WO2007043054A1

WO2007043054A1 - Treatment and monitoring disease state of liver cancer

Info

Publication number: WO2007043054A1
Application number: PCT/IL2006/001183
Authority: WO
Inventors: Pnina Fishman; Sara Bar-Yehuda
Original assignee: Can-Fite Biopharma Ltd.
Priority date: 2005-10-12
Filing date: 2006-10-15
Publication date: 2007-04-19

Abstract

The present invention 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 of A3AR agonists for example IB-MECA. The method of the invention is based on the determination of level of expression of A3 adenosine receptor agonist in white blood cells, the level being indicative of the disease state.

Description

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, TACE₅ 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 A₁, A_2A, A_2B and A₃. Among the different adenosine receptors, the A₃AR was found to mediate potent anti-tumor effect. High A₃AR 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 A₃AR compared to PBMNCs of healthy subjects.

Activation of the A₃AR by nanomolar (nM) concentrations of the A₃AR 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 A₃ adenosine receptor (A₃AR) 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 A₃AR in WBC from said patient; and

(b) comparing the level of expression of A₃AR 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 A₃AR agonist, or diagnosing cancer patients to determine whether their disease is of a kind that is treatable by an A₃AR agonist, comprising determining level of expression of A₃AR in WBC from said patients and selecting patients who have a higher level of A₃AR expression as compared to an average level ofA₃AR 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 A₃AR agonist to the patient, the method comprising determining level of expression of A₃AR 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 A₃AR expression in peripheral WBC from said patient and

(b) administering an A₃AR 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 A₃AR expression in

HCC patients tumor tissue; Fig. IA shows an RT-PCR blot of A₃AR expression in tumor tissue vs. normal tissue; Fig. IB shows a Western Blot (WB) of A₃AR 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

A₃AR 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 OfA₃AR agonists.

It is an object according to another aspect of the invention, to provide a therapeutic treatment for liver cancer patients that utilizes A₃AR 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 A₃AR 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 A₃AR 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 A₃ adenosine receptor (A₃AR) 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 A₃AR in WB C from said patient;

(b) comparing the level of expression of A₃AR 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 A₃AR agonist, or diagnosing cancer patients to determine whether their disease is of a kind that is treatable by an A₃AR agonist, the method comprises determining level of A₃AR expression in WBC from said patients and selecting patients who have a higher level of A₃AR expression as compared to an average level of A₃AR expression in liver cancer patients.

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

(a) determining the level of expression of A₃AR Ui WBC from said liver cancer patients;

(b) comparing the level of expression of A₃AR in the WBC with the level of prior determined or obtained standards; wherein, when said level of expression of A₃AR 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 A₃AR mRNA as well as the level of A₃AR protein or A₃AR 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 A₃AR 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 A₃AR 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 OfA₃AR 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 OfA₃AR 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 A₃AR expression in the WBC may be compared to a control level, the control level being the level OfA₃AR 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 A₃AR expression (which may be the level OfA₃AR 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 A₃AR expression level of between X₁ to X₂ per 1,000,000 cells may be defined as being indicative of grade 1 diseases, a higher protein content of Y₁ to Y₂ 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 A₃AR 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 A₃AR 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 A₃AR 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 A₃AR 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 A₃AR 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 OfA₃AR 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 A₃AR 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 A₃AR agonist.

Selection of liver cancer patients suitable for receiving an anti-cancer treatment that involves administration to the patients of an A₃AR agonist may be executed by detemήning the level of expression of A₃AR in a sample of WBC withdrawn from the patient before treatment. The patient may then be selected for treatment if the determined level OfA₃AR is above a predefined threshold. According to one embodiment, the threshold is a certain multiple of the level of A₃AR 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 A₃AR 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 A₃AR level may be

Phase I₃ 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 A₃ 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 A₃AR expression in peripheral WBC from said patient; and

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 OfA₃AR agonists. The characteristic of some adenosine A₃AR 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 A₃AR 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 R₁ is C₁-C₁₀ alkyl, C₁-C₁₀ hydroxyalkyl, C₁-C_1O carboxyalkyl or C₁- C₁₀ 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;

