HK1161116B - 3, 3' 4, 4' -tetrahydroxy-2,2'-bipyridine-n, n'-dioxides for the treatment of renal cell carcinoma - Google Patents

Description

3,3 ', 4, 4' -tetrahydroxy-2, 2 '-bipyridine-N, N' -dioxides for the treatment of renal cell carcinoma

Technical Field

The present invention relates generally to cancer therapy. More specifically, the present invention relates to the use of 3,3 ', 4, 4' -tetrahydroxy-2, 2 '-bipyridine-N, N' -dioxide, in particular 3,3 ', 4, 4' -tetrahydroxy-2, 2 '-bipyridine-N, N' -dioxide (Orellanine), for the treatment of renal cancer, in particular renal cell carcinoma derived from proximal renal tubule cells.

Background

Cancer occurs in more than 100 different forms, affecting almost every part of the body. Throughout life, healthy cells in the body divide, grow, and replace themselves in a controlled manner. Cancer develops when genes indicative of this cell division malfunction and the cells begin to proliferate and grow uncontrollably. The clumps or clusters of these abnormal cells are called tumors. Not all tumors are cancerous. Benign tumors, such as nevi, stop growing and do not spread to other parts of the body. However, cancerous or malignant tumors continue to grow and cover healthy cells, interfere with bodily functions, and draw nutrients from bodily tissues. Malignant tumors can spread to other parts of the body through a process called metastasis. Cells from the "parent tumor" detach, migrate depending on the tumor via the blood or lymphatic vessels, or within the chest, abdomen, or pelvis, and they eventually form new tumors elsewhere in the body.

Cancer of the kidney constitutes about 3% of all solid tumors. About 85% of renal tumors are classified as Renal Cell Carcinoma (RCC). Approximately 80% of the diagnosed RCC originates from epithelial cells lining the ureteral, proximal portion of the renal tubules of the kidney. Depending on its appearance under the microscope, this type of cancer is called renal clear cell carcinoma (RCCC, 65%) or renal papillary cell carcinoma (RPCC, 15%). While RCCC and RPCC constitute 80% of the RCC diagnosed, they are responsible for nearly 100% of deaths from renal cell carcinoma.

The most important factor in predicting prognosis is the period. Stage describes the size of the cancer and the depth to which it spreads beyond the kidneys. The staging system of the united states joint committee on cancer (AJCC) is known as the TNM system. The letter T followed by a number from 1 to 3 describes the size of the tumor and its spread to nearby tissues. Higher T numbers indicate larger tumors and/or more extensive spread to tissues near the kidney. The letter N followed by a number from 0 to 2 indicates whether the cancer has spread to lymph nodes near the kidney and, if so, how many lymph nodes are affected. The letter M followed by 0 or 1 indicates whether the cancer has spread to distant organs.

And (3) stage I:tumors were 7cm (about 23/4 inches) or less and were restricted to the kidneys. There was no spread to lymph nodes or distant organs.

And (2) in a stage II:tumors were larger than 7.0cm but still restricted to the kidney. There was no spread to lymph nodes or distant organs.

Stage III:including tumors of any size, with or without spread to adipose tissue surrounding the kidney, with or without spread into the great veins from the kidney to the heart, with spread to a nearby lymph node, but without spread to distant lymph nodes or other organs. Stage III also includes tumors that spread to the adipose tissue surrounding the kidney and/or to the large veins from the kidney to the heart, which do not spread to any lymph nodes or other organs.

Stage IV:this stage includes any cancer that has spread directly throughout the adipose tissue and fascia-ligament-like tissue surrounding the kidney. Stage IV also includes any cancer that has spread beyond one lymph node near the kidney, any lymph node not near the kidney, or any other organ (e.g., lung, bone, or brain).

Detailed definition of T, N, M class and stage group of renal cell carcinoma:

primary tumor (T):

TX: failure to assess primary tumors

T0: evidence of no primary tumor

T1: tumors 7cm or less, restricted to the kidney

T2: tumor greater than 7cm, restricted to kidney

T3: tumor expansion to major venous/adrenal/perirenal tissues; not exceeding Jes 'fascia (Gerota's fascica)

T3 a: tumor invasion of adrenal/perirenal fat

T3 b: tumor expansion into renal vein or inferior vena cava

T3 c: vena cava with tumor spread onto septum

T4: invasion of tumors beyond Jersey fascia

N-regional lymph node

NX: failure to assess regional lymph nodes

N0: absence of regional lymph node metastasis

N1: metastasis in a single regional lymph node

N2: metastasis of more than one regional lymph node

M-distant transfer

MX: failure to assess distant metastasis

M0: without remote transfer

M1: remote transfer

Empirically, stage I or II cancers are treated by surgical removal of the affected kidney and the prognosis is well-restored. In contrast, renal Cancer at stage III or IV is associated with very low survival rates, and the National Cancer Institute states on its website that "patients with renal cell carcinoma at stage IV are barely curable"

The national cancer society estimates that 49,096 new cases of renal cancer were diagnosed in the united states in 2009 (16/10)⁵Resident) 11,033 confirmed deaths (3.6/10)⁵Residents). The corresponding number (2006) of the European Union is 65,051 diagnoses (7.8/10)⁵Resident) and 27,326 deaths (3.3/10)⁵Resident) (European Cancer observer: http:// eu-cancer. iarc. fr/cancer-19-kidney. html, en). Worldwide, it is estimated (2006) that 209,000 diagnosed cases (3.2/10)⁵Residents) and 102,000 deaths (1.6/10)⁵Residents) (Gupta et al. cancer treat. rev.34, 193-; 2008). It appears that the seemingly higher incidence in the united states is due to the fact that NCI will report together cancers of the renal pelvis (which are relatively easy to treat) and with renal cell carcinoma. Lower global incidence and mortality may depend, at least in part, on excessively low diagnosis in large areas of the third world.

As described above, the main problem with conventional techniques is that the outcome of any patient diagnosed with renal cancer is largely determined by the timing of the diagnosis. The chance of survival is good if the disease is diagnosed before the tumor spreads outside the kidney, otherwise most patients die of the disease. The main reason for this is that renal cell carcinoma is refractory to all conventional therapies using cytostatic and cytotoxic drugs, such as cisplatin, carboplatin, docetaxel, paclitaxel, fluorouracil, capecitabine, gemcitabine, irinotecan, topotecan, etoposide, mitomycin, gefitinib, vincristine, vinblastine, doxorubicin, cyclophosphamide, celecoxib, rofecoxib and/or valdecoxib.

