Nitric oxide (NO) induces vasodilatatory, antiaggregatory, and antiproliferative effects in vitro. To delineate potential beneficial effects of NO in preventing vascular disease in vivo, we generated transgenic mice overexpressing human erythropoietin. These animals induce polyglobulia known to be associated with a high incidence of vascular disease. Despite hematocrit levels of 80%, adult transgenic mice did not develop hypertension or thromboembolism. Endothelial NO synthase levels, NO-mediated endothelium-dependent relaxation and circulating and vascular tissue NO levels were markedly increased. Administration of the NO synthase inhibitor N G -nitro-L-arginine methyl ester (L-NAME) led to vasoconstriction of peripheral resistance vessels, hypertension, and death of transgenic mice, whereas wild-type siblings developed hypertension but did not show increased mortality. L-NAMEtreated polyglobulic mice revealed acute left ventricular dilatation and vascular engorgement associated with pulmonary congestion and hemorrhage. In conclusion, we here unequivocally demonstrate that endothelial NO maintains normotension, prevents cardiovascular dysfunction, and critically determines survival in vivo under conditions of increased hematocrit.

ABSTRACT:The pharmacokinetics and metabolism of linagliptin (BI1356, 8-(3R-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methylquinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione) were investigated in healthy volunteers. Type 2 diabetes mellitus (T2DM) accounts for 90 to 95% of all diabetes cases and its incidence is increasing (Wild et al., 2004). The high frequency of complications associated with the disease leads to a significant reduction in life expectancy. One relatively new therapeutic option is the inhibition of the enzyme dipeptidyl peptidase-4 (DPP-4), which is responsible for the rapid degradation of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide. After food intake, both hormones augment the action of insulin (Holst and Gromada, 2004;Mari et al., 2005;Drucker and Nauck, 2006;Drucker, 2007). The plasma half-life of GLP-1 is limited to a few minutes because of its rapid proteolytic degradation by DPP-4 (Graefe-Mody et al., 2009). Inhibitors of DPP-4 prolong the half-life of GLP-1 and glucose-dependent insulinotropic polypeptide, which leads to increases in glucose-dependent insulin secretion, inhibition of endogenous glucose production, decreased blood glucose, and the induction of feelings of satiety (Drucker, 2002;Nauck et al., 2003;Holst and Gromada, 2004).Linagliptin is a novel, orally active, highly specific, and potent inhibitor of DPP-4 that is currently in clinical development for the treatment of T2DM (Eckhardt et al., 2007;Fuchs et al., 2009b). Early clinical studies with linagliptin suggested a reduction in the glycated hemoglobin levels in patients with T2DM while maintaining a placebo-like safety and tolerability profile (Heise et al., 2009;Retlich et al., 2009). The pharmacokinetics of linagliptin were previously shown to be nonlinear due to target-mediated, concentrationdependent changes in binding to DPP-4 (Hüttner et al., 2008; Thomas et al., 2008a,b;Fuchs et al., 2009a;Heise et al., 2009).We report here a series of in vivo and in vitro studies performed to further establish the pharmacokinetics and metabolism of linagliptin in humans after intravenous and oral administration.Article, publication date, and citation information can be found at http://dmd.aspetjournals.org. doi:10.1124/dmd.109.031476.ABBREVIATIONS: T2DM, type 2 diabetes mellitus; DPP-4, dipeptidyl peptidase-4; GLP-1, glucagon-like peptide-1; BI1355, 8-(3S-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione; CD1790, 7-but-2-ynyl-8-(3S-hydroxy-piperidin-1-yl)-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione; CD1789, 7-but-2-ynyl-8-(3R-hydroxy-piperidin-1-yl)-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione; CD10604, 7-but-2-yn-1-yl-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-8-(3-oxopiperidin-1-yl)-3, 7-dihydro-1H-purine-2,6-dione; LOQ, lower limit of quantification; LC-MS/MS, liquid chromatography-tandem mass spectrometry; HPLC, high-performance liquid chromat...

