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Abstract: Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N2-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N2-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram− bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5–10μg/ml), and were inactive against the Gram− organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice. [Copyright &y& Elsevier]

Objectives. This study sought to determine the rate of emergency department use among the elderly and examined whether that use is reduced if the patient has a principal-care physician. Methods. The Health Care Financing Administration's National Claims History File was used to study emergency department use by Medicare patients older than 65 years in Washington State during 1994. Results. A total of 18.1% of patients had 1 or more emergency department visits during the study year; the rate increased with age and illness severity. Patients with principal-care physicians were much less likely to use the emergency department for every category of disease severity. After case mix, Medicaid eligibility, and rural/urban residence were controlled for, the odds ratio for having any emergency department visit was 0.47 for patients with a generalist principal-care physician and 0.58 for patients with a specialist principal-care physician. Conclusions. The rate of emergency department use among the elderly is substantial, and most visits are for serious medical problems. The presence of a continuous relationship with a physician--regardless of specialty--may reduce emergency department use. (Am d Public Health. 2000;90:97-102) [ABSTRACT FROM AUTHOR]

Studies N2-(4-n-butylphenyl)-2'-deoxy-3,5'-cycloguanosine by one-dimensional 1H and 13C, two-dimensional TOCSY, HMQC and HMBC ang long range selective INEPT nuclear magnetic resonance experiments. Temperature and solvent dependence of equilibrium; Resonance assignments.

Introduction This paper describes a novel technique, Fluorosomes, applied to investigating the interaction of antimicrobials with proton driven microbial efflux transporters. These transporters remove toxic compounds from the cytoplasm, including antibiotics and are involved in antibiotic resistance. Methods To assess transporter activity we developed a methodology to generate a proton gradient across Fluorosome membranes into which selected purified fully active efflux transporters were reconstituted. The interior of the Fluorosome particle (a unilamellar liposome) contains a fluorescent drug sensing probe whose fluorescence is quantitatively quenched by transporter substrates. Using an injecting fluorescence plate reader to initiate a proton gradient and to monitor subsequent fluorescence change, real time transport kinetics can be followed and transport inhibition characterized. Results Fluorosomes containing the E scherichia coli EmrE efflux pump demonstrated transport of two known EmrE substrates, ethidium and methyl viologen upon creation of a proton gradient. For Fluorosomes containing the inactive EmrE mutant, E14Q, no transport was observed. When the gradient was fully collapsed by the addition of nigericin, full inhibition of substrate transport was observed. The IC 50 for nigericin inhibition of ethidium was shown to be 0.71 μM. Discussion We have for the first time prepared and validated a single bacterial efflux pump assay, Fluorosome- trans -EmrE, that faithfully mimics properties of the transporter in vivo. It is faster than whole cell screens, simple to use, amenable to robotics, and reports on very specific targets. We have demonstrated proof of principle with EmrE and have created the first of an intended series of proton driven Fluorosomes. [ABSTRACT FROM AUTHOR]

