TREATMENT AND MONITORING DISEASE STATE OF LIVER CANCER

FIELD OF THE INVENTION

This invention relates to the fields of treatment and monitoring disease state of liver cancer.

BACKGROUND OF THE INVENTION Liver cancer (hepatocellular carcinoma, HCC) is among the most deadly malignancies in the world. The highest frequency of liver cancer is in Southeast Asia and Africa, and recently it has become the most common cause of cancer death in Japan. In the USA it is the most rapidly increasing type of cancer. Hepatitis C virus (HCV) infection, alcohol use, nonalcoholic fatty liver diseases, and androgenic steroid use are the most common factors of cirrhosis which is the leading cause of liver cancer.

The current available treatments for liver cancer include liver transplantation, surgical restriction, TACE ₅ administration of intra-arterial iodine- 131-lipiodol, precutaneous treatment by ethanol injection or radiofrequency ablation, hormonal therapy (antiestrogen with tamoxifen or somatostatin analogue) and intra-hepatic chemotherapy. Unfortunately, no adjuvant or palliative treatment has been shown to prolong survival in liver cancer.

The increasing incidence rate and aggressive malignancy of liver cancer, as well as the subsequent dysfunction of the liver which limits the safety administration of chemotherapy and the lack of a satisfactory treatment are challenging factors to develop an effective systemic therapy for liver cancer.

Accumulative data have indicated that adenosine plays an important role in controlling tumor growth. Adenosine is involved in various cellular activities which are associated with cell growth, differentiation and death. Adenosine's effects on

cells are mediated via four protein associated cell surface receptor subclasses, the A ₁ , A _2A , A _2B and A ₃ . Among the different adenosine receptors, the A ₃ AR was found to mediate potent anti-tumor effect. High A ₃ AR mRNA, protein expression level and cell surface exhibition were reported in different tumor cell types, in comparison to normal adjacent tissues. It has been lately demonstrated that peripheral blood mononuclear cells (PBMNCs) derived from colorectal cancer patients have high expression levels of A ₃ AR compared to PBMNCs of healthy subjects.

Activation of the A ₃ AR by nanomolar (nM) concentrations of the A ₃ AR agonist 1 -Deoxy- 1 - [6-[[(3 -iodophenyl)methyl] amino] -9H-purme-9-yi] -N-methyl-b- D-ribofura-nuronamide (IB-MECA) or 2-chloro-N6-(3-iodobenzyl)-adenosine-5'- N-methyl-uronamide (CI-IB-MECA) inhibits the growth of melanoma, colon and prostate carcinoma tumor cell growth, both in vitro and in vivo. The mechanism of action includes deregulation of Wnt and NF-κB signal transduction pathways.

SUMMARY OF THE INVENTION In accordance with a first aspect, the present invention provides a method of determining disease state in a liver cancer patient, comprising detemiining level of expression of A ₃ adenosine receptor (A ₃ AR) in white blood cells (WBC) from said patient, wherein a high level of expression is indicative of an active disease state in the patient. In accordance with a second aspect, the present invention provides a method for determining severity of cancer in a liver cancer patient, comprising:

(a) determining level of expression of A ₃ AR in WBC from said patient; and

(b) comparing the level of expression of A ₃ AR in the WBC with a level of prior determined or obtained standards, to determine severity of the cancer state of said patient.

In accordance with a third aspect, the present invention provides a method for selecting liver cancer patients as candidates to receive a therapeutic treatment that makes use of an A ₃ AR agonist, or diagnosing cancer patients to determine whether their disease is of a kind that is treatable by an A ₃ AR agonist, comprising determining level of expression of A ₃ AR in WBC from said patients and selecting

patients who have a higher level of A ₃ AR expression as compared to an average level ofA ₃ AR expression in liver cancer patients.

In accordance with a fourth aspect, the invention provides a method for determining effectiveness of an anti-cancer therapeutic treatment of a patient having liver cancer, the treatment comprising administering an A ₃ AR agonist to the patient, the method comprising determining level of expression of A ₃ AR in WBC from the patient in two or more successive time points, at least one of which is during said anti-cancer therapeutic treatment, wherein a difference in the level between said two or more time points being indicative of effectiveness of said treatment. In accordance with yet a fifth aspect of the invention, there is provides a method for treating a liver cancer patient, comprising:

(a) determining level of A ₃ AR expression in peripheral WBC from said patient and

(b) administering an A ₃ AR agonist to said patient when said level is determined to be above prior determined or obtained standards.

BRIEF DESCRIPTION OF THE DRAWINGS

In order to understand the invention and to see how it may be carried out in practice, a preferred embodiment will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which: Figures IA-I C are blots and a respective analysis of A ₃ AR expression in

HCC patients tumor tissue; Fig. IA shows an RT-PCR blot of A ₃ AR expression in tumor tissue vs. normal tissue; Fig. IB shows a Western Blot (WB) of A ₃ AR expression as reflected in peripheral blood mononuclear cells (PBMNC) and Fig. 1C shows the respective analysis presented in the form of a bar graph of control vs. HCC patients.