Various methods are described in the prior art. Conventional chemotherapy for renal cell carcinoma is largely contraindicated due to low effectiveness and a large number of side effects. Alternative forms of treatment are thus sought, which can be divided into several categories:

1) anti-angiogenesis. In this method, the nutrients and oxygen of the tumor are cut off by the inhibition of the formation of blood vessels necessary to supply the tumor tissue. This can be achieved in several ways: 1a) inhibition of circulating growth factors, e.g., VEGF, PDGF and PIGF, by antibody treatment against these growth factors; 1b) blockade of angiogenic growth factor receptors on target cells using antibodies directed against the receptors; and 1c) treatment with smaller molecules that interfere with receptor function such that binding of angiogenic growth factors to its receptor does not elicit a physiological angiogenic effect.

2) And (4) immunoregulation treatment. This approach attempts to stimulate the endogenous immune system to recognize tumor cells as allogeneic and to begin fighting them. Immunostimulation as a treatment for renal cancer takes two main routes: 2a) treatment with interleukin 2 (IL-2); and 2b) interferon alpha (IFN alpha) treatment.

All of the above alternative treatment strategies significantly extend the life span of certain patients with renal cancer at an advanced stage. However, the effect is only on the order of a few months and treatment is associated with a number of serious side effects. Frequently, tumors adapt to treatment, and then treatment cannot continue, followed by accelerated tumor growth. Garcia et al ("Recent progress in the management of advanced secondary cell Carcinoma")CA Cancer.J.Clin.57(2): 112-25(2007)) and Atkins et al ("Innovations and changes in secondary cell carcinosa: a summary status from the Second Cambridge conference reference ″)Clin.Cancer.Res.13(2 Pt 2): 667s-670s (2007)) reviewed recent approaches to renal cancer treatment.

A review of the literature indicates that many therapeutic approaches derive from the identification of more or less specific cancer markers and the use of these markers for eliciting host immune responses against invasive tumor tissue. Thus, US2006134708 discloses several molecular markers of kidney and urinary bladder cancer, namely IGFBP-3 (insulin-like growth factor binding protein 3), ANGPTL4 (angiopoietin-like 4) and ceruloplasmin (cemloplasmin) for diagnostic purposes, as well as monoclonal antibodies against said markers for diagnostic use. The use of antisense compounds against the disclosed markers at the peptide and nucleic acid levels is described. Also, the use of monoclonal antibodies conjugated to cytotoxic agents, against the markers, is also contemplated as a therapeutic embodiment, linked to the severe side effects of less cytotoxic agents depending on the targeting provided by the antibody (also known as the "magic bullet" concept). A similar approach based on different tumor associated antigens was adopted in CN 1359941.

US6403373 discloses nucleic acid molecules associated with colon, kidney and stomach cancers, the peptide products of which elicit the production of antibodies in a host. The use of the peptides in vaccine methods is contemplated. EP0160250 discloses monoclonal antibodies for the diagnosis of kidney cancer and mentions the possibility of conjugating these antibodies to various cytotoxic agents.

WO2007059082 discloses the discovery of the antigen TIM-1(T cell, immunoglobulin or mucin domain 1) in ovarian and renal cancers, which is associated with cell proliferation. The use of antibodies raised against TIM-1 for the treatment of ovarian and renal cancers is taught, as well as the conjugation of therapeutic agents (toxins, radioisotopes, or chemotherapeutic agents) to such antibodies as a targeted killing means for tumor cells.

US6440663 discloses a number of genes, exemplified by kidney cancer cells, the products of which cause antibody production in the host. Various methods of eliciting or increasing an immune response in a host against a tissue indicative of the disclosed genes are described, including the priming of cytotoxic T cells and transfection of host cells with the disclosed genes or fragments thereof, followed by reintroduction of the cells into the host.

U.S. 2005261178 discloses the co-administration of a monoclonal antibody (G250) against an antigen (carbonic anhydrase IX) expressed on most renal cancers with the cytokine interleukin-2 or interferon-alpha. The cytokine is administered at a lower dose than that used when treated with the cytokine alone. Disease stabilization or "target response" for 22 weeks or more was achieved in about 30% of patients in the population with advanced renal cancer.

Other approaches are based on the use of known therapeutic substances in new treatment protocols. For example, WO2007044015 discloses the use of previously known dimethanesulfonate compounds, particularly NSC-281612, according to a new administration regimen to treat renal cancer. When tested against xenografts in nude mice, administration of NSC-281612 resulted in the apparent complete eradication of the tumor mass in some cases.

JP2001288110 discloses conjugates of interferon-alpha with polyethylene glycol (PEG) in an attempt to increase the circulating half-life and reduce the minimum therapeutically effective dose.

RU2188026 discloses a method of multiple chemotherapy using vincristine, adriamycin and depo-progesterone. This is claimed to improve relapse free time and reduce metastasis formation.

Finally, in a few cases, suggested therapies have been established for new starting materials. Thus, WO2004075887 discloses the use of 1- (2-chloroethane) -1-nitroso-3- (2-hydroxyethyl) urea (HECNU) for the treatment of many cancer types, including renal cancers. The main feature of HECNU is the improved water solubility compared to the previously known corresponding compound bis- (2-chloroethyl) -1-nitroso-urea (BCNU).

EP1712234 discloses the use of 4-picolyl-phthalazine derivatives as VEGF receptor inhibitors in the treatment of renal cancer, in particular for the inhibition of metastatic growth. It was found that co-administration of a 4-picolyl-phthalazine derivative with any of a variety of conventional chemotherapeutic agents has a synergistic effect, even if the tumor cells are refractory to chemotherapy alone. Further, combination therapy is associated with significantly fewer side effects.

Suthpin et al ("Targeting the Loss of the von Hippel-Lindau Tumor supressor Gene in Renal Carcinoma Cells", cancer. Res.67(12), 5896-. Chromomycin a3 significantly delayed tumor growth in xenografted nude mice without affecting normal kidney tissue expressing the VHL gene.

The present invention herein makes use of an orexin (formula I) which is a selective nephrotoxin present in relatively large amounts in several fungal species of the family myceliophthora (corticius). Poisoning by orexin after confusing myceliophthora fungi with edible fungi often occurs in europe, russia and north america. After ingestion of the fungus containing orexin, there is a period of days to 3 weeks with no symptoms or only mild, flu-like symptoms. The next stage, when medical assistance is generally sought, is characterized by uremia due to acute renal failure. Although much of the toxicity of Origan has been described in the scientific literature, other effects of Origan have not been reported except to mention only the toxicity of the kidney (Danel VC, Savicu PF, Garon D: Main features of corticus spp. posing: a lithium review. Toxicon 39, 1053-1060 (2001)). This selectivity is most probably due to the fact that orexin is specifically received by one cell type, namely, renal tubular epithelial cells, in particular proximal tubular epithelial cells (Prast H, Pfaller W: toxemic properties of the muscle Cortinarius oryzae (Fries) II. amplification of renal function in rates. Arch Toxicol 62, 89-96 (1988)). The toxic mechanism of orexin has not been elucidated and no treatment is available except to maintain dialysis while waiting to see if the kidneys recover. The final outcome depends largely on the amount of toxin ingested, and empirically, ingestion by one fungus creates a temporary problem, two fungi cause permanent loss of part of kidney function, and three or more fungi cause complete loss of kidney function and require lifelong dialysis or kidney replacement therapy.