X₁ and X₁ ¹ are same or different and are hydrogen, C₁-C_1O alkyl, R^aR^bNC(=O)- or HOR⁰-, wherein R^a and R^b may be the same or different and are selected from hydrogen, Ci-C₁₀ alkyl, amino, C₁-C₁O haloalkyl, C₁-Ci₀ aminoalkyl, Ci-Qo BOC-aminoalkyl, and C₃-C₁₀ cycloalkyl or are joined together to form a heterocyclic ring containing two to five carbon atoms, and R^c is selected from Ci-C₁₀ alkyl, amino, Ci-Cio haloalkyl, C₁-CiO aminoalkyl, C₁-C₁₀ BOC-arninoalkyl, and Ca-C_1O cycloalkyl, C₁-C₃ alkyl aminocarbonyl, C₁-Cs alkylthio C₁-C₃ alkyl, halo C₁-C₃ alkyl, hydrazinyl, hydroxy C₁-Cs alkyl, C₃-C₆ cycloalkylamino, hydroxylamino, and C₂-C₃ alkenyl or when Y is C(W)(W) X₁ or X₁' form therewith cyclopropyl ring; - X₂ is hydrogen, hydroxyl, C₁-C₁₀ alkylamino, C₁-C₁₀ alkylamido or

C₁-C₁₀ hydroxyalkyl, C₁-C₁₀ allcyl, R⁸¹R¹³NC(^O)- or HOR⁰-, wherein R^a and R^b may be the same or different and are selected from hydrogen, C₁-C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁₀ BOC-aminoalkyl, and C₃-C₁₀ cycloalkyl or are joined together to form a heterocyclic ring containing two to five carbon atoms, and R^c is selected from Ci-C₁₀ alkyl, amino, C₁-Ci₀ haloalkyl, C₁-Ci₀ aminoalkyl, Ci-C₁₀ BOC-aminoalkyl, and C₃-C₁₀ cycloalkyl, Ci-C₃ alkyl aminocarbonyl, Ci-C₃ alkylthio Ci-C₃ alkyl, halo Ci-C₃ alkyl, hydrazinyl, hydroxy C₁-C₃ alkyl, C₃-C₆ cycloalkylamino, hydroxylamino, and C₂-C₃ alkenyl; X₃ and X₄ 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 X₃ and X₄ are oxygen connected to >C=S to form a 5-membered ring, ureido, Ci-C₆ alkyl carbonylamino, hydroxy C₁-C₆ alkyl, hydrazinyl or X₂ and X₃ form the ring of formula (III):

where R' and R" are independently C₁-C₁₀ alkyl;

R₂ is selected from hydrogen, halo, C₁-C₁₀ alkylether, amino, hydrazido, C₁-C₁₀ alkylamino, C₁-Ci₀ alkoxy, C₁-Ci₀ thioalkoxy, pyridylthio, C₂-C₁₀ alkenyl; C₂-C₁₀ alkynyl, thio, and Ci-C₁₀ alkylthio, C₂-C₆ alkenyloxy,

C₂-C₆ alkynyloxy, C₃-C₈ cycloalkyl Ci-C₆ alkoxy, C₃-C₈ cycloalkenyloxy, C₇-Ci₂ bicycloalkyl Ci-C₆ alkoxy, C₇-Ci₂ bicycloalkenyl Ci-C₆ alkoxy, C₆- Ci₄ aryloxy, C₆-C_M aryl C₁-C₆ alkoxy, C₆-C₁₄ aryl C₃-C₆ cycloalkoxy, C₆-Ci₄ aryl Ci-C₆ alkylamino, C₆-Ci₄ aryl Ci-C₆ alkylthio, Ci-C₆ alkyl C₆-Ci₄ aryl C₁-C₆ alkoxy, C₁-C₆ alkoxy C₆-C₁₄ aryl C₁-C₆ alkoxy, halo C₆-C₁₄ aryloxy, halo C₆-C₁₄ aryl C₁-C₆ alkoxy, C₆-C₁₄ aryl C₁-C₆ alkylamino, C₁-C₆ dialkoxy C₆-C₁₄ aryl C₁-C₆ alkoxy, heterocyclyl C₁-C₆ alkoxy, hydrazinyl, and pyrazolyl, said pyrazolyl being optionally substituted with one or more substituents selected from the group consisting of C₁-C₆ alkyl, C₆-C₁₄ aryl

C₁-C₆ alkyl, C₆-C₁₄ haloaryl C₁-C₆ alkyl, aminocarbonyl, C₁-C₆ alkyl aminocarbonyl, C₁-C₆ alkoxyphenyl, C₆-CH₁₄ haloaryl, and heterocyclyl, and any combination thereof; and

