Anti-angiogenic and anti-tumour activities of Lignosus rhinocerus (Cooke) Ryvarden water extracts on HCT116 human colorectal carcinoma cells implanted in chick embryos.

ETHNOPHARMACOLOGICAL RELEVANCE: The sclerotium of Lignosus rhinocerus (Cooke) Ryvarden is used by the local communities in Southeast Asia and China to treat cancer, asthma, fever, and other ailments based on traditional knowledge. The sclerotial water extracts were previously reported to exhibit cytotoxic, apoptotic, and immunomodulatory activities - providing a scientific basis for its use in treating cancer; however, there is still a lack of evidence on its potential anti-angiogenic activity. AIM OF THE STUDY: This study aimed to investigate the toxicity, anti-angiogenic, and anti-tumour activities of the hot-water and cold-water extracts of L. rhinocerus using HCT116 human colorectal carcinoma cells implanted in the chick chorioallantoic membrane (CAM) model. MATERIALS AND METHODS: The toxicity of L. rhinocerus extracts towards the chick embryos was determined 24 h post-treatment. The anti-angiogenic activity of the extracts was then investigated at 0.1-10 µg/embryo (6.7-670 µg/mL) at targeted blood vessels. The anti-tumour effect of selected extracts against the HCT116 human colorectal carcinoma cells xenografted onto the chick embryos was also studied. RESULTS: The cold-water extracts of L. rhinocerus displayed strong in ovo toxicity (LC50: 1.2-37.7 µg/mL) while the hot-water extracts are non-toxic up to 670 µg/mL. Among the extracts, the hot-water extracts demonstrated the highest anti-angiogenic activity with 44.0 ± 17.7% reduction of capillary diameter (relative to the saline-treated control). Moreover, treatment of the HCT116 cells xenografted onto the chick embryos with the hot-water extracts resulted in smaller tumour size and lower number of blood vessels compared to the saline-treated control. CONCLUSIONS: The hot-water extracts of L. rhinocerus sclerotium demonstrated anti-angiogenic and anti-tumour activities but most of the cold-water extracts at similar concentrations were devoid of that. Our findings provide further scientific validation of the medicinal use of the sclerotium in treating cancer and thus, expanding our knowledge on the possible mechanism of its anti-cancer effect apart from direct cytotoxicity, induction of apoptosis and immunomodulation that have been studied thus far.

Rapeseed flower pollen bio-green synthesized silver nanoparticles: a promising antioxidant, anticancer and antiangiogenic compound.

Based on recent researches, bio synthesized silver nanoparticles (Ag-NPs) seem to have the potential in declining angiogenesis and oxidative stress. In the current study, rapeseed flower pollen (RFP) water extract was triggered to synthesize RFP-silver nanoparticles (RFP/Ag-NPs). Moreover, antioxidant, antiangiogenesis and cytotoxicity of the RFP/Ag-NPs against MDA-MB-231, MCF7 and carcinoma cell lines and normal human skin fibroblast HDF were compared. Results indicated that RFP/Ag-NPs have a peak at 430 nm, spherical shape and an average size of 24 nm. According to the results of FTIR, rapeseed pollen capped Ag-NPs. RFP/Ag-NPs have cytotoxicity on MDA-MB-231 and MCF7 cells and decrease cancerous cell viability (IC50 = 3 µg/ml and 2 µg/ml, respectively) in a dose- and time-dependent manner. The morphological data showed that the RFP/Ag-NPs increase the percentage of apoptotic cells compared to the control group and normal cells (human skin fibroblast cells). The apoptotic morphological change was also confirmed with a flow cytometric analysis. RFP/ Ag-NPs' antioxidant activity was evaluated by measuring their ability to scavenge ABTS and DPPH free radicals. The IC50 values were determined at 800 and 830 µg/ml for ABTS and DPPH tests, respectively. According to the results, green-synthesized RFP/Ag-NPs as a safe efficient apoptosis inducer and strong antioxidant compound have the potential to suppress breast cancer carcinogenesis by VEGF down-regulatiion and thus sensitizing them against apoptosis. However, further researches are required to clarify RFP/Ag-NPs' cell specificity and therapeutic doses in in vivo conditions.