No abstract

Hypoxia-inducible factor (HIF)-1␣ is the oxygen-sensitive subunit of HIF-1, a transcriptional master regulator of oxygen homeostasis. Oxygen-dependent prolyl hydroxylation targets HIF-1␣ for ubiquitinylation and proteasomal degradation. Unexpectedly, we found that exposing mice to elevated temperatures resulted in a strong HIF-1␣ induction in kidney, liver, and spleen. To elucidate the molecular mechanisms responsible for this effect, HepG2 hepatoma cells were exposed to different temperatures (34 -42°C) under normoxic (20% O 2 ) or hypoxic (3% O 2 ) conditions. Heat was sufficient to stabilize mainly a phosphatase-resistant, low molecular weight form of HIF-1␣ (termed HIF-1␣ a ). Heat-induced HIF-1␣ a accumulated in the nucleus but neither bound to DNA nor trans-activated reporter or target gene expression, demonstrating the need for post-translational modifications for these functions. The protein banding pattern of heat-induced HIF-1␣ in immunoblot analyses was clearly distinct from the HIF-1␣ pattern after prolyl hydroxylase inhibition (by hypoxia or iron chelation/replacement) or following proteasome inhibition, suggesting that heat stabilizes HIF-1␣ by a novel mechanism. Inhibition of the ATP-dependent chaperone activity of HSP90 by novobiocin or geldanamycin prevented heatinduced as well as hypoxia-induced HIF-1␣ accumulation, indicating a common role of the HSP90 chaperone activity in HIF-1␣ stabilization by these two environmental parameters.The hypoxia-inducible factor-1 (HIF-1) 1 is a heterodimeric transcription factor composed of the two subunits HIF-1␣ and HIF-1␤, both belonging to the basic helix-loop-helix (bHLH)-Per/arylhydrocarbon receptor (AhR) nuclear translocator (ARNT)/Sim (PAS) protein superfamily (1). HIF-1␤ is identical to the previously described ARNT protein. HIF-1 is a critical regulator of the physiological adaptive response to hypoxia, since it activates genes regulating, among other processes, angiogenesis, erythropoiesis, and glucose metabolism. HIF-1 has also been described to be involved in tumor angiogenesis and ischemic diseases such as myocardial ischemia or stroke (reviewed in Refs. 2 and 3). Whereas ARNT is constitutively expressed, HIF-1␣ expression is induced in hypoxic cells with an exponential increase in expression as cells are exposed to decreased oxygen partial pressures. As determined in HeLa cells, the highest HIF-1␣ protein levels are reached at 0.5% oxygen (4, 5). Recent studies have shown that HIF-1␣ is modified by oxygendependent prolyl hydroxylation (6 -9), allowing the binding of the von Hippel-Lindau protein (pVHL), which targets HIF-1␣ for ubiquitinylation and proteasomal degradation (10 -17). Further activation of HIF-1 involves nuclear translocation, dimerization with ARNT, DNA binding, and recruitment of transcriptional co-activators. These processes are regulated at least in part by posttranslational modifications. It has been demonstrated that phosphorylation of HIF-1␣ via the Ras/Raf-MEK-p42/44 and the phosphatidylinositol 3-kinase-PTEN-Akt-GSK3 kinase...

The most common cause of an increase of the hematocrit is secondary to elevated erythropoietin levels. Erythrocytosis is assumed to cause higher blood viscosity that could put the cardiovascular system at hemodynamic and rheological risks. Secondary erythrocytosis results from tissue hypoxia, and one can hardly define what cardiovascular consequences are caused by chronic erythrocytosis or hypoxia. Herein, a novel transgenic (tg) mouse line is characterized that is erythrocytotic because of chronic overexpression of the human erythropoietin gene. These mice grow up well, reaching a hematocrit of about 0.80 in adulthood. Blood volume of adult tg mice was markedly increased by 75%. Unexpectedly, blood pressure was not elevated and cardiac output was not decreased. Still, the adult tg mice showed features of cardiac dysfunction with increased heart weight. In vivo, high-frequency echocardiography revealed marked ventricular dilatation that was confirmed by histologic examination. Furthermore, by transmission electron microscopy, a prominent intracellular edema of the cardiomyocytes was seen. Exercise performance of the tg mice was dramatically reduced, unmasking the severity of their compromised cardiovascular function. In addition, life expectancy of the tg mice was significantly reduced to 7.4 months. Our findings suggest that severe erythrocytosis per se results in cardiac dysfunction and markedly reduced life span. (Blood. 2001;97:536-542)