Figures 2A-2D are WBs and respective analyses of the down regulation of

A ₃ AR expression level (Fig. 2A) and respective bar graph (Fig. 2B) in NlSl tumor bearing rats treated with Cl-IB-MECA; and the expression level as reflected in peripheral blood mononuclear cells (PBMNC, Fig. 2C) and the respective bar graph analysis (Fig. 2D).

Fig. 3 is a bar graph showing ³ [H]-Thymidine incoφoration in Cl-IB-MECA treated NlSl HCC cells reflecting the inhibitory effect of Cl-IB-MECA on the proliferation of said cells.

Figs. 4A-4C are an image showing the inhibitory effect of IB-MECA on the development of NlSl HCC tumors in rats treated with IB-MECA (Fig. 4A), or treated with vehicle only (Fig. 4B), where tumor area is circled; and a respective bar graph analysis (Fig. 4C).

Figs. 5A-5H are WB and respective analyses showing the de-regulating effect of IB-MECA on key signaling proteins involved with the NF-icB signal transduction pathway in Nl S 1 HCCC tumors.

Figs. 6A-6H are WB and respective analyses showing the de-regulating effect of IB-MECA on key signaling proteins involved with the Wnt signal transduction pathway in NlSl HCC tumors.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS It is an object of the invention, in accordance with one aspect thereof, to provide a method for determining and monitoring disease state in a patient having liver cancer, e.g. for determining the severity of the disease, for determining the effectiveness of an anti-cancer therapeutic treatment of a patient and for selecting patients to likely to benefit from an anti-cancer therapeutic treatment involving the use OfA ₃ AR agonists.

It is an object according to another aspect of the invention, to provide a therapeutic treatment for liver cancer patients that utilizes A ₃ AR agonists, particularly such patients selected in accordance with the invention as such having improved odds to benefit from such treatment. In the following the terms "liver cancer" and "hepatocellular carcinoma"

(or "HCC"), will be used interchangeably.

It was found in accordance with the invention that there is an increase in the level of A ₃ AR expression in the white blood cells (WBC) of a patient having liver cancer, as compared to the level of expression of same in WBC from a healthy subject. Furthermore, it was found that in animals with liver cancer there is an

increase in expression of A ₃ AR protein level in the tumor and in PBMNC vs. normal adjacent tissue and PBMNC from control animals, respectively.

By one embodiment of the invention, there is provided a method of determining disease state in a liver cancer patient, the method comprises determining level of expression of A ₃ adenosine receptor (A ₃ AR) in white blood cell (WBC) from said patient, wherein a high level of expression is indicative of an active disease state in the patient.

By another embodiment of the invention, there is provided a method for determining the severity of cancer in a liver cancer patient, the method comprises: (a) determining level of expression of A ₃ AR in WB C from said patient;

(b) comparing the level of expression of A ₃ AR in the WBC with a level of prior determined or obtained standards, to determine the severity of the cancer state of the patient.

By a third embodiment of -the invention, there is provided a method for selecting liver cancer patients that are candidates to receive a therapeutic treatment that makes use of an A ₃ AR agonist, or diagnosing cancer patients to determine whether their disease is of a kind that is treatable by an A ₃ AR agonist, the method comprises determining level of A ₃ AR expression in WBC from said patients and selecting patients who have a higher level of A ₃ AR expression as compared to an average level of A ₃ AR expression in liver cancer patients.

Selecting the patients as such candidates or said diagnosis, comprises:

(a) determining the level of expression of A ₃ AR Ui WBC from said liver cancer patients;

(b) comparing the level of expression of A ₃ AR in the WBC with the level of prior determined or obtained standards; wherein, when said level of expression of A ₃ AR in the WBC from a patient is above said standards, the patient is selected from anti-cancer therapeutic treatment.

The WBC may be obtained from said patients by drawing a blood sample comprising the WBC. The blood sample may be whole blood sample or may be a blood fraction that contains WBC. At times, it may be desired to use a fraction that includes a specific population of WBC such as mononuclear cells (MNC), sub- populations of MNC — monocytes or lymphocytes, or a sub-population of

lymphocytes, e.g. T-cells, B-cell or their sub-populations. A WBC-comprising sample may also at times be obtained from the lymphatic system, e.g. from lymph nodes.

The term "level of expression" as used herein includes one or both of the level of A ₃ AR mRNA as well as the level of A ₃ AR protein or A ₃ AR protein fragments in the sample. The level of expression may also be me determined through measuring the level of receptor exhibition , e.g. using immuno-histochemistry. This can be recorded in pathological slides or in blood smear from a patient.

The A ₃ AR level of expression in WBC in accordance with some embodiments of the invention may be used for a qualitative determination of the state or severity of the liver cancer, e.g. classification into "severe", "moderate" or

"light", hi accordance with other embodiments of the invention, the A ₃ AR level of expression may be used for quantitative determination of the severity of the disease.

The term "determining" or "determination" will be employed herein to refer to either or both quantitative or qualitative determination. When conducting a quantitative deteπnination of the level OfA ₃ AR expression, it is in accordance with a preferred embodiment of the invention that the difference in expression between the tested sample, i.e. the WBC-comprising sample obtained from a liver cancer bearing patient and the control/standards is a statistically significant difference. A statistically significant difference may be determined by any statistical test known in the art.

