Suppression of VEGF-induced angiogenesis by the protein tyrosine kinase inhibitor, lavendustin A.

1. Vascular endothelial growth factor (VEGF) is a heparin-binding angiogenic factor which specifically acts on endothelial cells via distinct membrane-spanning tyrosine kinase receptors. Here we used the rat sponge implant model to test the hypothesis that the angiogenic activity of VEGF can be suppressed by protein tyrosine kinase (PTK) inhibitors. 2. Neovascular responses in subcutaneous sponge implants were determined by measurements of relative sponge blood flow by use of a 133Xe clearance technique, and confirmed by histological studies and morphometric analysis. 3. Daily local administration of 250 ng VEGF165 accelerated the rate of 133Xe clearance from the sponges and induced an intense neovascularisation. This VEGF165-induced angiogenesis was inhibited by daily co-administration of the selective PTK inhibitor, lavendustin A (10 micrograms), but not its negative control, lavendustin B (10 micrograms). Blood flow measurements and morphometric analysis of 8-day-old sponges showed that lavendustin A reduced the 133Xe clearance of VEGF165-treated sponges from 32.9 +/- 1.5% to 20.9 +/- 1.6% and the total fibrovascular growth area from 62.4 +/- 6.1% to 21.6 +/- 6.8% (n = 12, P < 0.05). 4. Co-injection of suramin (3 mg), an inhibitor of heparin-binding growth factors, also suppressed the VEGF165-elicited neovascular response. In contrast, neither lavendustin A nor suramin produced any effect on the basal sponge-induced angiogenesis. 5. When given alone, low doses of VEGF165 (25 ng) or basic fibroblast growth factor (bFGF; 10 ng) did not modify the basal sponge-induced neovascularisation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Shikonin, acetylshikonin, and isobutyroylshikonin inhibit VEGF-induced angiogenesis and suppress tumor growth in lewis lung carcinoma-bearing mice

Lithospermum erythrorhizon has been used for treatment of inflammatory diseases and cancer as a folk remedy. Based on the evidences that anti-inflammatory agents frequently exert antiangiogenic activity, thus we examined comparatively the antiangiogenic activities of three naphthoquinone derivatives (shikonin, acetylshikonin, and isobutyroylshikonin) isolated from the plant. Three derivatives exhibited weak cytotoxicity against human umbilical vein endothelial cells (HUVECs) with IC50 of over 20 microM. Shikonin had more specific inhibitory effects on proliferation and vascular endothelial growth factor (VEGF) production by VEGF compared with different derivatives. All of derivatives significantly suppressed the migration of VEGF treated HUVECs at different optimal concentrations. Also, shikonin and acetylshikonin significantly disrupted VEGF-induced tube formation. Furthermore, three derivatives effectively downregulated the expression of urokinase-type plasminogen activator (uPA), but not its receptor uPAR. Additionally, shikonin significantly inhibited tumor growth in LLC-bearing mice, whereas its derivatives had relatively mild effects. Taken together, our findings suggest that shikonin and its derivatives exhibit the antiangiogenic and antitumorigenic effects by suppressing proliferation and angiogenic factors.     

Anti-inflammatory, analgesic and antipyretic activity of aqueous extract of fresh leaves of Coccinia indica

This study was aimed to evaluate both post- and pre-treatment anti-inflammatory activities of the aqueous extract of fresh leaves of Coccinia indica in rats using the carrageenan-induced paw oedema method at various dose levels. Analgesic and antipyretic properties were evaluated using tail flick model and yeast-induced hyperpyrexia, respectively. Ceiling effect of the extract was observed at 50 mg/kg in pre-treatment carrageenan test. In post-treatment studies, a dose-dependent anti-inflammatory effect was observed in the dose range of 25–300 mg/kg. The effect was equivalent to diclofenac (20 mg/kg) at 50 mg/kg but it was significantly pronounced at higher doses. Effectiveness of extract in the early phase of inflammation suggests the inhibition of histamine and serotonin release. The extract produced marked analgesic activity comparable to morphine at 300 mg/kg, which suggests the involvement of central mechanisms. A significant reduction in hyperpyrexia in rats was also produced by all doses of extract with maximum effect at 300 mg/kg comparable to paracetamol. In conclusion, this study has established the anti-inflammatory activity, analgesic and antipyretic activity of C. indica and, thus, justifies the ethnic uses of the plant.     

