EA044366B1 - COMPOUNDS FOR THE TREATMENT OF VON HIPPEL-LINDAU DISEASE - Google Patents

Abstract

The invention relates to the use of a selective β2-adrenergic receptor antagonist for the treatment and prevention of tumors in patients with von Hippel-Lindau syndrome. Specifically, the invention relates to the use of an alkanolamine derivative or a pharmaceutically acceptable acid addition salt thereof for the treatment and prevention of hemangioblastoma in patients with VHL syndrome.

Description

Field of technology to which the invention relates

The invention relates to the field of therapy and prevention, more precisely to the treatment and prevention of von Hippel-Lindau disease.

BACKGROUND OF THE INVENTION

Von Hippel-Lindau disease (VHL) is a rare type of cancer with an incidence of 1 in 36,000 individuals in the general population. VHL is an autosomal dominant inherited genetic disorder. The disease was first described separately by Hippel in 1911 and Lindau in 1926.

Clinical manifestations include multiple benign and malignant tumors that appear throughout the patient's life: retinal hemangioblastoma, central nervous system hemangioblastoma, clear cell renal cell carcinoma (CCRCC), pheochromocytoma, tumor of the islets of Langerhans, endolymphatic sac tumors, and cysts in the testes and broad ligament.

Tumor cells from VHL have lost von Hippel-Lindau protein function, leading to constitutive expression of hypoxia-inducible factor (HIF) even under normoxic conditions.

HIF is responsible for the activation of genes involved in angiogenesis, metabolism and apoptosis, which promote adaptation and survival in conditions of low oxygen concentration (hypoxia), which can be found in ischemic tissues and in most solid tumors.

Under normoxia, rapid degradation of HIF is controlled by the binding of pVHL to hydroxylated residues of HIF-α. Under hypoxic conditions, PHD fails to hydroxylate HIFa subunits, which then escape ubiquitin-mediated proteolysis, allowing HIFa to accumulate, translocate into the nucleus, and bind to HIFe. This heterodimer is capable of binding specific DNA sequences (hypoxia-responsive elements or HREs) to activate transcription of more than 200 target genes. Major genes regulated by pVHL are involved in tumor development, angiogenesis (vascular endothelial growth factor [VEGF] and platelet-derived growth factor [PDGF]), cell proliferation or survival (transforming growth factor [TGFa]), regulation of glucose uptake and metabolism (glucose transporter Glut-1) and erythropoiesis (EPO). Other target genes relevant to tumor biology are involved in extracellular matrix formation (matrix metalloproteinase 1 [MMP1]), chemotaxis (stromal cell factor 1 [SDF1] and its CXC chemokine receptor 4 [CXCR4], epidermal growth factor receptor [EGFR] ], hepatocyte growth factor receptor [HGFR, encoded by MET]), pH regulation (carbonic anhydrase IX and XII [CA9 and CA12]), and cell cycle (cyclin D1 [CCND1]).

Until now, therapeutic options for patients with VHL have come from surgery. Systemic therapy used for metastatic cancer has shown limited response in VHL pancreatic and renal tumors, whereas CNS tumors do not respond at all.

The VHL Spanish Alliance conducted a clinical trial with 7 patients with VHL for 12 months, from December 2014 to April 2016. Patients were treated for retinal hemangioblastomas with 120 mg propranolol per day (suboptimal dose, for many patients less than 2 mg/kg body weight per day). As a result, tumors did not grow in any cases during the clinical trial, and visual acuity improved in some cases. Even more interesting is that in 2 cases with exudates from hemangioblastomas, they completely disappeared between 3 and 6 months of treatment. Low blood pressure has been reported as a side effect (Albinana et al., 2017). Thus, the lack of therapies for recurrent disease means that there is an urgent need for effective drugs with reduced side effects for patients with VHL, especially those that stop tumor progression and delay surgical treatment.

Brief description of the invention

The present inventors unexpectedly discovered that ICI 118,551 reduces the viability of hemangioblastoma cells in VHL patients (Figs. 1 and 2) and inhibits hemangiosphere formation in VHL patients (Fig. 3). In addition, we showed that ICI 118,551 inhibits cell migration and angiogenesis of endothelial cells (Figs. 3 and 4) and that it inhibits hypoxia-induced HIF activity.

Thus, the invention relates to a selective _{β 2} -adrenergic receptor antagonist for use in the treatment and/or prevention of von Hippel-Lindau disease.

Detailed description of drawings

Fig. 1 - ICI 118,551 and propranolol reduce the viability of hemangioblastoma cells in patients with VHL. Propranolol (P) is a non-selective beta blocker that binds beta 1 and 2 adrenergic receptors. ICI 118,551 (I), is a selective beta blocker that binds only to the beta 2 receptor. Both beta blockers act by reducing the viability of primary hemangioblastoma cell cultures to 40-50% at 100 μM for 72 hours. FIG. Figure 1 shows a decrease in the viability of the primary HB11cib cell culture. Results are representative of 8 primary hemangioblastoma cell cultures treated in the same manner. Each measurement is based on triplicates and quantified using a fluorescent ATP detection kit (Cell titer Glo, from Promega).

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Fig. 2 - ICI 118,551 acts as a propranolol to reduce the viability of VHL hemangioblastoma cells. A. Primary culture of hemangioblastoma cells (sample from patient HB18), without (control) and after 4 days of treatment with ICI 118.551 at 50 and 100 µM. After treatment with ICI 118.551 100 µM we can see empty spaces on the plate compared to the control, which drains after 4 days of culture starting with the same number of cells. B. Comparison of 4 days of treatment with propranolol (P) and ICI 118,551 (I) at the same concentration of 100 μM primary culture of hemangioblastoma cells (sample from patient HB4).

Fig. 3 - treatment with ICI 118,551 causes cell apoptosis by stimulating the pro-apoptotic genes Bax and caspase 9 A. Relative levels of mRNA expression of BAX and CASP9 (genes involved in apoptosis), detected by RT-qPCR in the culture of hemangioblastoma cells HB26, after 72 hours 100 μM ICI 118.551 (1100). B. Relative expression levels of Bax and Casp9 mRNA (genes involved in apoptosis) detected by RT-qPCR in HB11 hemangioblastoma cell culture after 72 h with 100 μM ICI 118,551. Both Bax and Casp 9 genes are increased after ICI 118,551 treatment, so ICI 118,551 induces apoptosis in these cells.

