM with HLA-DR1 in the absence of Ii augmented responses of anti-PBMC T cell clones but inhibited allorecognition by T cell clones raised against DR+Ii-DM- transfectants. The conformational integrity of the class II molecules, as judged by serology, suggests that the patterns of reactivity of the T cell clones reflect specificity for different alloantigen-bound peptides. Hence, discordant regulation of expression of major histocompatibility complex class II, Ii, and HLA-DM molecules in vivo may lead to the display of novel self-peptides and possible interruption of self-tolerance.

Antigen-Presenting Cells↗
PubMed1997

Antigen presentation by interferon-gamma-treated thyroid follicular cells inhibits interleukin-2 (IL-2) and supports IL-4 production by B7-dependent human T cells.

The consequence of recognition of antigen on antigen-presenting cells that are induced to express major histocompatibility complex (MHC) class II molecules following an inflammatory process is still not clear. In this study, we have investigated the outcome of antigen presentation by epithelial cells and we have used as a model thyroid follicular cells (TFC) that are known to express MHC class II molecules in autoimmune thyroid diseases and acquire the capacity to present autoantigens to T cells infiltrating the thyroid gland. The result show that MHC class II-expressing TFC were unable to stimulate a primary T cell alloresponse, using CD4+ T cells from three HLA-mismatched responders. Phenotypic analysis showed that TFC, after incubation with interferon-gamma, do not express the costimulatory molecules B7-1 (CD80) and -2 (CD86). Addition of murine DAP.3 cells expressing human B7-1 (DAP.3-B7) to cultures containing peripheral blood CD4+ T cells and DR1-expressing TFC led to a proliferative response, suggesting that the failure of TFC to stimulate a primary alloresponse was due to a lack of co-stimulation. Similarly, HLA-DR-restricted, influenza-specific T cell clones dependent on B7 for co-stimulation did not respond to peptide presented by TFC; again the lack of response could be overcome by co-culture of TFC with DAP.3-B7. Furthermore, recognition of antigen on TFC inhibited interleukin-2 (IL-2) production in the B7-dependent T cells. In contrast, in T helper type 0 (Th0) T cells, IL-4 release was not affected by TFC presentation. In addition, antigen presentation by TFC favored IL-4 production relative to IL-2 production by B7-independent Th0 clones. These results suggest that antigen presentation by MHC class II+ TFC may induce tolerance in autoreactive Th1 cells but may simultaneously favors a Th2 response in uncommitted T cells, and thereby support autoantibody production.

Adult↗
PubMed1997

CD34 counts to predict the adequate collection of peripheral blood progenitor cells.

An essential prerequisite for successful procurement of sufficient autologous peripheral blood progenitor cells (PBPC) for engraftment is the optimal timing of collection. A number of surrogate markers of peripheral blood progenitor cells were analysed to identify a single test which could predict the optimum time to harvest, providing at least 2 x 10(6) CD34+ cells/kg patient body weight. The study comprised 95 patients undergoing varied mobilisation regimens with chemotherapy and G-CSF for both solid tumours and haematological malignancies. One hundred and fifty-seven PBPC harvests were collected. Full blood counts (FBC) and CD34+ cell enumeration was performed on blood samples taken during the mobilisation period and immediately prior to leucapheresis (pre-harvest). All PBPC collections were assayed for colony-forming cells and CD34+ cells in addition to a FBC. The white cell count on the day of harvest showed only weak correlation with the total number of CD34+ cells in the collection (r = 0.30). In contrast, the absolute number of circulating CD34+ cells strongly correlated with the CD34+ cell and CFU-GM yield of the corresponding apheresis product. Provided the mobilisation sample contained > or =20 x 10(6) CD34+ cells/ml, 94% of single collections, performed the following day, contained > or =2 x 10(6) CD34+ cells/kg.

Adolescent↗
PubMed1996

Antigen presentation by T cells inhibits IL-2 production and induces IL-4 release due to altered cognate signals.

