This application is a divisional application of patent application No. 008148007.
Summary of the invention
It has been found according to the present invention that adenosine A3 receptor agonists (A3RAg) have a dual effect in that they inhibit the proliferation of malignant cells on the one hand and they counteract the toxic side effects of chemotherapy on the other hand. The A3RAg compound can definitely inhibit the proliferation and growth of tumor cells, can reduce tumor load by the synergistic effect of the A3RAg compound and an anti-tumor cytotoxin medicament, can induce the proliferation and differentiation of bone marrow cells and leucocytes, and can offset the toxic and side effects of other medicaments, particularly chemotherapeutic medicaments. Furthermore, it has been found according to the present invention that A3RAg may exert these activities by a range of administration forms including parenteral administration, in particular oral administration. It has further been found in accordance with the present invention that some A3RAg activity can be mimicked by other agonists and antagonists of adenosine a1 or a2 receptors: an adenosine A1 receptor agonist (A1RAg) shares the activity of inducing G-CSF secretion with A3 RAg; an adenosine A2 receptor agonist (A2RAg) shares the activity of inhibiting malignant cell proliferation with A3 RAg; and adenosine A2 receptor antagonist (A2RAn) in combination with A3RAg, are responsible for the activity of counteracting toxic side effects of drugs, such as the treatment or prevention of leukopenia.
The invention relates in its broadest sense to the use of an active ingredient to obtain one of the following therapeutic/biological effects: inducing the production or secretion of G-CSF in the body; preventing or treating toxic side effects of a drug or preventing or treating leukopenia, especially drug-induced leukopenia; and inhibiting the growth and proliferation of abnormal cells. The active ingredient may be A3RAg or an agonist or antagonist of the adenosine receptor system which can produce these therapeutic effects, which effects can be obtained by using A3 RAg.
The present invention provides specific embodiments. The first embodiment, referred to as the "G-CSF-inducing embodiment", comprises the use of an active ingredient which may be an A3RAg or an A1RAg capable of causing secretion of G-CSF in the body of a subject. G-CSF is known to stimulate the proliferation and differentiation of hematopoietic cells and to control the functional activity of neutrophils and macrophages. Thus, G-CSF inducers such as those mentioned above may be of high therapeutic value, for example, in counteracting (i.e. preventing, reducing or ameliorating) toxicity in the bone marrow.
This embodiment is based on a method of inducing G-CSF secretion in a subject's body comprising administering to the subject an amount of an active ingredient comprising the group consisting of A3RAg, A1RAg, and a combination of A3RAg and A1 RAg.
Consistent with this embodiment, there is yet another method of therapeutic treatment which comprises administering to a subject an effective amount of the active ingredient required to achieve a therapeutic effect, the therapeutic effect comprising inducing production or secretion of G-CSF. Further provided by this embodiment is the use of said active ingredient for the manufacture of a pharmaceutical composition for inducing secretion of G-CSF. Also provided by this embodiment is a pharmaceutical composition for inducing G-CSF production or secretion in a body comprising a pharmaceutically acceptable load of an effective amount of the active ingredient.
According to another embodiment of the invention, referred to herein as the "leukopenia-prevention embodiment" or more specifically as the "neutropenia-phagocytosis-prevention embodiment", the active ingredient is used for the prevention or treatment of leukopenia resulting from bone marrow toxicity, said active ingredient may be A3RAg, or A2 Ran.
According to this embodiment, there may be provided a method of inducing proliferation or differentiation of bone marrow or leukocytes in a subject, the method comprising administering to the subject an effective amount of an active ingredient selected from the group consisting of A3RAg, adenosine A2Ran, and a combination of A3RAg or A2 Ran. Also provided by this embodiment is a method of preventing or treating leukopenia, the method comprising administering to a subject in need thereof an effective amount of said active ingredient. This embodiment further provides the use of said active ingredient for the manufacture of a pharmaceutical composition for inducing the proliferation or differentiation of bone marrow or leukocytes. This embodiment further provides the use of said active ingredient for the manufacture of a pharmaceutical composition for the prevention or treatment of leukopenia. The pharmaceutical composition can be used for preventing or treating leukopenia.