Fig. 4-propanolol and ICI 118,551 are antitumor agents that inhibit the formation of hemangiosphere from cells of VHL patients. A. Round, sphere-like structures with a distinct border appear as aggregates from VHL cell tumors. Two different hemangioblastomas were used for these experiments, HB2 and HB3. However, when cells were cultured for 7 days in the presence of 100 μM propranolol (P) or ICI 118,551 (I), the spheres appear disaggregated and the observed groups are irregular with few cells. Photographs were taken on the last day of treatment B. Same as panel A, but with two other hemangioblastomas from VHL patients. C. Same as panels A and B, but with HB23 from a VHL patient.

Fig. 5 - ICI 118,551 and propranolol inhibit endothelial cell migration. A. In vitro functional tests for angiogenesis involve wound healing or scratch plate testing to monitor the ability of endothelial cells to migrate over a short period of time. When a confluent monolayer is disrupted by a micropipette tip to create a rupture, endothelial cells typically migrate to cover the rupture and the wound heals within a short time, depending on the size of the wound, but typically less than 8 hours. This test measures migration by taking pictures at various times from creation wounds. Only the final picture after 6 hours is shown. The migration distance is quantified and the following graph shows the evolution. ICI 118,551 and propranolol delay cell migration and are therefore antiangiogenic. B. Migration distance of untreated and propranolol/ICI-treated 118,551 cells after 6 hours of scratching on the confluent layer of HUVEC (human umbilical vein endothelial cells).

Fig. 6 - ICI 118,551 and propranolol inhibit cellular angiogenesis of endothelial cells. Tubulogenesis is another in vitro test for angiogenesis. Endothelial cells cultured on Matrigel have the property of building cellular tubular structures that imitate the structure of blood vessels. A. Propranolol and ICI 118,551 at 100 µM significantly inhibit tubulogenesis. B. The number of closed cells (network density) is quantified and averaged over 5 different fields in each case. Histograms represent mean±SD.

Fig. 7 - Propranolol and ICI 118,551 inhibit HIF-mediated stimulation. Histograms show relative luciferase activity as a function of HIF-1 (hypoxia) in stable Hela cell transfectants for the HRE9x-luc reporter, which includes 9 tandem repeats of a hypoxia-responsive sequence element associated with HIF-1 fused to a luciferase reporter protein. Ct denotes control cells (normoxia and hypoxia). Deferroxamine (DFO) at 100 µM causes chemical hypoxia in treated cells. Treatment with propanolol or ICI 118,551 at 50 or 100 μM (P50, P100, I50, I100, respectively, for 24 h) inhibited DFO-induced hypoxia stimulation. It is noteworthy that the effect of ICI 118,551 is clearly superior to propranolol at an equal dose. Both DFO and drug therapy were administered simultaneously.

Fig. 8 - Propranolol and ICI 118.551 significantly reduced HIF-dependent transcription in HeLa cells, but not atenolol. Luciferase assays were performed in HRE-luc-stably transfected HeLa cells under hypoxic conditions and treated with propranolol, atenolol, and ICI 118.551. Propranolol and ICI (100 μM) prevented hypoxia stimulation in HeLa cells, as evidenced by decreased luciferase activity, by inhibiting hypoxia activation elements (HREs) by HIF. The results of atenolol were less significant than the others (propranolol and ICI).

Fig. 9 - ICI-118551 shows antitumor specific activity in VHL-/- cells. Viability (ATP content) of several cells (primary cultured and cell lines) exposed to various concentrations of mM ICI-118551 or propranolol for 72 h. Control wells did not contain ICI-118551 or propranolol. ICI-118551 (a specific beta-adrenergic receptor type 2 antagonist) and propranolol (a non-specific beta-adrenergic receptor type 1 and 2 antagonist) reduce the viability of VHL7 cells in a dose-dependent manner and with higher specificity. ATP content was measured using CellTiter-2 044366 assay

Glo® by Promega. A: HUVEC cell line, B: HMEC cell line, C: primary culture of hemangioblastoma 18, D: primary culture of hemangioblastoma 14, E: ccRCC Vhl -/- 786-O cell line.

Data shown are mean +/- SEM (n=3).

Fig. 10 - xenografts of human ccRCC 786-O Vhl -/- cells in mice. Male 7-8 week old NOD scid gamma (NSG) mice were injected into the posterior region with a single cell suspension of 106,786-O cells. When the tumor size reached 100 mm ³ , mice were randomly divided into 3 groups of 9/10 mice each. Two groups received 10 mg/kg body weight propranolol or ICI-118,551 intraperitoneally daily, respectively, and the third group received vehicle injection. Tumor size was measured with a caliper every 2–3 days, and volumes were calculated using the formula: smallest x largest x 0.52. Mice were sacrificed when the mean tumor volume of the control group reached the endpoint determined based on ethical procedures. The arrow marks the start of treatment.

Fig. 11 - ICI 118.551 differentially affects Vhl-/- cells and normal endothelial cells HUVEC and HMEC. Primary hemangioblastoma cultures from VHL, normal primary endothelial HUVECs, and the non-tumor microendothelial cell line HMEC-1 were cultured in the absence or presence of various concentrations of ICI 118.551 (50-250 μM) to measure viability. According to the viability curves, hemangioblastoma (HB) cells show only 20% viability at 100 μM, while non-tumor HUVEC or HMEC-1 cells show 80 and 55%, respectively, reduction in viability at 100 μM in both cultures.

Detailed Description of the Invention

The invention relates to a selective _{β 2} -adrenergic receptor antagonist for use in the treatment and/or prevention of von Hippel-Lindau disease.

Alternatively, the invention relates to the use of a selective _{β 2} -adrenergic receptor antagonist in the manufacture of a medicament for the treatment and/or prevention of von Hippel-Lindau disease.

Alternatively, the invention relates to a method of treating and/or preventing von Hippel-Lindau disease, comprising administering to said patient a therapeutically effective amount of a selective β ₂ -adrenergic receptor antagonist.

The term β ₂ -adrenergic receptor or e2AR as used herein refers to a class A G protein-coupled receptor (GPCR) that responds to diffusible hormones and neurotransmitters and is found predominantly in smooth muscle. There are two main groups of adrenergic receptors, α and β, with several subtypes:

α-receptors have subtypes α1 (Gq-coupled receptor) and _α2 (Gi-coupled receptor);

β receptors have subtypes β1, _β2 and _β3 . All three are associated with G _s proteins, which in turn are associated with adenylate cyclase. Binding of the agonist to these receptors causes an increase in the intracellular concentration of the second messenger cAMP.

Binding of the agonist to the _β2 -adrenergic receptor leads to relaxation of smooth muscles.

