EP1503752A1

EP1503752A1 - Use of 8-prenylflavanones for anti-angiogenesis therapy and for fibrinolytic therapy

Info

Publication number: EP1503752A1
Application number: EP03711961A
Authority: EP
Inventors: Wolf-Dieter Schleuning; Michael Pepper
Original assignee: Schering AG
Current assignee: Bayer Pharma AG
Priority date: 2002-05-10
Filing date: 2003-03-10
Publication date: 2005-02-09
Also published as: WO2003094910A1; EP1360959A1; AU2003218721A1; JP2005526122A

Abstract

The present invention provides the use of 8-Prenylflavanones of the general (formula I), wherein R1 designates a hydrogen atom, a hydroxyl group in position 2', 3' or 4', a methoxy group in position 2', 3' or 4' or an ethoxy group in position 3' or 4', R2 designates a hydrogen atom, a hydroxyl group in position 3', 4', 5' or 6', a methoxy group in position 3' or 4' or an ethoxy group in position 5', R3 designates a hydrogen atom, a hydroxyl group in position 4', 5' or 6' or a methoxy group in position 4', 5' or 6', R4 designates a hydrogen atom or a hydroxyl group, for the preparation of a pharmaceutical composition for the treatment or prevention of angiogenesis-related diseases and for the treatment or prevention of thrombotic disorders.

Description

Use of 8-Prenylflavanones for anti-angiogenesis therapy and for fibrinolytic therapy

The invention relates to the use of 8-Prenylflavanones in a pharmaceutical composition.

8-Prenylflavanones are found in a variety of plants. One member of this group of compounds is 8-PrenyInaringenin (5,7-Dihydroxy-2-(4-hydroxyphenyl)-8-(3-methylbut-2-enyl)-chroman-4- on) which occurs in e.g. Helichrysum hypocephalum (Bohlmann, F. et al. 1979, Phytochemistry 18, 1246-1247) and citrus plants (Ito, C. et al. 1988, Chem. Pharm. Bull. 36, 3292-3295). It has the following structure:

It has been shown to have estrogenic activity (Kitao a, M. JP 08-165238). It was also tested for cytotoxic activity against oral tumor cell lines, but the results indicated only a slightly higher cytotoxic activity against tumor cells compared to normal cells(Shirataki, Y. et al. 2001 , Anticancer Research 21 , 275-280).

Angiogenesis, the formation of new capillary blood vessels, is required for tissue growth, wound healing and female reproductive function, and is an important component of hemangiomas and the process of ocular neovascularization (reviewed by Pepper, 1997, Arterioscler. Thromb. Vase. Biol. 17: 605-619). It is also essential for the growth and persistence of solid tumors and their metastases. For solid tumors to grow beyond a critical size, they must recruit endothelial cells from the surrounding stroma to form their own endogenous microcirculation (Folkman, 1974). To stimulate angiogenesis, the production of positive regulators is upregulated. Positive regulators of angiogenesis include members of the vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) families. Endogenous negative regulators of angiogenesis, i.e. inhibitors of angiogenesis, include angiostatin, endostatin, thrombospondin, interferons, platelet factor IV, 16kDa N- terminal fragment of prolactin, IL-12, gro-β, proliferin-related protein, protease inhibitors, 2- methoxyestradiol and soluble cytokine. Most of these compounds are peptides or proteins.

For the treatment of angiogenesis-related diseases, it is desirable to have a low molecular weight compound which inhibits angiogenesis.

Fibrinolysis is controlled by the expression of plasminogen activator (tPA) and plasminogen activator inhibitor-1 (PAI-1). tPA or other plasminogen activators are used for the therapy of acute myocardial infarction. This therapy is, however, not applicable for the prevention or longterm treatment of thrombotic diseases. In this case, an endogenous increase of the fibrinolytic activity would be desirable. This could be achieved by increasing the plasminogen activator activity and by deceasing the activity of PAI-1. Substances have been described which either increase both activities (e.g. VEGF, Pepper et al. 1991 Arterioscler. Thromb. Vase. Biol. 17: 605-619; Mandriota et al. 1995, J. Biol. Chem. 270: 9709-9716 ) or have only very little effect on tPA although they increase the urokinase-type plasminogen activator (e.g. bFGF, Pepper et al. 1990, J. Cell Biol. 111 : 743-755).

Therefore, the problem is to find a substance which will increase the endogenous tPA activity and decrease the PAI-1 activity.