Inhibition of angiogenesis and tumor growth by beta and gamma-secretase inhibitors

The involvement of beta-secretase and gamma-secretase in producing the beta-amyloid component of senile plaques found in the brain of Alzheimer's patients has fueled a major research effort to design selective inhibitors of these proteases. Interestingly, gamma-secretase cleaves several proteins including Notch, E-cadherin, CD44 and ErbB-4 (erythroblastic leukemia viral oncogene homolog 4), which are important modulators of angiogenesis. The beta-amyloid precursor protein, which is cleaved by beta-secretase and gamma-secretase to produce beta-amyloid, is highly expressed in the endothelium of neoforming vessels suggesting that it might play a role during angiogenesis. These data prompted us to explore the effects of beta and gamma-secretase inhibitors of different structures on angiogenesis and tumor growth. Both the gamma and beta-secretase inhibitors tested reduce endothelial cell proliferation without inducing cellular toxicity, suppress the formation of capillary structures in vitro and oppose the sprouting of microvessel outgrowths in the rat aortic ring model of angiogenesis. Moreover, they potently inhibit the growth and vascularization of human glioblastoma and human lung adenocarcinoma tumors xenotransplanted into nude mice. Altogether these data suggest that the gamma and beta-secretases play an essential role during angiogenesis and that inhibitors of the beta and gamma-secretases may constitute new classes of anti-angiogenic and anti-tumoral compounds.     

梓醇通过调控肿瘤血管生成及能量代谢抑制肝癌生长

目的探讨梓醇对HepG2肝癌细胞增殖、侵袭、生长、血管生成和能量代谢的影响。方法细胞增殖实验研究梓醇对HepG2细胞体外增殖的影响;LDH试剂盒检测梓醇对HepG2细胞的毒性作用;构建人肝癌HepG2裸小鼠细胞移植瘤模型,研究梓醇对体内肝癌生长的影响;Transwell实验研究梓醇对HepG2细胞体外侵袭的影响;小管形成实验检测梓醇对HepG2细胞诱导的血管生成的影响;免疫组化研究梓醇对移植瘤中能量代谢关键指标单羧酸转运蛋白-1 (monocarboxylate transporter-1,MCT-1)、单羧酸转运蛋白-4(monocarboxylate transporter-4,MCT-4)和葡萄糖转运蛋白1(Glucose transporter 1,GLUT1)蛋白表达的影响。结果在一定浓度范围内,梓醇能够抑制HepG2细胞的体外增殖及侵袭,且具有一定的浓度依赖性;LDH检测显示其在50μmol·L~(-1)时对HepG2细胞表现出毒性作用;与对照组相比,梓醇能够显著抑制HepG2裸小鼠移植瘤的体内生长;HepG2细胞培养上清液能够显著刺激HUVEC细胞的小管形成,而梓醇能够在体外抑制HepG2细胞诱导的小管形成;梓醇能够抑制HepG2裸小鼠移植瘤组织中MCT-1,MCT4和GLUT1蛋白的表达。结论梓醇具有抑制肝癌细胞增殖、侵袭及生长的能力,其机制可能与抑制肝癌的血管生成及肿瘤能量代谢密切相关。     

A molecular connection of Pterocarpus marsupium,Eugenia jambolana and Gymnema sylvestre with dipeptidyl peptidase-4 in the treatment of diabetes

Context: Pterocarpus marsupium (PM) (Leguminosae), Eugenia jambolana (EJ) (Myrtaceae) and Gymnema sylvestre (GS) (Asclepiadaceae) are the most important medicinal plants in the Indian system of traditional medicine for the treatment of hyperglycemia.

Objectives: Dipeptidyl peptidase-4 (DPP-4) inhibitors are the emerging class of anti-diabetic agents. However, only few compounds are commercially available. Therefore, in the present study we tried to explore the naturally occurring PM, EJ and GS semi-standardized extracts for their potential DPP-4 inhibition in vitro and in vivo.