The term _{β 2} -adrenergic receptor antagonist as used herein refers to a compound that binds to the ₂ -adrenergic receptor and does not have any significant ability to activate the receptor itself. The term _β2 -adrenergic receptor antagonist includes both neutral antagonists and inverse agonists. A neutral antagonist is a compound that blocks the action of an agonist but does not affect intrinsic or spontaneous receptor activity. An inverse agonist is able to block the action of the agonist on the receptor and weaken the constitutive activity of the receptor. The term antagonist also includes competitive antagonists, which are drugs that bind to the same site as the natural ligand; non-competitive antagonists that bind to a different site on the receptor than the natural ligand; reversible antagonists that bind and unbind the receptor at rates determined by receptor-ligand kinetics; and irreversible antagonists, which permanently bind to the receptor either by forming a covalent bond to the active site or simply by binding so strongly that the rate of dissociation is effectively zero.

The term selective _{β 2} -adrenergic receptor antagonist as used herein means an antagonist that is selective for β ₂ -adrenergic receptors over β 1 -adrenergic receptors. In a specific embodiment, the selective _{β 2} -adrenergic receptor antagonist exhibits at least 10 times greater binding activity to β ₂ than to β 1 -adrenergic receptors, i.e., has a β ₂ /β 1 selectivity ratio of at least 10. More preferably, a selective _{β 2} receptor antagonist will have a β ₂ /β 1 selectivity ratio of at least 50. The affinity of various active agents for β ₂ and β 1 adrenergic receptors can be determined by assessing tissues containing the majority of β 2 receptors (eg, rabbit ciliary process, rat liver , feline choroid plexus or lung), tissues containing most β1 receptors (eg, cat and guinea pig heart), and tissues containing a mixture (eg, guinea pig trachea). Methods for determining relative

- 3044366 binding selectivities for these different tissue types are widely disclosed in O'Donnell and Wanstall,

Naunyn-Schmiedeberg's Arch. Pharmaco., 308, 183-190 (1979), Nathanson, Science. 204, 843-844 (1979), Nathanson, Life Sciences, 26, 1793-1799 (1980), Minneman et al., Mol. Pharmacol., 15, 21-33 (1979a), and Minneman et al., Journal of Pharmacology and Experimental Therapeutics, 211, 502-508 (1979).

There are a significant number of compounds known to have selective β ₂ adrenergic antagonist activity suitable for use in the present invention. In a specific embodiment, the selective β ₂ -adrenergic receptor antagonist is an alkanolamine derivative of formula I

where R ¹ represents an alkyl group with up to 6 carbon atoms, which branches at the α-carbon atom,

R ² represents alkyl with up to 3 carbon atoms,

R ³ is hydrogen, halogen or alkyl of up to three carbon atoms and n is 1 or 2, or a pharmaceutically acceptable acid addition salt thereof.

The term alkyl group as used herein refers to acyclic straight and branched groups derived from alkanes and having the formula --C _n H _2n+1 by removal of a hydrogen atom.

The term halogen as used herein refers to an atom selected from fluorine, chlorine, bromine and iodine.

R ¹ can be, for example, isopropyl or tert-butyl. In a specific embodiment, R ¹ is isopropyl.

R ² can be, for example, methyl or ethyl. In a specific embodiment, R ² is methyl.

R ³ can be, for example, hydrogen, chlorine, bromine, methyl or ethyl. In a specific embodiment, R ³ is methyl.

In a particular embodiment, n is 1.

In a specific embodiment, R ¹ is isopropyl and R ² is methyl. In a specific embodiment, R ¹ is isopropyl and R ³ is methyl. In a particular embodiment, R ¹ is isopropyl and n is 1. In a particular embodiment, R ² is methyl and R ³ is methyl. In a particular embodiment, R ² is methyl and n is 1. In a particular embodiment, R ³ is methyl and n is 1.

In a specific embodiment, R ¹ is isopropyl and R ² and R ³ are methyl. In another specific embodiment, R ¹ is isopropyl, R ² is methyl, and n is 1. In another specific embodiment, R ¹ is isopropyl, R ³ is methyl, and n is 1. In a specific embodiment, R ² and R ³ represents methyl and n is 1.

In a more specific embodiment, R ¹ is isopropyl, R ² and/or R ³ is methyl, and n is 1.

In an even more specific embodiment, the alkanolamine derivative has Formula II:

formula II

This compound of formula II is also known as ICI 118,551 and has the chemical name erythro-D, L-1(methylinden-4-yloxy)-3-isopropylaminobutan-2-ol. ICI 118,551 has a β ₂ /β 1 selectivity coefficient of at least 50, as determined and described in Life Sciences, 27,671 (1980) and Bilsky et al., J. Cardiovasc. Pharmacol., 5, 430-437 (1983).

It should be noted that the alkanolamine derivative of formula I has two asymmetric carbon atoms, namely the -CHOH- and -CHR2- groups, and that it can therefore exist in two racemic diastereomeric forms, threo and erythro, and four optically active forms that are (+) and (-) isomers of each of the racemic forms. It should be understood that the present invention covers any of these isomeric forms that have selective β2-adrenergic receptor antagonistic activity as defined above, since it is generally known how any particular isomer can be isolated and how any selective β2-adrenergic blocking activity can be measured. adrenergic receptor that it may possess.

It should be understood that, in general, the optical isomer which has the ^-absolute configuration of the -CHOH group is more active as a β2-adrenergic blocking agent than the corresponding isomer which has the ^-absolute configuration. It is also known that, in general, the erythro isomer is more β2-selective than the corresponding threo isomer, but that both the threo and erythro isomers of the compounds of the present invention have the required selectivity.

The term pharmaceutically acceptable acid addition salt refers to any acid addition salt that, when administered to a recipient, is capable of providing (directly or indirectly) a compound as described herein. Preferably, as used herein, the term pharmaceutically acceptable salt means one approved by a federal or state government regulatory agency or listed in the United States Pharmacopoeia or other generally accepted pharmacopoeia for use in animals and, in particular, in humans. The preparation of salts can be accomplished by methods known in the art. Illustrative non-limiting examples of a pharmaceutically acceptable acid addition salt of an alkanolamine derivative of Formula I include, for example, a salt derived from an inorganic acid, such as a hydrochloride, hydrobromide, phosphate, or sulfate, or a salt derived from an organic acid, such as an oxalate, lactate, tartrate, acetate, salicylate, citrate, benzoate, β-naphthoate, adipate or 1,1-methylene-bis-(2hydroxy-3-naphthoate), or a salt derived from an acidic synthetic resin, for example, sulfonated polystyrene resin. In a specific embodiment, the pharmaceutically acceptable acid addition salt is a hydrochloride. In a more specific embodiment, the selective _{β 2} -adrenergic receptor antagonist antagonist is the hydrochloride salt of a compound of Formula II.

In another specific embodiment, the selective _{β 2} -adrenergic receptor antagonist is selected from the list including the following compounds.

Butoxamine.