The term "WBC" used in accordance with the invention may include any of the known types of cells which make up the WBC group, hi particular, the term may preferably denote mononuclear cells (monocytes and/or lymphocytes or at times subpopulations of B and T lymphocytes or NK cells). The WBCs are typically PBMNCs. At times, the sample comprising the WBC may include in addition to any of the above, or in the alternative, granulocytes (neutrophils, eosinophils or basophils).

A high level of expression OfA ₃ AR may be employed as an indicator of the disease state in the patient. The term "high level" is to be understood as meaning a statistically significantly higher level of expression than in a control sample. For example, the level of the A ₃ AR expression in the WBC may be compared to a control level, the control level being the level OfA ₃ AR expression in normal WBC

of a healthy subject. Statistical significance may be determined by statistical tests as known to those versed in the art.

The determined expression level may also be compared to standards. The standards may be based on previously determined levels from healthy subjects or from liver cancer patients at different disease states. The standards may be provided, for example, in the form of discrete numeric values or, in case the assay method is colorimetric, in the form of a chart with different colors or shadings for healthy and different disease states; or they may be provided in the form of a comparative curve prepared on the basis of such standards. It is also possible, according to an embodiment of the invention, to have different standards for different populations such as: for different age groups; may be a gender-dependent standard; may be a standard adjusted according to parameters of disease history such as duration of disease or prior treatment; may be a standard dependent on other therapies received by the subject; may be a standard that is adjusted to personal properties such as weight; and others. The standard, according to some embodiments, may be an individual standard determined for the tested individual at an earlier time.

Such standards may be prepared by determining the level of A ₃ AR expression (which may be the level OfA ₃ AR protein, protein fragment, mRNA level, etc., as discussed above) present in WBC cells obtained from a plurality of patients positively diagnosed (by other means, for example by a physician, by various imaging techniques, by histological examination of biopsies, etc.) as having liver cancer at various disease states, hi another embodiment, the assay is carried out in parallel to a number of standards of healthy subjects and patients of different disease states and the level determined in the assayed WBC-comprising sample is then compared to such standards.

For example, the A ₃ AR expression level of between X ₁ to X ₂ per 1,000,000 cells may be defined as being indicative of grade 1 diseases, a higher protein content of Y ₁ to Y ₂ per 1,000,000 cells may be defined as being indicative of grade 2 disease, etc. After such standards are prepared, it is possible to compare the level of A ₃ AR expression obtained from a specific individual to the corresponding value of the standards, and thus obtain an assessment of the severity of the disease.

Determining the A ₃ AR level in WBC may also be used to determine the effectiveness of an anti-cancer therapeutic treatment of liver cancer patient that

makes use of A ₃ AR agonists as the active principle ingredient in a treatment regimen. Samples of WBC may be taken at various time points before, during and after cessation the treatment. For example, a first determination may be taken at a first time point prior to initiation of the treatment and a second determination may be taken at a time point during the treatment (the second determination may include one or more determination at sequential time points during the treatment). A significant decrease in the level of the A ₃ AR expression in the second determination time point as compared to the level determined in the first time point may be indicative that the treatment is effective. The degree of decrease could be indicative of the degree of effectiveness of the treatment, i.e. the correlation would be quantitative.

In another example, a first determination may be taken at a first time point during the treatment and a second (one or more) determinations may be taken at one or more time points during the treatment subsequent to the first time point. A significant decrease in the level of the A ₃ AR expression in the second sample as compared to the first sample would be indicative that the treatment is effective.

In accordance with one embodiment, two time points are taken for determining effectiveness of treatment. In accordance with another embodiment level of expression OfA ₃ AR in WBC is determined in more than two time points.

In a third example, a first determination may be taken at a first time point during the treatment and a second (one or more) determinations may be taken at a second (one or more) time point after the treatment has been discontinued. In this case, a significant increase in the level of the A ₃ AR expression in the second measurement as compared to the level determined at the first time point would be indicative that the treatment was effective. The above alternative methods may then provide a rationale for continuing the patient on a therapeutic treatment involving administration to the patients of effective amount of an A ₃ AR agonist.

Selection of liver cancer patients suitable for receiving an anti-cancer treatment that involves administration to the patients of an A ₃ AR agonist may be executed by detemήning the level of expression of A ₃ AR in a sample of WBC withdrawn from the patient before treatment. The patient may then be selected for treatment if the determined level OfA ₃ AR is above a predefined threshold.

According to one embodiment, the threshold is a certain multiple of the level of A ₃ AR expression in WBC of a healthy subject. According to another embodiment, the threshold is determined on the basis of the average expression level in patients having said liver cancer, and may be said average or a certain multiple or fraction thereof. By a further embodiment, the threshold is determined on the basis of clinical studies in human patients that are designed to determine the correlation between the level of expression and the response of the patients to the therapeutic treatment.

The selection method may also apply for selecting candidates for participating in clinical studies to test use of an A ₃ AR agonist in treatment of liver cancer. As appreciated by those versed in the art, a clinical study, is a scientific study in human volunteers to determine how a new medicine or treatment works in human subjects. Interventional trials determine whether experimental treatments or new ways of using known therapies are safe and effective under controlled environments. It is through clinical studies that physicians find new and better ways to prevent, detect, diagnose, control, and treat illnesses. The clinical studies for which patients are selected, in accordance with the invention, based on the A ₃ AR level may be

Phase I ₃ Phase II, Phase IE, Phase IV or any other type of clinical study.