Materials and methods: DPP-4 inhibition was evaluated by in vitro inhibitory assay, and enzyme kinetics were calculated using one-phase exponential decay equation. Glucose load (2?g/kg) was administered to control and diabetic rats 30?min following extract administration (100, 200 and 400?mg/kg) orally once, and blood samples were withdrawn at 0, 0.5, 1, 1.5, 2 and 3?h to measure plasma active glucagon-like peptide-1 (GLP-1) levels.

Results: PM and EJ inhibit DPP-4 potently with IC₅₀ values of 273.73?±?2.96 and 278.94?±?6.73?µg/mL, respectively, compared to GS (773.22?±?9.21?µg/mL). PM, EJ and GS exhibit long duration of action with enzyme inhibitory half-lives of 462.3, 317.2 and 153.8?min, respectively. Extracts significantly increase GLP-1 levels compared to negative control groups and peak GLP-1 level was observed at 2?h for PM and EJ, whereas for GS it was at 1.5?h

Discussion and conclusion: Taken together, results suggest the extracts may have potent DPP-4 inhibitory action, and their hypoglycemic action attributed through an increase in plasma active GLP-1 levels.     

Effects of Cordyceps militaris extract on angiogenesis and tumor growth

INTRODUCTIONAngiogenesis is the process of new blood vesselformation from existing blood vessels and a natural re-sponse of tissues to ischemia[1]. The steps of angio-genesis involve proteolytic degradation of extracelluarmatrix, endothelial cell-matrix adhesion, migration,proliferation, and differentiation[2]. This biological re-action is often associated with some diseases, such assolid tumor[3], diabetic retinopathy[4], and rheumatoidarthritis[5].Tumor formation requires the developm…     

大豆异黄酮抑制裸鼠移植瘤生长及其血管生成的实验研究

目的　探讨大豆异黄酮及其主要有效成分金雀黄素对人乳腺癌细胞裸鼠移植瘤的生长及其血管生成的影响。方法　选用人乳腺癌细胞株MCF 7裸鼠异种移植 ,观测移植瘤的重量、出瘤时间、成瘤率 ,同时应用FⅧ免疫组化染色 ,观测瘤组织内的新生血管。结果　FⅧ免疫组化染色结果表明 ,肿瘤组织内可见形态不规则 ,且无明显管腔的新生血管 ,血管分布以肿瘤组织的边缘处为多见。各组微血管记数结果金雀黄素组最少。肿瘤出瘤时间及成瘤率各防治组都明显延长和减少 ,以金雀黄素组最为明显 ,其次是高黄酮组和低黄酮组。结论　大豆异黄酮的主要有效成分金雀黄素能减少肿瘤的血管生成 ,从而抑制裸鼠移植瘤的体内生长。     

Suppression of basic fibroblast growth factor-induced angiogenesis by a specific chymase inhibitor,BCEAB, through the chymase-angiotensin-dependent pathway in hamster sponge granulomas

1. We investigated the profound involvement of mast cell chymase, an alternative angiotensin II-generating enzyme, in angiogenesis using a specific chymase inhibitor. We also studied the functional profiles of this novel inhibitor in basic fibroblast growth factor (bFGF)-induced angiogenesis. 2. In this study, angiogenesis was induced by daily injections of bFGF (0.3 micro g site(-1) day(-1)), angiotensin I (2 nmol site(-1) day(-1)) or angiotensin II (2 nmol site(-1) day(-1)) into sponges implanted to male hamsters subcutaneously for 7 days. Angiogenesis in the granulation tissue surrounding sponges was evaluated by measuring the haemoglobin (Hb) content and local blood flow as the parameters for angiogenesis. 3. A chymase inhibitor, BCEAB (4-[1-[[bis-(4-methyl-phenyl)-methyl]-carbamoyl]-3-(2-ethoxy-benzyl)-4-oxo-azetidine-2-yloxy]-benzoic acid), was simultaneously administered into the implanted sponges (2 or 5 nmol site(-1) day(-1), for 7 days) treated with bFGF and strongly suppressed the haemoglobin contents in sponge granulomas. In the studies using a laser doppler perfusion imager, BCEAB (5 nmol site(-1) day(-1)) also attenuated the bFGF-induced increase of local blood flow around the implanted sponge granuloma. 4. In bFGF-induced angiogenesis, chymase activity in sponge granulomas was substantially increased. It was also confirmed that the chymase activity increased by bFGF was significantly and dose-dependently inhibited by BCEAB (2, 5 nmol site(-1) day(-1)). 5. BCEAB inhibited the Hb contents and the expression of vascular endothelial growth factor (VEGF) mRNA induced by angiotensin I but not by angiotensin II. 6. These results suggest that the significance of chymase in bFGF-induced angiogenesis was confirmed, and a novel inhibitor, BCEAB, strongly suppresses the bFGF-induced angiogenesis through the chymase-angiotensin II-VEGF dependent pathway.     