Background: Emergence of drug-resistant Plasmodium falciparum has created an urgent need for new drug targets. DNA polymerase δ is an essential enzyme required for chromosomal DNA replication and repair, and therefore may be a potential target for anti-malarial drug development. However, little is known of the characteristics and function of this P. falciparum enzyme. Methods: The coding sequences of DNA polymerase δ catalytic subunit (PfPolδ-cat), DNA polymerase δ small subunit (PfPolδS) and proliferating cell nuclear antigen (PfPCNA) from chloroquine- and pyrimethamine-resistant P. falciparum strain K1% were amplified, cloned into an expression vector and expressed in Escherichia coli. The recombinant proteins were analysed by SDS-PAGE and identified by LC-MS/MS. PfPolδ-cat was biochemically characterized. The roles of PfPolδS and PfPCNA in PfPolδ-cat function were investigated. In addition, inhibitory effects of 1%1% compounds were tested on PfPolδ-cat activity and on in vitro parasite growth using SYBR Green I assay. Results: The purified recombinant protein PfPolδ-cat, PfPolδS and PfPCNA showed on SDS-PAGE the expected size of 1%4%3%, 5%7% and 3%4% kDa, respectively. Predicted amino acid sequence of the PfPolδ-cat and PfPolδS had 5%9%.2% and 2%4%.7% % similarity respectively to that of the human counterpart. The PfPolδ-cat possessed both DNA polymerase and 3%'-5%' exonuclease activities. It used both Mg2%+ and Mn2%+ as cofactors and was inhibited by high KCl salt (>2%0%0% mM). PfPolδS stimulated PfPolδ-cat activity threefolds and up to fourfolds when PfPCNA was included in the assay. Only two compounds were potent inhibitors of PfPolδ-cat, namely, butylphenyl-dGTP (BuPdGTP; IC5%0% of 3%8% μM) and 7%-acetoxypentyl-( 3%, 4% dichlorobenzyl) guanine (7%-acetoxypentyl-DCBG; IC5%0% of 5%5% μM). The latter compound showed higher inhibition on parasite growth (IC5%0% of 4%.1% μM). Conclusions: Recombinant PfPolδ-cat, PfPolδS and PfPCNA were successfully expressed and purified. PfPolS and PfPCNA increased DNA polymerase activity of PfPolδ-cat. The high sensitivity of PfPolδ to BuPdGTP can be used to differentiate parasite enzyme from mammalian and human counterparts. Interestingly, 7%-acetoxypentyl-DCBG showed inhibitory effects on both enzyme activity and parasite growth. Thus, 7%-acetoxypentyl-DCBG is a potential candidate for future development of a new class of anti-malarial agents targeting parasite replicative DNA polymerase. [ABSTRACT FROM AUTHOR]

Currently approved platelet adenosine diphosphate (ADP) receptor antagonists target only the platelet P2Y 12 receptor. Moreover, especially in patients with acute coronary syndromes, there is a strong need for rapidly acting and reversible antiplatelet agents in order to minimize the risk of thrombotic events and bleeding complications. In this study, a series of new P 1 ,P 4 -di(adenosine-5′) tetraphosphate (Ap 4 A) derivatives with modifications in the base and in the tetraphosphate chain were synthesized and evaluated with respect to their effects on platelet aggregation and function of the platelet P2Y 1 , P2Y 12 , and P2X1 receptors. The resulting structure–activity relationships were used to design Ap 4 A analogs which inhibit human platelet aggregation by simultaneously antagonizing both P2Y 1 and P2Y 12 platelet receptors. Unlike Ap 4 A, the analogs do not activate platelet P2X1 receptors. Furthermore, the new compounds exhibit fast onset and offset of action and are significantly more stable than Ap 4 A to degradation in plasma, thus presenting a new promising class of antiplatelet agents. [ABSTRACT FROM AUTHOR]