Non-toxic and non teratogenic extract of Thuja orientalis L. inhibited angiogenesis in zebra fish and suppressed the growth of human lung cancer cell line.

Lung cancer is a malignant tumour with minimal survival rate and the current treatments are not showing complete remission of tumour and have many side effects. Thus a natural herbal medicine with good anti-cancer properties is highly demanded. Thuja orientalis L. is a traditionally used medicine to cure cough, bronchitis, excessive menstruation, asthma, skin infection and premature baldness. In addition, recent studies have revealed that it has anti-proliferative and anti-cancer activity. Angiogenesis is the main reason for the propagation and metastasis of cancers. We therefore intended to study the effects of the leaf extract of Thuja orientalis L. on angiogenesis as well as lung cancer cell growth. We have tested the anti-angiogenesis efficiency by alkaline phosphatase assay and also analysed the in vivo toxicity and teratogenic effects of various concentration of Thuja orientalis L. extract by establishing an in vivo zebra fish (Danio rerio), a promising model for cancer research which share genetic structure similarity to that of human beings. Also we demonstrated an anti-cancer effect of leaf extract from Thuja orientalis L. on human lung cancer cell line (A549) by MTT and trypan blue assay. The results revealed that the Thuja orientalis L. extract is efficient in repressing lung tumour cell growth significantly (p ≤ 0.01) in all treatments (2.4 mg/ml to 0.3 mg/ml) except 0.15 mg/ml compared to the control. The in vivo toxicity assay has proven that it is non-toxic at concentrations 0.6 mg/ml, 0.3 mg/ml and 0.15 mg/ml in zebrafish. The teratogenic assays revealed the therapeutic index (TI) as 0.808 with 0.7029 mg/ml as LC50 concentration at 24 h which is within the desirable value (below 1) for drug administration. Noticeable inhibition of angiogenesis also was observed in treatment with 2.4 mg/ml to 0.3 mg/ml. Overall we found that Thuja orientalis L. plant leaf extract exhibits better anti-cancer properties as we have validated by in vitro and in vivo analysis.

Chemoembolization with Vascular Disrupting Agent CKD-516 Dissolved in Ethiodized Oil in Combination with Doxorubicin: A VX2 Tumor Model Study.

PURPOSE: To assess feasibility and efficacy of CKD-516, a vascular disrupting agent, in transarterial chemoembolization in a liver tumor model. MATERIALS AND METHODS: A VX2 carcinoma strain was implanted in rabbit liver (n = 40) and incubated for 2 weeks. After confirmation of tumor growth using computed tomography, transarterial chemoembolization was performed. CKD-516 was dissolved in ethiodized oil, and animals were allocated to 4 treatment groups (n = 10 in each): group A, ethiodized oil; group B, ethiodized oil/CKD-516; group C, ethiodized oil + doxorubicin; group D, ethiodized oil/CKD-516 + doxorubicin. To assess hepatic damage, serum aspartate transaminase and alanine transaminase levels were measured on day 1, 3, and 7 after delivery. To assess tumor necrosis, animals were euthanized on day 7, and explanted tumors were stained with hematoxylin and eosin and a terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling assay. Percentage areas of viable tumors were calculated using digitalized histopathologic specimen images. RESULTS: Tumor viability rates were 47.1% ± 11.4%, 27.5% ± 13.6%, 14.4% ± 12.5%, and 0.7% ± 1.0% in groups A, B, C, and D (P < .001). Liver enzyme levels were elevated after drug delivery but recovered during follow-up. Significant between-group differences were observed on days 1, 3, and 7 (aspartate transaminase and alanine transaminase: P = .0135 and P = .0134, P = .0390 and P = .0084, and P = .8260 and P = .0440). CONCLUSIONS: Treatment with a combination of CKD-516 and conventional transarterial chemoembolization showed therapeutic benefit in a liver tumor model.