According to a particular embodiment, referred to as a "toxicity prevention embodiment", the active ingredient described above (i.e., either A3RAg or A2Ran, and combinations thereof) is used to counteract toxic side effects of a drug, such as a chemotherapeutic or a nemoleptic drug.
The latter embodiment provides a method for preventing or treating a toxic side effect of a drug, comprising administering to a subject in need thereof an effective amount of an active ingredient selected from the group consisting of A3RAg, A2RAg and a combination of A3RAg and A2 RAg. This embodiment also provides a use of said active ingredient for the manufacture of a pharmaceutical composition for the prevention or treatment of drug-induced toxicity. This embodiment further provides a pharmaceutical composition for preventing or treating toxic side effects of a medicament comprising an effective amount of said active ingredient and a pharmaceutically acceptable carrier.
In general, to counteract drug-induced leukopenia or most of the drug-induced toxic side effects, it is sometimes desirable to prepare a drug having such toxic side effects for administration with both of the active ingredients. The invention therefore also provides a pharmaceutical composition comprising a drug capable of causing said toxic side effects in a patient to be treated and said active ingredient; the invention also provides the use of said active ingredient for the manufacture of a pharmaceutical composition as such. The active ingredient included in the composition is in an amount effective to prevent or treat the toxic side effects.
According to another embodiment, referred to as the "proliferation-inhibiting embodiment", the active ingredient may be used to selectively inhibit the growth of abnormal cells, such as tumor cells, and may be A3RAg, A2RAg, or a combination of both.
According to this embodiment there may be provided a method of inhibiting abnormal cell growth in a subject, the method comprising administering to the subject a therapeutic amount of an active ingredient selected from the group consisting of A3RAg, A2RAg and a combination of A3RAg and A2 RAg. This embodiment also provides a use of said active ingredient for the manufacture of a pharmaceutical composition for inhibiting abnormal cell growth. This embodiment further provides a pharmaceutical composition for inhibiting abnormal cell growth comprising said active ingredient and a pharmaceutically acceptable carrier.
In an embodiment of the invention, the active ingredients administered are intended to achieve a dual therapeutic effect: inhibiting the growth of abnormal cells and reducing the toxic side effects of drugs that can cause such side effects.
The preferred active ingredient of the present invention is A3 RAg. The preferred route of administration of the active ingredients of the present invention is oral. However, this preference does not exclude other active ingredients or routes of administration of active ingredients.
The dose of the active ingredient administered, particularly when the active ingredient therein is A3RAg, is preferably less than 100 μ g/kg body weight, typically less than 50 μ g and more desirably 1-10 μ g/kg body weight.
Detailed description of the invention
According to the present invention, certain active ingredients, particularly adenosine receptor agonists and antagonists, can provide novel therapeutic uses. In one embodiment, G-CSF-inducing embodiments, some such active ingredients are used to indirectly produce and secrete G-CSF from cells. In another embodiment, toxicity prevention embodiments, some of these active ingredients are used to counteract toxic side effects of drugs such as chemotherapy or nemoleptic drugs. In a further embodiment, leukopenia-preventing embodiments, some of such active ingredients are used to counteract leukopenia, particularly drug-induced leukopenia. Also in accordance with an embodiment of the present invention, a proliferation-inhibiting embodiment, some of these active ingredients are used to selectively inhibit the growth of abnormal cells.
The definition of "leukopenia" as used herein relates to a reduction in the number of circulating leukocytes. Although leukopenia is characterized by a reduction in the number of neutrophils phagocytes in the blood (neutropenia), it may sometimes be found that a reduction in the number of lymphocytes, monocytes, eosinophils or basophils occurs.