The applicant has recently disclosed the first study of the mode of action of orexin in healthy rats (Nilsson UA et al, the nutritional and biochemical assays oxidative stress and down-regulated cellular defects. free Rad. biol. Med. 44: 1562-9 (2008)). This study showed that increased oxidative stress in renal cortical tissue is accompanied by significantly reduced expression of several key antioxidant genes. During this work, it was recognized that the seemingly absolute specificity of orellan toxins for renal tubular epithelial cells could theoretically be extended to cover these cells after their transformation into cancer cells. If justified, such hypothesis would mean that orexin is a powerful weapon against renal cancers of epithelial origin, with the possibility of curing even at the stage of cancer progression and at metastases in other tissues.

Continuing this hypothesis, it was surprisingly found that orellan toxins are actually also accepted in human renal cancer cells, whether they originate from primary tumors or from metastatic tumor tissue, killing them with great efficacy. Cell death developed after a brief exposure to orexin, indicating that the toxin was actively accepted and maintained by the cells.

Disclosure of Invention

It is a primary object of the present invention to provide a method of treating renal cancer originating from epithelial cells, said method involving administering to a mammal in need of treatment at least one compound according to formula I.

Formula I

Other objects of the invention are to provide compounds according to formula I for use as medicaments, and to provide compounds of formula I for use in the treatment of renal cell carcinoma.

It is another object of the present invention to provide a pharmaceutical composition comprising at least one compound according to formula I, optionally comprising other agents with anti-cancer activity, together with carriers and any other excipients required to optimize the effectiveness of the composition.

It is a further object to provide a kit comprising the above composition in one or more separate compartments, together with diluents and/or solvents as required, whereby the composition can be easily prepared for use by a treating physician or nurse.

Other objects and advantages of the present invention will become apparent to those of ordinary skill in the art upon reading the specification and examples, and are intended to fall within the scope of the present invention.

Brief description of the drawings

FIG. 1:the results of viability of cells from 5 different human renal cell carcinomas (primary tumor and metastasis) were observed during 7 days after 24h exposure to 400 μ M orexin (100 μ g/ml medium). Viability was calculated by dividing the number of viable cells in the treated sample by the number of viable cells in the control sample (n-6). The grey area in the graph indicates the time of incubation of the orexin (24 h). Several days later, the cells were cultured in complete medium without orelbin. The medium was changed at each viability measurement.

FIG. 2:orexin toxicity to human renal cancer cells (strain 786-0) one week after orexin incubation according to the same parameters in figure 1. The micrograph on the left shows the cells exposed to vehicle and the micrograph on the right shows the cells exposed to 400 μ M orexin for 24 h. Both pictures were taken at the same magnification one week after the orelboxin/vehicle reproduction.

FIG. 3:culture of human renal cell carcinoma (786-0 and SKRC7)Dose/response effect of orexin at different concentrations in the nutrient. A clear correlation between the dose of orexin and cell death was seen in the concentration interval of 5-200. mu.g/ml.

FIG. 4:effects of repeated administration of low doses of orexin. Administration of a second dose (20 μ g/ml) at a low value of the dose/response interval resulted in a strong reduction in the number of viable cells 24h after the first dose, whereas administration of the second dose at 72h had a significantly smaller, but still significant effect. The first case corresponded to prolonged exposure of the cells (48h), while the cells in the second case were allowed to recover for two days in medium without orexin between the two doses. Viability was measured 96h after addition of the first orexin dose.

FIG. 5:effect of orellantoxin on the cell cycle (Western blot) in renal cell carcinoma (786-0 and SKRC-52). The protein level of cell cycle inhibitor p21 was gradually upregulated to the maximum of 6h of incubation. After 24h, the cell cycle stimulatory phosphorylation form of retinoblastoma protein (Rb) disappeared (two different phosphorylation sites are shown). These effects converge to stop the cell cycle at the GI/S checkpoint. At the same time, loss of the stimulatory factor cdc2 halted the cell cycle at checkpoint G2/M, preventing the initiation of cell division.

FIG. 6:induction of apoptosis of orexin in renal cancer cells (786-0): protein effect. The apoptotic p38 pathway was strongly upregulated after orellanin exposure (OR) (f-p38) and apoptosis was detected as cleaved caspase 3(cleaved caspase 3). Strong up-regulation of ERK1/2 (f-ERK1/2) after 24h was interpreted as an attempt of the cells to resist the apoptotic effects of orexin.

For ERK1/2 and p38, the ratio of phosphorylation (f) to total protein should be compared. Cleaved caspase 3 should be compared to loaded control alpha actin.

FIG. 7:ordotoxin in renal cancer cells (SKRC-52)Induction of apoptosis of elements: mRNA effect. RT-PCR analysis of mRNA expression of the apoptosis mediators PUMA, Fas ligand (FasL) and tumor necrosis factor alpha (TNF) showed significant up-regulation in all three cases. Only small changes in expression of the receptor mRNA were noted. Cells were compared to control cells incubated without orelbin.

FIG. 8:apoptosis in human clear cell carcinoma (SKRC52) during incubation with orexin. Typical symptoms of apoptosis, vacuole formation and cell shrinkage are already evident after 4h incubation and further aggravated after 24 h.

Detailed description of the preferred embodiments of the invention

The present invention provides pharmaceutical compositions comprising pyridine-N-oxide and bipyridine-N, N-dioxide compounds, and methods of treating renal cancer by administering the pharmaceutical compositions to a subject having or susceptible to renal cancer. The invention herein also includes kits for treating a patient suffering from or susceptible to renal cancer.

The present invention provides a method of treating a patient suffering from or susceptible to renal cell carcinoma, wherein said method comprises the step of administering to said patient a compound according to formula I, a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising said compound as defined earlier.

Compounds of formula I that are administered to patients include those wherein R1, R2, R3 and/or R4 do not substantially affect the cytotoxicity of orellanin (R1 ═ R2 ═ R3 ═ R4 ═ hydrogen). Thus, R1, R2, R3, and/or R4 include, but are not limited to, hydrogen, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 enolyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted with groups including, but not limited to, amino, mercapto, carboxyl, phosphoric acid, and halogen, including fluorine, chlorine, and bromine. In a preferred embodiment of the invention, the compound of formula I is orexin, i.e. R1 ═ R2 ═ R3 ═ R4 ═ hydrogen.