Polychlorinated biphenyls target Notch/Dll and VEGF R2 in the mouse placenta and human trophoblast cell lines for their anti-angiogenic effects.

The intrauterine environment is particularly vulnerable to environmental exposures. We previously established a mouse model that provided evidence for pregnancy complications and placental anti-angiogenesis in response to Aroclor 1254 (A-1254), a mixture of polychlorinated biphenyls (PCBs). Importantly, these effects were observed in IL-10-/-, but not wild type, mice, suggesting that IL-10 deficiency predisposes to pregnancy disruptive effects of environmental toxicants. However, the mechanisms by which PCBs cause anti-angiogenic effects are unclear. Here, we evaluated PCB-mediated anti-angiogenic effects by diverse but complementary approaches, including HUVEC-mediated trophoblast invasion in nude mice, in vitro three-dimensional capillary tube formation involving HUVEC and/or HTR8 trophoblasts, and aortic ring endothelial cell outgrowth/sprouting. Taken together, our data suggest that PCBs act as potent anti-angiogenic agents. Importantly, we show that treatment of pregnant IL-10-/- mice with A-1254 resulted in placental activation of the Notch/Delta-like ligand (Dll) pathway, a master regulator of cell-cell interaction and vascular patterning. Similar results were obtained with HUVEC and HTR8 trophoblasts. Rescue of A-1254-induced disruption of HUVEC-based tube formation by γ-secretase inhibitor L1790 confirmed the critical role of the Notch/Dll pathway. Our data suggest that PCBs impart pregnancy disruptive functions by activating the Notch/Dll pathway and by inducing anti-angiogenic effects at the maternal-fetal interface.

Pharmacokinetics of calycopterin and xanthmicrol, two polymethoxylated hydroxyflavones with anti-angiogenic activities from Dracocephalum kotschyi Bioss.

BACKGROUND: Recently flavonoids have attracted the attention of researchers in the fight against cancer. Calycopterin and xanthomicrol, are two polymethoxylated flavonoids found in the aerial parts of Dracocephalum kotschyi Bioss.. We have recently shown that these compounds possess antiangiogenic activity and may be of value as potential anticancer agents. In order to demonstrate putative in vivo antitumor effect of these compounds we needed preliminary information on both pharmacokinetics and toxicological properties of these two agents. METHOD: A new online SPE HPLC method for measurement of calycopterin and xanthomicrol in rat plasma was developed. Pharmacokinetic parameters of calycopterin and xanthomicrol, after i.v. administration in rats, were determined. RESULTS: The plasma half-life for both agents was around 4 h, however, the volume of distribution of calycopterin appeared to be about 8 times greater than xanthomicrol. This was probably due the greater hydrophobicity of the former which had other consequences such as much smaller maximum plasma concentration of calycopterin compared to its less methoxylated congener. Preliminary toxicological study of xanthomicrol failed to show any behavioral, histological and biochemical adverse effects after repeated administrations of high doses. Pharmacokinetics of xanthomicrol in rats.

Cissus quadrangularis attenuates the adjuvant induced arthritis by down regulating pro-inflammatory cytokine and inhibiting angiogenesis.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional medicine, Cissus quadrangularis has been used as a chief ingredient of many formulation for the treatment of inflammatory and bone disorders.. OBJECTIVE: The study was carried out to investigate the anti-arthritic activity of C. quadrangularis hydroalcoholic extract (CQHE) and to explore the plausible mechanism of action. MATERIALS AND METHODS: Arthritis was induced by sub plantar administration of formaldehyde (2% v/v) and 0.1ml of complete Freund's adjuvant. Joint swelling was measured on days 8, 9 and 10 in formaldehyde-induced arthritis and on 3, 7, 14 and 21 days in adjuvant induced arthritis (AIA) respectively. Serum and ankle joints of AIA rats were used for estimation of serum TNF-α level, oxidative stress markers and synovial expression of proinflammatory cytokines/cytokine receptor (IL-1ß, IL-6, TNF-R1), angiogenesis marker (VEGF) and matrix metalloproteinases (MMP-3& 9). An acute and 28-day oral toxicity was carried out to evaluate the safety of the test drug. RESULTS: CQHE produced a dose dependent inhibition of joint swelling in both formaldehyde-induced and adjuvant induced arthritis. CQHE treatment also reduced serum TNF-α level, oxidative stress and synovial expression of inflammatory and angiogenesis marker. In sub acute toxicity study of CQHE, chronic administration of CQHE did not produce any physiological and pathological changes as compared to normal rats. CONCLUSION: Our study demonstrated the anti-arthritic potential of C. quadrangularis and it validates its traditional use for the treatment of arthritis and other inflammatory disorders.

