Anginex
A potent angiogenesis inhibitor and anti-tumor peptide
Antiangiogenic therapies in endometriosis
Oral contraceptives, androgenic agents, progestins and gonadotropin-releasing
hormone analogues have all been successfully used in the treatment
of endometriosis. However, none of these drugs can eradicate
the disease. It is widely accepted that the growth of newly
formed blood vessels is essential for the establishment and
growth of endometriotic lesions; therefore, inhibition of angiogenesis
may offer a new option for treatment of this disorder. In this
paper, we reviewed anti-vascular endothelial growth factor agents
and other angiostatic drugs (i.e., TNP470, endostatin, anginex,
rapamycin) that have been studied in laboratory and animal models
of endometriosis. Although preliminary results are interesting,
further investigations are required before clinical trials can
be planned in humans. Ferrero S, Ragni N, Remorgida V.
Br J Pharmacol. 2006 Aug 7; [Epub ahead of print]
Cloning an artificial gene
encoding angiostatic anginex: From designed peptide to functional
recombinant protein
Anginex, a designed peptide 33-mer, is a potent angiogenesis
inhibitor and anti-tumor agent in vivo. Anginex functions by
inhibiting endothelial cell (EC) proliferation and migration
leading to detachment and apoptosis of activated EC's. To better
understand tumor endothelium targeting properties of anginex
and enable its use in gene therapy, we constructed an artificial
gene encoding the biologically exogenous peptide and produced
the protein recombinantly in Pichia pastoris. Mass spectrometry
shows recombinant anginex to be a dimer and circular dichroism
shows the recombinant protein folds with beta-strand structure
like the synthetic peptide. Moreover, like parent anginex, the
recombinant protein is active at inhibiting EC growth and migration,
as well as inhibiting angiogenesis in vivo in the chorioallantoic
membrane of the chick embryo. This study demonstrated that it
is possible to produce a functionally active protein version
of a rationally designed peptide, using an artificial gene and
the recombinant protein approach. Brandwijk RJ, et al. Biochem Biophys
Res Commun. 2005 Aug 12;333(4):1261-8
Anti-angiogenesis therapy can overcome endothelial
cell anergy and promote leukocyte-endothelium interactions and
infiltration in tumors
Tumor escape from immunity, as well as the failure of several
anti-cancer vaccination and cellular immunotherapy approaches,
is suggested to be due to the angiogenesis-mediated suppression
of endothelial cell (EC) adhesion molecules involved in leukocyte-vessel
wall interactions. We hypothesized that inhibition of angiogenesis
would overcome this escape from immunity. We investigated this
in vivo by means of intravital microscopy and ex vivo by immunohistochemistry
in two mouse tumor models. Angiogenesis inhibitors anginex,
endostatin, and angiostatin, and the chemotherapeutic agent
paclitaxel were found to significantly stimulate leukocyte-vessel
wall interactions by circumvention of EC anergy in vivo, i.e.,
by the up-regulation of endothelial adhesion molecules in tumor
vessels. This was confirmed by in vitro studies of cultured
EC at the protein and mRNA levels. The new angiostatic designer
peptide anginex was most potent at overcoming EC anergy; the
enhanced leukocyte-vessel interactions led to an increase in
the numbers of tumor infiltrating leukocytes. While anginex
inhibited tumor growth and microvessel density significantly,
the amount of infiltrated leukocytes (CD45), as well as the
number of CD8+ cytotoxic T lymphocytes, was enhanced markedly.
The current results suggest that immunotherapy strategies can
be improved by combination with anti-angiogenesis. Dirkx AE, et al. FASEB J. 2006
Apr;20(6):621-30.
Anginex synergizes with radiation therapy
to inhibit tumor growth by radiosensitizing endothelial cells
We have demonstrated that the designed peptide anginex displays
potent antiangiogenic activity. The aim of our study was to
investigate the effect of anginex on established tumor vasculature
as an adjuvant to radiation therapy of solid tumors. In the
MA148 human ovarian carcinoma athymic mouse model, anginex (10
mg/kg) in combination with a suboptimal dose of radiation (5
Gy once weekly for 4 weeks) caused tumors to regress to an impalpable
state. In the more aggressive SCK murine mammary carcinoma model,
combination of anginex and a single radiation dose of 25 Gy
synergistically increased the delay in tumor growth compared
to the tumor growth delay caused by either treatment alone.
Immunohistochemical analysis also demonstrated significantly
enhanced effects of combined treatment on tumor microvessel
density and tumor or endothelial cell proliferation and viability.
