The Fucosylation Inhibitor, 2-Fluorofucose, Inhibits Vaso-Occlusion, Leukocyte-Endothelium Interactions and NF-ĸB Activation in Transgenic Sickle Mice

<div><p>2-Fluorofucose (2FF) blocks the fucosylation and the tethering of sialyl-Lewis^x tetrasaccharide and structural variants on leukocytes and red blood cells to P- and E-selectins on activated endothelial cell surfaces. Because P- and E-selectin are required for vaso-occlusion in murine sickle cell disease (SCD), we investigated whether 2FF would inhibit vaso-occlusion in SCD mice. Microvascular stasis was measured in subcutaneous venules in NY1DD and HbSS-Townes SCD mice with dorsal skin-fold chambers after infusion of hemoglobin or exposure to hypoxia/reoxygenation. 2FF in drinking water or administered by gavage inhibited stasis in sickle mice in a dose-responsive manner. Significant inhibitory effects on stasis were seen 1 day post-treatment. 2FF treatment of SCD mice also significantly reduced leukocyte rolling and adhesion along the vessel walls of SCD mice and the static adhesion of neutrophils and sickle red blood cells isolated from 2FF-treated SCD mice to resting and activated endothelial cells. Total white blood cell counts increased in response to 2FF. NF-ĸB activation and VCAM-1 and E-selectin expression were inhibited in the livers of SCD mice consistent with an overall decrease in vascular inflammation and ischemia-reperfusion physiology. Pretreatment with 2FF completely eliminated heme-induced lethality in HbSS-Townes mice, consistent with the observed anti-inflammatory and anti-adhesive properties of 2FF in SCD mice. These data suggest that 2FF may be beneficial for preventing or treating vaso-occlusive crises in SCD patients.</p></div

2FF prevents heme-induced lethality in HbSS-Townes mice.

<p>All HbSS-Townes mice (n = 6 mice/treatment) were given a bolus infusion of heme (32 μmols/kg) at time zero. Mice were given water or 100 mM 2FF in drinking water for 7 days prior to heme infusion. Time of death after heme infusion was recorded.</p

2FF inhibits microvascular stasis and leukocyte rolling and adhesion in sickle mice <i>in vivo</i>.

<p>(<b>A</b>) NY1DD transgenic sickle mice (n = 4/group) were given the indicated concentration of 2FF in their drinking water for 7 days. Microvascular stasis was measured on day 7, one hour after the infusion of stroma-free Hb (3.2 μmols heme/kg). <b>*</b>P≤.05 for all pairwise comparisons. (<b>B</b>) NY1DD sickle mice were gavaged with water or 150 mg/ml 2FF (.01 ml/g, twice per day) for 1 or 3 days. After the indicated 2FF treatment period, stroma-free Hb (3.2 μmols heme/kg body weight) was infused intravenously and microvascular stasis was measured 1 hour later. <b>*</b>P≤.01 for water vs 2FF. (<b>C</b>) NY1DD and HbSS-Townes sickle mice (n = 3/group) were given 100 mM 2FF in their drinking water for 7 days. Stasis was measured on day 7 after exposure to 1h of hypoxia and 1h of reoxygenation (H/R). <b>*</b>P<.001 for water vs 100 mM 2FF. (<b>D</b>) Leukocyte rolling flux was measured in venules in the DSFC windows of NY1DD mice before (baseline) and 1h after infusion of Hb (3.2 μmol/kg). Half of the mice (n = 4) were treated with 2FF (100 mM in drinking water X 7 days) prior to baseline measurements. Control mice (n = 4) were given drinking water without 2FF. The rolling flux was determined as the total number of leukocytes rolling through a given section of vessel per minute. Values are means + SD. ^<b>#</b>P<.001 baseline vs Hb and <b>*</b>p<.001 for water vs 2FF. (<b>E</b>) Leukocyte adhesion was measured in the same venules as described in (D). Values are mean number of adherent cells per 100 μm + SD. ^<b>#</b>P<.001 for baseline vs Hb and <b>*</b>p<.001 for water vs 2FF. (<b>F</b> and <b>G</b>) PMNs (<b>F</b>) and SS-RBCs (<b>G</b>) were isolated from HbSS-Townes mice (n = 2 mice twice) given drinking water or 100 mM 2FF in drinking water for 7 days. Fluorescently labeled PMNs or SS-RBCs were incubated with resting and activated HUVEC (4 wells/treatment) for 30 minutes. Activated HUVEC, which express P-selectin on their surface, were prepared by incubating HUVEC with 10 μM heme for 30 minutes. Values are mean % area of HUVEC cell surface covered by PMNs (<b>F</b>) or SS-RBCs (<b>G</b>) + SD after HUVEC washing. ^<b>#</b>P<.001 for resting vs activated and <b>*</b>p<.001 for water vs 2FF.</p

2FF increases the total, neutrophil (PMN) and lymphocyte white blood cell (WBC) counts in sickle mice.

<p>2FF increases the total, neutrophil (PMN) and lymphocyte white blood cell (WBC) counts in sickle mice.</p

2FF inhibits NF-ĸB activation and adhesion molecule expression in livers of sickle mice.

<p>After 7 days administration of water or 100 mM 2FF in drinking water and 4 hours after infusion of stroma-free Hb, livers from NY1DD sickle mice were removed and flash frozen. NF-kB phospho- and total-p65 were measured by western blot using liver nuclear extracts. Nuclear NF-ĸB phospho-p65 is a marker of NF-ĸB activation. VCAM-1, E-selectin and GAPDH were measured by western blot using liver microsomes.</p

Orally Active Fumagillin Analogues: Transformations of a Reactive Warhead in the Gastric Environment

Semisynthetic analogues of fumagillin, <b>1</b>, inhibit methionine aminopeptidase-2 (MetAP2) and have entered the clinic for the treatment of cancer. An optimized fumagillin analogue, <b>3</b> (PPI-2458), was found to be orally active, despite containing a spiroepoxide function that formed a covalent linkage to the target protein. In aqueous acid, <b>3</b> underwent ring-opening addition of water and HCl, leading to four products, <b>4–7</b>, which were characterized in detail. The chlorohydrin, but not the diol, products inhibited MetAP2 under weakly basic conditions, suggesting reversion to epoxide as a step in the mechanism. In agreement, chlorohydrin <b>6</b> was shown to revert rapidly to <b>3</b> in rat plasma. In an ex vivo assay, rats treated with purified acid degradants demonstrated inhibition of MetAP2 that correlated with the biochemical activity of the compounds. Taken together, the results indicate that degradation of the parent compound was compensated by the formation of active equivalents leading to a pharmacologically useful level of MetAP2 inhibition