For clinical studies a threshold according to the invention may also be of an abnormal (higher than normal) level of expression of A ₃ AR. An abnormal level may be defined based on considerations known to those experienced in clinical studies to be a suitable threshold for selecting such candidates.

According to another aspect of the invention there is provided a method for treating a liver cancer patient, comprising: (a) determining level of A ₃ AR expression in peripheral WBC from said patient; and

(b) administering an A ₃ AR agonist to said patient when said level is determined to be above prior determined or obtained standards.

In accordance with one embodiment of the above method, the peripheral WBC comprises or consist primarily of peripheral blood mononuclear cells (PBMNC).

High level of expression refers in particular to a level of expression higher than a certain predetermined threshold as defined above. The determination may be carried out in a manner as described above.

The therapeutic treatment may make use of a wise variety OfA ₃ AR agonists. The characteristic of some adenosine A ₃ AR agonists and methods of their preparation are described in detail in, inter alia, US 5,688,774; US 5,773,423;

US 5,573,772; US 5,443,836; US 6,048,865; WO 95/02604; WO 99/20284;

WO 99/06053; and WO 97/27173, all of which are incorporated herein by reference.

According to one embodiment of the invention, the active ingredient is an A ₃ AR agonist which exerts its prime effect through the A3 adenosine receptor and is a purine derivative falling within the scope of the general formula (I):

wherein R ₁ is C ₁ -C ₁₀ alkyl, C ₁ -C ₁₀ hydroxyalkyl, C ₁ -C _1O carboxyalkyl or C ₁ - C ₁₀ cyanoalkyl or a group of the following general formula (II):

in which:

Y is oxygen, sulfur atom or CH CH _1-2 or , C(W)(W) where W and W could be same or different and are H or F or valence bond;

X ₁ and X ₁ ¹ are same or different and are hydrogen, C ₁ -C _1O alkyl, R ^a R ^b NC(=O)- or HOR ⁰ -, wherein R ^a and R ^b may be the same or different and are selected from hydrogen, Ci-C ₁₀ alkyl, amino, C ₁ -C ₁ O haloalkyl, C ₁ -Ci ₀ aminoalkyl, Ci-Qo BOC-aminoalkyl, and C ₃ -C ₁₀ cycloalkyl or are joined together to form a heterocyclic ring containing two to five carbon atoms, and R ^c is selected from Ci-C ₁₀ alkyl, amino, Ci-Cio haloalkyl, C ₁ -CiO aminoalkyl,

C ₁ -C ₁₀ BOC-arninoalkyl, and Ca-C _1O cycloalkyl, C ₁ -C ₃ alkyl aminocarbonyl, C ₁ -Cs alkylthio C ₁ -C ₃ alkyl, halo C ₁ -C ₃ alkyl, hydrazinyl, hydroxy C ₁ -Cs alkyl, C ₃ -C ₆ cycloalkylamino, hydroxylamino, and C ₂ -C ₃ alkenyl or when Y is C(W)(W) X ₁ or X ₁ ' form therewith cyclopropyl ring; - X ₂ is hydrogen, hydroxyl, C ₁ -C ₁₀ alkylamino, C ₁ -C ₁₀ alkylamido or

C ₁ -C ₁₀ hydroxyalkyl, C ₁ -C ₁₀ allcyl, R ⁸¹ R ¹³ NC(^O)- or HOR ⁰ -, wherein R ^a and R ^b may be the same or different and are selected from hydrogen, C ₁ -C ₁₀ alkyl, amino, C ₁ -C ₁₀ haloalkyl, C ₁ -C ₁₀ aminoalkyl, C ₁ -C ₁₀ BOC-aminoalkyl, and C ₃ -C ₁₀ cycloalkyl or are joined together to form a heterocyclic ring containing two to five carbon atoms, and R ^c is selected from Ci-C ₁₀ alkyl, amino, C ₁ -Ci ₀ haloalkyl, C ₁ -Ci ₀ aminoalkyl, Ci-C ₁₀ BOC-aminoalkyl, and C ₃ -C ₁₀ cycloalkyl, Ci-C ₃ alkyl aminocarbonyl, Ci-C ₃ alkylthio Ci-C ₃ alkyl, halo Ci-C ₃ alkyl, hydrazinyl, hydroxy C ₁ -C ₃ alkyl, C ₃ -C ₆ cycloalkylamino, hydroxylamino, and C ₂ -C ₃ alkenyl; X ₃ and X ₄ each independently are hydrogen, hydroxyl, amino, amido, azido, halo, alkyl, alkoxy, carboxy, nitrilo, nitro, triiluoro, aryl, alkaryl, thio, thioester, thioether, -OCOPh, -OC(=S)OPh or both X ₃ and X ₄ are oxygen connected to >C=S to form a 5-membered ring, ureido, Ci-C ₆ alkyl carbonylamino, hydroxy C ₁ -C ₆ alkyl, hydrazinyl or X ₂ and X ₃ form the ring of formula (III):

where R' and R" are independently C ₁ -C ₁₀ alkyl;