Leukopenia resulting from decreased neutrophil production or excessive spleen segregation may be caused by genetic or congenital diseases. However, leukopenia has been observed mainly after treatment with drugs such as cytopenic (cytoreductive) cancer drugs, antithyroid drugs, phenothiazines, anticonvulsants, penicillins, sulfonamides and chloramphenicol. Leukopenia caused by some antineoplastic agents is a predictable side effect.
Hereinafter, the reduction in the number of leukocytes or the number of neutrophils phagocytosed due to the drug will be defined herein as "drug-induced leukopenia" or "drug-induced neutropenia". And whenever cytopenia is mentioned, it should be understood that "neutropenia" is specifically concerned.
Furthermore, the definition "prevention and treatment of leukopenia" is to be understood as meaning that the process of reduction of the number of leukocytes which otherwise may occur is reduced, a complete prevention or a manipulation of increasing the number of leukocytes if such a reduction has already taken place. Leukopenia may be manifested by a range of side effects, such as an increased likelihood of infection with some important and other infectious agents. The definition "prophylaxis or treatment of leukopenia" may also be understood as meaning the improvement of such parameters representing the outcome of leukopenia.
The pharmaceutically or therapeutically effective amount herein is determined by a number of factors which may be known in the art. The amount must be effective to achieve the desired therapeutic effect, depending on the type and method of treatment. It will be apparent to the skilled artisan that the amount is an amount effective to improve survival, allow the patient to heal quickly, provide symptom relief or elimination, or other suitable indicator selected by one of skill in the art. For example, when the active ingredient is administered to induce the production of G-CSF, an effective amount of the active ingredient can be an amount that produces and secretes G-CSF from peripheral blood mononuclear cells, endothelial cells, or fibroblasts in which G-CSF is produced, e.g., to stimulate the conversion of mature granulocyte progenitor particles into mature neutrophil phagemids. In the case where the active ingredient is administered to counteract drug-induced leukopenia, the effective amount of the active ingredient may be an amount that protects the individual from a drug-induced reduction in the number of leukocytes, particularly neutrophilic granulocytes; may be an increase in the amount of active ingredient that has reduced such cells, e.g., returned to normal levels or sometimes higher than normal levels; and so on. In the case where the active ingredient is administered for reducing the toxic side effects of the drug, the amount of the active ingredient may be, for example, an amount effective to recover the body weight loss due to the administration. In the case where the active ingredient is administered for the purpose of inhibiting abnormal cell growth, as described in detail hereinafter, the effective amount may be an amount that inhibits such cell proliferation and even eliminates tumors in the subject. In the case where the active ingredient is administered to enhance the effect of an anti-cancer chemotherapeutic agent, the effective amount may be an amount that enhances the cancer specific drug toxicity of the chemotherapeutic treatment; or to achieve the desired effect but reduce the amount of chemotherapeutic agent or pharmaceutical composition required, i.e., reduce tumor burden; and so on. An effective amount of the embodiment is one in which the A3RAg is administered in an amount of less than 100 μ g/kg body weight per day, typically less than 50 μ g/kg body weight and preferably less than 10 μ g/kg body weight, e.g., about 3-6 μ g/kg body weight. Such A3RAg amounts are still representative of single daily doses, although such daily doses may sometimes be divided into several doses for administration within a day or the doses for several days combined into a single dose for administration to a patient every few days, particularly in the case of sustained release formulations.
According to the invention, the active ingredient is preferably A3 RAg. A3RAg is a non-selective agonist (any agonist) which binds to the A3 receptor and then activates the A3 receptor to produce a therapeutic effect of the present invention. It should be noted that sometimes the A3 receptor also interacts with other receptors such as the a1 and a2 receptors. However, the A3RAg used according to the invention exerts a major effect via the A3 receptor (i.e. may exert some minor effect by interacting with other adenosine receptors).
By way of embodiment, the active ingredient on which the invention is based is a nucleoside derivative. The term "nucleoside" means any compound comprising a sugar, preferably ribose or deoxyribose, or a purine or pyrimidine base or a combination of a sugar and a purine or pyrimidine base, preferably linked via an N-glycosyl bond. The definition of "nucleoside derivative" herein means a natural nucleoside, a synthetic nucleoside or a nucleoside obtained by the insertion, deletion or chemical modification of the groups outside and inside the ring or a derivative having a desired biological effect obtained by conformational modification thereof as defined above.