Preliminary safety evaluation of a taurocholate-conjugated low-molecular-weight heparin derivative (LHT7): a potent angiogenesis inhibitor.

In our previous studies, taurocholic acid (TA)-conjugated low-molecular-weight heparin derivative (LHT7) has been proven to be a potent anti-angiogenic agent by demonstrated successful blockage capability of vascular endothelial growth factors (VEGF). Preliminary safety evaluations were conducted based on its mechanism of action and chemical behavior. For this purpose, acute toxicity study, and hematological and serological evaluations were carried out. Additionally, in order to evaluate mechanism-related side effects, both blood pressure and the occurrence of proteinuria were measured using a treatment regime of multiple high doses of LHT7 in a biodistribution study. LD50 values for LHT7 in female and male mice were 56.9 and 64.7 mg kg(-1) doses, respectively. There were no vital fluctuations in the serological and hematological parameters, except for the elevated levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) at 100 and 200 mg kg(-1) doses of LHT7, representing vital changes in the liver function. Moreover, the results of mechanism-related studies showed that blood pressure at 50 mg kg(-1) did not change but showed elevated levels of protein in urine. In the biodistribution study, a slight accumulation of LHT7 in the kidney and the liver were observed at the 50 mg kg(-1) repeated dose owing to the presence of bile acid. No fatal damage was observed in this study; most observations were related to the chemical composition or the mechanism of action of the material.

Preclinical investigations of intravitreal ziv-aflibercept.

BACKGROUND AND OBJECTIVE: To investigate the retinal safety of intravitreal (IVT) ziv-aflibercept in rabbits. MATERIALS AND METHODS: Eighteen rabbits were given an IVT injection of ziv-aflibercept (25 mg/mL) or aflibercept (40 mg/mL) and examined by funduscopy, electroretinography (ERG), optical coherence tomography (OCT), light microscopy, and transmission electron microscopy (TEM). Serum, aqueous, and vitreous were obtained afterward for osmolarity analysis. The effect of ziv-aflibercept on human retinal cultured cells (ARPE-19) was assessed by the MTT cell viability assay. RESULTS: All eyes showed normal funduscopy, OCT, and ERG findings at baseline and 24 hours or 7 days after the procedure. Median baseline serum, vitreous, and aqueous osmolarity remained unchanged. Histology and TEM showed no major anatomic signs of toxicity. No cytotoxic effect was observed in ARPE-19 cells exposed to ziv-aflibercept. CONCLUSION: IVT injection ziv-aflibercept at a concentration of 25 mg/mL proved to be safe for the rabbit retina.

A novel poly-naphthol compound ST104P suppresses angiogenesis by attenuating matrix metalloproteinase-2 expression in endothelial cells.