In one embodiment of the method of treating a patient suffering from or susceptible to renal cancer according to the invention, the compound of formula I administered to the patient is a pharmaceutically acceptable salt, hydrate or solvate. As used herein, a pharmaceutically acceptable salt is an acid or base salt, which is generally recognized in the art as suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include basic residues such as mineral and organic acid salts of amines. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, oxalic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxyethylsulfonic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid (pamoic acid), succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids such as acetic acid, HOOC- (CH-CH) and₂)_n-COOH, wherein n is 0-4, etc.

In the methods of treating renal cancer provided herein, the compound of formula I can be administered in a single dose, a series of daily doses, or in an intermittent dosing regimen (e.g., administration of multiple doses or a sequence of doses at an interval between 1 day and about 30 days, an interval between 1 day and about 14 days, or an interval between 1 day and about 7 days). In certain methods, the administration regimen and the compound of formula I are selected to provide at least a 50% reduction in tumor size, or more preferably at least a 75%, 90%, or 95% reduction in tumor size after completion of the administration regimen, while in certain other methods, the administration regimen and the compound of formula I are selected to cause a 95% reduction in tumor size, a 99% reduction in tumor size, or substantially complete elimination of the tumor. In those methods of treatment comprising a single dose administration regimen, between about 1mg/kg and about 100mg/kg of a compound according to formula (I) or an equivalent molar amount of a pharmaceutically acceptable salt thereof is administered to the patient, while preferred single doses comprise between about 2mg/kg and about 25mg/kg, most preferably about 5mg/kg to about 15mg/kg of a compound of formula I or an equivalent molar amount of a pharmaceutically acceptable salt thereof.

In certain other therapeutic methods of treating renal cancer, a compound of formula I or a pharmaceutically acceptable salt thereof is administered to a patient having or susceptible to renal cancer in two or more doses. Typically, the dose is administered daily or intermittently (e.g., at least one non-administration day separating consecutive doses). In certain methods, wherein the compound of formula I or a pharmaceutically acceptable salt thereof is administered in multiple doses, each dose comprises from about 0.5mg/kg to about 10mg/kg of the compound, more preferably, each dose comprises from about 1mg/kg to about 5mg/kg, or more preferably, about 2mg/kg of the compound or salt of formula I.

In certain methods of intermittently administering successive doses, the successive doses are administered at intervals between two days and seven days, in other methods involving intermittent administration of a compound or salt of formula I, the compound is administered to the patient in three, four, five, or six or more doses, wherein each dose is administered at intervals between three and five days, and in yet other methods, the patient is administered four, five, or six or more doses at intervals between three days and four days, wherein each dose contains from about 1mg/kg to about 20mg/kg of a compound of formula I or a pharmaceutically acceptable salt thereof, preferably from 2 to 10mg/kg, and most preferably about 5 mg/kg. In certain other therapeutic methods of treating renal cancer, a patient is administered a daily dose of a compound of formula I, or a pharmaceutically acceptable salt thereof, for at least two days. Typical daily doses for administration to a patient are between 0.1 and 10mg/kg, preferably between 1 and 5mg/kg, most preferably about 2 mg/kg. The treatment regimen generally comprises daily administration of a compound of formula I, or a pharmaceutically acceptable salt thereof, for between 5 and about 30 days, or preferably between 10 and 20 days, or most preferably for about 14 days.

In certain instances, it may be desirable to perform multiple intermittent administration regimens, as described above, daily administration regimens, or combinations thereof, in combination with a rest and/or recovery period. Thus, in certain instances, it may be desirable to administer a compound of formula I or a pharmaceutically acceptable salt thereof according to the daily or intermittent administration methods provided herein, measure the tumor response to treatment, and then subsequently administer treatment daily or intermittently as needed to eliminate or further reduce the size of the renal cancer tumor. Such administration strategies are well known to those of ordinary skill in the oncology arts.

In a particularly preferred embodiment of the invention, a patient suffering from renal cell carcinoma is treated with a substance according to formula 1 of the invention by daily injection of about 0.5-5mg of orexin/kg b.w. (by weight), most preferably about 2mg of orexin/kg b.w., for about 7-21 days, most preferably for about 14 days. 1 to 5 hours after each daily injection of a compound according to formula 1, most preferably about 2 hours after such injection, the patient is subjected to hemodialysis for 1-5h, most preferably about 2h, to eliminate the compound according to formula 1 that has not been received into the tumor tissue, thereby minimizing any undesirable side effects that may occur in the intercellular space.

The preferred dosage and mode of administration as described above is based on a human being with a 70kg body weight, renal cell carcinoma with a tumor burden of about 1 kg. However, as readily appreciated by a person of ordinary skill in the cancer medicine art, such preferred dosages and modes of administration are to a large extent regulated by patient characteristics, e.g., age, sex, weight, general condition and especially tumor burden of the individual patient and response to treatment. As before, the ultimate responsibility for selecting the appropriate dosage and treatment strategy is the physician in charge of the patient.

The present invention provides methods of treating a patient suffering from or susceptible to renal cell carcinoma. In certain methods, the tumor to be treated is located in one or both of the patient's kidneys. In certain other methods, the renal cell carcinoma has metastasized, e.g., at least one renal cell carcinoma tumor is present in at least one non-renal tissue. In general, the methods provided herein are applicable to the treatment of patients having or susceptible to a renal cell carcinoma tumor, which is present in kidney, non-kidney tissue, or a combination thereof. In a preferred embodiment, the tumor is present in non-renal tissue, or a combination of renal and non-renal tissue. The therapeutic methods provided herein contemplate any route of administration that provides a therapeutically effective dose of the compound of formula I to the vicinity of the tumor. In certain preferred methods of treatment provided herein, a compound of formula I, or a pharmaceutical composition comprising a compound of formula I, is administered intravenously, subcutaneously, or intraperitoneally. Typically, a compound of formula I or a pharmaceutical composition comprising a compound of formula I is administered intravenously.

In another aspect, the present invention provides a compound of formula I, wherein R1, R2, R3 and/or R4 do not substantially affect the cytotoxicity of orexin (R1 ═ R2 ═ R3 ═ R4 ═ hydrogen), for use as a medicament. The invention also provides the use of a compound of formula I wherein R1, R2, R3 and/or R4 do not substantially affect the cytotoxicity of orellanin (R1 ═ R2 ═ R3 ═ R4 ═ hydrogen) as a medicament. R1, R2, R3, and/or R4 include, but are not limited to, hydrogen, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 enolyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted with groups including, but not limited to, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine. In a preferred embodiment of the invention, the compound of formula I is orexin, i.e. R1 ═ R2 ═ R3 ═ R4 ═ hydrogen. Other preferred embodiments of this aspect of the invention will be apparent from the apparent description.