A preferred embodiment of the active ingredient of the present invention is A3 RAg.
According to an embodiment of the present invention, the active ingredient is a nucleoside derivative represented by the following general formula (I):
wherein R is1Is C1-C10Alkyl radical, C1-C10Hydroxyalkyl radical, C1-C10Carboxyalkyl or C1-C10Cyanoalkyl or a group represented by the following general formula (II):
wherein:
-Y is an oxygen, sulfur or carbon atom;
-X1is H, C1-C10Alkyl radical, RaRbNC (═ O) -or HORc-, wherein RaAnd RbPossibly identical or different radicals selected from hydrogen, C1-C10Alkyl, amino, C1-C10Haloalkyl, C1-C10Aminoalkyl radical, C1-C10BOC-aminoalkyl, and C3-C10Cycloalkyl or are linked to each other to form a heterocyclic ring containing 2 to 5 carbon atoms, and RcIs selected from C1-C10Alkyl, amino, C1-C10Haloalkyl, C1-C10Aminoalkyl radical, C1-C10BOC-aminoalkyl and C3-C10A cycloalkyl group; -X2Is H, hydroxy, C1-C10Alkylamino radical, C1-C10Alkylamido or C1-C10A hydroxyalkyl group;
-X3and X4Respectively hydrogen, hydroxy, amino, amido, azido, halogen, alkyl, alkoxy, carboxyl, nitrilo, nitro, trifluoro, aryl, alkaryl, mercapto, thioester, thioether, -OCOPh, -OC (═ S) OPh or X3And X4Are all oxygen which is linked to > C ═ S to form a 5-membered ring, or X2And X3Forming a ring of formula (III):
wherein R 'and R' are each C1-C10An alkyl group;
-R2selected from hydrogen, halogen, C1-C10Alkyl ether, amino, hydrazide group, C1-C10Alkylamino radical, C1-C10Alkoxy radical, C1-C10Thioalkoxy, thiopyridyl, C2-C10An alkenyl group; c2-C10Alkynyl, mercapto and C1-C10A thioalkyl group; and
-R3is-NR4R5Radical, R4Is hydrogen or is selected from alkyl, substituted alkyl or aryl-NH-C (Z) -, Z is O, S, or NRa，RaThe meaning of (a) is as above,
-and R5At R4In the case of hydrogen, R5Selected from the substituents unsubstituted or taken at one or more positionsSubstituted cis-and trans-1-phenylethyl, benzyl, phenylethyl or acylanilino groups, said substituents being selected from C1-C10Alkyl, amino, halogen, C1-C10Haloalkyl, nitro, hydroxy, acetylamino, C1-C10Alkoxy and sulfonic acids or their salts; or R4Is benzodioxane methyl, furan methyl, L-propylalanylaminobenzyl, beta-alanylamino-benzyl, T-BOC-beta-alanylamino-benzyl, phenylamino, carbamoyl, phenoxy or C1-C10A cycloalkyl group; or R5Is a group of the formula:
or a suitable salt of a compound as defined above, such as the triethylamine salt; or when R is4When selected from the group consisting of alkyl, substituted alkyl, or aryl-NH-C (Z) -R5Is selected from substituted or unsubstituted heteroaryl-NRa-C (Z) -, heteroaryl-C (Z) -, alkylaryl-NRaThe groups of-C (Z) -, alkylaryl-C (Z) -, aryl-NR-C (Z) -, and aryl-C (Z) -; wherein Z is as defined above.
According to this embodiment of the invention, the active ingredient is preferably a nucleoside derivative represented by the general formula (IV):
wherein X1、R2And R4The definition of (A) is as above.