Angiogenesis, the process of neovascularization, plays an important role in physiological and pathological conditions. ST104P is a soluble polysulfated-cyclo-tetrachromotropylene compound with anti-viral and anti-thrombotic activities. However, the functions of ST104P in angiogenesis have never been explored. In this study, we investigated the effects of ST104P in angiogenesis in vitro and in vivo. Application of ST104P potently suppressed the microvessels sprouting in aortic rings ex vivo. Furthermore, ST104P treatment significantly disrupted the vessels' development in transgenic zebrafish in vivo. Above all, repeated administration of ST104P resulted in delayed tumor growth and prolonged the life span of mice bearing Lewis lung carcinoma. Mechanistic studies revealed that ST104P potently inhibited the migration, tube formation and wound closure of human umbilical endothelial cells (HUVECs). Moreover, ST104P treatment inhibited the secretion and expression of matrix metalloproteinase-2 (MMP-2) in a dose-dependent manner. Together, these results suggest that ST104P is a potent angiogenesis inhibitor and may hold potential for treatment of diseases due to excessive angiogenesis including cancer.

Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models.

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.

Antitumor and anti-angiogenic activities of Scutellaria barbata extracts in vitro are partially mediated by inhibition of Akt/protein kinase B.

The Akt pathway is considered a pivotal player in regulating cell survival, growth, migration and angiogenesis. Disruption of normal Akt/PKB/PTEN signaling frequently occurs in numerous types of human cancers. Therefore, this signaling pathway is regarded as an important target for effective cancer therapeutic strategies. In the present study, methanol extracts from Scutellaria barbata (S. barbata) were determined to be Akt/protein kinase B inhibitory, after screening a panel of 40 traditional Chinese herbs with the Fast Activated Cell-based ELISA (FACE) assay. S. barbata extracts were found to suppress the phosphorylation levels of Akt. This inhibition was Akt kinase-specific as it had no effect on PI3K, the upstream kinase of Akt, whereas the levels of phosphorylated Bad and FHKR, the two downstream targets of Akt, changed as the levels of Akt changed. S. barbata extracts also exhibited cytotoxicity against LoVo and human umbilical vein endothelial cells (HUVECs). Furthermore, this extract inhibited the process of in vitro angiogenesis of HUVECs on Matrigel. S. barbata may be a suitable alternative source with which to isolate small molecules for use as Akt kinase inhibitors.

Reproductive toxicity of Rhizoma Sparganii (Sparganium stoloniferum Buch.-Ham.) in mice: mechanisms of anti-angiogenesis and anti-estrogen pharmacologic activities.

ETHNOPHARMACOLOGICAL RELEVANCE: Indications and preliminary studies of Rhizoma Sparganii (RS) suggest its pharmacological mechanism is involved with endocrine/angiogenesis functions. We therefore studied its potential toxicity on reproduction in mice. MATERIALS AND METHODS: Reproductive toxicity of 100, 200 and 400 mg/kg RS extract were studied in pregnant ICR mice and its offspring. The embryos' fibroblast growth factor-1 (FGF-1), vascular endothelial growth factor (VEGF) and estrogen receptor-α (ER-α) were evaluated as targets of endocrine/angiogenesis by immunohistochemical test. RESULTS: The offspring of treated mice (100, 200 and 400 mg/kg RS extract) during their pregnancy had various pathological conditions, suggesting an abnormal FGF signaling phenomenon during pregnancy. Embryos from the 400 mg/kg group had significantly depressed levels of FGF-1 (P < 0.01) and VEGF (P < 0.05) expression levels as compared to controls by immunohistochemical test. Dysplasia in the heart (12.9%), craniofacial region (18.3%) and vertebrae (32.5%) presented in embryos of the 400 mg/kg group. Furthermore, the ER-α expression was inversely proportional to FGF-1 levels in the same embryo (P < 0.01). CONCLUSIONS: These results implicate a FGF signaling abnormality in vivo and indicate that RS has anti-angiogenesis and anti-estrogen toxicity effects in pregnant rodents.

Broad spectrum antiangiogenic treatment for ocular neovascular diseases.