In a further aspect, the present invention provides a compound of formula I wherein R1, R2, R3 and/or R4 do not substantially affect the cytotoxicity of orexin (R1 ═ R2 ═ R3 ═ R4 ═ hydrogen) for use in the treatment of renal cell carcinoma. The invention also provides the use of a compound of formula I wherein R1, R2, R3 and/or R4 do not substantially affect the cytotoxicity of orexin (R1 ═ R2 ═ R3 ═ R4 ═ hydrogen) for the manufacture of a medicament for the treatment of renal cell carcinoma. R1, R2, R3, and/or R4 include, but are not limited to, hydrogen, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 enolyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted with groups including, but not limited to, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine. In a preferred embodiment of the invention, the compound of formula I is orexin, i.e. R1 ═ R2 ═ R3 ═ R4 ═ hydrogen. Other preferred embodiments of this aspect of the invention will be apparent from the apparent description.

In another aspect, the present invention provides a pharmaceutical composition comprising at least one pharmaceutically acceptable carrier and a compound according to formula (I), wherein R1, R2, R3 and/or R4 do not substantially affect the cytotoxicity of orexin (R1 ═ R2 ═ R3 ═ R4 ═ hydrogen). Thus, R1, R2, R3, and/or R4 are exemplified by, but not limited to, hydrogen, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 enolyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted with groups including, but not limited to, amino, mercapto, carboxyl, phosphate, and halogen, including fluorine, chlorine, and bromine. In a preferred embodiment of the invention, the compound of formula I is orexin, i.e. R1 ═ R2 ═ R3 ═ R4 ═ hydrogen.

In certain other pharmaceutical compositions, the compounds of formula I are incorporated into the composition as pharmaceutically acceptable salts, hydrates, or solvates. As used herein, a pharmaceutically acceptable salt is an acid or base salt, which is generally recognized in the art as suitable for use in contact with the tissues of humans or animals without excessive toxicity, irritation, allergic response, or other problem or complication. Such salts include minerals and organic acid salts of basic residues, such as amines. Specific pharmaceutical salts include, but are not limited to, salts of acids such as hydrochloric acid, phosphoric acid, hydrobromic acid, malic acid, oxalic acid, fumaric acid, sulfuric acid, sulfamic acid, sulfanilic acid, formic acid, toluenesulfonic acid, methanesulfonic acid, benzenesulfonic acid, ethanedisulfonic acid, 2-hydroxydisulfonic acidIsethionic acid, nitric acid, benzoic acid, 2-acetoxybenzoic acid, citric acid, tartaric acid, lactic acid, stearic acid, salicylic acid, glutamic acid, ascorbic acid, pamoic acid (pamoic acid), succinic acid, fumaric acid, maleic acid, propionic acid, hydroxymaleic acid, hydroiodic acid, phenylacetic acid, alkanoic acids, e.g. acetic acid, HOOC- (CH)₂)_n-COOH, wherein n is 0-4, etc.

The pharmaceutical compositions provided herein are suitable for any route of administration contemplated by the method of treatment using the composition. In the methods of the invention, the compounds of the invention according to formula I and pharmaceutical compositions thereof may be administered to a subject by a variety of routes including parenteral (including intravenous, subcutaneous, intramuscular, and intradermal), topical (including buccal, sublingual), oral, nasal, and the like. In certain preferred pharmaceutical compositions provided herein, the pharmaceutical compositions are formulated for administration by intravenous, subcutaneous, or intraperitoneal injection. Typically, the pharmaceutical composition is formulated for administration by intravenous injection.

In certain parenteral routes of administration, the pharmaceutical composition is a sterile saline solution containing from about 0.1mg/ml to about 25mg/ml of a compound of formula I or a pharmaceutically acceptable salt thereof. Certain preferred pharmaceutical compositions for parenteral administration comprise from about 0.5mg/ml to about 10mg/ml of a compound of formula I, or a pharmaceutically acceptable salt thereof, in a saline solution, optionally comprising one or more pharmaceutically acceptable additives.

In certain preferred pharmaceutical compositions, the composition comprises between about 25mg to about 5000mg, or between about 5mg to about 2500mg of a compound according to formula (I) or an equimolar amount of a pharmaceutically acceptable salt thereof. In certain other pharmaceutical compositions of the invention, the composition comprises between about 1mg to about 1500mg of a compound according to formula (I) or an equimolar amount of a pharmaceutically acceptable salt thereof. Still other pharmaceutical compositions are formulated comprising about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, or about 100mg of a compound of formula I or an equimolar amount of a pharmaceutically acceptable salt thereof.

In certain methods of treating a patient having or susceptible to cancer, administration of a compound according to formula I to a patient having or susceptible to cancer reduces tumor size by at least 50% or more preferably by at least about 60%, 70%, 80%, 90% or about 95%. In certain other methods of treating a patient having cancer, administration of a compound according to formula I to a patient having cancer reduces the tumor size by at least 99%, or reduces the tumor size until no detectable tumor remains.

Certain preferred methods of treating patients with cancer include the treatment or prevention of cancer or other neoplastic disorders in mammalian patients, including domestic animals, companion animals (dogs, cats, horses, etc.), primates, and humans.

The treatment methods of the present invention generally comprise administering to a patient a therapeutically effective amount of one or more compounds of formula I. In current treatment methods, a therapeutically effective amount is sufficient to reduce the size of or eliminate a renal cell carcinoma tumor present in the patient. Suitable patients include those subjects having a disorder or disease identified herein. Typical patients to be treated according to the invention include mammals, in particular primates, in particular humans. Other suitable subjects include domesticated companion animals, e.g., dogs, cats, horses, etc., or livestock animals, e.g., cows, pigs, sheep, etc.

Preferred methods of the invention include identifying and/or selecting a subject (e.g., a mammal, particularly a human) having a condition disclosed herein, particularly a subject having one or more cancers. The pharmaceutical compositions of the invention can also be packaged with instructions for treatment of cancer (e.g., written, e.g., a written foldout) disclosed herein, e.g., instructions for treating a subject having cancer.

The compounds of the invention are suitably administered to a subject in a water-soluble form, e.g., following appropriate chemical transformationThe pharmaceutically acceptable salts obtained as organic or inorganic acids, for example, hydrochloride, sulfate, hemisulfate, phosphate, nitrate, acetate, oxalate, citrate, maleate, methanesulfonate, and the like. Also, when an acidic group is present on the compound, a pharmaceutically acceptable salt of an organic or inorganic base, for example, an ammonium salt, a salt of an organic amine, a salt of an alkali metal or alkaline earth metal such as potassium, calcium or sodium salt, may be employed. Particularly suitable pharmaceutically acceptable salts include those with non-toxic cations, preferably alkali metal cations such as K or Na, alkaline earth metal cations such as Mg or Ca, other non-toxic metal cations such as Al or Zn or non-toxic non-metal cations such as NH₄ ⁺Piperazine or 2-hydroxyethylamine. Certain preferred compounds suitable for use in the methods of the present invention are sufficiently water soluble in the neutral form in such a way that they can be delivered without prior production of pharmaceutically acceptable salts.