萝卜硫素对A375黑色素瘤增殖、生长、凋亡和血管生成的影响及其机制

目的研究萝卜硫素(sulforaphane,SFN)对A375黑色素瘤增殖、生长、凋亡和血管生成的影响及其作用机制。方法细胞增殖实验研究SFN对A375细胞增殖的影响;裸小鼠皮下移植瘤模型研究SFN对黑色素瘤体内生长的影响;流式细胞仪检测萝卜硫素对A375黑色素瘤细胞凋亡的影响;小管形成实验检测SFN对A375人黑色素瘤血管生成的影响;蛋白质印迹法检测SFN对A375细胞中调控增殖、凋亡及血管生成相关蛋白表达的影响,如Bax、Bcl-2、VEGF、b FGF、cleaved PARP、p-P38、t-P38、p-Erk、t-Erk。结果 SFN对A375细胞的增殖具有明显的抑制作用;SFN能够明显抑制A375裸鼠皮下移植瘤的体内生长;SFN能显著抑制肿瘤细胞诱导的血管生成;SFN对A375黑色素瘤细胞的凋亡无明显影响。机制研究发现,SFN对VEGF、b FGF、p-P38及p-Erk的表达具有抑制作用,但对Bcl-2、Bax及cleaved PARP蛋白的表达没有明显影响。结论萝卜硫素具有抗黑色素瘤体内生长的能力,其作用机制与调控黑色素瘤细胞的凋亡无关,而可能是通过抑制黑色素瘤的血管生成。     

Discovery of PG545: a highly potent and simultaneous inhibitor of angiogenesis, tumor growth, and metastasis

Increasing the aglycone lipophilicity of a series of polysulfated oligosaccharide glycoside heparan sulfate (HS) mimetics via attachment of a steroid or long chain alkyl group resulted in compounds with significantly improved in vitro and ex vivo antiangiogenic activity. The compounds potently inhibited heparanase and HS-binding angiogenic growth factors and displayed improved antitumor and antimetastatic activity in vivo compared with the earlier series. Preliminary pharmacokinetic analyses also revealed significant increases in half-life following iv dosing, ultimately supporting less frequent dosing regimens in preclinical tumor models compared with other HS mimetics. The compounds also displayed only mild anticoagulant activity, a common side effect usually associated with HS mimetics. These efforts led to the identification of 3β-cholestanyl 2,3,4,6-tetra-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-α-d-glucopyranosyl-(1→4)-2,3,6-tri-O-sulfo-β-d-glucopyranoside, tridecasodium salt (PG545, 18) as a clinical candidate. Compound 18 was recently evaluated in a phase I clinical trial in cancer patients.     

Inhibition of tumor angiogenesis by antibodies, synthetic small molecules and natural products

Cancer remains one of the major causes of death worldwide. The switch to pathological angiogenesis is a key process in the promotion of cancer and consequently provides several new and promising targets to anticancer therapy. Thus, antagonizing angiogenesis cuts off the tumor's oxygen and nutrition supply. This review focuses on angiogenesis inhibitors as option for cancer treatment. Modes of action, adverse effects, mechanisms of resistance as well as new developments are highlighted. One approach in angiogenesis inhibition is intermitting the further VEGF (vascular endothelial growth factor) signal pathway with monoclonal antibodies. Bevacizumab is a highly specific recombinant humanized monoclonal IgG antibody targeting VEGF-A. An efficient antitumor therapy demands more specific antibodies that affect other signal molecules besides VEGF-A, which is in the focus of current research. In addition to antagonizing VEGF, there are also small molecules that inhibit receptor tyrosine kinases (RTKs). Many RTK inhibitors have been described, which exhibit different specificity profiles. The question, whether highly specific antagonists are necessary remains open, because other affected RTKs may also represent growth factor receptors that are essential for tumor growth. Therefore their inhibition may also contribute to anticancer activity. Secondary plant metabolites represent templates for the development of new small molecules. The identification of new drugs from plants has a long and successful history. There is convincing evidence for the beneficial effect of phytochemicals on cancer-related pathways, particularly with regard to anti-angiogenesis. Plant phenolics are the most important category of phytochemicals, including flavanoids. Prominent phytochemicals affecting different pathways of angiogenesis are green tea polyphenols (epigallocatechin gallate) and soy bean isoflavones (genistein).     