The present invention solves the two problems by providing the use of 8-Prenylflavanones of the general formula I:

wherein

RT designates a hydrogen atom, a hydroxyl group in position 2', 3' or 4', a methoxy group in position 2', 3' or 4' or an ethoxy group in position 3' or 4',

R₂ designates a hydrogen atom, a hydroxyl group in position 3', 4', 5' or 6', a methoxy group in position 3' or 4' or an ethoxy group in position 5',

R₃ designates a hydrogen atom, a hydroxyl group in position 4', 5' or 6' or a methoxy group in position 4', 5' or 6',

R₄ designates a hydrogen atom or a hydroxyl group, for the preparation of a pharmaceutical composition for the treatment and prevention of angiogenesis-related diseases and for the treatment and prevention of thrombotic disorders.

The preferred compound of formula I is 8-Prenylnaringenin wherein R1 , R2 and R4 designate a hydrogen atom and R3 designates a hydroxyl group in position 4'.

8-Prenylflavanones show an inhibitory effect on angiogenesis. 8-Prenylnaringenin inhibits angiogenesis induced by various positive regulators, e.g. bFGF and VEGF and also the synergistic effect of bFGF and VEGF in combination. Treatment of the chicken embryo chorioallantoic membrane (CAM) with 8-prenylnaringenin resulted in significant reductions in blood vessel growth (Table and figure 6).

One or more 8-Prenylflavanones of the general formula I can be used for the preparation of a pharmaceutical composition for the prevention and treatment of angiogenesis-related diseases. Angiogenesis-related diseases are e.g. angiogenesis-dependent cancers like solid, malignant and metastasing tumors, benign tumors, rheumatoid arthritis, psoriasis, ocular angiogenesis disease, plaque neovascularization, atherosclerosis and scleroderma.

The 8-Prenylflavanones of the general formula I increase the expression of tPA, measured by determining the amount of the corresponding mRNA, and the activity of tPA, measured by zymography. The amount of RNA can be determined by a Northern blot analysis as described in example 6 and the zymography can be performed as described in example 5. In addition, the 8-Prenylflavanones of the general formula I decrease the activity of PAI-1 , measured by reverse zymography (see example 5) and increases the expression of uPA, measured by determining the amount of the corresponding mRNA, and the activity of uPA. Genistein, an structurally unrelated phytoestrogen, has previously been shown to decrease uPA (Fotsis et al. 1993, Cancer Res. 57: 2916-2921). An object of the invention is the use of one or more 8-Prenylflavanones of the general formula I for the preparation of a pharmaceutical composition for the treatment and prevention of thrombotic disorders. Thrombotic disorders are e.g. coronary artery thrombosis (acute mycardial infarction), venous thrombosis, cerebral artery thrombosis (ischemic stroke), thrombangitis obliterans (Burger's disease), arteriitis temporalis (Horton's disease), arteriitis Takayashu and APC-resistance.

In a further object, the present invention concerns a pharmaceutical composition comprising one or more 8-Prenylflavanones and at least one pharmaceutically acceptable excipient for the treatment of angiogenesis-related diseases and for the treatment and prevention of thrombotic disorders. These excipients may be carriers, diluents, absorption enhancers, preservatives, buffers, agents for adjusting the osmotic pressure, tablet disintegrating agents and other ingredients which are conventionally used in the art. Examples of solid carriers are magnesium carbonate, magnesium stearate, dextrin, lactose, sugar, talc, gelatin, pectin, tragacanth, methyl cellulose, sodium carboxymethyl cellulose, low melting waxes and cocoa butter. Liquid compositions include sterile solutions, suspensions and emulsions. A composition for transdermal administration of 8-Prenylnaringenin may be provided in the form of a patch and a composition for nasal administration may be provided in the form of a nasal spray in liquid or powder form.

Usually, not more than 1000 mg, preferably not more than 100 mg, and in some preferred instances not more than 10 mg of one or more 8-Prenylflavanones is to be administered to a human per day.

Examples

Example 1 : Synthesis of 8-Prenylflavanones

8-Prenylflavanones are synthesized by the methods described in JP 08-165238.

8-Prenylnaringenin was synthesized as described in Satoshi Tahara, Phytochemistry 36, 1261-1271 , 1994.