Compounds suitable for use in the process of the present invention include any and all of the various single pure isomers as well as mixtures of two or more isomers. The term isomer is intended to include diastereomers, enantiomers, regioisomers, structural isomers, rotational isomers, tautomers, and the like. For compounds containing one or more stereogenic cores, e.g., chiral compounds, the methods of the invention can be performed with enantiomerically enriched compounds, racemates, or mixtures of diastereomers. Preferred enantiomerically enriched compounds have an enantiomeric excess of 50% or more, more preferably the compounds have an enantiomeric excess of 60%, 70%, 80%, 90%, 95%, 98% or 99% or more. The compounds of the invention according to formula I used in the methods of the invention may be employed alone, or in combination with one or more other therapeutic agents, as pharmaceutical compositions in admixture with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier materials suitable for the desired route of administration, which do not deleteriously react with the active compound nor are they deleterious to the recipient thereof. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, vegetable oils, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidone, and the like. The pharmaceutical preparations can be sterilized and, if desired, mixed with auxiliary agents which do not deleteriously react with the active compounds, such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorants, flavors and aromatic substances and the like.

For parenteral applications, solutions, preferably oil or aqueous solutions, as well as suspensions, emulsions or implants, including suppositories, are particularly suitable. Ampoules are convenient unit doses.

For enteral applications, particularly suitable are tablets, dragees or capsules having talc and/or carbohydrate carrier binders and the like, preferably lactose and/or corn starch and/or potato starch. Syrups, elixirs and the like may be employed wherein a sweetening excipient is employed. Sustained release compositions can be formulated, including compositions in which the active ingredient is protected with various degradable coatings, e.g., microcapsules, multilayer coatings, and the like. Tablets, capsules and syrups or other fluids are generally preferred for oral administration.

It is to be understood that in addition to the ingredients explicitly mentioned above, the formulations of the invention may include other agents conventional in the art having regard to the type of formulation in question, for example, flavoring agents may be included as appropriate for oral administration.

According to certain embodiments, the compounds of formula I may be administered in combination with other compounds, including, for example, chemotherapeutic agents, anti-inflammatory agents, anti-psychotic agents, radiosensitizing agents, radioprotective agents, urologic agents, antiemetics, and/or anti-dysentery agents, e.g., cisplatin, carboplatin, docetaxel, paclitaxel, fluorouracil, capecitabine, gemcitabine, irinotecan, topotecan, etoposide, mitomycin, gefitinib, vincristine, vinblastine, doxorubicin, cyclophosphamide, celecoxib, rofecoxib, valdecoxib, ibuprofen, naproxen, ketoprofen, dexamethasone, prednisone, prednisolone, hydrocortisone, acetaminophen, misonidazole, amifostine, tamsulosin, phenazopyridine, ondansetron, granisetron, alosetron, palonosetron, promethazine, prochlorperazine, prochloraz, and combinations thereof, Trimethobenzamide, aprepitant, diphenoxylate with atropine, and/or loperamide. In a preferred embodiment, the compound according to formula I is administered in combination with an anti-angiogenic drug, including, for example, monoclonal antibodies directed against Vascular Endothelial Growth Factor (VEGF) and placental growth factor (P1 GF); and inhibitors of VEGF and P1GF receptors, including, for example, bevacizumab (bevacizumab), sorafenib (sorafenib), PTK78, SU11248, AG13736, AEE788, and ZD 6474. In another embodiment, the compound according to formula I is administered in combination with an immunomodulatory drug, including, for example, interleukin 2(IL-2) and interferon alpha (IFN α). In yet another embodiment, the compound according to formula I is administered in combination with a drug that interferes with cell growth signaling, including an inhibitor of mammalian target of rapamycin (mTOR), for example.

In yet other embodiments of the invention, the compound according to formula I is chemically bound to a molecule that enhances target selectivity even further by specifically targeting the compound of formula I to cancer cells. Examples of such molecules include (a) polyclonal and monoclonal antibodies to markers that occur specifically or in greater numbers on target cells than normal kidney tissue, and (B) ligands for receptors that occur specifically or in greater numbers on target cells than normal kidney tissue. Such guide molecules and the techniques for conjugating them to compounds according to formula I are known in the art, and the coupling reaction can be performed by the skilled person without undue experimentation. The kit of the invention herein comprises at least one pharmaceutically acceptable carrier and 50 to 3,500mg of a compound according to formula I, or an equimolar amount of a pharmaceutically acceptable salt thereof as discussed above. In said kit, the compound according to formula I or an acceptable salt thereof and the pharmaceutically acceptable carrier are preferably located in separate compartments. The compounds according to formula I are preferably present as solids. For administration, the compound according to formula I or a pharmaceutically acceptable salt thereof is preferably combined with a carrier such that it is completely or substantially dissolved in the carrier. The kit may comprise between about 100mg and about 1,500mg, most preferably between about 200mg and about 500mg of a compound according to formula I or an equivalent amount of a pharmaceutically acceptable salt thereof.

The foregoing description of the invention is merely illustrative, and it is understood that variations and modifications can be made without departing from the spirit and scope of the invention as set forth in the following claims. Each of the documents mentioned herein is incorporated by reference into the disclosure of the present application.

Examples

Example 1: extraction and isolation of orexin from a myceliophthora mushroom

A. Polarity method: 2g of dried mushrooms of the Neisseria species were pulverized and then extracted with 50% methanol at 25 ℃ for 24 hours. The mixture was centrifuged and the supernatant removed to give a final volume of 5 ml. Upon repeated addition of 5vol of cold methanol, a precipitate formed which was discarded until a clear solution formed. The solvent was evaporated and the residue was dissolved in water and the non-polar material was removed by extraction with petroleum ether. The polar phase was loaded onto a Sephadex column (Sephadex) and eluted with 50% ethanol. The resulting fractions were chromatographed on thin-layer cellulose, eluting with butanol: acetic acid: water (3: 1). Origanin (Orellanin) was identified as a fluorescent band under Rf 0.68.

