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<p>Recombinant human erythropoietin (rHuEPO) is used effectively in the treatment of various anemic disorders. Belgrade rat is a useful animal model of anemia caused by defect in iron utilization. The objective of the present study was to investigate the dynamics of erythropoietic biomarkers in Belgrade rats receiving rHuEPO. Pharmacokinetics of rHuEPO was evaluated in Belgrade rats and normal rats after intravenous administration of single doses of the drug (100 and 1350 IU/kg). Pharmacodynamic biomarkers included levels of red blood cells, hemoglobin, and reticulocytes following administration of a single intravenous dose of rHuEPO (100 IU/kg). Red blood cell survival was assessed after treatment with rHuEPO (450 IU/kg), three times a week for 2 weeks. It was found that rHuEPO exhibited non-linear pharmacokinetics in both Belgrade and control rats. At the low dose, plasma concentrations and AUC (area under the curve) were significantly lower while clearance and volume of distribution were higher in Belgrade rats (p < 0.05). At the higher dose, there was no difference in pharmacokinetics between the two groups. Erythropoietic effect of rHuEPO was negligible in Belgrade rats at the dose of 100 IU/kg whereas all studied erythropoietic biomarkers were increased in normal rats. The levels of red blood cells, hemoglobin were significantly lower whereas the percentage of reticulocytes was higher in Belgrade rats compared to that in normal rats (p < 0.05). RHuEPO increased red blood cell survival in both animal groups. In conclusion, rHuEPO effect on erythropoietic biomarkers was stronger in normal rats than Belgrade rats at the studied doses. The findings from this study may provide further insights into understanding of anemic disorders resulting from mutations in the divalent metal transporter.</p

Data_Sheet_1.DOCX

<p>Recombinant human erythropoietin (rHuEPO) is used effectively in the treatment of various anemic disorders. Belgrade rat is a useful animal model of anemia caused by defect in iron utilization. The objective of the present study was to investigate the dynamics of erythropoietic biomarkers in Belgrade rats receiving rHuEPO. Pharmacokinetics of rHuEPO was evaluated in Belgrade rats and normal rats after intravenous administration of single doses of the drug (100 and 1350 IU/kg). Pharmacodynamic biomarkers included levels of red blood cells, hemoglobin, and reticulocytes following administration of a single intravenous dose of rHuEPO (100 IU/kg). Red blood cell survival was assessed after treatment with rHuEPO (450 IU/kg), three times a week for 2 weeks. It was found that rHuEPO exhibited non-linear pharmacokinetics in both Belgrade and control rats. At the low dose, plasma concentrations and AUC (area under the curve) were significantly lower while clearance and volume of distribution were higher in Belgrade rats (p < 0.05). At the higher dose, there was no difference in pharmacokinetics between the two groups. Erythropoietic effect of rHuEPO was negligible in Belgrade rats at the dose of 100 IU/kg whereas all studied erythropoietic biomarkers were increased in normal rats. The levels of red blood cells, hemoglobin were significantly lower whereas the percentage of reticulocytes was higher in Belgrade rats compared to that in normal rats (p < 0.05). RHuEPO increased red blood cell survival in both animal groups. In conclusion, rHuEPO effect on erythropoietic biomarkers was stronger in normal rats than Belgrade rats at the studied doses. The findings from this study may provide further insights into understanding of anemic disorders resulting from mutations in the divalent metal transporter.</p

Pentacyclic Nitrofurans with <i>In Vivo</i> Efficacy and Activity against Nonreplicating <i>Mycobacterium tuberculosis</i>

<div><p>The reductively activated nitroaromatic class of antimicrobials, which include nitroimidazole and the more metabolically labile nitrofuran antitubercular agents, have demonstrated some potential for development as therapeutics against dormant TB bacilli. In previous studies, the pharmacokinetic properties of nitrofuranyl isoxazolines were improved by incorporation of the outer ring elements of the antitubercular nitroimidazole OPC-67683. This successfully increased stability of the resulting pentacyclic nitrofuran lead compound Lee1106 (referred to herein as <b>9a</b>). In the current study, we report the synthesis and antimicrobial properties of <b>9a</b> and panel of <b>9a</b> analogs, which were developed to increase oral bioavailability. These hybrid nitrofurans remained potent inhibitors of <i>Mycobacterium tuberculosis</i> with favorable selectivity indices (>150) and a narrow spectrum of activity. <i>In vivo</i>, the pentacyclic nitrofuran compounds showed long half-lives and high volumes of distribution. Based on pharmacokinetic testing and lack of toxicity <i>in vivo,</i><b>9a</b> remained the series lead. <b>9a</b> exerted a lengthy post antibiotic effect and was highly active against nonreplicating <i>M. tuberculosis</i> grown under hypoxia. <b>9a</b> showed a low potential for cross resistance to current antitubercular agents, and a mechanism of activation distinct from pre-clinical tuberculosis candidates PA-824 and OPC-67683. Together these studies show that <b>9a</b> is a nanomolar inhibitor of actively growing as well as nonreplicating <i>M. tuberculosis</i>.</p></div

Murine model of acute tuberculosis infection.

<p>Log₁₀ reduction provided by compound <b>9a</b> in lungs (black bars) and spleen (grey bars) after 9 days of daily oral administration of 300 mg/kg was determined by calculating the difference between bacillary loads in organs from the untreated group and <b>9a</b> dissolved in (<b>1</b>) 0.5% methylcellulose in DI-H₂O (<b>2</b>) 30% captisol in DI-H₂O (<b>3</b>) 10% vitamin E TPGS in DI-H₂O (<b>4</b>) 0.5% Tween 80 in DI-H₂O (<b>5</b>) 20% cyclodextrin in DI-H₂O or (<b>6</b>) cold PEG (50∶35∶15 H₂O:PEG300:PG). Error bars indicate SEM within treatment groups of 5–7 mice per group.</p

In vitro activity against NRP bacteria grown under hypoxic conditions.

<p>Viability of hypoxic <i>M. tuberculosis</i> cultures was assessed using the rapid anaerobic dormancy model.</p

Known nitroaromatic antimicrobial drugs and nitrofurans explored in this study.

<p>Known nitroaromatic antimicrobial drugs and nitrofurans explored in this study.</p

Cross Resistance Profiles for Compounds 9a Resistant Clones.

<p>Abbreviations: S, susceptible; <b>R</b>, resistant.</p

MIC and MBCs for select nitrofurans and controls.

<p>Treatments were considered cidal when the MBC_99.9 for <i>M. tuberculosis</i> H37Rv was less than 4X the MIC.</p

Nutrient starvation model of nonreplicating persisters.

<p>The viability of mid-log phase (black bars) or nutrient-starved (gray bars) after exposure to DMSO carrier (1% v/v), 1 µg/mL of isoniazid (INH), or 1 µg/mL of <b>9a</b>. Averaged results and SEM from two biologically independent experiments are presented.</p

Synthesis of pentacyclic nitrofurans.

<p>Reagents and conditions: a) MsCl, Et₃N, CH₂Cl₂, RT, 2 h, 94%; b) substituted phenol, <i>n</i>Bu₄NCl, H₂O, 100°C, 12 h, 86–90%; c) TFA, CH₂Cl₂, RT, 1 h, 92–95%; d) aryl bromide, 2-(di-<i>tert</i>-butylphosphino)biphenyl, NaO<i>t</i>Bu, Pd(OAc)₂, toluene, 100°C, 3 h, 50–70%; e) <b>8</b>, Et₃N, CHCl₃, RT, 3 h, 50–68%.</p