TREATMENT OF PORTAL HYPERTENSION AND RELATED CONDITIONS BY COMBINED INHIBITION OF THE VEGF AND PDGF SIGNALLING PATHWAYS

The present invention relates to the field of medicine. More specifically the invention relates to methods for the therapeutic and/or prophylactic treatment 5 of portal hypertension and manifestations related thereof.

BACKGROUND ART

Portal hypertension is the most important complication of chronic liver 0 diseases and is a leading cause of mortality and liver transplantation worldwide. A characteristic feature of the portal hypertensive syndrome is the development of a hyperdynamic splanchnic circulation with an increase of the blood flow in splanchnic organs draining into the portal vein and a subsequent increase in portal venous inflow. 5

Such an increased portal venous inflow represents a significant factor maintaining and worsening the increase of the portal pressure. The rising of portosystemic collateral vessels and gastroesofageal varices are major problems in portal hypertension, because these varicose veins can cause o haemorrhages in the upper digestive tract which can threat the survival of the patient. 50% of patients suffering portal hypertension have a life expectancy shorter than a year if they are not urgently treated.

Current treatments of portal hypertension include both surgical and drug5 therapies. Surgical methods, e.g. shunts and liver transplantation, are intended to be a solution in the case of advanced stages of portal hypertension wherein the condition become an imminent threat to the survival of the patient. Drug treatments, e.g. vasopressin, glypressin, and somatostatin, are focused to patients with developed gastroesophageal 0 varices suffering from gastroesophageal bleeding crises. These treatments are to be administered clinically and supervised by medical staff. Side effects encompassing those treatments, e.g. alterations in systemic circulation and arterial ischemia, make them inappropriate for most hypertensive and cardiac patients, including portal hypertensive patients, and for patients in the earlier 5 stages of the syndrome.

Vasopressin is a peptide hormone that shows a vasoconstrictor effect,

improving the resistance of peripheral blood vessels and also increasing arterial blood pressure. It has been used for the treatment of acute variceal bleeding. Vasoconstrictor effect of vasopressin is not limited to the splachnic zone, also affecting systemic blood flow, increasing arterial pressure and lowering coronary blood flow and cardiac frequency. Said systemic effects cause several cardiovascular problems to cardiac and/or hypertensive patient.

Vasopressin treatments have to be clinically administered since vasopressin is used in the case of a bleeding crisis being administered in the form of intravenous continued perfusion for a period of more than 24 hours. Despite the combined administration of vasopressin and nitroglicerine shows a decrease of the secondary effects, the treatment still have to be interrupted in the 25% of the patients due to secondary effects.

Glypressin is a synthetic derivative of vasopressin with less secondary effects and a lasting therapeutic effect. However, glypressin side effects on systemic blood pressure and the risk of arterial ischemia are still significant, and it is inappropriate for most hypertensive and cardiac patients, including portal hypertensive patients.

Somatostatin treatment is able to decrease portal hypertension without many of the side effects of above treatments and to improve their effectiveness. However, it is still indicated only as a clinical treatment in response to gastroesophageal bleeding crises.

Recently, the present inventors have found that an increase in the splanchnic vascular bed size mediated by a vascular endothelial growth factor (VEGF)-dependent angiogenic process significantly contributes to increase overall blood flow in splanchnic tissues of portal hypertensive animals and to increase the formation of collateral vessels (see, Fernandez et al. 'Inhibition of VEGF receptor-2 decreases the development of hyperdynamic splanchnic circulation and portal-systemic collateral vessels in portal hypertensive rats' <L Hepatol. 2005 vol. 43, pgs. 98-103; and Fernandez et al. ' Anti-VEGF Receptor-2 Monoclonal Antibody Prevents Portal-Systemic Collateral Vessel Formation in Portal Hypertensive Mice' Gastroenterology 2004 vol.126, pgs, 886-894).

There is the need in the art for methods to prevent, reduce or eliminate portal hypertension and related manifestations thereof, eligible for the wider number of patients and without being impaired by harmful side effects.

SUMMARY OF THE INVENTION

The present inventors have surprisingly found that the combined inhibition of VEGF and platelet-derived growth factor (PDGF) signalling pathways has a therapeutic and prophylactic effect on portal hypertension beyond an anti-angiogenic action. Experimental data show that the treatment of the simultaneous inhibition of VEGF and PDGF signalling pathways not only prevent but also revert the manifestations associated with portal hypertension without affecting systemic blood flow. The method of treatment of the invention have also shown a preventive and reversal effect on the formation of portosystemic collateral vessels, thus impairing the rising of gastroesophageal varices and lowering the risk of gastroesophageal bleeding, as well as on other manifestations related to portal hypertension.