The chemical name for butoxamine is DL-erythro-α-(2,5-dimethoxyphenyl)-β-tert-butylaminopropanol hydrochloride. Determination of the beta ₂ selectivity of butoxamine is described in O'Donnell and Wanstall, Naunyn-Schmiedeberg's Arch.Pharmaco., 308, 183-190 (1979), which describes that the selectivity coefficient beta ₂ /beta ₁ is at least 17.

H35/25.

The chemical name for H35/25 is 1-(4'-methylphenyl)-b 2,2-1isopropylaminopropanol.

Prenalterol.

Prenalterol is a compound with the formula

Selective antagonistic activity at _{the 2} -adrenergic receptor has been described by Johansson and Waldeck, J. Pharm. Pharmacol., 1988, 32(9), 659-660.

Various 4- and 5-[2-hydroxy-3-(isopropylamino)propoxy]benzimidazoles as described by Crooks et al., J. Med. Chem., 22(2), 210-214 (1979).

1-(tert-Butylamino-3-ol-2-propyl)oxymino-9 fluorene as described by Imbs et al., Br. J. Pharmacol. 60(3), 357-362 (1977).

Various 2-(alpha-hydroxyarylmethyl)-3,3-dimethylaziridines as described by Jain et al., J. Med. Chem., 21(1), 68-72 (1978).

The term treatment, as used herein, refers to any process, act, application, therapy or the like wherein a subject (or patient), including a human, is provided with medical care for the purpose of improving the subject's condition, directly or indirectly, or slowing the progression of the condition or a disorder in the subject, or alleviation of at least one symptom of the disease or disorder being treated.

The term prophylaxis, as used herein, refers to the administration of a compound of the invention at an initial or early stage of a disease or also to prevent its occurrence.

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The term von Hippel-Lindau disease or VHL disease or von Hippel-Lindau disease or VHL disease or VHL syndrome or von Hippel-Lindau syndrome as used herein refers to a rare disease caused by a mutation in the Von Hippel-Lindau tumor suppressor (VHL ), resulting in the absence of VHL protein or an aberrant nonfunctional VHL protein. More than 370 inherited mutations in the VHL gene have been identified in people with von Hippel-Lindau disease (http://www.umd.be/VHL/). The VHL gene mutations associated with this condition either prevent the production of any VHL protein or result in the production of an abnormal version of the protein. VHL disease is characterized by the formation of multiple benign and malignant tumors and fluid-filled sacs (cysts) in many different parts of the body, including: retinal hemangioblastoma, central nervous system hemangioblastoma, clear cell renal cell carcinoma (CCRCC), pheochromocytoma, islet tumors of Langerhans, endolymphatic sac tumors, and testicular cysts and broad ligament.

The term patient or subject as used herein refers to any animal, preferably a mammal, and includes, but is not limited to, domestic and farm animals, primates and humans, e.g., humans, non-human primates, cows, horses, pigs , sheep, goats, dogs, cats or rodents. In a preferred embodiment, the subject is a person of any age or race. In the present invention, the patient suffers from von Hippel-Lindau disease.

The term von Hippel-Lindau disease patient as used herein means that the patient has been diagnosed with VHL disease. VHL disease can be diagnosed according to the following diagnostic criteria (Frantzen et al., Von Hippel-Lindau Syndrome, GeneReviews®):

In an individual without a family history of VHL disease, VHL disease is diagnosed if the patient has two or more characteristic lesions:

two or more hemangioblastomas of the retina, spine, or brain, or one hemangioblastoma combined with a visceral manifestation (eg, multiple renal or pancreatic cysts).

Renal cell carcinoma.

Adrenal pheochromocytomas and extra-adrenal pheochromocytomas.

Less commonly, tumors of the endolymphatic sac, papillary cystadenoma of the epididymis or broad ligament, or neuroendocrine tumors of the pancreas.

In an individual with a positive history of VHL disease, VHL disease is diagnosed when one or more of the following disease manifestations are present:

retinal angioma;

spinal or cerebellar hemangioblastoma;

adrenal pheochromocytomas and extra-adrenal pheochromocytomas;

renal cell carcinoma;

multiple cysts of the kidneys and pancreas.

In any case where a heterozygous terminal VHL pathogenic variant is identified by molecular testing.

In a specific embodiment, VHL disease occurs with the appearance of one or more tumors.

In a specific embodiment, the invention relates to a selective β ₂ adrenergic receptor antagonist for use in the treatment and/or prevention of a tumor in a patient with von Hippel-Lindau disease.

The term tumor or cancer, as used herein, refers to a broad group of diseases involving unregulated cell growth and which are also called malignancies. The term is usually applied to a disease characterized by uncontrolled cell division (or increased survival or resistance to apoptosis) and the ability of these cells to invade other nearby tissues (invasion) and spread to other areas of the body where cells are not normally found (metastasis) through the lymphatic and circulatory systems. vessels, circulate through the bloodstream, and then penetrate normal tissues in other parts of the body. In a specific embodiment, the cancer appears as a benign tumor, that is, tumors that cannot spread through invasion or metastasis, that is, they grow only locally. In another specific embodiment, the cancer manifests itself as a malignant tumor, that is, a tumor that is capable of spreading through invasion and metastasis.

Illustrative non-limiting examples of tumors include hemangioblastoma, pheochromocytoma, endolymphatic sac tumor, lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, cancer of the anus, anal canal or anorectum, eye cancer, intrahepatic bile duct cancer, joint cancer, cancer of the neck, gall bladder or pleura, cancer of the nose, nasal cavity or middle ear, vulvar cancer, chronic lymphocytic leukemia, chronic myeloid cancer, cervical cancer, glioma, Hodgkin's lymphoma, pituitary cancer,

- 6 044366 kidney cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin lymphoma, ovarian cancer, cyst in the broad ligament, abdominal cancer, omentum and mesentery, pharyngeal cancer, prostate cancer gland cancer, rectal cancer, kidney cancer including renal cell cancer, skin cancer, soft tissue cancer, testicular cancer, testicular cysts, thyroid cancer, ureteral cancer, bladder cancer and gastrointestinal cancer such as e.g. esophageal cancer, gastrointestinal cancer, pancreatic cancer, including tumor of the islets of Langerhans, stomach cancer, small intestinal cancer, gastrointestinal carcinoid tumor, oral cancer, colorectal cancer and hepatobiliary cancer.