Background Based on resistance of currently used anti-malarials, a new anti-malarial drug target against Plasmodium falciparum is urgently needed. Damaged DNA cannot be transcribed without prior DNA repair; therefore, uracil-DNA glycosylase, playing an important role in base excision repair, may act as a candidate for a new anti-malarial drug target. Methods Initially, the native PfUDG from parasite crude extract was partially purified using two columns, and the glycosylase activity was monitored. The existence of malarial UDG activity prompted the recombinant expression of PfUDG for further characterization. The PfUDG from chloroquine and pyrimethamine resistant P. falciparum strain K1 was amplified, cloned into the expression vector, and expressed in Escherichia coli. The recombinant PfUDG was analysed by SDS-PAGE and identified by LC-MS/MS. The three dimensional structure was modelled. Biochemical properties were characterized. Inhibitory effects of 12 uracilderivatives on PfUDG activity were investigated. Inhibition of parasite growth was determined in vitro using SYBR Green I and compared with results from human cytotoxicity tests. Results The native PfUDG was partially purified with a specific activity of 1,811.7 units/mg (113.2 fold purification). After cloning of 966-bp PCR product, the 40-kDa hexa-histidine tagged PfUDG was expressed and identified. The amino acid sequence of PfUDG showed only 24.8% similarity compared with the human enzyme. The biochemical characteristics of PfUDGs were quite similar. They were inhibited by uracil glycosylase inhibitor protein as found in other organisms. Interestingly, recombinant PfUDG was inhibited by two uracilderived compounds; 1-methoxyethyl-6-(p-n-octylanilino)uracil (IC50 of 16.75 μM) and 6- (phenylhydrazino)uracil (IC50 of 77.5 μM). Both compounds also inhibited parasite growth with IC50s of 15.6 and 12.8 μM, respectively. Moreover, 1-methoxyethyl-6-(p-noctylanilino) uracil was not toxic to HepG2 cells, with IC50 of > 160 μM while 6- (phenylhydrazino)uracil exhibited cytoxicity, with IC50 of 27.5 μM. Conclusions The recombinant PfUDG was expressed, characterized and compared to partially purified native PfUDG. Their characteristics were not significantly different. PfUDG differs from human enzyme in its size and predicted amino acid sequence. Two uracil derivatives inhibited PfUDG and parasite growth; however, only one non-cytotoxic compound was found. Therefore, this selective compound can act as a lead compound for anti-malarial development in the future. [ABSTRACT FROM AUTHOR]

Background: Diadenosine tetraphosphate (Ap4A), a constituent of platelet dense granules, and its P1,P4-dithio and/or P2,P3-chloromethylene analogs, inhibit adenosine diphosphate (ADP)-induced platelet aggregation. We recently reported that these compounds antagonize both platelet ADP receptors, P2Y1 and P2Y12. The most active of those analogs, diadenosine 5′,5″″-P1,P4-dithio-P2,P3-chloromethylenetetraphosphate, (compound 1), exists as a mixture of 4 stereoisomers. Objective: To separate the stereoisomers of compound 1 and determine their effects on platelet aggregation, platelet P2Y1 and P2Y12 receptor antagonism, and their metabolism in human plasma. Methods: We separated the 4 diastereomers of compound 1 by preparative reversed-phase chromatography, and studied their effect on ADP-induced platelet aggregation, P2Y1-mediated changes in cytosolic Ca2+, P2Y12-mediated changes in VASP phosphorylation, and metabolism in human plasma. Results: The inhibition of ADP-induced human platelet aggregation and human platelet P2Y12 receptor, and stability in human plasma strongly depended on the stereo-configuration of the chiral P1- and P4-phosphorothioate groups, the SPSP diastereomer being the most potent inhibitor and completely resistant to degradation in plasma, and the RPRP diastereomer being the least potent inhibitor and with the lowest plasma stability. The inhibitory activity of SPRP diastereomers depended on the configuration of the pseudo-asymmetric carbon of the P2,P3-chloromethylene group, one of the configurations being significantly more active than the other. Their plasma stability did not differ significantly, being intermediate to that of the SPSP and the RPRP diastereomers. Conclusions: The presently-described stereoisomers have utility for structural, mechanistic, and drug development studies of dual antagonists of platelet P2Y1 and P2Y12 receptors. [ABSTRACT FROM AUTHOR]