UNLABELLED: Pathological neovascularization is a hallmark of late stage neovascular (wet) age-related macular degeneration (AMD) and the leading cause of blindness in people over the age of 50 in the western world. The treatments focus on suppression of choroidal neovascularization (CNV), while current approved therapies are limited to inhibiting vascular endothelial growth factor (VEGF) exclusively. However, this treatment does not address the underlying cause of AMD, and the loss of VEGF's neuroprotective can be a potential side effect. Therapy which targets the key processes in AMD, the pathological neovascularization, vessel leakage and inflammation could bring a major shift in the approach to disease treatment and prevention. In this study we have demonstrated the efficacy of such broad spectrum antiangiogenic therapy on mouse model of AMD. METHODS AND FINDINGS: Lodamin, a polymeric formulation of TNP-470, is a potent broad-spectrum antiangiogenic drug. Lodamin significantly reduced key processes involved in AMD progression as demonstrated in mice and rats. Its suppressive effects on angiogenesis, vascular leakage and inflammation were studied in a wide array of assays including; a Matrigel, delayed-type hypersensitivity (DTH), Miles assay, laser-induced CNV and corneal micropocket assay. Lodamin significantly suppressed the secretion of various pro-inflammatory cytokines in the CNV lesion including monocyte chemotactic protein-1 (MCP-1/Ccl2). Importantly, Lodamin was found to regress established CNV lesions, unlike soluble fms-like tyrosine kinase-1 (sFlk-1). The drug was found to be safe in mice and have little toxicity as demonstrated by electroretinography (ERG) assessing retinal and by histology. CONCLUSIONS: Lodamin, a polymer formulation of TNP-470, was identified as a first in its class, broad-spectrum antiangiogenic drug that can be administered orally or locally to treat corneal and retinal neovascularization. Several unique properties make Lodamin especially beneficial for ophthalmic use. Our results support the concept that broad spectrum antiangiogenic drugs are promising agents for AMD treatment and prevention.

Comparative evaluation of apoptotic activity in photoreceptor cells after intravitreal injection of bevacizumab and pegaptanib sodium in rabbits.

PURPOSE: To evaluate quantitatively the apoptotic activity after intravitreal injections of pegaptanib sodium and bevacizumab in the rabbit retina. METHODS: Different doses of bevacizumab (0.25, 0.625, 1.25, and 2.5 mg) and pegaptanib sodium (0.15, 0.3, and 0.6 mg) were injected intravitreally in 48 rabbits. The eyes were enucleated at different times for early studies at day 14 and for late studies at 3 months after a single injection or at 3 months, with 1 injection in each of the 3 months (day 90). The time course and dose-response of photoreceptor cells in the rabbit retina after intravitreal injection of bevacizumab or pegaptanib sodium were examined by histologic analysis with hematoxylin and eosin (H&E) staining, caspase-3 and -9 immunostaining, and in situ terminal-deoxynucleotidyl transferase-mediated biotin-deoxyuridine triphosphate nick-end labeling (TUNEL) of DNA fragments of paraffin-embedded sections. RESULTS: No sign of retinal toxicity was seen in H&E stained histologic sections of eyes that had received bevacizumab or pegaptanib sodium. Nuclear DNA fragmentation in the outer retinal layers shown by the TUNEL method was evident in the high-dose groups (55.3% with 1.25 mg and 64.5% with 2.5 mg bevacizumab, and 48.5% with 0.6 mg pegaptanib sodium) at 14 days and also in the clinical dose groups (49.8% with three injections [1 each month] of 0.625 mg bevacizumab and 44.3% with 0.15 mg pegaptanib sodium) at 90 days. The ratios of TUNEL-positive cells in physiologic saline and the sham-control groups were 32.3% and 21%, respectively. CONCLUSIONS: Intravitreal injection of bevacizumab and pegaptanib sodium caused a significant increase in apoptotic activity in rabbit photoreceptor cells. However, although bevacizumab caused increasing apoptotic activity at higher doses, similar dose-dependent adverse effects were not evident for pegaptanib sodium.

Taspine isolated from Radix et Rhizoma Leonticis inhibits growth of human umbilical vein endothelial cell (HUVEC) by inducing its apoptosis.