Enhanced suppression of prostate tumor growth by combining C-CAM1 gene therapy and angiogenesis inhibitor TNP-470

We have previously shown that C-CAM1-based gene therapy effectively suppressed prostate tumor growth in nude mice xenograft models. In this study, we examined the effects of combining C-CAM1-based therapy and TNP-470, a potent angiogenesis inhibitor, on prostate cancer in a xenografted tumor model. The direct cytotoxic effects of Ad-C-CAM1 (recombinant adenovirus containing C-CAM1 cDNA) and TNP-470 on DU145 cells in vitro were determined by microculture tetrazolium assay. The in vivo antitumor effects of either agent alone were studied in a DU145 xenografted tumor model. Cells were infected with Ad-C-CAM1 or the control virus at multiplicities of infection (m.o.i.) of 5 or 10 and then inoculated onto nude mice 48 h later. TNP-470 (0, 17 or 35 mg/kg) was given 15, 17 and 19 days after inoculation. Combined treatments in vivo were carried out to determine whether there were synergistic antitumor effects. Both Ad-C-CAM1 and the control virus were minimally toxic to DU145 in vitro. There was evident dose-dependent suppression of xenografted tumor growth by either Ad-C-CAM1 or TNP-470. By the median-effect analysis, combination of the two agents generated strong synergistic antitumor effects as shown by marked tumor suppression as compared to either treatment alone. The novel strategy may have clinical implications for the treatment of prostate cancer.     

Multiple biological activities of lactic acid in cancer: influences on tumor growth, angiogenesis and metastasis

High rate of glycolysis is a metabolic hallmark of cancer. While anaerobic glycolysis promotes energy production under hypoxia, aerobic glycolysis, the Warburg effect, offers a proliferative advantage through redirecting carbohydrate fluxes from energy production to biosynthetic pathways. To fulfill tumor cell needs, the glycolytic switch is associated with elevated glucose uptake and lactic acid release. Altered glucose metabolism is the basis of positron emission tomography using the glucose analogue tracer [18F]- fluorodeoxyglucose, a widely used clinical application for tumor diagnosis and monitoring. On the other hand, high levels of lactate have been associated with poor clinical outcome in several types of human cancers. Although lactic acid was initially considered merely as an indicator of the glycolytic flux, many evidences originally from the study of normal tissue physiology and more recently transposed to the tumor situation indicate that lactic acid, i.e. the lactate anion and protons, directly contributes to tumor growth and progression. Here, we briefly review the current knowledge pertaining to lactic acidosis and metastasis, lactate shuttles, the influence of lactate on redox homeostasis, lactate signaling and lactate-induced angiogenesis in the cancer context. The monocarboxylate transporters MCT1 and MCT4 have now been confirmed as prominent facilitators of lactate exchanges between cancer cells with different metabolic behaviors and between cancer and stromal cells. We therefore address the function and regulation of MCTs, highlighting MCT1 as a novel anticancer target. MCT1 inhibition allows to simultaneously disrupt metabolic cooperativity and angiogenesis in cancer with a same agent, opening a new path for novel anticancer therapies.     