Some of the results and conclusions included in the present invention have been published by the inventors in Hepatoloqy, 2007 46(4), pgs. 1208-1217, firstly published on-line on 24.07.2007, which is included herein by reference.

DETAILED DESCRIPTION OF THE INVENTION

Combined treatments with VEGF and PDGF inhibitors have been disclosed in the art for treating different types of cancer and other proliferative diseases.

WO 200404644 A2 discloses a combination of imatinib and rapamycin in the treatment of imatinib-resistant Chronic Myeloid Lymphoma.

WO 2005027972 A2 relates to the combined use of chemotherapeutic agents and VEGF inhibitors for the treatment or the prevention of tumours and hyperplasia as well as fibrotic diseases triggered by persistent angiogenesis. Implicitly, it describes a combination of imatinib and VEGF inhibitors to be administered on a chemotherapeutic treatment.

WO 2005049021 A1 discloses combinations of mTOR and PDGF-R inhibitors for the prevention and treatment of conditions triggered by neointimal hyperplasia, as well as other fibroproliferative vasculopathies. More specifically, said patent application discloses a combination of rapamycin and imatinib for the treatment of neointimal hyperplasia.

WO 2002070019 A1 discloses the use of nucleic acid expression constructs for the overexpression (and not the inhibition) of VEGF and PDGF to induce angiogenesis within the cirrhotic liver tissue. Said document discloses that the induction of angiogenesis in the cirrhotic liver improves liver regeneration and, thus, portal hypertension is alleviated.

The object of the invention is a method for the treatment and/or prevention of portal hypertension and manifestations related thereof, comprising the administration of a pharmaceutically effective amount of one or more pharmaceutical active substances for the inhibition of both platelet-derived growth factor and vascular endothelial growth factor signalling pathways.

The inventors have found that the inhibition of such signalling pathways contributes significantly to ameliorate and prevent portal hypertension. As known by the skilled in the art, the inhibition of the signalling pathway of a receptor can be the effect of targeting directly the receptor of interest, as well as targeting any other member of such pathway, e.g. ligands, enzymes, and tyrosine kinases, by other routes related to the interesting pathway (see, e.g. www.sigmaaldrich.com/Area_of_lnterest/Life_Science/PathFinde r/Pathway_M apsA/EGF_Pathway.html for VEGF signalling pathway, www.sigmaaldrich.com/Area_of_lnterest/Life_Science/PathFinde r/Pathway_M aps/PDGF_Pathway.html for PDGF signalling pathway, www.sigmaaldrich.com/Area_of_lnterest/Life_Science/PathFinde r/Pathway_M aps/mTOR_Pathway.html for mTOR pathway scheme). Thus, the treatment of the invention is directed to ameliorate the manifestations of portal hypertension. These include: increased portal blood flow, formation of portosystemic collateral vessels, increased splanchnic blood flow, increased splanchnic neovascularization, and splanchnic vascular resistance. When these manifestations of portal hypertension are ameliorated, the conditions causing portal hypertension are also ameliorated.

Further, complications and conditions resulting from portal hypertension are prevented by the treatment of the invention. Such conditions related to portal hypertension comprise ascites, i.e. free fluid in the peritoneal cavity, usually leading to bacterial peritonitis, wherein portal hypertension plays an important role by raising capillary hydrostatic pressure within the splanchnic bed; hepatorenal syndrome, a usually fatal condition rising by alterations in blood flow and blood vessel tone of the splanchnic circulation system, e.g. portal hypertension; splenomegaly, i.e. the congestion of the spleen impairing splenic function, with consequent sequestration therein of red blood cells, white blood cells, and platelets; and a number of conditions arisen by angiogenic processes due to portal hypertension.

Portal hypertension usually leads to the development of collateral vessels that directly connect the portal blood vessels to the general circulation, bypassing the liver. Because of this bypass, substances (e.g. toxins) that are normally removed from the blood by the liver can pass into the general circulation. These toxic substances can lead to hepatic encephalopathy when they reach the brain without being modified or purified. Hepatic encephalopathy encompasses impaired cognition, a flapping tremor (i.e. asterixis), and a decreased level of consciousness including coma, cerebral edema, and, ultimately, death.