Conflicting results of the effects of Ag presentation by MHC class II-expressing T cells have been described. In some studies class II-expressing T cells have been shown to act as effective APCs, while others have reported that the recognition of Ag on the surface of another T cell inactivates IL-2 production. In this study we have investigated the mechanisms involved in Ag presentation by T cells. The results obtained suggest that 1) lack of costimulation is not responsible for the inhibitory effects of T cell Ag presentation on IL-2 production; the provision of costimulation by immobilized anti-CD28 Ab or by the addition of accessory cells failed to reverse the effects of T cell Ag presentation, but restored the response to immobilized anti-CD3; 2) T cell Ag presentation induced a minimal increase in intracellular Ca2+ compared with that induced by antigen-pulsed B cells; this difference in the calcium response is not explained by quantitative differences in ligand density between B cells and T cells; and 3) despite the weak calcium signal, T cell presentation supported IL-4 release in the absence of IL-2 production. Taken together these data suggest that T cell Ag presentation leads to altered TCR/CD3-transduced signals, which biases the T cell towards a Th2 phenotype.

Animals↗
PubMed1996

When to harvest peripheral-blood stem cells after mobilization therapy: prediction of CD34-positive cell yield by preceding day CD34-positive concentration in peripheral blood.

PURPOSE: To evaluate whether the CD34+ yield from a single peripheral-blood stem-cell (PBSC) harvest could be predicted by measurement of the patient's circulating WBC and CD34+ cell concentrations on the day before harvest. PATIENTS AND METHODS: Thirty-nine patients with hematologic or nonhematologic malignancy underwent 41 stem-cell mobilization episodes with cytotoxic chemotherapy and/or granulocyte colony-stimulating factor (G-CSF), and a total of 63 leukapheresis procedures were performed. Peripheral-blood samples were analyzed for WBC and CD34+ cell concentration both on the day before and the day of leukapheresis. RESULTS: The median WBC and CD34+ concentrations on the day preceding leukapheresis were 10.0 x 10(9)/L (range, 0.4 to 44.4) and 24.9 x 10(6)/L (range, 0.1 to 349.4), respectively. On the day of harvest, the corresponding figures were 15.1 x 10(9)/L (range, 1.5 to 52.6) and 29.3 x 10(6)/L (range, 0.1 to 543.1), respectively. The median CD34+ cell number collected in a single leukapheresis was 2.6 x 10(6)/kg body weight (range, 0.1 to 26.1). Both the preceding day (r = .84, P < .001) and harvest day (r = .95, P < .001) CD34+ circulating concentrations correlated significantly with the number of CD34+ cells per kilogram collected at leukapheresis. The correlation between CD34+ cells per kilogram collected and harvest day WBC count was also significant (r = .43, P <.001), but with the preceding day WBC count was nonsignificant. CONCLUSION: The number of CD34+ cells harvested in a single leukapheresis can be predicted by measurement of the preceding day peripheral-blood circulating CD34+ concentration, and on the basis of these data a table of probable CD34+ cell yield has been constructed. This correlation may facilitate the efficient organization of leukapheresis procedures.

Antigens, CD34↗
PubMed1995

DNase I hypersensitivity mapping and promoter polymorphism analysis of human C4.

Human complement component C4 is encoded by two structurally distinct loci in the major histocompatibility complex (MHC) class III region. The two isotypes, C4A and C4B, differ at only four residues in the C4d fragment, but C4 constitutes the most polymorphic of the complement components. It is not known, however, whether the regions involved in the regulation of C4 expression also display polymorphic variation. By using the technique of DNase I hypersensitivity mapping, we established that the only area of transcriptional activity for C4 in the hepatocyte cell line, HepG2, occurs approximately 500 base pairs upstream of the transcriptional start site. This region was found to be remarkably constant in sequence when analyzed in the context of differing MHC haplotypes including HLA B57, C4A6, C4B1, DR7, which has been correlated with reduced expression of the C4A isotype. Similarly, polymerase chain reaction followed by single-strand conformation polymorphism analysis failed to demonstrate any promoter polymorphisms in 103 individuals comprising 52 systemic lupus erythematosus patients and 51 healthy controls.

Base Sequence↗