R ₂ is selected from hydrogen, halo, C ₁ -C ₁₀ alkylether, amino, hydrazido, C ₁ -C ₁₀ alkylamino, C ₁ -Ci ₀ alkoxy, C ₁ -Ci ₀ thioalkoxy, pyridylthio, C ₂ -C ₁₀ alkenyl; C ₂ -C ₁₀ alkynyl, thio, and Ci-C ₁₀ alkylthio, C ₂ -C ₆ alkenyloxy,

C ₂ -C ₆ alkynyloxy, C ₃ -C ₈ cycloalkyl Ci-C ₆ alkoxy, C ₃ -C ₈ cycloalkenyloxy, C ₇ -Ci ₂ bicycloalkyl Ci-C ₆ alkoxy, C ₇ -Ci ₂ bicycloalkenyl Ci-C ₆ alkoxy, C ₆ - Ci ₄ aryloxy, C ₆ -C _M aryl C ₁ -C ₆ alkoxy, C ₆ -C ₁₄ aryl C ₃ -C ₆ cycloalkoxy, C ₆ -Ci ₄ aryl Ci-C ₆ alkylamino, C ₆ -Ci ₄ aryl Ci-C ₆ alkylthio, Ci-C ₆ alkyl C ₆ -Ci ₄ aryl

C ₁ -C ₆ alkoxy, C ₁ -C ₆ alkoxy C ₆ -C ₁₄ aryl C ₁ -C ₆ alkoxy, halo C ₆ -C ₁₄ aryloxy, halo C ₆ -C ₁₄ aryl C ₁ -C ₆ alkoxy, C ₆ -C ₁₄ aryl C ₁ -C ₆ alkylamino, C ₁ -C ₆ dialkoxy C ₆ -C ₁₄ aryl C ₁ -C ₆ alkoxy, heterocyclyl C ₁ -C ₆ alkoxy, hydrazinyl, and pyrazolyl, said pyrazolyl being optionally substituted with one or more substituents selected from the group consisting of C ₁ -C ₆ alkyl, C ₆ -C ₁₄ aryl

C ₁ -C ₆ alkyl, C ₆ -C ₁₄ haloaryl C ₁ -C ₆ alkyl, aminocarbonyl, C ₁ -C ₆ alkyl aminocarbonyl, C ₁ -C ₆ alkoxyphenyl, C ₆ -CH ₁₄ haloaryl, and heterocyclyl, and any combination thereof; and

R ₃ is a -NR ₄ R ₅ group with R ₄ being hydrogen or a group selected from alkyl, substituted alkyl or aryl-NH-C(Z)-, with Z being O, S, or NR ^a , and when R ₄ is hydrogen, R ₅ being selected from C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, hydroxy, C ₃ -C ₈ cycloalkyl, C ₃ -C ₈ cycloalkyl C ₁ -C ₆ alkyl, C ₃ -C ₈ dicycloalkyl C ₁ -C ₆ alkyl, C ₇ -C ₁₂ bicycloalkyl C ₁ -C ₆ _ _alkyl, _{. .} C ₇ -C ₁₄ . tricycloalkyl C ₁ -C ₆ alkyl, C ₆ -C ₁₄ aryl, C ₆ -C ₁₄ aryl C ₁ -C ₆ alkyl, C ₆ -C ₁₄ diaryl

C ₁ -C ₆ alkyl, C ₆ -C ₁₄ aryl C ₁ -C ₆ alkoxy, heterocyclyl C ₁ -C ₆ alkyl, heterocyclyl, 4-[[[4-[[[(2-amino C ₁ -C ₆ alkyl) amino]-carbonyl]-Ci-C ₆ alkyl] aniline] carbonyl] C ₁ -C ₆ alkyl] C ₆ -C ₁₄ aryl, and C ₆ -C ₁₄ aryl C ₃ -C ₈ cycloalkyl, wherein the aryl or heterocyclyl portion of R4 is optionally substituted with one or more substituents selected from the group consisting of halo, amino,

C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₆ -C ₁₄ aryloxy, hydroxy C ₁ -C ₆ alkyl, hydroxy C ₂ - C ₆ alkenyl, hydroxy C ₂ - C ₆ alkynyl, aminocarbonyl C ₁ -C ₆ alkoxy, and C ₆ -C ₁₄ aryl C ₁ -C ₆ alkoxy, and any combination thereof; and the alkyl or cycloalkyl portion of Rl is optionally substituted with one or more substituents selected from the group consisting of halo, hydroxy, amino, and any combination thereof; or (R)- and (5)-l-phenylethyl, benzyl, phenylethyl or anilide groups, each said groups being unsubstituted or substituted in one or more positions with a substitαent selected from C ₁ -C ₁₀ alkyl, amino, halo, C ₁ -C ₁₀ haloalkyl, nitro, hydroxyl, acetoamido, C ₁ -C ₁₀ alkoxy, and sulfonic acid or a salt thereof; or R ₅ is benzodioxanemethyl, fururyl, L-propylalanyl- aminobenzyl, β-alanylamino- benzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl, phenoxy or C ₁ -C ₁₀ cycloalkyl; or R ₅ is a group of the following formula (TV):