Abstract: Introduction: P-Glycoprotein (ABCB1, MDR1) is a multidrug efflux pump that is a member of the ATP-binding cassette (ABC) superfamily. Many drugs in common clinical use are either substrates or inhibitors of this transporter. Quantitative details of P-glycoprotein inhibition by pharmaceutical agents are essential for assessment of their pharmacokinetic behavior and prevention of negative patient reactions. Cell-based systems have been widely used for determination of drug interactions with P-glycoprotein, but they suffer from several disadvantages, and results are often widely variable between laboratories. We aimed to demonstrate that a novel liposomal system employing contemporary biochemical methodologies could measure the ability of clinically used drugs to inhibit the P-glycoprotein pump. To accomplish this we compared results with those of cell-based approaches. Methods: Purified transport-competent hamster Abcb1a P-glycoprotein was reconstituted into a unilamellar liposomal system, Fluorosome-trans-pgp, whose aqueous interior contains fluorescent drug sensors. This provides a well-defined system for measuring P-glycoprotein transport inhibition by test drugs in real time using rapid fluorescence-based technology. Results: Inhibition of ATP-driven transport by Fluorosome-trans-pgp employed a panel of 46 representative drugs. Resulting IC50 values correlated well (r2 =0.80) with Kd values for drug binding to purified P-glycoprotein. They also showed a similar trend to transport inhibition data obtained using LLC-MDR1 cell monolayers. Fluorosome-trans-pgp IC50 values were in agreement with published results of digoxin drug–drug interaction studies in humans. Discussion: This novel approach using a liposomal system and fluorescence-based technology is shown to be suitable to study whether marketed drugs and drug candidates are P-glycoprotein inhibitors. The assay is rapid, allowing a 7-point IC50 determination in <6min, and requires minimal quantities of test drug. The method is amenable to robotics and offers a cost advantage relative to conventional cell-based assays. The well-defined nature of this assay also obviates many of the inherent complications and ambiguities of cell-based systems. [Copyright &y& Elsevier]

Abstract: Introduction: Diadenosine 5'',5''''''-P1,P4- tetraphosphate (Ap4A) is stored in platelet dense granules, but its effects on platelet function are not well understood. Methods and Results: We examined the effects of Ap4A on platelet purinergic receptors P2Y1, P2Y12 and P2X1. Flow cytometry was used to measure the effects of Ap4A in the presence or absence of ADP on: a) P2Y12-mediated decrease in intraplatelet phosphorylated vasodilator stimulated phosphoprotein (VASP), b) P2Y1-mediated increase in platelet cytosolic Ca2+, and c) P2X1-mediated intraplatelet entry of extracellular Ca2+. ADP-stimulated platelet shape change (P2Y1-mediated) and aggregation (P2Y1- and P2Y12-mediated) were measured optically. Ap4A inhibited 3μM ADP-induced: a) platelet aggregation (IC50 9.8±2.8μM), b) P2Y1-mediated shape change, c) P2Y1-mediated increase in platelet cytosolic Ca2+ (IC50 40.8±12.3μM), and d) P2Y12-mediated decrease in VASP phosphorylation (IC50 >250μM). In the absence of added ADP, Ap4A had agonist effects on platelet P2X1 and P2Y12, but not P2Y1, receptors. Conclusion: Ap4A, a constituent of platelet dense granules, is a) an antagonist of platelet P2Y1 and P2Y12 receptors, where it inhibits the effects of ADP, and b) an agonist of platelet P2X1 and P2Y12 receptors. [Copyright &y& Elsevier]

Background: Black-white disparities exist in receipt of recommended medical care, including colorectal cancer treatment. This retrospective cohort study examines the degree to which health systems (e.g., physician, hospital) factors explain black-white disparities in colon cancer care.Methods: Data from the Surveillance, Epidemiology, and End Results program; Medicare claims; the American Medical Association Masterfile; and hospital surveys were linked to examine chemotherapy receipt after stage III colon cancer resection among 5294 elderly (> or = 66 years of age) black and white Medicare-insured patients. Logistic regression analysis was used to identify factors associated with black-white differences in chemotherapy use. All statistical tests were two-sided.Results: Black and white patients were equally likely to consult with a medical oncologist, but among patients who had such a consultation, black patients were less likely than white patients (59.3% versus 70.4%, difference = 10.9%, 95% confidence interval [CI] = 5.1% to 16.4%, P < .001) to receive chemotherapy. This black-white disparity was highest among patients aged 66-70 years (black patients 65.7%, white patients 86.3%, difference = 20.6%, 95% CI = 10.7% to 30.4%, P < .001) and decreased with age. The disparity among patients aged 66-70 years also remained statistically significant in the regression analysis. Overall, patient, physician, hospital, and environmental factors accounted for approximately 50% of the disparity in chemotherapy receipt among patients aged 66-70 years; surgical length of stay and neighborhood socioeconomic status accounted for approximately 27% of the disparity in this age group, and health systems factors accounted for 12%.Conclusions: Black and white Medicare-insured colon cancer patients have an equal opportunity to learn about adjuvant chemotherapy from a medical oncologist but do not receive chemotherapy equally. Little disparity was explained by health systems; more was explained by illness severity, social support, and environment. Further qualitative research is needed to understand the factors that influence the lower receipt of chemotherapy by black patients. [ABSTRACT FROM AUTHOR]

