The uptake and degradation of cholesterol-rich remnant lipoproteins, referred to as are shown in the present study to be mediated by LDL receptors (apoB,E(LDL) Immunoblots of monocyte-macrophage extracts with these antibodies revealed a single protein in human macrophages with an apparent molecular weight identical to that of the apoB,E(LDL) receptor found on human fibroblasts. Like receptors on cultured human fibroblasts, the apoB,E(LDL) receptors on monocyte-macrophages responsible for '25I-j-VLDL and 125I-LDL uptake were efficiently down regulated by preincubation of the cells with fl-VLDL or LDL. Finally, monocyte-macrophages from seven homozygous familial hypercholesterolemia subjects were unable to metabolize 6-VLDL or LDL, but demonstrated normal uptake of acetoacetylated LDL. The classic apoB,E(LDL) receptors on human monocyte-macrophages thus mediate the uptake of I-VLDL by these cells.

The inhibition of key receptor tyrosine kinases (RTKs) that are implicated in tumor vasculature formation and maintenance, as well as tumor progression and metastasis, has been a major focus in oncology research over the last several years. Many potent small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) kinases have been evaluated. More recently, compounds that act through the complex inhibition of multiple kinase targets have been reported and may exhibit improved clinical efficacy. We report herein a series of potent, orally efficacious 4-amino-3-benzimidazol-2-ylhydroquinolin-2-one analogues as inhibitors of VEGF, PDGF, and fibroblast growth factor (FGF) receptor tyrosine kinases. Compounds in this class, such as 5 (TKI258), are reversible ATP-competitive inhibitors of VEGFR-2, FGFR-1, and PDGFRbeta with IC(50) values <0.1 microM. On the basis of its favorable in vitro and in vivo properties, compound 5 was selected for clinical evaluation and is currently in phase I clinical trials.

In hepatocytes isolated from fasted rats, 2,5-anhydromannitol inhibits gluconeogenesis from lactate plus pyruvate and from substrates that enter the gluconeogenic pathway as triose phosphate. This fructose analog has no effect, however, on gluconeogenesis from xylitol, a substrate that enters the pathway primarily as fructose 6-phosphate. The sensitivity of gluconeogenesis to 2,5-anhydromannitol depends on the substrate metabolized; concentrations of 2,5-anhydromannitol required for 50% inhibition increase in the order lactate plus pyruvate < dihydroxyacetone < glycerol < sorbitol < fructose. The inhibition by 2,5-anhydromannitol of gluconeogenesis from dihydroxyacetone is accompanied by an increase in lactate formation and by two distinct crossovers in gluconeogenic-glycolytic metabolite patterns i.e., increases in pyruvate concentrations with decreases in phosphoenolpyruvate and increases in fructose-1,6-bisphosphate concentrations with little change in fructose 6-phosphate. In addition, 2,5-anhydromannitol blocks the ability of glucagon to stimulate gluconeogenesis and inhibit lactate production from dihydroxyacetone. 2,5-Anhydromannitol decreases cellular fructose 2,6-bisphosphate content in hepatocytes; therefore the effects of the fructose analog are not mediated by fructose 2,6-bisphosphate, a naturally occurring allosteric regulator. 2,5-Anhydromannitol also inhibits gluconeogenesis in hepatocytes isolated from fasted diabetic rats, but higher concentrations of the analog are required.2,5-Anhydro-D-mannitol (2,5-AM-ol), an analog of P-D-fructose locked in the furan ring structure, is phosphorylated by fructokinase to form 2,5-AM-ol-l-P (1, 2).Because 2,5-AM-ol is symmetrical, the monophosphate product can be considered an analog of both fructose-i-P and fructose-6-P (3). Because of the stability of its ring structure, 2,5-AMI-ol monophosphate cannot be cleaved bv aldolase in a manner similar to that of fructose-l-P, nor can it act as a substrate for phosphoglucoisomerase and be converted to glucose-6-P in a manner similar to that of fructose-6-P. 2,5-AM-ol monophosphate is a substrate for phosphofructokinase 1 (4, 5). The resulting product, 2,5-AM-ol bisphosphate, is an analog of 8-fructose-1,6-P2 rather than a-fructose-1,6-P2 and, as such, is not hydrolyzed readily by fructose-1,6-bisphosphatase, which prefers the a anomer (6). The bisphosphate compound, thus, should accumulate within the cell. In vitro experiments have shown that 2,5-AM-ol bisphosphate is a competitive inhibitor of fructose-1,6-bisphosphatase (7,8). In view of the described findings, the potential of 2,5-AM-ol to act as a regulator of gluconeogenesis and glycolysis was examined in isolated rat hepatocytes.METHODS AND MATERIALS Synthesis of 2,5-AM-ol. 2,5-AM-ol was prepared as in ref.9 except the crystallization step was omitted. The crude 2,5-AM1-ol, in 5 mM ammonium borate (pH 9), was purified on Dowex-I-X-8 (borate) (10). The unretained material, after deionization and repeated concentration by evaporation of methanol, showe...