Example 2: Cell culture

Bovine adrenal cortex-derived microvascular endothelial cells (BME cells, Furie et al., 1984, J. Cell Biol. 98, 1033-1041) were grown in minimal essential medium, alpha modification (α- MEM - Gibco BRL), supplemented with heat-inactivated donor calf serum (DCS - Flow Laboratories), penicillin (110U/ml) and streptomycin (110μg/ml). Bovine aortic endothelial (BAE) cells, isolated from scrapings of adult bovine thoracic aortas and cloned by limiting dilution as previously described (Pepper et al., Am J. Physiol. 262: C1246-C1257, 1992), were cultured in low glucose Dulbecco's modified minimal essential medium (DMEM, Gibco) supplemented with 10% DCS and antibiotics. Cell lines were maintained in 1.5% gelatin- coated tissue culture flasks (Falcon Labware, Becton-Dickinson Company) and subcultured at a split ratio of 1/3 or 1/4. The endothelial nature of the cells has previously been confirmed by Dil-Ac-LDL (Paesel and Lorei) uptake and immunostaining with a rabbit polyclonal antiserum against human von Willebrand factor (Nordic Immunology).

Example 3:

8-Prenylnaringenin inhibits BME and BAE cell in vitro angiogenesis

bFGF and VEGF induce BME and BAE cells to invade three-dimensional collagen gels, and this is accompanied by the formation of capillary like tubular structures (Montesano et al., Proc. Natl. Acad. Sci. USA 83: 7297-7301, 1986; Pepper et al., Biochem. Biophys. Res. Comm. 189: 824-831 , 1992; Pepper et al., Exp. Cell Res. 204: 356-363, 1993, Pepper et al., J. Cell Sci. 108: 73-83, 1995). In addition, bFGF and VEGF induce a synergistic invasive response in BME and BAE cells (Pepper et al., 1992, Biochem. Biophys. Res. Commun. 189: 824-831 ; Pepper et al 1995, J. Cell Sci. 108: 73-83).

In vitro angiogenesis assay:

Three-dimensional collagen gels were prepared as described (Montesano et al., Cell 42:

469-477, 1985): 8 volumes of a solution of type I collagen from rat tail tendons (approximately 1.5mg/ml) were mixed with 1 volume of 10x minimal essential medium and 1 volume of sodium bicarbonate (11.76mg/ml) on ice, dispensed into 18mm tissue culture wells, and allowed to gel at 37^eC for 10 minutes. BME cells were then seeded onto the collagen gels at 1.0 x 10⁵ cells/well in 500μl α-MEM supplemented with 5% DCS and were grown to confluence (3-4 days), at which point serum was reduced to 2% and treatment with bFGF (155 amino acid form) and/or VEGF (165-amino acid homodimeric species -

VEGF-J65 from Peprotech) and 8-Prenylnaringenin was begun. Medium and treatments were renewed after 2 and 4 days. Three randomly selected fields measuring 1.0 mm x 1.4 mm were photographed in each well at a single level beneath the surface monolayer by phase contrast microscopy using a Nikon Diaphot TMD inverted photomicroscope. Invasion was quantitated by determining the total additive length of all cellular structures which had penetrated beneath the surface monolayer either as apparently single cells or in the form of cell cords (Pepper et al., Biochem. Biophys. Res. Comm. 189: 824-831 , 1992). Results are expressed as the mean total additive sprout length in μm ± s.e.m., and are from three separate experiments per condition. In the case of (±)-δ-Prenylnaringenin, cells treated with bFGF (10ng/ml) or bFGF + VEGF were photographed after 2-3 days, whereas cells treated with VEGF (30ng/ml) alone were photographed after 4-7 days. It has previously been shown that the kinetics of VEGF-induced invasion are slower than those for bFGF or bFGF + VEGF (Pepper et al., J. Cell Physiol. 177: 439-452, 1998).

Results:

(±)-δ-Prenylnaringenin inhibits in vitro angiogenesis induced by bFGF, VEGF or a combination of the bFGF and VEGF, in a dose-dependent manner (Figure 1). A quantitative analysis revealed that 8-Prenylnaringenin inhibited BME cell invasion with an IC₅₀ of between 3-1 OμM, irrespective of the angiogenic stimulus (Figure 1 ).