Background: Black-white disparities exist in receipt of recommended medical care, including colorectal cancer treatment. This retrospective cohort study examines the degree to which health systems (e.g., physician, hospital) factors explain black-white disparities in colon cancer care. Methods: Data from the Surveillance, Epidemiology, and End Results program; Medicare claims; the American Medical Association Masterfile; and hospital surveys were linked to examine chemotherapy receipt after stage III colon cancer resection among 5294 elderly (⩾66 years of age) black and white Medicare-insured patients. Logistic regression analysis was used to identify factors associated with black-white differences in chemotherapy use. All statistical tests were two-sided. Results: Black and white patients were equally likely to consult with a medical oncologist, but among patients who had such a consultation, black patients were less likely than white patients (59.3% versus 70.4%, difference = 10.9%, 95% confidence interval [CI] = 5.1% to 16.4%, P<.001) to receive chemotherapy. This black-white disparity was highest among patients aged 66-70 years (black patients 65.7%, white patients 86.3%, difference = 20.6%, 95% CI = 10.7% to 30.4%, P<.001) and decreased with age. The disparity among patients aged 66-70 years also remained statistically significant in the regression analysis. Overall, patient, physician, hospital, and environmental factors accounted for approximately 50% of the disparity in chemotherapy receipt among patients aged 66-70 years; surgical length of stay and neighborhood socioeconomic status accounted for approximately 27% of the disparity in this age group, and health systems factors accounted for 12%. Conclusions: Black and white Medicare-insured colon cancer patients have an equal opportunity to learn about adjuvant chemotherapy from a medical oncologist but do not receive chemotherapy equally. Little disparity was explained by health systems; more was explained by illness severity, social support, and environment. Further qualitative research is needed to understand the factors that influence the lower receipt of chemotherapy by black patients. [ABSTRACT FROM AUTHOR]

Objective: To evaluate the influence of community- and family-level factors on racial/ethnic disparities in the uptake of nongroup (individual) health insurance.Data Sources: Responses to the 1996-1997 Community Tracking Study Household Survey plus community-level descriptors from several sources including census data, the Area Resource File, and community and migrant health center Medicare cost reports.Study Design: Logistic regression was used to compare families in which at least one person had nongroup health insurance to families without nongroup insurance in which at least one person was uninsured. Sequential models were constructed examining family- and community-level factors.Results: Twenty-three percent of families with otherwise-uninsured persons purchased nongroup insurance, ranging from 11% to 41% among the 60 communities sampled. Disadvantaged minority group members, especially Spanish-speaking Hispanics, had half or less the odds of whites of purchasing nongroup insurance. Education had a weaker association with purchasing nongroup insurance among minority group members than among whites. Community-level factors had minimal effect on disparities in uptake, although greater housing segregation was associated with lower uptake among blacks.Conclusions: Minority group members are much less likely to purchase nongroup insurance than whites. Family income and community factors do not explain this gap. Programs aimed at stimulating voluntary insurance purchase will continue to underenroll disadvantaged minorities if nonfinancial barriers to acquiring insurance coverage, including the interplay between race/ethnicity and education, are not better understood and addressed. [ABSTRACT FROM AUTHOR]