(IV)

or, when R ₄ is alkyl, substituted alkyl, or aryl-NH-C(Z)-, then, R ₅ is selected from the group consisting of substituted or uiisubstituted heteroaryl-NR ^a - C(Z)-, heteroaryl-C(Z)-, alkaryl-NR ^a -C(Z)- ₅ alkaryl-C(Z)-, aryl-NR-C(Z)- and aryl-C(Z)-; or the A ₃ AR agonist is a xanthine-7-riboside derivative of the following general formula (V):

wherein:

X is O or S;

R ₆ is R ^a R ^b NC(=O)- or HOR ^C -, wherein - R ^a and R ^b may be the same or different and are selected from hydrogen, C ₁ -

C ₁₀ alkyl, amino, C ₁ -C ₁₀ haloalkyl, C ₁ -C ₁₀ aminoalkyl, and C ₃ -C _1O cycloalkyl, or are joined together to form a heterocyclic ring containing two to five carbon atoms; and

R ^c is selected from C ₁ -C ₁₀ alkyl, amino, C ₁ -C ₁₀ haloalkyl, C ₁ -C ₁₀ aminoalkyl, C ₁ -C ₁ O BOC-aminoalkyl and C ₃ -C ₁₀ cycloalkyl; - R ₇ and R ₈ may be the same or different and are selected from C ₁ -C ₁₀ alkyl,

C ₁ -C ₁₀ cycloalkyl, (R)- or (S)-l-phenylethyl, an unsubstituted benzyl or anilide

group, and a phenylether of ben2yl group substituted in one or more positions with a substituent selected from Ci-C ₁₀ alkyl, amino, halo, C ₁ -C ₁₀ haloalkyl, nitro, hydroxyl, acetamido, C ₁ -Ci ₀ alkoxy, and sulfonic acid;

R ₉ is selected from the group consisting of halo, benzyl, phenyl, C ₃ -C ₁₀ cyclalkyl, and C ₁ -C ₁₀ alkoxy; or a suitable salt of any of the compounds defined above.

According to a more preferred embodiment, the A ₃ AR agonist is a nucleoside derivative of the general formula (VII):

wherein X ₁ , R ₂ and R ₄ are as defined above.

Preferred A ₃ AR agonists in accordance with formula (VII) include, although not exclusively, N ⁶ -benzyladenosine-5'-uronamide derivatives. Some preferred N ⁶ - benzyladenosine-5'-uronamide derivatives are N ⁶ -2-(4-aminophenyl)ethyladenosine (APNEA), N ⁶ -(4-amino-3- iodobenzyl) adenosine-5'-(N-methyluronamide) (AB- MECA) and l-deoxy-l-{6- [({3-iodoρhenyl} methyl)amino]- 9H-purine-9-yl}-N- methyl-β-D-ribofuranuronamide (IB-MECA), 2-chloro-N ⁶ -(3- iodobenzyl)adenosine- 5'-N-methlyuronamide (Cl-IB-MECA).

According to another preferred embodiment, the A ₃ AR agonist is a nucleoside derivative of the general formula (VIII):

wherein Y, X ₁ ', R ₂ and R ₄ are as defined above.

According to yet another preferred embodiment, the A ₃ AR agonist is a nucleoside derivative of the general formula (IX):

wherein Y, X ₂ , R ₂ and R ₄ are as defined above.

Preferred agonists of A ₃ AR in accordance with formulae (VIII) and (IX) include, although not exclusively, IB-MECA and Cl-IB-MECA derivatives, such as 4 ¹ -(2-chloro-6-{[(3-iodophenyl)methyl]amino}ρurin-9-yl)-2',3' - dihydroxybicyclo[3.1.0]hexyl]-N-methylcarboxamide (bicyclo-Cl-IB-MECA), 4 ^r -(6- { [(3 -iodophenyl)methyl]amino}purin-9-yl)-2',3 '-dihydroxybicy clo [3.1.OJhexyl] -N- methylcarboxamide (bicyclo-IB-MECA) and 2-[2-chloro-6-

(iodobenzylamino)purin-9-yl]-3,4-dihydroxytetrahydrothiop hene-2-carboxylic acid

metlαyl amide (thio-Cl-IB-MECA) and 2-[2-6-(iodobenzylamino)purin-9-yl]-3,4- dihydroxytetrahydrothiophene-2-carboxylic acid methyl amide (thio-IB-MECA).

According to another embodiment, the A ₃ AR agonist is N ⁶ -benzyl- adenosine-S'-alkyluronamide-N^oxide or N ⁶ -benzyladenosrne-5'-N-dialyl- uronamide-NWide.

The A ₃ AR agonist is administered in an effective amount, being an amount that is effective in achieving the desired therapeutic effect, which may be manifested by a slow-down of tumor growth, tumor shrinkage, amelioration of disease symptoms, etc. The therapeutic effect may be an increase in time to tumor progression, a partial response (namely partial tumor shrinkage) or a complete response. It may also be manifested through decrease in tumor-related effects such as loss of appetite, cahexia and others. The effective amount may be determined in dose-finding clinical studies in cancer patients or through extrapolation from animals using one of many such extrapolation methods readily known to those of skill in the art of clinical studies. The effective amount may depend on factor known per se such as weight, body surface, gender, disease history and status, concomitant medicines taken by the patient, severity of disease, frequency of administration, drug residence time in the plasma or blood, extent of binding of the drug by blood proteins and others. The effective

NON-LIMITING EXEMPLARY EMBODIMENTS

In order to understand the invention, experimental results of specific embodiments will now be described. As will be appreciated, this specific embodiment is meant to be illustrative and the invention is not limited thereto.