The present study was to evaluate the effects of taspine isolated from Radix et Rhizoma Leonticsi on the growth and apoptosis of human umbilical vein endothelial cell (HUVEC) line by MTT and flow cytometer, respectively. At the same time, a series of changes were observed in HUVEC treated by taspine, including microstructure, protein expression of bax, bcl-2 and VEGF. The change of microstructure was observed by transmission electron microscope (TEM). The protein expression of bax and bcl-2 was detected by immunohistochemistry (IHC), and VEGF protein secreted was determined by enzyme-linked immunosorbent assay (ELISA). The results showed taspine could inhibit growth and induce apoptosis of HUVEC in a dose-dependent manner. Cell cycle was significantly stopped at the S phase. Under electronic microscope, the morphology of HUVEC treated with taspine showed nuclear karyopycnosis, chromatin agglutination and typical apoptotic body. Bcl-2 and VEGF expressions were decreased and bax expression was increased. All these results demonstrate that taspine has an inhibitory effect on growth of HUVEC and can induce its apoptosis.

Preclinical safety evaluation of intravitreal injection of full-length humanized vascular endothelial growth factor antibody in rabbit eyes.

PURPOSE: To evaluate the preclinical safety of intravitreal bevacizumab, which is a full-length humanized monoclonal antibody against the vascular endothelial growth factor (VEGF), in rabbit eyes over a short-term period. METHODS: Twenty-four rabbits were divided into two groups, each with two subgroups. The first group (groups 1 and 2) received 1.25 mg (0.05 mL) intravitreal bevacizumab, and the second group (groups 3 and 4) received 3.00 mg (0.12 mL) intravitreal bevacizumab. The right eyes were designated as the study eyes, and the left eyes served as a control and received the same volume of saline intravitreally. Groups 1 and 3 were labeled as early groups and scheduled to be terminated at 14 days. Groups 2 and 4, labeled as late groups, were scheduled to be terminated at 28 days. Besides electroretinography (ERG) and visually evoked potentials (VEP), central corneal thickness, intraocular pressure, fundus photography, and anterior segment imaging were performed at baseline and scheduled time points. Enucleated eyes were preserved for light and electron microscopic investigation. RESULTS: No anterior segment inflammation was observed, except in one eye in group 1 which showed a uveitic reaction. No evidence of retinal toxicity was seen with intravitreal bevacizumab at doses of 1.25 and 3.00 mg, by either ERG or light microscopy. Electron microscopic assessment revealed mitochondrial damage in the inner segments of photoreceptors. Immunohistochemical staining with bax and caspase-3 and -9 showed intensive apoptotic protein expression in all study sections and minimal expression in the control eyes. CONCLUSIONS: Although electrophysiologic investigation and light microscopy showed normal retinal function and structure, mitochondrial disruption in the inner segments of photoreceptors was detected by electron microscopy, and apoptotic expression was detected after the injection of intravitreal bevacizumab.

Preclinical safety of anecortave acetate.

A number of preclinical safety pharmacology and toxicity studies have been performed on the angiostatic cortisene anecortave acetate in various species and using different routes of administration (oral, intravenous, subcutaneous, topical ocular, intraocular injection, posterior juxtascleral) and a wide range of doses (0-1,000 mg/kg). Anecortave acetate did not interact with a broad panel of pharmacological receptors and had no apparent pharmacological effects on major organ systems including the central nervous, gastrointestinal, renal, cardiovascular, and respiratory systems. Oral, topical ocular, and posterior juxtascleral administration of anecortave acetate had no significant ocular or systemic side effects or toxicity. In addition, there was no significant carcinogenic or reproductive/developmental toxicity associated with anecortave acetate in genotoxicity, carcinogenicity, and reproductive toxicity studies.

Sorafenib in advanced melanoma: a Phase II randomised discontinuation trial analysis.