Chylomlcron remnant catabollsm appears to be mediated by apolipoprotein (apo) E binding to hepatic llpoprotein receptors. Previously, the apo B.E(LDL) receptor and a unique apo E-blndlng protein (referred to as the apo E receptor) were Isolated from solublllzed canine and human livers. In the present study, the apo E-blndlng fraction was further characterized and found to contain at least three proteins, all of which bind apo E-contalnlng llpoprotelns with high affinity. The 56-kDa band was found to contain the a-and B-subunlts of F,-ATPase, presumably derived from mltochondrlal membranes. In addition, an apo E-blndlng protein with an apparent M r =-59000 was identified. The 59-kDa protein displays calcium-Independent binding on llgand blots, but displays both calcium-dependent and -Independent binding In assays performed with detergent-solubilized protein. The 59-kDa protein recognized llpld-free as well as llpld-bound apo E In llgand blots, and also bound apo E-2, apo E-3, and apo E-4 in a comparable way. Monoclonal antibodies produced against the 59-kDa protein did not react with the 56-kDa proteins. Normal human liver, as well as the liver of a patient lacking the apo B,E(LDL) receptor, possessed the 56-kDa and 59-kDa proteins. These data Indicate that liver cells possess at least three proteins, in addition to the apo B.E(LDL) receptor, that bind apo E-contalnlng llpoprotelns with high affinity. The physiological role of these proteins In apo E metabolism remains to be determined. (LDL) at a much reduced rate. In addition, Watanabe Heritable Hyperlipidemic (WHHL) rabbits, which express less than 5% of the normal number of apo B,E(LDL) receptors, 6 are still able to catabolize chylomicron remnants at normal rates.7 Furthermore, down-regulation of hepatic apo B.E(LDL) receptors by cholesterol feeding results in retarded clearance of LDL but has no effect on the hepatic uptake of remnant lipoproteins. 8In vivo observations have shown that apo E on the chylomicron remnant surface is required for hepatic clearance of these lipoproteins. 49 -13 Competitive binding studies in the perfused rat liver have shown that both chylomicron remnants and apo E HDL C [the cholesterol-induced high density lipoproteins (HDL) that contain apo E] are taken up by the same process. 4 Furthermore, chylomicron remnants without apo E are removed by the perfused rat liver at a slow rate. 11 The importance of apo E in chylomicron remnant metabolism is also evident from studies of type III hyperiipoproteinemic (dysbetalipoproteinemic) subjects. The apo E-2 isolated from these patients is defective, in comparison with the normal apo E-3, in promoting hepatic uptake of model lipoproteins. 13 In addition, chylomicron remnants accumulate in the plasma of patients who have a marked deficiency in plasma apo E.

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