Materials and Methods Drugs and Antibodies

The A ₃ AR agonists l-Deoxy-l-[6-[[(3-iodophenyl)methyl]amrno]-9H- purine-9-yl]-N-methyl-b-D-ribofura-nuronamide (IB-MECA) and 2-cbloro-N ⁶ -(3- iodobenzyl)-adenosine-5'-N-methyl-uronamide (Cl-IB-MECA), both described hi US patent 5,773,423, were used. A stock solution of 10 niM was prepared in DMSO and further dilutions in culture medium or PBS were performed to reach the desired concerntraion.

Rabbit polyclonal antibodies against A ₃ AR and the cell growth regulatory proteins PKB/Akt, IKK, NF-κB, GSK-3β, LEF-I, β-catenin, c-myc and β-actin were purchased from Santa Cruz Biotechnology Inc., Ca, USA

Tumor cells and proliferation assay NlSl, rat HCC cell line (American Type Culture Collection, Manassas,

Virginia) was grown in RPMI 1640 penicillin, streptomycin, 2 mM. L-glutamine and 10% fetal bovine serum (FBS). The cells were maintained in T-75 flasks at 37 ⁰ C in a 5% CO ₂ incubator and transferred to a freshly prepared medium twice weekly. For the in vitro studies serum starved cells were used. FBS was omitted from the cultures for 18 hours and the experiment was carried out on monolayers of cells in RPMI medium supplemented with 1% FBS in a 37°C, 5% CO ₂ incubator.

³ [H] -thymidine incorporation assay was used to evaluate cell growth. NlSl cells (1.5xlO ⁴ /ml) were incubated with an A ₃ AR agonist (10-100OnM) in 96-well microtiter plates for 24 hours. For the last 18h of incubation, each well was pulsed with lμCi ³ [H]-thymidine. Cells were harvested and the [ ³ H] -thymidine uptake was determined in an LKB liquid scintillation counter (LKB, Piscataway, NJ, USA). These experiments were repeated 3 times

In vivo studies

Male Sprague-Dawley rats, weighing an average of 20Og were obtained from Harlan Laboratories, Jerusalem, Israel, and maintained on a standardized pelleted diet and supplied with tap water. Experiments were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at

Can-Fite BioPharma, Petah Tikva, Israel. A subxyphoid laparatomy was performed and NlSl cells (5xl0 ⁶ /50μL saline) were injected to the left hepatic lobe. The rats were divided randomly to two groups: Control group (which received vehicle only) and the A ₃ AR treated group. The treatment started 24 hours after tumor inoculation and lasted for 15 days. Body weight was monitored twice weekly. At the end of the study, the liver was weighed and the width [W] and length [L] were measured and the tumor size was calculated according to the following formula: Tumor Size = [(W) ² x L]/2.

RT-PCR analysis of formalin-fixed paraffin-embedded tissue slides

Tissue sections (5μm thick) from liver of patients with HCC were mounted on slides that were stained by H&E and were observed by a pathologist. The neoplastic area and the normal area were detected and marked each one separately. In each marked area cells were counted. Non-stained sequential slides were marked for neoplastic and normal tissue based on the stained slides. Tissue sections on slides were deparaffinized in xylen and rehydrated by washing in serial dilutions of ethanol. Slides were used immediately or stored at -8O ⁰ C until used. After rehydration, 20 μl of solution A [1.25XPCR buffer (200 Mm Tris-HCl, 500 niM KCl) ₅ 6.25 mM MgCl ₂ , 5 U RNasin (Promega, Madison, WI), 2mM DTT, 1 U RQl RNase-free DNase (Promega)] was directly applied to the marked area. The marked area was completely scraped off the slide using a pipette tip and the neoplastic tissue or the normal tissue were collected to different microcentrifuge tubes. The samples were treated with proteinase K at a final concentration of 0.1 mg/ml. The samples were incubated at 37°C for Ih to allow for DNA digestion. Cells lysate were heated to 95 ⁰ C for 15 min in order to inactivate DNase and proteinase K. Following centrifugation at 14,000 RPM for 5 min, 17μl of the supernatant was transferred to separate tube and 4 μl of RT mixure [ 5mM dNTPs, 2.5 μM random hexamer, 5 U RNasin, 100 U Superscript. One Step RT-PCR with Platinum Taq (Invitrogene) was carried out with the following primers for A ₃ AR: 5'-ACGGTGAGGTACCACAGCTTGTG (SEQ ID NO.l), and

3'-ATACCGCGGGATGGCAGACC (SEQ ID NO:2). The RT reaction was performed at 45 ⁰ C for 45 min., followed by heating to 99 ⁰ C for 5 min. 50 cycles of 94°C for 30s, 59°C for 45s and 73°C for 45s were performed. Products were elecrtophoresed on 2% agarose gels, stained with ethidium bromide and visualized with UV illumination. The specificity of the RT- PCR reaction was confirmed by size determination on agarose gels in comparison to a positive control, from RNA extracted using standard techniques and by sequencing the RT-PCR product and comparing the sequences to that of the known sequences (ADORA3-L77729, L77730). The optical density of the bands (Et-Br) was quantified using an image analysis system. To quantitate A ₃ AR mRNA expression, the optical density value was normalized against the cell number in each marked area (tumor or adjacent tissue).