The effects of sorafenib--an oral multikinase inhibitor targeting the tumour and tumour vasculature--were evaluated in patients with advanced melanoma enrolled in a large multidisease Phase II randomised discontinuation trial (RDT). Enrolled patients received a 12-week run-in of sorafenib 400 mg twice daily (b.i.d.). Patients with changes in bi-dimensional tumour measurements <25% from baseline were then randomised to sorafenib or placebo for a further 12 weeks (ie to week 24). Patients with > or =25% tumour shrinkage after the run-in continued on open-label sorafenib, whereas those with > or =25% tumour growth discontinued treatment. This analysis focussed on secondary RDT end points: changes in bi-dimensional tumour measurements from baseline after 12 weeks and overall tumour responses (WHO criteria) at week 24, progression-free survival (PFS), safety and biomarkers (BRAF, KRAS and NRAS mutational status). Of 37 melanoma patients treated during the run-in phase, 34 were evaluable for response: one had > or =25% tumour shrinkage and remained on open-label sorafenib; six (16%) had <25% tumour growth and were randomised (placebo, n=3; sorafenib, n=3); and 27 had > or =25% tumour growth and discontinued. All three randomised sorafenib patients progressed by week 24; one remained on sorafenib for symptomatic relief. All three placebo patients progressed by week-24 and were re-started on sorafenib; one experienced disease re-stabilisation. Overall, the confirmed best responses for each of the 37 melanoma patients who received sorafenib were 19% stable disease (SD) (ie n=1 open-label; n=6 randomised), 62% (n=23) progressive disease (PD) and 19% (n=7) unevaluable. The overall median PFS was 11 weeks. The six randomised patients with SD had overall PFS values ranging from 16 to 34 weeks. The most common drug-related adverse events were dermatological (eg rash/desquamation, 51%; hand-foot skin reaction, 35%). There was no relationship between V600E BRAF status and disease stability. DNA was extracted from the biopsies of 17/22 patients. Six had V600E-positive tumours (n=4 had PD; n=1 had SD; n=1 unevaluable for response), and 11 had tumours containing wild-type BRAF (n=9 PD; n=1 SD; n=1 unevaluable for response). In conclusion, sorafenib is well tolerated but has little or no antitumour activity in advanced melanoma patients as a single agent at the dose evaluated (400 mg b.i.d.). Ongoing trials in advanced melanoma are evaluating sorafenib combination therapies.

Cartilage dysplasia and tissue mineralization in the rat following administration of a FGF receptor tyrosine kinase inhibitor.

PD176067 is a reversible and selective inhibitor of fibroblast growth factor receptor tyrosine kinase, and was in preclinical development as an angiogenesis inhibitor for the treatment of solid tumors. A 14-day oral toxicity study of PD176067 in young female rats (7 weeks old) was conducted at doses of 2.5, 5, and 10 mg/kg/day (15, 30, and 60 mg/m(2), respectively). Skeletal changes, and vascular and soft tissue mineralization were observed as primary drug-related toxicities. To determine if these changes are specific to young, rapidly growing animals with increased vascular and osseous development, PD176067 was administered to mature (11 months old) rats. Female rats received PD176067 by gavage for 14 days at doses of 2.5, 5, and 10 mg/kg/day and necropsied on day 15. Clinical signs of toxicity were seen at > or =5 mg/kg and one death occurred at 10 mg/kg. Physeal dysplasia (distal femur, proximal tibia, sternum) occurred in all drug-treated animals and was characterized by dose-related increased thickness of the zones of chondrocyte proliferation and hypertrophy, and marked thickening of the zone of ossification. Cartilage hyperplasia was characterized by proliferation of chondrocytes along margins of the synchondrosis and subperiosteum of sternebrae. Serum phosphorus levels increased 47% and 166% at 5 and 10 mg/kg, respectively. Mineralization of cardiac myocytes, aorta, various arteries, renal tubules, and gastric mucosa and muscularis was seen at 10 mg/kg, and consistent with the presence of calcium-phosphorus deposition. Physeal changes occurred at similar plasma PD176067 exposures in young and mature rats (AUC > or = 4.83 microg.hr/mL). PD176067 produced morphologically similar lesions in young and adult rats.