R₃ is a -NR₄R₅ group with R₄ 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 R₄ is hydrogen, R₅ being selected from C₁-C₆ alkyl, C₁-C₆ alkoxy, hydroxy, C₃-C₈ cycloalkyl, C₃-C₈ cycloalkyl C₁-C₆ alkyl, C₃-C₈ dicycloalkyl C₁-C₆ alkyl, C₇-C₁₂ bicycloalkyl C₁-C₆ _ _alkyl,_{. .} C₇-C₁₄. tricycloalkyl C₁-C₆ alkyl, C₆-C₁₄ aryl, C₆-C₁₄ aryl C₁-C₆ alkyl, C₆-C₁₄ diaryl

C₁-C₆ alkyl, C₆-C₁₄ aryl C₁-C₆ alkoxy, heterocyclyl C₁-C₆ alkyl, heterocyclyl, 4-[[[4-[[[(2-amino C₁-C₆ alkyl) amino]-carbonyl]-Ci-C₆ alkyl] aniline] carbonyl] C₁-C₆ alkyl] C₆-C₁₄ aryl, and C₆-C₁₄ aryl C₃-C₈ 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,

C₁-C₆ alkyl, C₁-C₆ alkoxy, C₆-C₁₄ aryloxy, hydroxy C₁-C₆ alkyl, hydroxy C₂- C₆ alkenyl, hydroxy C₂- C₆ alkynyl, aminocarbonyl C₁-C₆ alkoxy, and C₆-C₁₄ aryl C₁-C₆ 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 C₁-C₁₀ alkyl, amino, halo, C₁-C₁₀ haloalkyl, nitro, hydroxyl, acetoamido, C₁-C₁₀ alkoxy, and sulfonic acid or a salt thereof; or R₅ is benzodioxanemethyl, fururyl, L-propylalanyl- aminobenzyl, β-alanylamino- benzyl, T-BOC-β-alanylaminobenzyl, phenylamino, carbamoyl, phenoxy or C₁-C₁₀ cycloalkyl; or R₅ is a group of the following formula (TV):

(IV)

or, when R₄ is alkyl, substituted alkyl, or aryl-NH-C(Z)-, then, R₅ 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 A₃AR agonist is a xanthine-7-riboside derivative of the following general formula (V):

wherein:

X is O or S;

R₆ is R^aR^bNC(=O)- or HOR^C-, wherein - R^a and R^b may be the same or different and are selected from hydrogen, C₁-

C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, and C₃-C_1O cycloalkyl, or are joined together to form a heterocyclic ring containing two to five carbon atoms; and

R^c is selected from C₁-C₁₀ alkyl, amino, C₁-C₁₀ haloalkyl, C₁-C₁₀ aminoalkyl, C₁-C₁O BOC-aminoalkyl and C₃-C₁₀ cycloalkyl; - R₇ and R₈ may be the same or different and are selected from C₁-C₁₀ alkyl,

C₁-C₁₀ 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-C₁₀ alkyl, amino, halo, C₁-C₁₀ haloalkyl, nitro, hydroxyl, acetamido, C₁-Ci₀ alkoxy, and sulfonic acid;

R₉ is selected from the group consisting of halo, benzyl, phenyl, C₃-C₁₀ cyclalkyl, and C₁-C₁₀ alkoxy; or a suitable salt of any of the compounds defined above.

According to a more preferred embodiment, the A₃AR agonist is a nucleoside derivative of the general formula (VII):

wherein X₁, R₂ and R₄ are as defined above.

Preferred A₃AR agonists in accordance with formula (VII) include, although not exclusively, N⁶-benzyladenosine-5'-uronamide derivatives. Some preferred N⁶- benzyladenosine-5'-uronamide derivatives are N⁶-2-(4-aminophenyl)ethyladenosine (APNEA), N⁶-(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-N⁶-(3- iodobenzyl)adenosine- 5'-N-methlyuronamide (Cl-IB-MECA).

According to another preferred embodiment, the A₃AR agonist is a nucleoside derivative of the general formula (VIII):

wherein Y, X₁', R₂ and R₄ are as defined above.

According to yet another preferred embodiment, the A₃AR agonist is a nucleoside derivative of the general formula (IX):

wherein Y, X₂, R₂ and R₄ are as defined above.

Preferred agonists of A₃AR in accordance with formulae (VIII) and (IX) include, although not exclusively, IB-MECA and Cl-IB-MECA derivatives, such as 4¹-(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-dihydroxytetrahydrothiophene-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 A₃AR agonist is N⁶-benzyl- adenosine-S'-alkyluronamide-N^oxide or N⁶-benzyladenosrne-5'-N-dialyl- uronamide-NWide.