Collateral vessels are also developed at the lower end of the esophagus and at the upper part of the stomach. There, the vessels become varicose veins in the esophagus (i.e. esophageal varices) or stomach (i.e. gastric varices).

These engorged vessels are fragile and prone to bleeding, sometimes seriously and occasionally with fatal results. Other collateral vessels may be developed on the abdominal wall and at the rectum.

The present inventors have surprisingly found a noticeable synergistic effect between the inhibition of VEGF and PDGF signalling pathways when treating portal hypertension. As shown in the results of Example 1 , using rapamycin and imatinib as VEGF and PDGF inhibitors respectively, the effect on portal hypertension of the simultaneous inhibition of VEGF and PDGF is not a mere addition of the separate effect of the inhibition of each signalling pathway. In fact, the treatment based on the inhibition of PDGF pathway alone does not result in a significant effect, but the effect of the combined inhibition of both

pathways is shown to be superior to the sum of the effects of the treatment by the inhibition of one of the pathways.

The term "effective amount" as used herein, means an amount of an active ingredient high enough to deliver the desired benefit, but low enough to avoid serious side effects within the scope of medical judgment.

Studies have been performed over an animal model of established portal hypertension syndrome (see Example 1 -Therapeutic Studies) and resulted in a significant decrease of abnormally high portal vein blood pressure and splanchnic blood flow. Therefore, the risk of arising haemodynamic conditions, e.g. ascites, hepatorenal syndrome, and splenomegaly, is significantly decreased. The treatment of the invention also reverted the splanchnic neovascularization and the formation of portosystemic collateral vessels. Thus, the risk of suffering angiogenic conditions related to portal hypertension, e.g. hepatic encephalopathy, gastric and esophageal varices, also decreases when the abnormal blood circulation parameters are therapeutically treated. In the animal model, splanchnic vascular resistance was also improved, therefore lowering the risk of bleeding from the new developed vessels.

The present inventors have found that the simultaneous inhibition of both pathways reverses and prevents the development of portal hypertension and the manifestations related thereof by the decrease of hyperdynamic splanchnic circulation and/or portal pressure in portal hypertensive patients.

The prophylactic use of rapamycin and imatinib as a combination of a VEGF and PDGF inhibitors according to the invention, e.g. in the case of patients suffering of a condition that could lead to develop portal hypertension, e.g. cirrhosis of the liver, schistosomiasis, hepatic vein obstruction, portal vein obstruction, or splenic vein obstruction, results in the inhibition of the increase of hyperdynamic splanchnic circulation and neovascularization is significantly detained. Experimental results using an animal model (Example 1 , Prophylactic Studies) showed no significant variation on portal pressure in portal hypertensive animals prophylactically treated according to the invention with regard to portal hypertensive control group. As disclosed by the inventors in previous studies, (see, Fernandez et al. Hepatology, 2007 46(4), pgs. 1208-1217) the prophylactic treatment of portal hypertension according to the

present invention results in a noticeable decrease of hyperdynamic splanchnic circulation and the inhibition of the formation of portosystemic collateral vessels. Therefore, in the scenario of a decreased splanchnic circulation and an inhibited formation of new portosystemic vessels, an increased portal pressure does not results in the development of conditions typically related to portal hypertension syndrome.

In a preferred embodiment, the method of treatment of the invention is used to treat portal hypertension and manifestations thereof arisen from a non-cirrhotic condition.

In a particular embodiment, the invention relates to a method of treatment according to the present invention, which prevents or reduces the formation of portosystemic blood vessels.

A further particular embodiment of the invention relates to a method of treatment according to the present invention which prevents or reduces abnormally increased portal vein blood pressure.

In another particular embodiment of the invention the method of treatment comprises the administration of at least two pharmaceutical active substances, each of them inhibiting one of the PDGF or VEGF signalling pathways. There are a number of compounds known in the art for inhibit mainly one single target. These pharmaceutical active substances direct to PDGF or VEGF, but in addition, they can have other effects by directing to other signalling pathways. The method of treatment of the invention comprises the combined administration of at least two of these single target inhibitors, i.e. at least one for the inhibition of each of PDGF and VEGF signalling pathways. Preferably, the pharmaceutical active substance which inhibits the VEGF signalling pathway is selected from the group consisting of