In a specific embodiment, the tumor is not a pancreatic tumor. The term pancreatic tumor as used herein includes both exocrine and endocrine tumors. Exocrine pancreatic tumors include adenocarcinoma, acinar cell carcinoma, intraductal papillary mucinous tumor (IPMN), mucinous cystadenocarcinoma, among others. Endocrine tumors of the pancreas, also called islet cell tumors, include gastrinoma (Zollinger-Ellison syndrome), glucagoma, insulinoma, somatostatinoma, VIPoma (Werner-Morrison syndrome), atrophic pancreatic islet tumor, and multiple endocrine neoplasia type-1 (MEN1) among the others. In a more specific embodiment, the tumor is not an exocrine pancreatic tumor. In an even more specific embodiment, the tumor is not pancreatic adenocarcinoma.

In another specific embodiment, the tumor is selected from hemangioblastoma, clear cell renal cell carcinoma (CCRCC), pheochromocytoma, islet tumor of Langerhans, endolymphatic sac tumor, and cysts in the testes and broad ligament.

The term hemangioblastoma as used herein refers to a benign, highly vascular tumor that can arise in the central nervous system (brain or spinal cord) and the retina. In a more specific embodiment, the tumor is a hemangioblastoma. In an even more specific embodiment, the tumor is a retinal hemangioblastoma or a central nervous system hemangioblastoma.

The term clear cell renal cell carcinoma or ccRCC as used herein refers to a renal cortical tumor typically characterized by malignant epithelial cells with clear cytoplasm and a compact alveolar or acinar growth pattern with venous vasculature.

The term pheochromocytoma or PCC as used herein refers to a neuroendocrine tumor of the adrenal medulla or extrarenal chromaffin tissue formed in involute after birth that secretes large amounts of catecholamines, mainly norepinephrine and, to a lesser extent, epinephrine.

The term endolymphatic sac tumor as used herein refers to a papillary epithelial neoplasm arising in the endolymphatic sac or endolymphatic duct.

The term cyst as used herein refers to a cluster of cells grouped together to form a sac. The distinguishing feature of a cyst is that the cells that form the lining of such a sac are clearly abnormal (both in appearance and behavior) compared to all surrounding cells for that location. It may contain air, liquids or a semi-solid. In a specific embodiment, the cysts affect the testicles or broad ligament.

For its administration to a patient, the selective β2-adrenergic receptor antagonist of the invention, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, will be formulated in a pharmaceutical composition.

The term pharmaceutical composition as used herein refers to a composition containing a therapeutically effective amount of a selective β ₂ adrenergic receptor antagonist of the invention, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof and at least at least a pharmaceutically acceptable excipient or carrier.

The term therapeutically effective amount as used herein refers to an amount of a compound sufficient to provide the desired effect and will generally be determined, among other reasons, by the characteristics of the compound itself and the therapeutic effect achieved. This will also depend on the subject being treated, the severity of the disease from which said subject is suffering, the dosage form chosen, the route of administration, and the like. For this reason, the doses mentioned in the present invention should be considered only as a guide for one skilled in the art, who should adjust the doses depending on the above-mentioned variables.

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Even though individual needs vary, determination of optimal ranges for therapeutically effective amounts of compounds for use in accordance with the invention is within the general experience of those skilled in the art. In general, the dosage required to provide effective treatment, which may be adjusted by one skilled in the art, will vary depending on age, health, fitness, sex, nutrition, weight, degree of receptor alteration, frequency of treatment, nature and condition injury, the nature and extent of the disorder or disease, the health status of the subject, the route of administration, pharmacological criteria such as activity, efficacy, the pharmacokinetic and toxicological profile of the particular compound used, if systemic drug delivery is used, and if the compound is administered as part of a drug combination. The amount of a compound for use in accordance with the invention that is therapeutically effective for the prevention and/or treatment of ischemia/reperfusion injury in a subject can be determined by conventional clinical methods (see, for example, The Physician's Desk Reference, Medical Economics Company, Inc., Oradell, NJ, 1995, and Drug Facts and Comparisons, Inc., St. Louis, MO, 1993).

In a specific embodiment, the therapeutically effective amount causes a reduction in one or more symptoms of the VHL disease. In a specific embodiment, a selective β2-adrenergic receptor antagonist, preferably an alkanolamine derivative of Formula I, more preferably an alkanolamine derivative of Formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered at a dose of from about 0.2 mg/kg/day to about 5 mg/kg /day, preferably from about 0.5 mg/kg/day, about 0.7 mg/kg/day, about 1 mg/kg/day, about 1.5 mg/kg/day to about 1.7 mg/kg/ day, approximately 1.9 mg/kg/day, approximately 2.1 mg/kg/day, approximately 2.2 mg/kg/day, approximately 2.5 mg/kg/day, approximately 2.7 mg/kg/day day, about 2.9 mg/kg/day, about 3.1 mg/kg/day, about 3.3 mg/kg/day, to about 4.9 mg/kg/day, about 4.8 mg/kg /day, approximately 4.7 mg/kg/day, approximately 4.6 mg/kg/day, approximately 4.5 mg/kg/day. In a more specific embodiment, a selective _{β 2} -adrenergic receptor antagonist, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered at a dose of 3.3 mg/kg body weight/day to 4. 5 mg/kg body weight/day. In another more specific embodiment, a selective _{β 2} -adrenergic receptor antagonist, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered at a dose of 0.5 mg/kg body weight/day to 1 mg/kg body weight/day. In an even more specific embodiment, a selective _{β 2} -adrenergic receptor antagonist, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered at a dose of 0.81 mg/kg body weight/day.

Dosages of the compounds of the invention can be expressed either in mg of antagonist per kg of body weight or in mg of antagonist per square meter of body surface. An experienced practitioner will know how to determine the dose for a particular animal, particularly the human dose, from doses experimentally tested in mice. For example, an article from Reagan-Shaw S. et al. (Reagan-Shaw S. et al. Dose translation from animal to human studies revisited. FASEB J 2008, 22(3):659-661), provides standard conversion factors used to convert mg/kg to mg/ ^m2 .

Dose (mg/kg) x K _m = dose (mg/m ² ).

The article also explains that this conversion is the basis for converting a dose in a first animal species to a dose in a second animal species (allometric dose conversion). Thus, an animal dose (AD) in mg/kg can be converted to a human equivalent dose (HED) in mg/kg using the following formula:

HED (mg/kg) = AD (mg/kg) x (animal Km/human K _m ), where K _m for each species is shown in table. 1 (data taken from Reagan-Shaw S. et al. Dose translation from animal to human studies revisited. FASEB J 2008, 22(3):659-661).

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Thus, experiments with doses of 10 mg/kg in mice correspond to total doses in humans of 0.8 mg/kg.