Example 4: 8-Prenylnaringenin inhibits BME cell proliferation

Proliferation assay:

BME cells were seeded into gelatin-coated wells of 24 well plates at 10O00 cells per well in α-MEM supplemented with 5% DCS. 24 hours later, fresh medium was added together with 8-Prenylnaringenin at the indicated concentrations or TGF-β1 (1ng/ml), and bFGF (10ng/ml) was added to half the wells. 2 days later, medium and compounds were renewed, and after a further 3 days (i.e. 5 day assay), cells were trypsinized and counted in a FACScan Analyzer. Results are shown as % inhibition calculated from the mean of duplicate wells. Results:

In the 5 day proliferation assay in the presence of 5% DCS, 8-Prenylnaringenin reduced BME cell number in a dose dependent manner (Figure 2). This effect was observed whether or not bFGF was co-added to the medium. Inhibition occurred with an IC₅₀ of about 10μM, and a maximal inhibitory effect was observed with 30μM 8-Prenylnaringenin (67 and 75% inhibition in the absence and presence of bFGF respectively). For comparison, TGF-β1 inhibited BME cell proliferation by 90 and 92% in the absence or presence of bFGF respectively (Figure 2).

Example 5: Zymography and reverse zymography

Assay:

Confluent monolayers of BME or BAE cells in 35mm gelatin-coated tissue culture dishes were washed twice with serum-free medium, and were treated with (+)-8-Prenylnaringenin in the absence or presence of bFGF or VEGF in serum-free medium containing Trasylol (200 Kallikrein Inhibitory Units/ml). 15 hours later, cell extracts were prepared and analyzed by zymography and reverse zymography as previously described (Vassalli et al., 1984, J. Exp. Med. 159: 1653-1668).

Results:

By zymography, we observed that (±)-δ-Prenylnaringenin induced a dose-dependent increase in uPA and tPA activity in BME cells (Figure 1). This was accompanied by a reduction in PAI-1 activity as detected by reverse zymography, which in all likelihood is due to sequestering of PAI-1 into a tPA/PAI-1 complex. (Addition of Trasylol to the serum-free culture medium retains uPA and tPA in a single-chain form; however, unlike uPA, tPA in this form is able to bind to PAI-1 , a situation which cannot be avoided under the culture conditions employed). We have previously reported that bFGF markedly increases uPA with very little effect on tPA in BME cells (Pepper et al., 1990, J. Cell Biol. 111 : 743-755). Here we have observed that bFGF potentiates the induction of tPA activity by (±)-8-Prenylnaringenin, with a variable concentration-dependent effect on uPA (Figure 9). We have previously reported that VEGF induces uPA, uPAR, tPA and PAI-1 in BME cells (Pepper et al., 1991 , Arterioscler. Thromb. Vase. Biol. 17: 605-619.; Mandriota et al., 1995, J. Biol. Chem. 270: 9709-9716). In the present studies we have observed that VEGF and (±)-8-Prenylnaringenin synergize in the induction of tPA activity in BME cells (data not shown). In the experiments described above, in which cells were incubated for 15 hours in the absence of serum for zymographic and reverse zymographic analysis, frank toxicity was observed with 30μM (±)-8- Prenylnaringenin. This is in contrast to toxicity seen at 100μM when (+)-8-Prenylnaringenin was added to BME cells in the presence of 2% serum.

By zymography, both uPA and tPA activity were increased in BAE cells by (±)-8- Prenylnaringenin; this was accompanied by a decrease in PAI-1 , as detected by reverse zymography (Figure 10). As described above for BME cells, the reduction of PAI-1 activity is in all likelihood due to sequesterinh of PAI-1 in a tPA/PAI-1 complex. We have previously reported that bFGF increases uPA and tPA in BAE cells (Mandriota et al., 1995, J. Biol. Chem. 270: 9709-9716). Here we show that in BAE cells, bFGF potentiates the induction of tPA activity by (±)-8-Prenylnaringenin, and that (+)-8-Prenylnaringenin reduces bFGF- induced uPA activity (Figure 10).

Example 6: RNA preparation, in vitro transcription, and Northern blot hybridization