B. A nonpolar method: 4g of powdered Mucor mushroom was refluxed in diethyl ether for 24 hours, and the solvent was discarded. The residue was refluxed in methanol, followed by solvent evaporation, washed with 20ml of water (6h, 4 ℃). They were then dissolved in 50% aqueous ethanol (pH 7.0). The mixture was loaded onto a sephadex column and eluted with 50% ethanol. The resulting fractions were chromatographed on thin-layer cellulose, eluting with butanol: acetic acid: water (3: 1). Orexin was identified as a fluorescent band under rf0.68.

Example 2: synthesis of Origancin

Orexin is synthesized from commercially available 3-hydroxypyridine essentially as described by others. (Tiecco M, Tingoli M, Testaferi L, Chianelli D and Wenkert E: Total synthesis of orellanine, the lethal toxin of corticois orellanus Fries from tetrahedron 42, 1475-

Example 3: origanoxin has specific toxic effects on human renal cell carcinoma in vitro.

Background and methods

Cells harvested from 5 different human renal cell carcinomas (SKRC-52, 786-0, SKRC-17, SKRC-7, and SKRC-21), representing the maternal tumor and metastatic growth, were cultured under standard conditions. When approximately 70% confluent and rapidly growing, the cells were exposed to medium containing orellanin (400 μ M) for 24 hours. The medium was then changed back to conventional complete medium and the cells were observed for an additional six days.

Results and description

The effect of the described orexin treatment of the cells is illustrated in figure 1. As is evident from the figures, orexin is highly toxic for all cell types tested. Furthermore, toxicity is not affected by removal of orexin from the culture medium after initial exposure. This indicates that orexin accumulates in the cells and remains therein, even in the absence of extracellular orexin. Figure 2 shows the appearance of cells before and after 24 hours orelbin exposure.

Example 4: dose-response effects of Origansin

Background and methods

Cells harvested from 2 different human renal cell carcinomas (SKRC-7, 786-0), representing maternal and metastatic growth, were cultured under standard conditions. When approximately 70% confluent and rapidly growing, the cells were exposed to media containing different concentrations of orexin (400 μ M) for 24 hours. The medium was then changed back to conventional complete medium and the cells were observed for an additional six days.

Results and description

As shown in fig. 3, there was a clear correlation between the exposure concentration and the fraction of dead cells. The dose response interval for a single 24h exposure of the orexin is between about 5 and 200 μ g/ml.

Example 5: effect of repeated administration of Origanoxin at smaller doses

Background and methods

Cells harvested from human renal cell carcinoma 786-0 were cultured under standard conditions. When approximately 70% confluent and rapidly growing, the cells were exposed to medium containing low concentrations of orexin (20. mu.g/ml) for 24 hours. The medium was then changed to a new medium containing 20. mu.g/ml of orexin for 24h (middle column) or changed back to conventional complete medium for 48h followed by another 24h in the presence of 20. mu.g/ml of orexin (right column).

Results and description

Repeated exposure to orexin, even at doses at the lower end of the single exposure response interval, produces a further, significant toxic effect on renal cancer cells.

Example 6: effect of Origansin on other cell types

Background and methods

Cell lines and primary cells derived from a number of human tissues (glomerular epithelium, podocytes and mesangial cells from kidney tissue, fibroblasts, macrophages, aortic endothelium, microvascular and umbilical cord endothelium, intestinal epithelium (duodenal, jejunal, ileal and colonic) and chondrocytes) were cultured under standard conditions. When approximately 70% confluent and at stable growth, the cells were exposed to medium containing the selected concentration of orexin to achieve a dose response for 24 h. The medium was then changed back to conventional complete medium and the cells were observed for an additional six days, followed by viability assays. To compensate for any growth inhibition due to the dilution of the culture medium with the added large volume of orexin solution, all cells were supplemented with an equal volume of orexin buffer.

Results and description

When the orelboxin exposure according to example 4 is at a concentration of up to 1,000 μ g/ml, which is the highest concentration achievable, none of the tested cell types exhibit any effect on viability.

Example 7: origanoxin induces growth retardation by stopping the cell cycle

Background and methods

Renal cancer cells cultured essentially as described in example 3 were treated with orellanin (100 μ g/ml medium) for 24 hours. During this period, cells were harvested after 0, 2, 6 and 24h of exposure. Western blotting of the harvested antibody-bearing material was performed using a protein directed against A) the kinase inhibitor p21, B) phosphorylated retinoblastoma protein (growth hormone), and C) cdc2 protein which allows cells to enter the M phase of the cell cycle, where the cells divide.

Results and description

The effect of orexin exposure on protein expression of p21, retinoblastoma protein and cdc2 is shown in figure 5. The intracellular level of the cell cycle inhibitor p21 increased to a maximum at about 6h, while the cell cycle stimulatory phosphorylated form of the retinoblastoma protein was completely absent as measured at 24 h. In a similar manner, the cdc2 protein, which allows cells to enter the cell division phase, was significantly down-regulated at 24 h. This clearly demonstrates that orelbin has profound inhibitory effects on the cell cycle, acting at least two important checkpoints.

Example 8: oreotoxin increases the activity of several apoptosis-inducing pathways leading to cancer cell death

Background and methods

Kidney cancer was cultured and exposed to orexin according to example 7, harvested at various times up to 24 h. The p38 MAPK system, the p53 system, the Fas ligand, tumor necrosis factor alpha (TNF) and cleaved caspase 3 are key factors in the apoptotic pathway leading to cell death. Western blots were used to determine intracellular levels of A) p38, B) cleaved caspase 3, and C) phosphorylated ERK1/2, a proliferation factor. Quantitative PCR was used to determine D) a p53 up-regulated regulator of apoptosis (PUMA), which mediates essentially all of the apoptotic effects of the p53 pathway, E) Fas ligand and F) mRNA expression of TNF.

Results and description

The results are summarized in FIGS. 6-8. There was a steady increase in phosphorylated (activated) p38 throughout the 24h observation period, increasing the apoptotic signal intensity of the cells (figure 6). (Simultaneous upregulation of growth stimulator phosphorylated ERK1/2 was interpreted by the inventors as an attempt by the cells to "fight" the apoptotic effects of orexin).

At the mRNA level, the extreme up-regulation of PUMA and the death receptor ligands FasL and TNF constitute a strong apoptotic stimulus (fig. 7). Finally, the number of cleaved caspase 3, the major effector of apoptosis, increased significantly 24h after exposure to orexin (fig. 6).

Together with the images of progressively more apoptotic cells presented in figure 8, the above results clearly indicate that apoptosis is the primary mode of action of orexin in renal cancer cells.

Example 9: origansin eradicates human renal cell carcinoma growth in athymic rats

Background and methods

Athymic T-cell deficient rats (RNU, Charles River Laboratories, FRG) were used as a system for in vivo growth of human renal cell carcinoma. The lack of T cell-based immune defenses in these animals makes them tolerant to xenografts. One week after arrival at the animal facility, 10 animals received a 5Gy dose of X-ray radiation to suppress their B-cell mediated responses.