In another specific embodiment, a selective β2-adrenergic receptor antagonist, preferably an alkanolamine derivative of Formula I, more preferably an alkanolamine derivative of Formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered to a human at a dose where each administration is from 0.2 to 5 mg/m ² . preferably from about 0.2 mg/kg/day, about 0.25 mg/kg/day, about 0.3 mg/kg/day, about 0.35 mg/kg/day, about 0.4 mg/kg/ day, approximately 0.45 mg/kg/day, approximately 0.50 mg/kg/day, approximately 0.55 mg/kg/day, approximately 0.6 mg/kg/day, approximately 0.65 mg/kg/day day, approximately 0.7 mg/kg/day, approximately 0.75 mg/kg/day, approximately 0.8 mg/kg/day, approximately 0.85 mg/kg/day, approximately 0.90 mg/kg/day day, about 0.95 mg/kg/day, about 1 mg/kg/day, about 1.2 mg/kg/day, about 1.4 mg/kg/day, about 1.6 mg/kg/day, about 1.8 mg/kg/day, about 2 mg/kg/day about 2.2 mg/kg/day, about 2.4 mg/kg/day, about 2.6 mg/kg/day, about 2, 8 mg/kg/day, approximately 3 mg/kg/day, approximately 3.2 mg/kg/day, to approximately 3.4 mg/kg/day, approximately 3.6 mg/kg/day, approximately 3.8 mg/kg/day, approximately 4 mg/kg/day, approximately 4.5 mg/kg/day, approximately 5 mg/kg/day. In a more preferred embodiment, a selective β2-adrenergic receptor antagonist, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered at a dose of 0.7 mg/kg body weight/day to 1 mg/day. kg body weight/day. In an even more preferred embodiment, a selective _{β 2} -adrenergic receptor antagonist, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered at a dose of 0.81 mg/kg body weight/day.

In another specific embodiment, a selective _β2 -adrenergic receptor antagonist, preferably an alkanolamine derivative of Formula I, more preferably an alkanolamine derivative of Formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered daily, preferably once daily, twice daily, three times daily. day. In a more preferred embodiment, it is administered once daily. In another specific embodiment, a selective β2-adrenergic receptor antagonist, preferably an alkanolamine derivative of Formula I, more preferably an alkanolamine derivative of Formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered for 10 days, 15 days, 20 days, 25 days, 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months or more than 12 months, preferably within 25 days.

The terms pharmaceutically acceptable excipient or pharmaceutically acceptable carrier refer to any compound or combination of compounds that is substantially non-toxic to a subject at the dosage and concentration used and is compatible with the other components of the pharmaceutical composition. Thus, the excipient is an inactive substance formulated together with the active ingredient (i.e., a selective β2-adrenergic receptor antagonist, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof) of a pharmaceutical composition, with the purpose of increasing the volume of compositions that contain these active ingredients. The increase in volume allows for convenient and accurate dosing of the medicinal substance during the manufacture of the dosage form. Excipients may also serve various therapeutic purposes, such as facilitating the absorption or solubility of a compound (drug) or other pharmacokinetic criteria. Excipients may also be useful during the manufacturing process to aid handling of the respective active substance, for example to improve flowability

- 9 044366 powder or non-stick properties in addition to providing in vitro stability such as preventing denaturation during the expected shelf life. The selection of appropriate excipients depends on the route of administration and dosage form, as well as the active ingredient and other factors. The excipient may be a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or any conventional type of auxiliary composition. Illustrative, non-limiting examples of excipients or carriers include water, saline (brine) solutions, alcohol, dextrose, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil , monoglycerides and diglycerides of fatty acids, esters of fatty acids, petroesters, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

The selective β2-adrenergic receptor antagonist of the invention, preferably an alkanolamine derivative of Formula I, more preferably an alkanolamine derivative of Formula II, or a pharmaceutically acceptable acid addition salt thereof, can be administered by any suitable route of administration, such as, but not limited to, parenteral, oral, topical, nasal , rectal, intravitreal route. In a specific embodiment, the selective β ₂ adrenergic receptor antagonist of the invention, preferably an alkanolamine derivative of Formula I, more preferably an alkanolamine derivative of Formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered intraperitoneally, intravenously, subcutaneously, intradermally, intramuscularly, or intravitreally. In a preferred embodiment of the invention, the selective β2-adrenergic receptor antagonist of the invention, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered orally, intravenously or intravitreally. In a more preferred embodiment, the selective β2-adrenergic receptor antagonist of the invention, preferably an alkanolamine derivative of formula I, more preferably an alkanolamine derivative of formula II, or a pharmaceutically acceptable acid addition salt thereof, is administered intraperitoneally.

The invention is described below with the help of the following examples, which are to be considered only as illustrative and not limiting the scope of the invention.

Examples

Ways.

Cell culture.

HeLa 9XHRE cells were stably transfected with an HREluc reporter carrying nine copies in tandem of the hormone response element (HRE) followed by the luciferase gene and cultured in DMEM (Dulbecco's modified Eagle's medium, Gibco, Grand Island, NY, USA) supplemented 10% fetal bovine serum (FBS; Gibco). To induce hypoxic conditions, HeLa cells were cultured with 100 μM desferrioxamine (DFO) (chemical hypoxia). When necessary, HeLa cells were treated with propranolol (a non-selective beta blocker that binds both beta 1 and beta 2 receptors), ICI 118.551 (specific for beta 2) and atenolol (specific for beta 1) (100 μM). Primary cultures of CNS hemangioblastoma were prepared as previously described by Albinana et al., Orphanet J Rare Diseases, 2015, 10: 118. When necessary, primary cultures of CNS hemangioblastoma were treated with propranolol and ICI (100 μM).

Real-time RT-PCR.

Total cellular RNA was isolated from hemangioblastoma cells using the Nucleo Spin RNA kit (Macherey-Nagel, Duren, Germany). One microgram of total RNA was reverse transcribed in a final volume of 20 μl using the First Strand cDNA Synthesis Kit (Roche, Mannheim, Germany) using random primers. The SYBR Green PCR system (BioRad, Hercules, CA, USA) was used to perform real-time PCR with the iQ5 system. The primers used for qPCR were:

table 2

Primers used for qPCR

GENE Direct Reverse

5’- CTCAACACGGGAAACCTCAC - 5’-CGCTCCACCAACTAAGAACG 18S 3’ 3’.

VAC 5CACTCCCGCCACAAAGAT - 3 ' 5CAAGACCAGGGTGGTTGG - 3 '

CASP9 5’-CCCAAGCTCTTTTTCATCCA-3’ 5 -TTACTGCCAGGGGACTCGT - 3’

Luminescent cell viability assay.

Hemangioblastoma and HeLa 9X HRE cell viability was measured using the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI, USA). This is a homogeneous method for determining the number of viable cells in a culture based on the quantification of ATP present, which signals the presence of metabolically active cells. In general

- 10 044366 complexity 10,000 cells were seeded in 96-well plates and cultured with propranolol/ICI

118.551 in 100 µl of medium. After treatment, plates were equilibrated to room temperature for 30 min before adding 100 μl of Cell Titer-Glo reagent (lysis buffer, Ultra-Glo recombinant luciferase, luciferin and Mg ²⁺ ). Luminescence was measured using a multi-detection system

Glomax (Promega).