Method: (±)-δ-Prenylnaringenin or genistein were added to confluent BME cell monolayers to which fresh medium containing 5% DCS had been added 24 hours previously. Total cellular RNA was prepared after the indicated times using Trizol reagent (Life Technologies AG, Basel, Switzrland) according to the manufacturer's instructions. Northern blots, UV cross-linking and methylene blue staining of filters, in vitro transcription, hybridization and post-hybridization washes were as previously described (Pepper et al., 1990, J. Cell Biol. 111: 743-755). [³²PJ- labelled cRNA probes were prepared from bovine urokinase-type plasminogen activator (u- PA) (Kratzschmar et al., 1993, Gene 125: 177-183), bovine u-PA receptor (u-PAr) (Kratzschmar et al., 1993, Gene 125: 177-183), human tissue-type PA (t-PA) (Fisher et al., 1985, J. Biol. Chem. 260: 11223-11230) and bovine PA inhibitor-1 (PAI-1) (Pepper et al., 1990, J. Cell Biol. 111: 743-755) cDNAs as previously described (Pepper et al., 1990, J. Cell Biol. 111 : 743-755). Filters were exposed to Kodak X-OMAT XAR-5 Scientific Imaging Film (Eastman Kodak Co., Rochester, NY). Autoradiograms were scanned and quantitated using ImageQuant (Molecular Dynamics, Sunnyvale, CA). Results: By northern blot analysis, we found that (±)-δ-PrenyΙnaringenin increased steady state levels of uPA, uPAR and tPA mRNAs in BME cells (Figures 7 and δ). The maximal effect of (+)-δ- Prenylnaringenin was observed after 9 hours for uPA (7.5-fold increase), uPAR (6.4-foId increase) and tPA (9.6-fold increase). (±)-δ-Prenylnaringenin had no significant effect on the levels of PAI-1 mRNA. In contrast to BME cells, (±)-3-Prenylnaringenin had virtually no effect on uPA or uPAR mRNA levels in BAE cells, but did induce a 4.6-fold increase in tPA levels after 24 hours (Figure δ). Example 7: Effects of 8-prenylnaringenin in the early chicken embryo chorioallantoic membrane (CAM) assay

Method: • Fertilized chicken eggs were cracked outside the shell on Day 3 of incubation into an ex ovo apparatus.

• Incubation was continued to Day 4 in a 37°C humidified incubator.

• On Day 4 the embryos were photographed using a dissecting microscope (SZ-CTV Olympus) with digital camera attached (Panasonic GP-KR222), and images captured with image analysis software (Video Pro, Leading Edge Pty Ltd, Australia).

• The CAM areas were measured, and the embryos divided into groups of similar size, with a vehicle control per group.

• Treatments were applied on methylcellulose discs that had been dried overnight in a container under vacuum. The discs were 5-8mm in diameter, and larger than the CAMs at the time of application ie. substance was diffused over the entire surface of the CAM.

Dose levels used were 62.5, 125, 250 and 500 nmol for the δ-prenylnaringenin; 62.5, 125 and 250 nmol for the genistein; and 62.5 nmol of 8-prenylnaringenin or 62.5 nmol of genistein were added to the discs either alone or together for the combination treatment group.

• Embryos were incubated for a further 18-24 hours.

• Skim milk plus contrast was injected into the CAM using a 30G needle. Images were immediately captured at increasing levels of magnification. A 63x image was also taken of the major vein branch/es as they exited the left side of the CAM.

• Vein and artery lengths and CAM areas were measured from the five times image of each CAM using image analysis. Relative vessel lengths were calculated as the vessel length/ CAM area.

• Vessel diameter was measured using the 63x image of the major vein branch/es.

• Statistics were performed using SigmaStat for Windows Version 2.03 (SPSS Inc.). Oneway ANOVA was used, with pairwise multiple comparisons using Student-Newman-Keuls method. Where data were non-normally distributed, ANOVA on Ranks was performed followed by Dunn's test for multiple pairwise comparisons. Significance was designated at p<0.05. Results:

Treatment of the chicken embryo chorioallantoic membrane (CAM) with 8-prenylnaringenin resulted in significant reductions in blood vessel growth (Table). Vessel lengths were maximally decreased at 125 and 500 nmol of 8-prenylnaringenin, vein length was 66% of control and artery length 74% of control in these groups. Total vessel length was reduced by 500 nmol of δ-prenylnaringenin to 69% of control (p<0.05). There were only minor effects on growth of the CAM, with an increase in CAM area of δ6% of control in the 125 nmol group. Relative vein and artery lengths were also decreased; relative vein length was 70% of control in the 500 nmol group (p<0.05). Vein diameter was reduced more than vein length, and showed a reduction to 54% of control in the 500 nmol group. The vehicle treated CAM has a well developed vasculature, with highly organised interdigitating vessels. In the δ-prenylnaringenin treated CAM the vein and artery branches are poorly developed, and the major vein branch is tortuous and thin. Fine capillaries in a superficial level of the CAM were present.

The diameter of the main vein was reduced to a greater extent than the vessel length measurements. Blood flow is far more dependent on vessel diameter than vessel length, as it is proportional to the fourth power of the vessel diameter. Approximately 50% reductions in vein diameter were seen in the treatment groups, which would represent blood flow of only 6% of control. Functionally this means that the δ-prenylnaringenin are having dramatic effects on the flow of blood within the CAM.