The A₃AR 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 A₃AR agonists l-Deoxy-l-[6-[[(3-iodophenyl)methyl]amrno]-9H- purine-9-yl]-N-methyl-b-D-ribofura-nuronamide (IB-MECA) and 2-cbloro-N⁶-(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 A₃AR 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 37⁰C in a 5% CO₂ 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% CO₂ incubator.

³ [H] -thymidine incorporation assay was used to evaluate cell growth. NlSl cells (1.5xlO⁴/ml) were incubated with an A₃AR 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 [³H] -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 (5xl0⁶/50μL saline) were injected to the left hepatic lobe. The rats were divided randomly to two groups: Control group (which received vehicle only) and the A₃AR 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 -8O⁰C until used. After rehydration, 20 μl of solution A [1.25XPCR buffer (200 Mm Tris-HCl, 500 niM KCl)₅ 6.25 mM MgCl₂, 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 95⁰C 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 A₃AR: 5'-ACGGTGAGGTACCACAGCTTGTG (SEQ ID NO.l), and

3'-ATACCGCGGGATGGCAGACC (SEQ ID NO:2). The RT reaction was performed at 45⁰C for 45 min., followed by heating to 99⁰C 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 A₃AR 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 A₃AR protein expression level as well as the expression level of key signaling proteins downstream to A₃A. 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 4⁰C. 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 A₃AR 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 A₃AR 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 OfA₃AR was high in comparison to the receptor level in PBMNC derived from healthy volunteers

In addition, up-regulation in the A₃AR 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 A₃AR 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 A₃AR 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 ICs₀ 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 A₃AR 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).

Claims

CLAIMS:

1. A method of determining disease state in a liver cancer patient, comprising determining level of expression of A₃ adenosine receptor (A₃AR) in white blood cells (WBC) from said patient, wherein a high level of expression is indicative of an active disease state in the patient.

2. A method for determining severity of cancer in a liver cancer patient, comprising:

(a) determining level of expression of A₃AR in WBC from said patient;

3. A method for selecting liver cancer patients to receive a therapeutic treatment that makes use of an A₃AR agonist, comprising determining level of expression of A₃AR in WBC from said patients and selecting patients who have a higher level of A₃AR expression as compared to an average level of A₃AR expression in liver cancer patients.

4. A method for diagnosing liver cancer patients to determine whether their disease is of a kind that is treatable by an A₃AR agonist, comprising determining level of expression of A₃AR in WBC from said patients and selecting patients who have a higher level of A₃AR expression as compared to an average level of A₃AR expression in liver cancer patients.

5. A method according to Claim 3, wherein said selecting comprises:

(a) determining the level of expression of A₃AR in WBC from said liver cancer patients;

(b) comparing said level of expression of A₃AR in the WBC with a level of prior determined or obtained standards; wherein when said level of expression OfA₃AR in WBC from a patient is above the standards, the patient is selected for anti-cancer therapeutic treatment.

6. The method of any one of Claims 1 to 5, wherein said level of expression of A2AR in WBC is compared to a control level, the control level being the level of A₃AR expression in WBC of a healthy subject, or being a standard reference level for A₃AR expression which is indicative of a non-cancerous state.

7. A method for determining effectiveness of an anti-cancer therapeutic treatment of a patient having liver cancer, the treatment comprising administering an A₃AR agonist to the patient, the method comprising detemήning level of expression OfA₃AR 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.

8. The method of claim 7, wherein a first time point is prior to initiation of the treatment and one or more subsequent time points are during the treatment, wherein a decrease in the level of the A₃AR expression in the one or more subsequent time points as compared to the first time point is indicative that the treatment is effective.

9. The method of claim 7 wherein a first time point is during the treatment and one or more subsequent time points are during the treatment subsequent to the first time point, wherein a decrease in the level of the A₃AR expression in the one or more subsequent time points as compared to the first time point is indicative that the treatment is effective.

10. The method of claim 7, wherein a first time point is during the treatment and one or more subsequent time points are after treatment has been discontinued, wherein a increase in the level of the A₃AR expression in the one or more subsequent time points as compared to the first time point is indicative that the treatment is effective.

11. A method for treating a liver cancer patient, comprising:

12. The method of any one of Claims 1 to 11, wherein said WBC comprises mononuclear cells.

13. The method of Claim 12, wherein said mononuclear cells are peripheral blood mononuclear cells (PBMNC).

14. The method of any one of Claims 11 to 13 , wherein said A₃AR agonist is IB- MECA or Cl-IB-MECA. 13-01. txt SEQUENCE LISTING

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