Novel antiangiogenic strategies with complementary mechanisms are needed to maximize efficacy and minimize resistance to current angiogenesis inhibitors. We explored the therapeutic potential and mechanisms of alphaPlGF, an antibody against placental growth factor (PlGF), a VEGF homolog, which regulates the angiogenic switch in disease, but not in health. alphaPlGF inhibited growth and metastasis of various tumors, including those resistant to VEGF(R) inhibitors (VEGF(R)Is), and enhanced the efficacy of chemotherapy and VEGF(R)Is. alphaPlGF inhibited angiogenesis, lymphangiogenesis, and tumor cell motility. Distinct from VEGF(R)Is, alphaPlGF prevented infiltration of angiogenic macrophages and severe tumor hypoxia, and thus, did not switch on the angiogenic rescue program responsible for resistance to VEGF(R)Is. Moreover, it did not cause or enhance VEGF(R)I-related side effects. The efficacy and safety of alphaPlGF, its pleiotropic and complementary mechanism to VEGF(R)Is, and the negligible induction of an angiogenic rescue program suggest that alphaPlGF may constitute a novel approach for cancer treatment.

The clinical syndromes of thromboembolism are evoked by an excessive stimulation of the coagulation cascade. In this context, the serine protease thrombin plays a key role. Considerable efforts have therefore been devoted to the discovery of safe, orally active inhibitors of this enzyme. On the basis of the X-ray crystal structure of the peptidelike thrombin inhibitor NAPAP complexed with bovine thrombin, we have designed a new structural class of nonpeptidic inhibitors employing a 1,2,5-trisubstituted benzimidazole as the central scaffold. Supported by a series of X-ray structure analyses, we optimized the activity of these compounds. Thrombin inhibition in the lower nanomolar range could be achieved although the binding energy mainly results from nonpolar, hydrophobic interactions. To improve in vivo potency, we increased the overall hydrophilicity of the molecules by introducing carboxylate groups. The very polar compound 24 (BIBR 953) exhibited the most favorable activity profile in vivo. This zwitterionic molecule was converted into the double-prodrug 31 (BIBR 1048), which showed strong oral activity in different animal species. On the basis of these results, 31 was chosen for clinical development.

The effects of plasminogen activator inhibitor-l (PAI-1) gene inactivation on hemostasis, thrombosis and thrombolysis were studied in homozygous PAI-i-deficient (PAT-I-) mice, generated by homologous recombination in D3 embryonic stem cells. Diluted (10-fold) whole blood clots from PAI-1i--and from PAI-1 wild type (PAI-i 1+) mice underwent limited but significantly different (P < 0.001 ) spontaneous lysis within 3 h (6±1 vs 3±1%, respectively). A 25-A1I '25-fibrin-labeled normal murine plasma clot, injected into a jugular vein, was lysed for 47±5, 66±3, and 87±7% within 8 h in PAI-1 +/+, heterozygous PAT-i-deficient (PA-i +1-), and PAT-i -mice, respectively (P = 0.002 for PAI-i 1+ vs PAI-1-i-mice). Corresponding values after pretreatment with 0.5 mg/kg endotoxin in PA-i +'+ and PAT-I-mice, were 35±5 and 91±3% within 4 h, respectively (P < 0.001). 11 out of 26 PA-i +'+ but only 1 out of 25 PAI-1-i-mice developed venous thrombosis (P = 0.004) within 6 d after injection of 10 or 50 ,ug endotoxin in the footpad. Spontaneous bleeding or delayed rebleeding could not be documented in PA -I -mice after partial amputation of the tail or of the caecum.Thus, disruption of the PAI-i gene in mice appears to induce a mild hyperfibrinolytic state and a greater resistance to venous thrombosis but not to impair hemostasis. (J. Clin. Invest. 1993Invest. . 92:2756Invest. -2760

BACKGROUND RGD-containing peptides are able to prevent binding of ligands to certain integrins such as alpha IIb beta 3 (glycoprotein IIb/IIIa) and alpha v beta 3 and as such are inhibitors for platelet aggregation and smooth muscle cell migration, both of which are involved in neointima formation. METHODS AND RESULTS Hamster carotid arteries were damaged, and neointima formation was determined at different time points. G4120, a cyclic RGD-containing peptide, was administered continuously intravenously by an implanted osmotic pump. Neointima formation was inhibited dose dependently. The inhibition was strongest when treatment was started before the vascular injury and continued for the full observation period. Treatment started after the damage and maintained until neointima assessment or started before and stopped earlier was less effective. CONCLUSIONS Inhibition of integrin function by an RGD-containing peptide results in reduction of the development of a neointima. This effect is due both to an early event, which could be due to inhibition of secretion of PDGF by the platelets with blocked alpha IIb beta 3, and to a late event, possibly by interference with smooth muscle cell alpha v beta 3.

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