Table

Effects of δ-prenylnaringenin in the early CAM assay expressed as a percentage of the vehicle treated control in each group.

(n= 9 except for vein diameter where n=6; data are mean ± SEM)

^* Relative vessel length = absolute vessel length (pixels)/ CAM area (pixels) ¹: p<0.05 vs Vehicle ^A P=0.052

Description of the figures

Figure 1

8-Prenylnaringenin inhibits angiogenesis in vitro. BME cells were treated for 3 days with bFGF (10ng/ml), VEGF (30ng/ml) or a combination of bFGF and VEGF, either in the absence or presence of 8-Prenylnaringenin at the indicated concentrations. Results are expressed as the mean additive sprout length ± s.e.m. from at least three separate experiments per condition.

Figure 2

8-Prenylnaringenin reduces endothelial cell number in a 5 day proliferation assay. BME cells were seeded into 24 well plates at 10O00 cells per well in medium containing 5% DCS. 24 hours later, cells were treated with 8-Prenylnaringenin at the indicated concentrations or with TGF-β1 (1 ng/ml), and bFGF (10ng/ml) was added to half the wells. Results are shown as % inhibition calculated from the mean of duplicate wells.

Figure 3 (±)-8-prenylnaringenin increases steady-state levels of uPA, uPAR and tPA mRNAs in BME cells.

Replicate filters containing total cellular RNA (5 μg/lane) prepared from confluent monolayers of BME cells incubated in the absence (Control) or presence of 10μM (±)-8-prenylnaringenin for the indicated times, were hybridized with ³²P-labelled cRNA probes. RNA integrity and uniformity of loading were determined by staining the filters with methylene blue after transfer and cross-linking (lower panel of the figure); 28S and 18S ribosomal RNAs are shown.

Figure 4

Effect of (±)-δ-prenylnaringenin on uPA, tPA and PAI-1 activity in BME cells. Cell extracts (CE) and culture supernatants (Sup) prepared from BME cells incubated with (+)-8-prenylnaringenin at the indicated concentrations in the absence or presence of bFGF (10ng/ml) for 15 hours, were subjected to zymography (uPA, tPA and tPA/PAI-1 complex activities) and reverse zymography (PAI-1 activity)

Figure 5

Effect of (±)-δ-prenylnaringenin on uPA, tPA and PAI-1 activity in BAE cells. Cell extracts (CE) and culture supernatants (Sup) prepared from BAE cells incubated with (±)-8-prenylnaringenin at the indicated concentrations in the absence or presence of bFGF (3ng/ml) for 15 hours, were subjected to zymography (uPA, tPA and tPA/PAI-1 complex activities) and reverse zymography (PAI-1 activity). The second panel marked CE is a longer incubation of the gel shown in the top panel.

Figure 6

Effects of 8-prenylnaringenin on CAM vessel length The experiments were performed as described in example 7. The vessel length of veins and arteries were measured. The total of both measurements are also shown. For each set of experiments and the total, the first column indicates the control in ethanol, the second the addition of 62.5 nmol 8-Prenylnaringenin, the third, fourth and fifth column the addition of 125 nmol, 250 nmol and 500 nmol 8-Prenylnaringenin, respectively.

Claims

Use of 8-Prenylflavanones of the general formula I:

wherein

R₄ designates a hydrogen atom or a hydroxyl group,

for the preparation of a pharmaceutical composition for the treatment or prevention of angiogenesis-related diseases and for the treatment or prevention of thrombotic disorders.

2. Use according to claim 1 wherein the 8-Prenylflavanone is 8-Prenylnaringenin.

3. Use according to claim 1 wherein the angiogenesis-related disease is cancer.

4. A pharmaceutical composition comprising one or more 8-Prenylflavanones of the general formula I and at least one pharmaceutically acceptable excipient for the prevention and treatment of angiogenesis-related diseases.

5. Pharmaceutical composition according to claim 4 wherein the 8-Prenylflavanone is 8- Prenylnaringenin

6. Pharmaceutical composition according to claim 4 or 5 wherein the angiogenesis- related disease is cancer.

7. A pharmaceutical composition comprising one or more 8-Prenylflavanones of the general formula I and at least one pharmaceutically acceptable excipient for the prevention and treatment of thrombotic disorders.

8. Pharmaceutical composition according to claim 7 wherein the 8-Prenylflavanone is 8- Prenylnaringenin