DC101 (from ImClone), semaxanib (SU5416, from Sugen), avastin (from Genentech), AZD2171 (from AstraZeneca), CP-547632 (from Pfizer), RPI.4610 (Angiozyme ^® , from Ribozyme Pharmaceuticals), VEGF-Trap (Regeneron Pharmaceuticals), ZD6474 (from AstraZeneca), YM359445, HuMV833 (Toagosei Protein Design Laboratories), GFB-116, NM3 (from ILEX Oncology), ZD4190 (from AstraZeneca), CGP 41251 (Novartis Pharma), CEP-5214, VGA1102, gefitinib (Iressa ^® ), AEE788 (from Novartis), lestaurtinib

(from Cephalon), CEP-7055 (from Cephalon), pegaptanib (Nextar Pharmaceuticals), Neovastat (AE941 AEterna Laboratories), IMC1C11 (Inclone Systems) and rapamicyn, or prodrugs, solvates, hydrates, and pharmaceutically acceptable salts thereof. More preferably, the pharmaceutical active substance which inhibits the VEGF signalling pathway is rapamycin, a prodrug, solvate, hydrate, or a pharmaceutically acceptable salt thereof.

Rapamycin reduces VEGF production by the inhibition of the mTOR pathway. Rapamycin is marketed in the US as Rapamune ^® by Wyeth Pharms Inc. for the prophylaxis of organ rejection in patients receiving renal transplants. Besides its antibiotic and immunosupressor action, rapamycin has been reported to inhibit downstream signalling from the mammalian target of rapamycin (mTOR) proteins which functions in a signalling pathway reported to promote tumor growth. The use of rapamycin as an inhibitor of the imatinib-resistance of tumor cells is disclosed in WO 200404644 A2 for the treatment of imatinib-resistant Chronic Myeloid Lymphoma.

Also preferably, the pharmaceutical active substance which mainly inhibits the PDGF signalling pathway is selected from the group consisting of leflunomide (Arava ^® , from Sanofi-Aventis), CDP860, CP673451 , GFB-111 , AG1296, 7d-6, AMN107 (from Novartis), BMS-354825 (from Bristol-Myers Squibb), MLN518 (from Millennium), and imatinib, or prodrugs, solvates, hydrates, and pharmaceutically acceptable salts thereof. More preferably, the pharmaceutical active substance which inhibits the PDGF signalling pathway is imatinib mesylate.

Imatinib is an inhibitor of PDGF receptor, marketed in the US by Novartis as Gleevec ^® as a chemotherapeutic agent for the treatment of chronic myeloid leukemia (CML). The commercial form of Imatinib is imatinib mesylate.

Further, the preferred combination of VEGF and PEGF inhibitors is rapamycin and imatinib mesylate.

In an embodiment of the invention, the substance to be administered in the method of treament of the present invention is a multitarget drug which inhibits both the PDGF and the VEGF signalling pathways. There are a

number of known drugs which inhibits a number of targets. The present invention includes the administration of multitarget drugs inhibiting both VEGF and PDGF signalling pathways. Preferably, the multitarget drug is selected from the group consisting in sunitinib (Sutent ^® as sunitinib maleate, from Pfizer), midostaurin (from Novartis), vatalanib (from Novartis/Schering), AG-013736 (from Pfizer), SU6668 (from Sugen), ABT-869 (from Abbot), AMG706 (from Amgen), BAY 57-9352 (from Bayer), CHIR-258 (from Chiron), OSI-930 (from OSI Pharmaceuticals), OSI-817 (from OSI Pharmaceuticals), SU014813 (from Pfizer), XL999 (from Exelis) and sorafenib, or prodrugs, solvates, hydrates, and pharmaceutically acceptable salts thereof. More preferably, the multitarget drug inhibiting VEGF and PDGF signalling pathway is sorafenib, a prodrug, a solvate, a hydrate, or a pharmaceutically acceptable salt thereof.

Sorafenib is an inhibitor of VEGF and PDGF receptors, marketed in the US as Nexavar ^® by Bayer and Onyx Pharmaceuticals. Nexavar ^® is approved by the FDA for the treatment of advanced renal cell carcinoma and it is being developed for the treatment of other cancers. Sorafenib targets several serine/threonine and receptor kinases in both tumor cells and tumor vasculature. These kinases include RAF kinase, angiogenic related kinases as VEGFR-2 and VEGFR-3, cell growth related kinases as PDGFR-β and KIT, and leukemia cell proliferation kinase FLT-3.