In assessing physiologic effects of anginex, we observed a reduction
in tumor perfusion and tumor oxygenation in SCK tumors after
5-7 daily treatments with anginex with no reduction in blood
pressure. To test anginex as a radiosensitizer, additional studies
using SCK tumors were performed. Three daily i.p. injections
of anginex were able to enhance the effect of 2 radiation doses
of 10 Gy, resulting in 50% complete responses, whereas the known
antiangiogenic agent angiostatin did not enhance the radiation
response of SCK tumors. Mechanistically, it appears that anginex
functions as an endothelial cell-specific radiosensitizer because
anginex showed no effect on in vitro radiosensitivity of SCK
or MA148 tumor cells, whereas anginex significantly enhanced
the in vitro radiosensitivity of 2 endothelial cell types. This
work supports the idea that the combination of the antiangiogenic
agent anginex and radiation may lead to improved clinical outcome
in treating cancer patients. Dings RP, et al. Int J Cancer.
2005 Jun 10;115(2):312-9.
Figure 1 amino acid sequence and structure of anginex and
CP analogues
The amino acid sequence of anginex is shown conformed as
an anti-parallel b-sheet on the left-hand side of the Figure.
The N-terminus is at the right bottom as labelled. CP analogues
have only the first 27 residues and therefore are devoid
of C-terminal residues Arg-Glu-Lys-Ser-Lys-Asp. The new
C-terminal residue has its backbone carboxylate group amidated.
The amino acid sequence for the control linear peptide 27mer
(no cysteine residues) CP-1 is given on the right-hand side
of the Figure. CP-2 to CP-6 [i.e. CP-2 (Cys6–Cys25),
CP-3 (Cys8–Cys23), CP-4 (Cys10–Cys21), CP-5
(Cys12–Cys19), CP-6 (Cys13–Cys18)] have the
same sequence as CP-1, with disulphide bridges positioned
between b-strand 1 and b-strand 2 as indicated. CP-7 [Cys13–Cys19]
and CP-8 [Cys6–Cys26] are control peptides that alter
the alignment of b-strands 1 and 2. Single-letter amino-acid
notation is used. Ruud P. M. DINGS et al. Biochem. J. (2003)
373 (281–288)
Anginex causes significant tumor growth inhibition. The
mean tumor growth of human epithelial ovarian carcinoma
cell line MA148 is shown in athymic mice treated with a
dose range of anginex administered by minipumps implanted
in the left flank of animals (,
5 mg/kg/day, n = 14; ,
10 mg/kg/day, n = 16; ,
20 mg/kg/day, n = 8). Controls ()
contained PBS with BSA (n = 13) and PBS with 5
mg/kg/day ßpep28 (,
n = 8) and 10 mg/kg/day ßpep28 (,
n = 4), which did not differ from each other. The
treatment period was initiated on the day of tumor inoculation
(day 0) and lasted for 28 days as indicated by the arrow.
Data from three independent studies are shown and represent
the mean tumor volume in mm3 (±SE). Ruud P. M. Dings et al. Cancer
Research 63, 382-385, January 15, 2003
The mean tumor growth curves
in a human ovarian carcinoma model treated with anginex, carboplatin,
angiostatin, or a combination treatment. A, groups
shown are defined as follows: ,
vehicle containing BSA (n = 11); ,
anginex (10 mg/kg/day, n = 11); x, carboplatin (n
= 12); , a combination group (n = 12). Carboplatin
was given in a suboptimal dosage (32.5 mg/kg) once every 3
days i.p. B, groups shown are defined as follows:
,
vehicle containing BSA (n = 11); ,
angiostatin (20 mg/kg/day, n = 11); ,
anginex (10 mg/kg/day, n = 11); ,
a combination group (n = 12). In both experiments,
treatment was given for 28 days starting on day 7 postinoculation.
The vehicle and anginex were given by osmotic minipump implanted
s.c. in the flank, and angiostatin was given daily by s.c.
injections in the neck (9) . The data are shown as means of
tumor burden. Error bars, SEs. The tumor volumes
were determined three times a week. The insets in
both A and B show body weights of mice during
treatment as an indirect measurement of toxicity. Ruud P. M. Dings et al. Cancer
Research 63, 382-385, January 15, 2003
,
5 mg/kg/day, n = 14; 
,
10 mg/kg/day, n = 16; 
,
20 mg/kg/day, n = 8). Controls (
)
contained PBS with BSA (n = 13) and PBS with 5
mg/kg/day ßpep28 (
,
n = 8) and 10 mg/kg/day ßpep28 (
,
n = 4), which did not differ from each other. The
treatment period was initiated on the day of tumor inoculation
(day 0) and lasted for 28 days as indicated by the arrow.
Data from three independent studies are shown and represent
the mean tumor volume in mm3 (±SE).
,
angiostatin (20 mg/kg/day, n = 11); 