Evaluation of Some Pharmacological Activities of ethanol extracts of seeds, pericarp and leaves of Eugenia Jamolana in Rats

The present study investigated the effect of ethanol extracts of seeds, pericarp and leaves of Eugenia Jamolana (E. Jamolana) on inflammation, gastric ulcer, anti-oxidants and hepatoprotective in rats. The acute inflammation was induced by intra-plantar injection of carrageenan (100 μl of 1 %) in the rat hind paw. Gastric ulcer was evoked by indomethacin (25 mg/kg) oral administration. Liver damage was induced by given CCL₄ (2.5 ml/kg) orally. The median lethal (LD₅₀) of the ethanol extract of both seeds and pericarp were determined and revealed that the investigated extracts of seeds and pericarp were non toxic up to 5g/kg. The anti-inflammatory results showed that the oral administration of ethanol extract of E. Jamolana seeds (250,500 mg/kg) showed significant inhibition of oedema formation in dose-dependent manner by −27.86, −41.23, −44.73, −51.78 % and by −63.16, −37.77, −47.04, −55.36 % at 1, 2, 3 and 4 h at 1, 2, 3 and 4 h, respectively. While the pericarp given at dose (500 mg/kg) exhibited significant inhibition of the oedema formation by −34.64, −21,8, 19.23 and −33.47 % at 1, 2, 3 and 4 h, respectively post carrageenan injection as compared with saline control group. E. Jamolana leaves fraction 1 given orally at dose of 25 mg/kg, induced non significant change on oedema, while the oedema response was significantly inhibited by −25.14, −33.4, −20.57 and −26.46 % at 1, 2, 3 and 4 h, respectively in group of rats that received leaves fraction 2 at the same dose. Rats were given leaves fraction 3 extract showed inhibition of oedema formation by −4.48 % at 1^st h post- carrageenan injection, while at 2^nd, 3^rd and 4^th h showed non significant change on oedema formation. The acute gastric mucosal lesions was significantly reduced by given ethanol extract of E. Jamolana seeds, pericarp (250, 500 mg/kg) and leaves fractions 1, 2 and 3 (25 mg/kg) respectively in dose dependent manner, as compared with indomethacin treated group (control group). All tested extracts showed significant reduction in elevated serum ALT, AST and ALP levels as compared with CCl ₄ treated group. The ethanol extract of E. Jamolana seeds, pericarp and leaves fractions 1, 2, 3 showed significant elevation of blood GSH level and significant reduction in elevated plasma lipid peroxides (MDA) as compared with CCl₄ treated group. In conclusion we can see that the ethanol extracts of E. Jamolana of seeds, pericarp and leaves fractions showed anti-inflammatory, anti-ulcer, hepatoprotective and anti-oxidants activity. Received 15 June 2008; accepted 11 November 2008     

Antioxidant,antimicrobial and cytotoxic potential of silver nanoparticles synthesized using flavonoid rich alcoholic leaves extract of Reinwardtia indica

The present work discusses the establishment of a green route for the rapid synthesis of silver nanoparticles (AgNPs) using an alcoholic extract of Reinwardtia indica (AERI) leaves which act as a reducing as well as a capping agent. The change in color from yellowish green to dark brown confirmed the synthesis of AgNPs. A characteristic surface plasmon resonance (SPR) band at 436?nm advocated the presence of AgNPs. The synthesis process was optimized using one factor at a time approach where 1.0?mM AgNO₃ concentration, 5?mL 0.4% (v/v) of AER inoculum dose and 30?min of sunlight exposure were found to be the optimum conditions. The synthesized AgNPs was characterized by several characterizing techniques such as HR- TEM, SAED, HR-SEM, EDX, XRD, FTIR and AFM analysis. For evaluation and comparison of AgNPs with AERI used human pathogen E. coli, P. aeurogenosa, S. aeurus and C. albicans for antimicrobial, for cytotoxicity study SiHa cell line at concentration of (10, 50, 100, 250 and 500?µg mL^?1) and for enzymatic assay superoxide dismutase, catalase, malondialdehyde and glutathione peroxidase method were used. The size of nanoparticle in the range of 3–15?nm was confirmed TEM, spherical shape by SEM and crystal lattice nature by XRD. AFM results revealed the 2?D and 3?D pattern of particle scatter nature on the surface. This protocol as simple, rapid, one step, eco-friendly, nontoxic and AgNPs showed strong antimicrobial activity as well as cytotoxic potential in comparison to AERI.     