Analysis of wound healing and tubule formation.

In vitro scratches were prepared by scraping confluent HUVEC-1 monolayers in the wells of a P-24 plate with sterile disposable pipette tips. The remaining cells were washed with PBS and incubated with EGM-2 (Lonza) in the absence or presence of propranolol/ICI 118,551 100 µM for up to 6-8 hours. For tubule formation assays, HUVECs were plated as before, but on a Matrigel plate (BD Biosciences, Bedford, MA, USA), and incubated at 37°C without and with propranolol/ICI 118,551 (100 μM) treatment. Tubule formation was monitored for up to 8 h.

Formation and culture of the hemangiosphere.

VHL-derived hemangioblastoma cells were grown in suspension in phenol red-free DMEM:F-12 with GlutaMAX supplemented with B27, 20 ng/ml EGF, 20 ng/ml bFGF and 1% penicillin/streptomycin at 37°C. in 5% CO ₂ . Single cell suspensions were seeded at a density of 50,000 cells/ml in 75 ^cm2 ultra-low attachment flasks (Corning). Cultures were left untreated or treated with propranolol/ICI 118,551 at the indicated concentrations.

Measuring cell viability.

Endothelial cell lines (HUVEC and HMEC), the human ccRCC cell line Vhl/- 786-O, and primary patient-derived hemangioblastoma cultures (Hb14 and 18) were seeded in triplicate in a 96-well plate (2x103 per well). The next day, cells were treated with vehicle or various concentrations of mM propranolol or ICI-118,551 for 72 h. Cell viability (cell proliferation, ATP content) was determined using the CellTiter-Glo Luminescent Cell Viability Assay Kit (Promega) according to the supplier's instructions. Cell Titer-Glo reagent ((lysis buffer, recombinant Ultra-Glo luciferase, luciferin and Mg ²⁺ ). Cell lysis was induced in an orbital shaker for 2 min, and then the plates were incubated at room temperature for 10 min to stabilize the luminescent signal Luminescence was measured using the GlomaxMultidetection system (Promega) and was normalized to control (100%).

Xenografts of human ccRCC 786-O cells in mice.

Male 7-8 week old NOD scid gamma (NSG) mice were injected into the posterior region with a single cell suspension of 106,786-O cells. When the tumor size reached 100 mm ³ , mice were randomly divided into 3 groups of 9/10 mice each. Two groups received 10 mg/kg body weight propranolol or ICI-118,551 intraperitoneally daily, respectively, and the third group received vehicle injection. Tumor size was measured with a caliper every 2–3 days, and volumes were calculated using the formula: smallest ² x largest x 0.52. Mice were sacrificed when the mean tumor volume of the control group reached the endpoint determined based on ethical procedures.

Results.

ICI 118,551 reduces hemangioblastoma viability and inhibits hemangiosphere formation in patients with VHL.

The results shown in the examples demonstrate that ICI 118,551, a selective beta 2 blocker, not only mirrors the results obtained with propranolol (a non-selective beta blocker that binds beta 1 and beta 2 receptors) (see Albinana et al., Orphanet J of are diseases., 10:118 (2015)), but also shows superior results compared to propranolol. Specifically, in the case of VHL tumor-derived cells, ICI 118,551 reduces viability by increasing cell apoptosis, inhibits hemangiosphere formation, which is a specific property of tumors containing undifferentiated stem cells, and is an antiangiogenic agent by inhibiting HIF-stimulated transcription of its genes. targets.

Propranolol and ICI 118,551 reduced the viability of hemangioblatoma cells from a primary culture derived from a VHL tumor by at least 55-60% compared to the same untreated cells (Fig. 1). In most cases, the effect of ICI 118,551 is superior to that of propranolol when both are used at 100 µM, after 72 h of treatment. The reduction in viability ranges from 60 to 40% depending on different tumors, patients and culture time.

In fig. Figure 2 shows the remaining cells in cultures after 72 hours of treatment with P (propranolol 100 μM) and ICI 118,551 50 μM (HB 18) and 100 μM in (HB 4) compared to the control (untreated cells of different hemangioblastomas from 2 different patients). There is a significant reduction in the number of cells up to 30-40% compared to untreated ones. The reduction occurs due to apoptosis, as shown for ICI 118,551 in Fig. 3. In FIG. Figure 3 shows the amount of mRNA from two proapoptotic genes, Bax and protease-caspase 9, which was significantly increased after cells were treated with ICI 118,551 at 100 μM.

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One of the characteristic features of tumor cells in culture is the ability to form organized spheres around the nucleus of undifferentiated stem cells, and these spheres have a clearly defined boundary surrounded by an ordered surface of contact with the environment. Antineoplastic agents inhibit or disrupt sphere formation by cell disaggregation. In fig. 4 shows hemangiosphere cultures from different hemangioblastomas without treatment and after treatment with 100 μM either propranolol or ICI 118,551 for 7 days in these specific cases shown in these FIGS. 4 and 5. As shown in 5 different tumor cultures, hemangiospheres are destroyed by beta-blocker treatment, which is especially noticeable in the case of hemangioblastoma 4 (Fig. 4B) and 23 (Fig. 4C) after 100 μM ICI 118,551.

Angiogenesis is the property of endothelial cells to form vessels from existing ones. Angiogenesis requires migration of endothelial cells (including destruction of the cell matrix) and tubulogenesis or the formation of new tubules (vessels). These properties can be studied in vitro using the wound healing test (Fig. 5) and tubulogenesis on Matrigel (Fig. 6).

If confluent monolayers of endothelial cells are disrupted due to scratches on their surface, endothelial cells tend to migrate and fill the gap. This so-called wound healing test can be performed over time to monitor cell migration. In fig. Figure 5A shows how ICI 118,551 and propranolol at 100 μM delay gap closure compared to untreated cells (control). Migration of untreated cells occurs faster, as quantified in the graph in FIG. 5B, while ICI 118,551 and propranolol significantly affect the migration process.

In fig. Figure 6A shows a characteristic network of tubules mimicking the capillary network formed by endothelial cells in Matrigel after 3 hours. However, if the cells are pretreated with ICI 118,551 or propranolol (100 μM), the number of closed structures decreases sharply. The average quantification of gated cells in five different fields is shown in Fig. 6B. It is clear that propranolol and ICI 118,551 act as inhibitors of angiogenesis.