Objective. To evaluate the influence of community– and family–level factors on racial/ethnic disparities in the uptake of nongroup (individual) health insurance. Data Sources. Responses to the 1996–1997 Community Tracking Study Household Survey plus community–level descriptors from several sources including census data, the Area Resource File, and community and migrant health center Medicare cost reports. Study Design. Logistic regression was used to compare families in which at least one person had nongroup health insurance to families without nongroup insurance in which at least one person was uninsured. Sequential models were constructed examining family– and community–level factors. Results. Twenty–three percent of families with otherwise–uninsured persons purchased nongroup insurance, ranging from 11% to 41% among the 60 communities sampled. Disadvantaged minority group members, especially Spanish–speaking Hispanics, had half or less the odds of whites of purchasing nongroup insurance. Education had a weaker association with purchasing nongroup insurance among minority group members than among whites. Community–level factors had minimal effect on disparities in uptake, although greater housing segregation was associated with lower uptake among blacks. Conclusions. Minority group members are much less likely to purchase nongroup insurance than whites. Family income and community factors do not explain this gap. Programs aimed at stimulating voluntary insurance purchase will continue to underenroll disadvantaged minorities if nonfinancial barriers to acquiring insurance coverage, including the interplay between race/ethnicity and education, are not better understood and addressed. [ABSTRACT FROM AUTHOR]

OBJECTIVE Our goal was to compare the quality of diabetic care received by patients in rural and urban communities. STUDY DESIGN We performed a retrospective analysis of claims data captured by the Medicare program. POPULATION We included all fee-for-service Medicare patients 65 years and older living in the state of Washington who had 2 or more physician encounters for diabetes care during 1994. OUTCOME MEASURES The outcomes were the extent to which patients received 3 specific recommended services: glycated hemoglobin determination, cholesterol measurement, and eye examination. RESULTS A total of 30,589 Medicare patients (8.4%) were considered to have diabetes; 29.1% lived in rural communities. Generalists provided most diabetic care in all locations. Patients living in small rural towns received almost half their outpatient care in larger communities. Patients living in large rural towns remote from metropolitan areas were more likely to have received the recommended tests than patients in all other groups. Patients who saw an endocrinologist at least once during the year were more likely to have received the recommended tests. CONCLUSIONS Large rural towns may provide the best conditions for high-quality care: They are vibrant, rapidly growing communities that serve as regional referral centers and have an adequate--but not excessive--supply of both generalist and specialist physicians. Generalists provide most diabetic care in all settings, and consultation with an endocrinologist may improve adherence to guidelines. [ABSTRACT FROM AUTHOR]

Dual antiplatelet therapy with aspirin and an adenosine diphosphate (ADP) P2Y12 receptor antagonist reduces ischemic events in patients with acute coronary syndrome. Previous evidence from our group, obtained in a preclinical model of recurrent platelet-mediated thrombosis, demonstrated that GLS-409, a diadenosine tetraphosphate derivative that inhibits both P2Y1 and P2Y12 ADP receptors, may be a novel and promising antiplatelet drug candidate. However, the salutary antiplatelet effects of GLS-409 were accompanied by a trend toward an unfavorable increase in bleeding. The goals of this study were to: 1) provide proof-of-concept that the efficacy of GLS-409 may be maintained at lower dose(s), not accompanied by an increased propensity to bleeding; and 2) establish the extent and kinetics of the reversibility of human platelet inhibition by the agent. Lower doses of GLS-409 were identified that inhibited in vivo recurrent coronary thrombosis with no increase in bleeding time. Human platelet inhibition by GLS-409 was reversible, with rapid recovery of platelet reactivity to ADP, as measured by platelet surface activated GPIIb-IIIa and platelet surface P-selectin. These data support the concept that GLS-409 warrants further, larger-scale investigation as a novel, potential therapy in acute coronary syndromes. [ABSTRACT FROM AUTHOR]