Western Blot Analysis

To separate PBMNC from naϊve and tumor bearing rats as well as from HCC patients, heparinized peripheral blood was subjected to a density gradient centrifugation (Ficoll/Histopaque 1077 g/ml). Tumor lesions were removed upon study termination in order to evaluate A ₃ AR protein expression level as well as the expression level of key signaling proteins downstream to A ₃ A. Cells (NlSl and PBMNC) and tissue samples were rinsed with ice-cold PBS and transferred to ice- cold lysis buffer (TNN buffer, 5OmM Tris buffer pH=7.5, 15OmM NaCl, NP 40 0.5% for 20 min). Cell debris was removed by centrifugation for 10 min, at 14,000 RPM. The supernatants were utilized for Western Blot analysis. Protein concentrations were determined using the Bio-Rad protein assay dye reagent. Equal amounts of the sample (50μg) were separated by SDS-PAGE, using 12% polyacrylamide gels. The resolved proteins were then electroblotted onto nitrocellulose membranes (Schleicher & Schuell, Keene, NH, USA). Membranes were blocked with 1 % bovine serum albumin and incubated with the desired primary antibody (dilution 1:1000) for 24 h hour at 4 ⁰ C. Blots were then washed and incubated with a secondary antibody for Ih at room temperature. Bands were recorded using BCIP/NBT color development kit (Promega, Madison, WI, USA). The optical density of the bands was quantified using an image analysis system and corrected by the optical density of the corresponding actin bands. Data presented in the different figures are representative of at least three different experiments.

Statistical analysis.

Results were analyzed by Wilcoxon signed rank test, with statistical significance at p<0.05.

Results

A3 AR expression level in PBMNC reflects the receptor expression status in the HCC tumor. mRNA was extracted from formalin fixed paraffin embedded HCC tissue sections derived from HCC patients. Neoplastic and normal regions were scraped from the marked areas on the slides and collected separately to two different tubes for the RT-PCR reaction. Higher A ₃ AR mRNA expression was noted in the tumor in comparison to the adjacent normal tissue in 70% of the patients. Fig. IA depicts a

representative blot. The high expression of the A ₃ AR in the tumor tissue was reflected in the PBMNC derived from HCC patients, as can be seen in Figs. 1B-1C. In Western Blot (WB) analysis of protein extract derived from PBMNC of the patients the expression level OfA ₃ AR was high in comparison to the receptor level in PBMNC derived from healthy volunteers

In addition, up-regulation in the A ₃ AR expression level was noted in rat NlSl hepatoma tumor tissues in comparison to normal liver tissues. In tumor tissues derived from hepatoma bearing rats treated with Cl-IB-MECA, down- regulation of A ₃ AR expression was noted shortly after treatment (Figs. 2A-2B), demonstrating that receptor activation took place. This was also reflected in the PBMNC protein extracts i.e., high expression Of A ₃ AR in PBMNC derived from tumor bearing rats in comparison to low expression in Naive animals and in Cl-IB-MECA treated tumor bearing rats (Figs. 2C-2D).

Effect of IB-MECA on the growth of NlSl hepatoma cells The effect of Cl-IB-MECA on the proliferation of NlSl hepatoma cells was examined in vitro utilizing ³ [H] -Thymidine incorporation assay. IB-MECA inhibited the proliferation of the NlSl cells in an inverse dose dependent manner, with an ICs ₀ presented at the concentration of 1OnM (Fig. 3).

In vivo, NlSl cells were inoculated into the liver and oral treatment with lOOμg/kg of IB-MECA, twice daily was initiated 24 hours after tumor inoculation. The treatment lasted for 15 days. The tumor size in the vehicle and IB-MECA treated group is presented in Fig. 4A. IB-MECA was efficacious in inhibiting the growth of the NlSl tumors by 60% of inhibition as shown in Fig. 4B.

Effect of Cl-IB-MECA on signal transduction pathways down-stream to A3 AR activation

Protein extracts of NlSl tumor tissues derived from Cl-IB-MECA and vehicle treated animals were subjected to WB analysis. The data revealed that expression levels of key signaling proteins downstream to A ₃ AR activation were modulated upon treatment with Cl-IB-MECA. De-regulation of PKB/Akt was noted in protein extracts derived from Cl-IB-MECA treated NlSl tumor bearing

animals. Consequently, the expression levels of proteins playing a major role in the Wnt and the NF-IcB signaling pathways were de-regulated. IKKα/ β, NF-kB, and TNF-α expression level was down-regulated upon treatment with Cl-IB- MECA, demonstrating the involvement of the NF-IcB signaling pathway (Fig. 5A- 5H). In Addition, the expression level of GSK-3β, a protein playing a key role in the Wnt signaling pathway, was up-regulated, resulting in decreased level of the down-stream proteins LEF/TCF, β-catenin and c-myc (Fig. 6A-6H).