The following day, all animals were equipped with indwelling catheters for Peritoneal Dialysis (PD). PD treatment will reset kidney function, which is lost as a side effect when orexin is administered.

One day later, 5 animals were inoculated approximately 10X 10 subcutaneously in the shoulder area⁶Human renal cancer cells (SKRC-52). The 5 remaining animals received the same number of cells by intravenous injection. In the subcutaneous group, localized tumors, 1X 2cm, were evident under the skin of the animals after 2-4 weeks. At this point, 2 animals in each group (control) were injected with physiological saline solution i.p., and the other 3 animals received 10mg of orexin/kg b.w.i.p.

Results and description

Two weeks after the first injection of saline/orexin, the tumor size in the control animals in the subcutaneous injection group nearly doubled, while the tumor in the animals injected with orexin collapsed to less than 25% of the size recorded at the time of injection. At this time, another dose of 5mg of orexin/kg b.w. was injected into the tumor site of 3 animals previously receiving orexin in the subcutaneous group, and the control animals received 10mg of orexin/kg b.w. into the tumor site. After another 2 weeks, no signs of tumor were evident in the animals injected twice with orexin, the former reduced the tumor size by more than 75% in the control animals.

One and two weeks after the first saline/orexin injection, 5 animals in the intravenous group received an i.v. injection of 5mg/kg orexin or saline, respectively. After another week, animals were sacrificed and their abdominal and thoracic cavities were scanned for tumor growth. The identification of tumor volumes showed that the animals treated with orexin had a 10% less tumor burden than the control animals.

This clearly demonstrates the tumor killing activity of orexin in vivo systems.

Example 10: safety of i.v. orexin during long-term treatment in pigs and dogs

5 pigs of the Gottingen mini-pig line and 5 dogs of mixed breeding (body weight 10-15kg) were hemodialysis using equipment designed for children and infants. Animals received a starting dose of 10mg of orexin/kgb.w. and after 24h, the animals underwent a dialysis period of approximately 3 h. After dialysis, animals were injected with 5mg of orexin/kg b.w. The dialysis/re-injection process was repeated 3 times a week (monday, wednesday, friday) for 8 weeks. Evaluation of general condition of animals was performed once a week. At the end of the experiment, all animals were sacrificed and samples for histopathological evaluation were taken from heart, lung, kidney, liver, spleen, small intestine, large intestine, brain, muscle and skin.

The behavior and general condition of the animals remained normal throughout the experimental period. Histopathological examination revealed no tissue damage other than the kidney, with a large area of tubular damage present, leading to complete renal failure.

The results show that long-term treatment with high doses of orelbin is safe from the point of view of renal cancer patients, with negligible side effects in non-renal tissues.

Example 11: treatment of human patients with advanced renal cell carcinoma with orexin

Patients in need of treatment of renal cancer are given a series of intravenous injections of orexin 10 times a day. The initial tumor burden of the patient was determined to be about 2 kg. Based on this value, a suitable daily dose was determined to be 280mg (4mg/kg b.w.70kg). Prior to the first injection, the patient is prepared for hemodialysis or peritoneal dialysis, as orexin treatment inevitably destroys healthy renal epithelial tissue while killing cancer cells, thus depriving the patient of renal function. Hemodialysis was started 2 hours after each injection and continued for 2 hours. This procedure, with repeated administration of a smaller amount of orexin, has the advantage of causing a gradual accumulation of orexin to the level of death of the tumour tissue, which actively receives the substance, while the extracellular concentration of toxin remains below the level causing the side effects. Optionally, if the disease is unilateral, the unaffected kidney may be surgically removed and stored during treatment, with re-implantation attempted after the end of treatment. The patient's progress is monitored for one month, after which additional continuous administrations of orexin are given as needed to inhibit the growth of renal cancer. During the treatment, the mass of the tumor tissue in the patient is reduced and at the end of the treatment, the renal cancer is completely destroyed, exhibiting the efficacy of orelbin against renal clear cell carcinoma.

Claims (11)

1. The use of a compound according to formula I or a pharmaceutically acceptable salt thereof in the manufacture of a medicament for treating a patient suffering from or susceptible to renal cell carcinoma,

wherein:

r1, R2, R3 and R4 are selected from: hydrogen, amino, mercapto, carboxyl, phosphate, fluorine, chlorine, bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 alkenylyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted by amino, mercapto, carboxyl, phosphate, fluorine, chlorine, bromine;

the compound is administered in a single dose of 1mg/kg to 100mg/kg, or the compound is administered in two or more doses, wherein each dose comprises between 1mg/kg to 20mg/kg of the compound.

2. Use according to claim 1, wherein R₁、R₂、R₃And R₄Is hydrogen.

3. Use according to claim 1 or 2, wherein successive doses are administered at intervals of between two and seven days.

4. The use of claim 3, wherein the compound is administered daily.

5. The use of claim 4, wherein the compound is administered intravenously, subcutaneously or intraperitoneally.

6. A pharmaceutical composition for treating renal cell carcinoma, comprising at least one pharmaceutically acceptable carrier and 50mg to 3,500mg of a compound according to formula (I):

wherein:

R₁、R₂、R₃and R₄Selected from: hydrogen, amino, mercapto, carboxyl, phosphate, fluorine, chlorine, bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 alkenylyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted by amino, mercapto, carboxyl, phosphate, fluorine, chlorine, bromine,or an equimolar amount of a pharmaceutically acceptable salt thereof.

7. The pharmaceutical composition of claim 6, wherein R₁、R₂、R₃And R₄Is hydrogen.

8. The pharmaceutical composition of claim 6 or 7, wherein the composition is formulated for intravenous, subcutaneous, or intraperitoneal administration to a patient.

9. A kit for treating a patient suffering from renal cell carcinoma, the kit comprising at least one pharmaceutically acceptable carrier and 50mg to 3,500mg of a compound according to formula (I):

wherein:

R₁、R₂、R₃and R₄Selected from: hydrogen, amino, mercapto, carboxyl, phosphate, fluorine, chlorine, bromine, C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 alkanolyl, C1-C6 alkenylyl, C1-C6 alkoxy, C1-C6 alkenyloxy, each of which may be further substituted with amino, mercapto, carboxyl, phosphate, fluorine, chlorine, bromine, or an equimolar amount of a pharmaceutically acceptable salt thereof.

10. The kit of claim 9, wherein the compound according to formula (I) or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier are administered in combination such that the compound according to formula (I) or a pharmaceutically acceptable salt thereof is completely or substantially dissolved in the carrier.

11. The kit of claim 9 or 10, wherein the compound is formulated for intravenous, subcutaneous, or intraperitoneal administration to a patient.