The pharmacokinetics and metabolism of BIBF 1120, an oral triple angiokinase inhibitor targeting vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR), and fibroblast growth factor receptor (FGFR), were studied in healthy male volunteers (n = 8) who had received a single oral dose of 100 mg [(14)C]-radiolabelled BIBF 1120 administered as solution. BIBF 1120 was well-tolerated and rapidly absorbed; median time to reach maximum plasma concentrations was 1.3 h and gMean terminal half-life was 13.7 h. A relatively high apparent total body clearance and volume of distribution possibly indicated a high tissue distribution. Plasma concentrations of BIBF 1120 plus carboxylate metabolite BIBF 1202 were lower than the total [(14)C]-radioactivity in plasma, indicating presence of additional metabolites. Total recovery in excreta was 94.7% 1 week post-dose; mass balance was considered complete after 96 h. BIBF 1120 and metabolites were mainly excreted via faeces. The major metabolic pathway for BIBF 1120 was methyl ester cleavage to BIBF 1202. Subsequently, the free carboxyl group of BIBF 1202 was glucuronidated to 1-O-acylglucuronide. Pathways of minor importance were oxidative N-demethylation to yield BIBF 1053, and oxidation of the piperazine moiety and conjugation. Glucuronidation of the parent drug and formylation of the secondary aliphatic amine of the piperazine ring played a minor role.

We have generated a transgenic mouse line that reaches a hematocrit concentration of 0.85 due to constitutive overexpression of human erythropoietin in an oxygen-independent manner. Unexpectedly, this excessive erythrocytosis did not lead to thrombembolic complications in all investigated organs at any age. Thus, we investigated the mechanisms preventing thrombembolism in this mouse model. Blood analysis revealed an age-dependent elevation of reticulocyte numbers and a marked thrombocytopenia that matched the reduced megakaryocyte numbers in the bone marrow. However, platelet counts were not different from wild-type controls, when calculations were based on the distribution (eg, plasma) volume, thereby explaining why thrombopoietin levels did not increase in transgenic mice. Nevertheless, bleeding time was significantly increased in transgenic animals. A longitudinal investigation using computerized thromboelastography revealed that thrombus formation was reduced with increasing age from 1 to 8 months in transgenic animals. We observed that increasing erythrocyte concentrations inhibited profoundly and reversibly thrombus formation and prolonged the time of clot development, most likely due to mechanical interference of red blood cells with clot-forming platelets. Transgenic animals showed increased nitric oxide levels in the blood that could inhibit vasoconstriction and platelet activation. Finally, we observed that plasmatic coagulation activity in transgenic animals was significantly decreased. Taken together, our findings suggest that prevention of thrombembolic disease in these erythrocytotic transgenic mice was due to functional consequences inherent to increased erythrocyte concentrations and a reduction of plasmatic coagulation activity, the cause of which remains to be elucidated. (Blood. 2003;101:4416-4422)

The primary function of the glycoprotein hormone erythropoietin (Epo) is to promote red cell production by inhibiting apoptosis of erythrocytic progenitors in hemopoietic tissues. However, functional Epo receptors (Epo-R) have recently been demonstrated in various nonhemopoietic tissues indicating that Epo is a more pleiotropic viability and growth factor. Herein, in vitro and in vivo effects of Epo in the brain and the cardiovascular system are reviewed. In addition, the therapeutic impact of Epo in oncology is considered, including the question of whether Epo might promote tumor growth. Convincing evidence is available that Epo acts as a neurotrophic and neuroprotective factor in the brain. Epo prevents neuronal cells from hypoxia-induced and glutamate-induced cell death. Epo-R is expressed by neurons and glia cells in specific regions of the brain. Epo supports the survival of neurons in the ischemic brain. The neuroprotective potential of Epo has already been confirmed in a clinical trial on patients with acute stroke. With respect to the vasculature, Epo acts on both endothelial and smooth muscle cells. Epo promotes angiogenesis and stimulates the production of endothelin and other vasoactive mediators. In addition, Epo-R is expressed by cardiomyocytes. The role of Epo as a myocardial protectant is at the focus of present research. Epo therapy in tumor patients is practiced primarily to maintain the hemoglobin concentration above the transfusion trigger and to reduce fatigue. In addition, increased tumor oxygenation may improve the efficacy of chemotherapy and radiotherapy. However, tumor cells often express Epo-R. Therefore, careful studies are required to fully exclude that recombinant human Epo (rHuEpo) promotes tumor growth.