Thrombospondin-1 mimetic peptide inhibitors of angiogenesis and tumor growth: design, synthesis, and optimization of pharmacokinetics and biological activities

The heptapeptide 1, NAc-Gly-Val-DIle-Thr-Arg-Ile-ArgNHEt, a structurally modified fragment derived from the second type-1 repeat of thrombospondin-1 (TSP-1), is known to possess antiangiogenic activity. However, therapeutic utility could not be demonstrated because this peptide has a very short half-life in rodents. To optimize the PD/PK profile of 1, we initiated a systematic SAR study. The initial structural modifications were performed at positions 5 and 7 of peptide 1 and at the N- and C-termini. Out of several hundred peptides synthesized, the nonapeptide 5 (ABT-526) emerged as a promising lead. ABT-526 inhibited VEGF-induced HMVEC cell migration and tube formation in the nanomolar range and increased apoptosis of HUAEC cells. ABT-526 showed acceptable PK in rodents, dog, and monkey. ABT-526, when incorporated in an angiogenic pellet implanted in the rat cornea at 10 microM, reduced neovascularization by 92%. Substitution of DalloIle in place of DIle in ABT-526 provided nonapeptide 6 (ABT-510), which was 30-fold less active than ABT-526 in the EC migration but 20-fold more active in the tube formation assay. In comparison to ABT-526, ABT-510 has increased water solubility and slower clearance in dog and monkey. Radiolabeled ABT-510 demonstrated saturable binding to HMVEC cells at 0.02-20 nM concentrations and was displaceable by TSP-1. ABT-510 and ABT-526 were shown to significantly increase apoptosis of HUAEC cells. ABT-510 was effective in blocking neovascularization in the mouse Matrigel plug model and inhibited tumor growth in the mouse Lewis lung carcinoma model. Previous studies had shown that ABT-510 was effective in inhibiting the outgrowth of murine melanoma metastases in syngeneic mice and in blocking the growth of human bladder carcinoma implanted in nude mice. It had been also shown that ABT-510 could regress tumor lesions in pet dogs or cause unexpected stabilization of the disease in advanced canine cancer. ABT-526 and ABT-510 are the first compounds in the class of potent inhibitors of angiogenesis that mimic the antiangiogenic function of TSP-1. ABT-510 is currently in phase II clinical studies.     

Rhodostomin, a snake venom disintegrin, inhibits angiogenesis elicited by basic fibroblast growth factor and suppresses tumor growth by a selective alpha(v)beta(3) blockade of endothelial cells

Angiogenesis consists of the proliferation, migration, and differentiation of endothelial cells, although angiogenic factor and integrin-extracellular matrix interaction modulate this process. We report here that a snake venom-derived disintegrin, rhodostomin, inhibited distinct steps in angiogenesis elicited by basic fibroblast growth factor (bFGF), and also suppressed in vivo murine melanoma tumor growth. Rhodostomin dose-dependently inhibited bFGF-induced human umbilical vein endothelial cell (HUVEC) proliferation as examined by cell number count, metabolic activity, and BrdU incorporation assays with submicromolar IC(50) values. However, it apparently did not affect the viability of murine B16F10 melanoma cells, even up to 50 microM. Rhodostomin also inhibited HUVEC migration and invasion evoked by bFGF, and tube formation of bFGF-treated HUVECs in Matrigel. Moreover, rhodostomin selectively inhibited bFGF-, but not vascular endothelial growth factor-associated angiogenesis in the chick chorioallantoic membrane model. Furthermore, rhodostomin blocked both bFGF- and B16F10-induced neovascularization in murine Matrigel plug model and suppressed the growth of subcutaneously inoculated B16F10 solid tumor, leading to a prolonged survival of the rhodostomin-treated C57BL/6 mice. The antiangiogenic effect of rhodostomin on bFGF-treated HUVECs is related to the integrin alpha(v)beta(3) blockade, as evidenced by its selective inhibition on the binding of 7E3, a monoclonal antibody (mAb) raised against alpha(v)beta(3,) but not that of P1F6, an alpha(v)beta(5) mAb toward both naive and bFGF-primed HUVECs. Moreover, 7E3 specifically blocked fluorescein isothiocyanate-conjugated rhodostomin binding to HUVEC, whereas P1F6 and anti-integrin alpha(2), alpha(3), alpha(4), or alpha(5) mAbs did not.