When employed in the method of treatment of the invention, sorafenib showed a significant effect on the treatment of portal hypertension and manifestations related thereof. Sorafenib treatment resulted in a significant decrease on splanchnic blood flow, in the formation of portosystemic collateral vessels and a noticeable decrease of neovascularization, while the splanchnic vascular resistance was increased (see, FIG.1 , FIG. 2, and Example 2).

Furthermore, the present invention covers all possible combinations of particular and preferred groups described hereinabove.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skilled in the art to which this invention belongs. Methods and materials similar or

equivalent to those described herein can be used in the practice of the present invention. Throughout the description and claims the word "comprise" and variations of the word, such as "comprising", is not intended to exclude other technical features, additives, components, or steps. Additional objects, advantages and features of the invention will become apparent to those skilled in the art upon examination of the description or may be learned by practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to limit the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of sorafenib administration on the splanchnic neovascularization of rats with prehepatic portal hypertension. Representative histological images of mesentery sections stained with H&E (original magnification x40) of rats treated with Sorafenib (n=6) or vehicle (n=6), and quantitative analysis of vascular areas are shown.

FIG. 2 shows the effect of sorafenib administration on the splanchnic neovascularization of rats with intrahepatic portal hypertension and liver cirrhosis. Representative histological images of mesentery sections stained with H&E (original magnification x40) of rats treated with Sorafenib (n=6) or vehicle (n=6), and quantitative analysis of vascular areas are shown.

EXAMPLES

Animals

Adult male Sprague-Dawley rats or male C57BL/6J mice (8-week-old) were studied. All experiments were approved by the "Laboratory Animal Care and Use Committee" of the University of Barcelona, and were conducted in accordance with the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH publication 86-23, revised 1985).

Induction of portal hypertension by partial portal vein ligation

While each animal was under anaesthesia (80 mg/kg body weight ketamine plus 12 mg/kg body weight xylacin, intramuscularly), the portal vein was isolated and a calibrated constriction was performed using a single ligature of silk (3-0 silk in rats, and 5-0 silk in mice) tied around the portal vein and a blunt-tipped needle (20-gauge needle in rats and 27-gauge needle in mice). The needle was then removed, leaving a calibrated constriction of the portal vein. In sham-operated control animals, the portal vein was isolated and similarly manipulated, but not ligated.

Measurement of mean arterial pressure, portal pressure and heart rate

Under anaesthesia, the trachea was cathetehzed (PE-240 tubing) for maintenance of airway patency. A polyethylene catheter (PE-50 in rats and PE-10 in mice) was introduced into a femoral artery to measure mean arterial pressure (MAP, mmHg). A midline abdominal incision was made, and the portal vein was cannulated through an ilecolic vein with a PE-50 catheter. After verifying that free reflux of blood had been achieved, the catheter was fixed to the mesentery with cyanocrylate glue. This catheter was used for portal pressure measurements (PP, mmHg). All catheters were connected to highly sensitive pressure transducers, and blood pressure was recorded on a multichannel computer-based recorder (PowerLab, ADInstruments, Colorado Springs, CO). Heart rate was measured by counting the number of beats per minute. The external zero reference point was placed at the midportion of the animal.

Measurement of splanchnic blood flow by flowmetry

Under anaesthesia, a non-constrictive perivascular transit-time ultrasonic flowprobe (Transonic Systems Inc., Ithaca, New York, USA) was placed around the superior mesenteric artery, close to its aortic origin, and connected to a flowmeter to measure superior mesenteric artery blood flow (SMABF, ml-min ^"1 -100 g ^"1 ), as an index of splanchnic blood flow. Superior mesenteric artery resistance (SMAR, mmHg-mr ¹ -min ^"1 -100 g ^"1 ) was calculated as: (MAP-PP)/SMABF.

Determination of the extent of portosystemic collateral formation

The extent of portosystemic collateral vessels was quantified using 5 ¹ Cr-labeled radioactive microspheres (Perkin-Elmer, Boston, Massachusetts, USA), injected into the spleen of animals. The animals were then sacrificed and the radioactivity in the liver and lungs was determined in a γ-scintillation counter. This allows quantifying the degree of collateral formation in a 0 to 100% scale, by the equation:

Collateralization (%)=[lungs radioactivity / (lungs radioactivity + liver radioactivity)] x 100

Approximately 30,000 ⁵¹ Cr-labeled microspheres (diameter 15 ± 3 μm; specific activity 41 mCi/g) were injected into the splenic pulp of each animal.