Angiogenesis is largely dependent on the HIF transcriptional program, triggered in the presence of a hypoxic condition or, as in the case of von Hippel-Lindau hemangioblastoma cells, due to the absence of the VHL protein responsible for targeting the HIF protein for its proteasomal processing under normoxic conditions in wild-type cells . A particular distinguishing feature of hemangioblastoma cells and clear cell renal cell carcinoma cells is that they constitutively express the HIF protein, which is active and translocates into the nucleus, where it can bind and activate its gene targets, such as: VEGF, PDGF, EPO, endoglin, several metalloproteases, carbonic anhydrase, Glut-1 and so on. To test that propranolol and ICI 118,551 are inhibitors of HIF transcriptional activity, a luciferase reporter system was used under the control of a nine-copy hormone response element (HRE) in tandem followed by luciferase. HeLa 9XHRE cells were stably transfected with the HREluc reporter. To induce hypoxic conditions, HeLa cells were cultured with 100 μM desferrioxamine (DFO) (chemical hypoxia). These hypoxic cells were either untreated or treated with 100 μM propranolol or ICI 118,551, and luciferase activity was measured by luminometry. In fig. Figure 7 shows how luciferase expression is triggered under hypoxic conditions and how this up-regulation is attenuated by propranolol and ICI 118,551. ICI 118,551 is clearly superior to propranolol in completely eliminating hypoxia-induced luciferase activity, so ICI 118,551 targets HIF transcriptional activity, which is reduced to almost 0.

Propranolol and ICI 118,551 affect the hypoxia pathway primarily through blockade of beta 2 adrenergic receptors.

When HeLa cells carrying the HRE-luciferase hypoxia reporter are treated with DFO (deferroxyamine), HIF translocates into the nuclei and binds to the HRE (hormone response element) fused to the luciferase reporter, and stimulation of the hypoxia target can be quantified by luminescence. However, hypoxia stimulation is extremely reduced in the presence of ICI 118,551 and propranolol at 100 μM. Since both are beta 2 adrenergic blockers, atenolol, a specific beta 1 adrenergic blocker, was also used. As shown in FIG. 8, atenolol, almost does not reduce the stimulation of hypoxia. Accordingly, it appears that ICI 118,551 mimics propranolol by targeting the HIF-induced pathway and acts by blocking the beta 2 receptor type. VHL-derived tumors have constitutive expression of HTF, so the therapeutic effect of ICI 118,551, a specific beta 2 blocker, in relation to von Hippel-Lindau disease is explained by decreased stimulation of the HIF gene program. Thus, ICI 118,551 mimics the effects of propranolol on the hypoxia pathway, primarily through blockade of beta 2 adrenergic receptors.

ICI 118,551 preferentially targets Vhl-/- tumor cells compared to non-tumor cells such as HUVEC and HMEC.

When Vhl-/- cells, primary cultures of hemangioblastoma cells from patients with VHL, including the Vhl-/- kidney cancer cell line 786-0, are treated at various doses from 0 to 250 μM ICI 118,551, they

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Claims (10)

more sensitive than non-tumor cells such as HUVEC (human umbilical vein endothelial cells) and HMEC (human microvascular endothelial cells). While the LD50 for VHL hemangioblastomas is 50 to 100 µM, ICI 118.551, at 786-O is 100 µM, in the case of HUVEC and HMEC it is around 150 µM. This is critical to selectively targeting Vhl−/− cells at a dose of ICI 118,551 below the toxic range for non-Vhl−/− cells. See fig. 9. Thus, ICI 118,551 preferentially targets Vhl-/- tumor cells compared to non-tumor cells. ICI 118,551 and propranolol act to reduce tumor growth of ccRCC Vhl-/786-O xenograft in NSG mouse model in vivo. NSG mice treated with propranolol or ICI 118,551 (beta blockers) inhibited tumor growth, as shown by the significant differences found between tumor sizes 50-60 days after cell inoculation (Figure 10). There were no significant differences between propranolol and ICI 118,551 treatments, while these treatments showed a significant reduction of approximately 30% in tumor size compared to vehicle-treated mice. No toxicity or side effects were observed during treatment. Thus, ICI 118,551 acts to reduce tumor growth of ccRCC Vhl-/-786-O xenografts in an in vivo NSG mouse model. ICI 118.551 differentially affects Vhl-/- cells and normal endothelial cells HUVEC and HMEC. Primary hemangioblastoma cultures from VHL, normal primary endothelial HUVECs, and the non-tumor microendothelial cell line HMEC-1 were cultured in the absence or presence of various concentrations of ICI 118.551 (50-250 μM) to measure viability. According to the viability curves, hemangioblastoma (HB) cells show only 20% viability at 100 μM, while non-tumor HUVEC or HMEC-1 cells show 80 and 55%, respectively, reduction in viability at 100 μM in both cultures. Propranolol and ICI 118,551 have a dual mode of action: on the one hand, they stimulate apoptosis and thus arrest cell growth and cause cell death in a programmed manner. On the other hand, they suppress the transcriptional activation of the HIF-induced program, thereby reducing the expression of target genes such as VEGF, EPO, endoglin, metalloproteases, and so on. CLAIM 1. The use of a selective _{β 2} -adrenergic receptor antagonist for the treatment and/or prevention of von Hippel-Lindau disease (VHL), wherein the selective _{β 2} -adrenergic receptor antagonist is an alkanolamine derivative of the formula or a pharmaceutically acceptable salt thereof. 2. Use according to claim 1, wherein the VHL disease occurs with the appearance of one or more tumors. 3. Use according to claim 1 or 2, wherein the tumor is a hemangioblastoma. 4. Use according to claim 3, wherein the hemangioblastoma is a retinal hemangioblastoma or a central nervous system hemangioblastoma. 5. Use according to any one of claims 1 to 4, wherein the pharmaceutically acceptable acid addition salt is a hydrochloride. 6. Use according to any one of claims 1 to 4, wherein the alkanolamine derivative or a pharmaceutically acceptable acid addition salt thereof is administered at a dose of 0.2 to 5 mg/kg body weight/day. 7. Use according to claim 6, wherein the alkanolamine derivative or a pharmaceutically acceptable acid addition salt thereof is administered at a dose of 0.5 to 1 mg/kg body weight/day. 8. Use according to claim 7, wherein the alkanolamine derivative or a pharmaceutically acceptable acid addition salt thereof is administered at a dose of 0.8 mg/kg body weight/day. 9. Use according to any one of claims 1 to 8, wherein the alkanolamine derivative or a pharmaceutically acceptable acid addition salt thereof is administered intraperitoneally. 10. Use according to any one of claims 1 to 9, wherein the alkanolamine derivative or a pharmaceutically acceptable acid addition salt thereof is administered daily for 25 days. -