Western blot analysis

Tissue samples were excised, immediately snap-frozen in liquid nitrogen, and stored at -80 °C for protein analysis. Tissues were homogenized in ice-cold lysis buffer containing 50 mM Tris-HCI (pH 7.4), 0.1 mM EGTA, 0.1 mM

EDTA, 2 mM leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 % Nonidet P40, 0.1 % sodium dodecyl sulfate, and 0.1 % deoxycholate (all these reagents were purchased from Sigma Chemical, St. Louis, Missouri, USA). The resultant lysates were centrifuged at 10000 g for 30 minutes at 4 °C. The supernatants were collected and protein concentration was quantified using a colorimetric assay (Bio-Rad Laboratories, Hercules, California, USA). Proteins (100 or 200 micrograms) were mixed with double-strength sample buffer (250 mM Tris-HCI, 4% sodium dodecyl sulfate, 10% glycerol, 2% β-mercaptoethanol, and 0.006% bromophenol blue [pH 6.8]) (all these reagents were purchased from Sigma Chemical, St. Louis, Missouri, USA).

Samples were then boiled for 5 minutes, and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins were transferred to nitrocellulose membranes (Bio-Rad Laboratories, Hercules, California, USA), and stained with Ponceau S solution to check for equal loading of protein in each lane. Membranes were blocked in Tris-buffered saline (TBS) with 0.05% polyoxyethylenesorbitan monolaureate (Tween 20; TBS-T buffer) containing 5% (wt/vol) nonfat dry milk. The membranes were

then incubated, overnight at 4 °C, with rabbit polyclonal antibodies against (rat or mice) VEGF, VEGF receptor-2, CD31 , PDGF, PDGFR-β, α-SMA or GAPDH (1 :200 or 1 :500 dilution in TBS-T with 5% milk) (Santa Cruz Biotechnology, Santa Cruz, California, USA). Blots were subsequently washed in TBS-T and incubated with horseradish peroxidase-conjugated antibody against rabbit IgG (1 :10000 dilution in TBS-T with 5% milk) (Stressgen, Sidney, Canada), for 30 minutes at room temperature. Immunoreactive bands were detected using the enhanced chemiluminescence western blotting system (Santa Cruz Biotechnology, Santa Cruz, California, USA). Loading accuracy was evaluated by membrane rehybridization with monoclonal antibodies against α-tubulin or GAPDH (1 :2000 dilution) (Santa Cruz Biotechnology, Santa Cruz, California, USA). Quantification of protein signals was performed using computer-assisted densitometry. Pre-stained molecular mass marker proteins were used as standards (Bio-Rad Laboratories, Hercules, California, USA). The Western blot analysis was repeated to confirm reproducibility using the samples collected from four individual animals of each experimental group.

lmmunohistochemistry

lmmunohistochemical detection of CD31 and α-SMA was carried out on sections from paraffin-embedded mesenteric tissues fixed in 10% neutral-buffered formalin solution. Endogenous peroxidase activity was blocked using methanol containing 0.3% H ₂ O ₂ . Antigen retrieval for CD31 detection was performed using citrate buffer. Non-specific antibody binding was prevented by incubation with 10% normal horse serum. Sections were incubated with primary antibodies against CD31 (diluted 1 :200), and α-SMA (diluted 1 :500) (Santa Cruz Biotechnology, Santa Cruz, California, USA), and then the bound antibodies were visualized using diaminobenzidine (DAB) as the chromogen (Sigma Chemical, St. Louis, Missouri, USA). For the negative control, phosphate buffered saline was used instead of the primary antibody.

Measurement of vascular areas

Tissue samples were fixed in 10% neutral-buffered formalin, and embedded in paraffin. Tissues were sectioned (2-μm thickness), and slides were stained with hematoxylin and eosin (H&E) or immunostained for CD31 and α-SMA.

Quantitative analysis of angiogenesis was performed with the assistance of ImageJ 1.37v software (NIH, USA) by scanning of the entire tissue section, and counting the areas occupied by vascular structures. A value from each tissue section (1 section per rat) was obtained and then the results were averaged from individual sections in each experimental group (3-5 sections per group). Group values reflect the average readings from all sections in the group. The results of neovascularization measurements are expressed as the mean area (± SEM; expressed in square micrometers) occupied by vascular structures within a tissue field measuring 1 mm ² .

Measurement of cardiac output and regional blood flows

Cardiac output and regional blood flows were quantified using the radioactive microsphere reference organ technique. Briefly, radioactive microspheres labeled with ⁸⁵ Sr (diameter 15 ± 3 μm; specific activity 19 mCi/g) were injected into the left ventricle, through a PE-50 catheter introduced in the right carotid artery, over a 10-seconds period. At the same time, a reference blood sample from a femoral artery catheter was taken at a flow rate of 1 ml/min using a continuous-withdrawal pump starting 15 seconds before microsphere injection for a total of 75 seconds. This reference blood sample was weighed to estimate the volume of blood collected, and counted by a γ-scintillation counter to calculate the radioactivity (in counts per minute, cpm).

Animals were sacrificed at the end of the experiments by overdose of ketamine. Splanchnic organs, heart, kidneys, and testes were dissected, blot-dried, weighed, and cut into small pieces, and the radioactivity (cpm) of each organ was determined in a γ-scintillation counter. Regional blood flow to the left and right kidneys and testes was determined to assess adequacy of microsphere mixing. A >10% difference in regional flow between kidneys or testes was a criterion for removing an animal from analysis.

Haemodynamic parameters were calculated as follows:

Cardiac output (CO; ml/min) was calculated as: [Injected radioactivity (cpm) x Reference sample blood flow (ml/min)]/Reference sample blood radioactivity (cpm)

Cardiac index (Cl; ml • min ^"1 • 100 g ^"1 ) was calculated as: CO (ml/min)/[body wt (g) x 100].

Regional blood flows (ml /min) were calculated as: [Organ radioactivity (cpm) x Reference sample blood flow (ml/min)]/Reference sample blood radioactivity (cpm)

Portal venous inflow (PVI), which represents the total blood flow entering the portal venous system, was calculated as the sum of the blood flow to the stomach, spleen, intestine, colon, pancreas, and mesentery.

Resistances in the vascular systems (mmHg-ml ^"1 -min ^"1 -100 g ^"1 ) were calculated from the ratio between perfusion pressure (P) and blood flow (Q) of each vascular territory.

Systemic vascular resistance was: mean arterial pressure (MAP) / CO

Splanchnic arteriolar resistance was: (MAP-PP)/PVI

Portal-collateral resistance was: PP/Collateral blood flow

Statistical analysis

Results are presented as mean ± SEM. Data were normally distributed, and, therefore, parametric statistical procedures were used (Student ^' s t-test for unpaired data, and two-way ANOVA followed by the Tukey-Kramer test for multiple comparisons). A probability value of <0.05 was considered statistically significant.

Example 1

Treatment of Portal Hypertension Syndrome by rapamvcin and/or imatinib

Therapeutic studies

In a first protocol (therapeutic studies), rats were treated with rapamycin (2 mg kg ^"1 day ^"1 ; intraperitoneal^), imatinib (20 mg kg ^"1 day ^"1 ; intraperitoneal^), rapamycin plus imatinib, or vehicle (700 μl of NaCI 0.9%), over a 2-week period, starting one week after partial portal vein ligation (or sham operation in controls), when portal hypertension is fully established. Results are shown in Table 1.

Table 1. Rapamvcin and/or imatinib - Therapeutic Studies

Prophylactic studies

In an additional protocol (prophylactic studies), rats were treated with a combination of VEGF signaling inhibitor rapamycin (2 mg kg ^"1 day ^"1 ; intraperitoneally) (Wyeth Europe, Berkshire, United Kingdom) and the PDGF signaling inhibitor imatinib (20 mg kg ^"1 day ^"1 ; intraperitoneally) (Novartis, West Sussex, United Kingdom), or vehicle (700 μl of NaCI 0.9%) for 5 days, starting immediately after the initial surgery (partial portal vein ligation or sham operation). Results are shown in Table 2.

Table 2. Rapamycin and imatinib - Prophylactic Studies

Synergy

As shown above, there was a noticeable synergistic effect between the inhibition of VEGF and PDGF signalling pathways when treating portal hypertension. The combined effect of the simultaneous treatment of portal hypertension with rapamycin and imatinib was not a mere addition of the separate effect of each drug. In fact, the treatment with imatinib alone did not result in a significant effect, but the effect of the combined administration of both drugs is shown to be superior to the sum of the effects of both drugs when administered alone.

Synergy was shown between rapamycin and imatinib effects on four of the studied parameters: portal pressure, blood flow in upper mesenteric artery (Splanchnic blood flow index), upper mesenteric artery vascular resistance (Splanchnic vascular resistance index), intestinal expression of CD31 (Splanchnic neovascularization index), and the formation of portosystemic collateral vessels.

The effect of the treatment using the combination of rapamycin and imatinib was superior to the effect of the treatment with one separate drug. Results are shown in percentage with regard to the group treated with vehicle (n=12).

Treatments administered when portal hypertension was fully established (therapeutic treatments) led to a very important decrease of splanchnic neovascularization, splanchnic blood flow and to the increase of splanchnic vascular resistance. In this case, the splanchnic blood flow decrease resulted

in a significant decrease of portal hypertension, up to 40% after the rapamycin and imatinib combined treatment. Porto-systemic collateral vessels did not revert with anti-angiogenic treatment. However, the decrease of portal hypertension was so significant that even with the presence of collateral vessels, the probability of massive haemorrhages (a major death cause for cirrhotic patients) was clearly diminished.

Example 2

Effect of Sorafenib over Portal Hypertension Syndrome

Non-cirrhotic portal hypertensive rats

Rats were treated with Sorafenib (2 mg kg ^"1 day ^"1 ; by gavage) (Bayer Pharmaceuticals, West Haven, Connecticut, USA), or vehicle (0.9% sodium chloride), over a 2-week period, commencing one week after partial portal vein ligation (or sham operation in controls), when portal hypertension was fully established.

As shown on Table 3 below, treatment of portal hypertensive rats using Sorafenib led to a significant decrease of the splanchnic blood flow and a significant increase of splanchnic vascular resistance. Similarly, the index of formation of portosystemic collateral vessels significantly decreased after Sorafenib treatment. That results are consistent with those previously obtained using a rapamycin and imatinib combination to inhibit VEGF and PDGF signalling pathways. Control rats subjected to a fictitious operation were also studied: Sorafenib did not show any significant effect over control rats in comparison with the group treated with vehicle.

Table 3. Sorafenib

(continued)

FIG. 1 shows the effect of sorafenib administration on the splanchnic neovascularization of portal hypertensive rats.

Cirrhotic portal hypertensive rats

The previous Examples were performed over a model of portal hypertension induced by partial portal vein ligation. This is a model of prehepatic portal hypertension that does not develop liver cirrhosis; the liver remains actually normal. This model has the advantage of being rapid (portal hypertension is completely established in one week after induction of portal hypertension), very homogeneous, and develops all the hemodynamic and circulatory disturbances associated with portal hypertension, including increased splanchnic angiogenesis and portosystemic collateral vessels.

The model of portal hypertension induced by common bile duct ligation is a model of intrahepatic portal hypertension with secondary biliary cirrhosis. In addition to all the hemodynamic and circulatory disturbances associated with portal hypertension, this model also develops liver cirrhosis and an increased resistance in the intrahepatic vascular bed. This model can therefore be used to assess the effect of the treatment of the invention over portal hypertension arisen from cirrhotic conditions.

Portal hypertension was inducted by common bile duct ligation. While each animal was under anesthesia (80 mg/kg body weight ketamine plus 12 mg/kg body weight xylacin, intramuscularly), the common bile duct was exposed by median laparotomy, and occluded by double ligature with 5-0 silk thread. The first ligature was made below the junction of the hepatic ducts; the second was made above the entrance of the pancreatic ducts. The common bile duct was then resected between the two ligatures. The abdominal incision was closed and the animals were allowed to recover. Two days after surgery, dark-brown urine was observed, indicating successful ligation. Vitamin K (8 mg/kg) was given by intramuscular injection once every week, starting the day of common bile duct ligation. In addition, ampicillin (10 mg/kg) was administered intramuscularly, just before the bile duct ligation. In sham-operated control animals, the common bile duct was isolated but not ligated. Subsequent studies were performed 4 weeks after the initial surgery. Rats were treated with sorafenib (1 mg kg ^"1 day ^"1 ; by gavage) (Bayer Pharmaceuticals, West Haven, Connecticut, USA), or vehicle (0.9% sodium chloride), over a 2-week period, commencing two weeks after common bile duct ligation (or sham operation in controls), when portal hypertension is already established.

Table 4

FIG. 2 shows the vascular area quantification results in the mesentery of cirrhotic rats treated with vehicle (n=6) or sorafenib (n=6).