Preferred active ingredients according to this embodiment of the invention are N6-benzyladenosine-5' -uronamide and derivatives thereof having A3-selective adenosine receptor agonist function in general. Examples of such derivatives are N6-2- (4-aminobenzene)Yl) Ethyl adenosine (APNEA), N6- (4-amino-3-iodobenzyl) adenosine-5' - (N-methylguronamide) (AB-MECA) and 1-deoxy-1- {6- [ ({ 3-iodophenyl } methyl) amino]-9H-purin-9-yl } -N-methyl- β -D-ribofuranose-amide, the latter also known in the art as N63-iodobenzyl-5 '-methylcarboxyamidoadenosine, N6- (3-iodobenzyl) adenosine-5' -N-methylguronamide and chlorinated derivatives herein before and hereinafter abbreviated to IB-MECA or IB-MECA (R)2Ci), the latter being referred to herein as Cl-IB-MECA, IB-MECA and Cl-IB-MECA being particularly preferred in the present invention.
According to another embodiment of the invention, the active ingredient may be what is generally referred to as N6-benzyl-adenosine-5' -alkyluronamide-N-oxide or N6-benzyladenosine-5' -N-dialkylfurfural-amide-N1-adenosine derivatives of oxides.
Further, the active ingredient may be a xanthine-7-ribonucleoside derivative represented by the following general formula (V):
wherein X is O or S;
R6is RaRbNC (═ 0) -or HOPc-, wherein
-RaAnd RbMay be the same or different and is selected from hydrogen, C1-C10Alkyl, amino, C1-C10Haloalkyl, C1-C10Aminoalkyl and C3-C10Cycloalkyl, or linked together to form a heterocyclic ring containing 2 to 5 carbon atoms; and
-Rcis selected from C1-C10Alkyl, amino, C1-C10Haloalkyl, C1-C10Aminoalkyl radical, C1-C10BOC-aminoalkyl and C3-C10A cycloalkyl group;
-R7and R8May be the same or different and is selected from alkyl, C1-C10Cycloalkyl, cis-or trans-1-phenylethyl, unsubstituted benzyl or acylanilino and phenyl ethers in which the phenyl radical is substituted in one or more positions by substituents selected from the group consisting of C1-C10Alkyl, amino, halogen, C1-C10Haloalkyl, nitro, hydroxy, acetylamino, C1-C10Alkoxy and sulfonic acid;
-R9optional groups include halogen, benzyl, phenyl, C3-C10Cycloalkyl and C1-C10An alkoxy group;
or salts of such compounds, such as their triethylamine salts.
Some of the above defined compounds and their synthesis are described in detail in US 5,688,744; US 5,773,423, US 6,048,865, WO 95/02604, WO 99/20284 and WO 99/06053, which are listed herein by reference.
The active ingredient in the case of a GSF-inducing embodiment may be A1 Rag. It is a representative adenosine derivative of the formula
-R1Represents lower alkyl, cycloalkyl, preferably C3-C8Cycloalkyl (including the well-known cyclohexyl and cyclopentyl derivatives, denoted as CPA and CHA, respectively), which may be substituted, e.g., by hydroxy or lower alkyl; r1May also represent hydroxy or hydroxyalkyl; phenyl, anilide, or lower alkylphenyl, all of which may be substituted by one or moreSubstituted by substituents, e.g. halogen, lower alkyl, haloalkyl such as trifluoromethyl, nitro, cyano, - (CH)2)mCO2Ra、-(CH2)mCONR2RaRb、-(CH2)mCORaWherein m represents an integer from 1 to 6; -SORc、-SO2Rc、-SO3H、-SO2NRaRb、-ORa、-SRa、-NHSO2Rc、-NHCORa、-NRaRbor-NHRaCO2Rb(ii) a Wherein
-RaAnd RbRespectively represents hydrogen, lower alkyl, alkanoyl, phenyl or naphthyl (the latter may be partially saturated), the alkyl group may be optionally substituted by substituted or unsubstituted phenyl or phenoxy; or when R is1represents-NRaRbWhen said R isaAnd RbTogether with the nitrogen atom, form a 5-or 6-membered heterocyclic ring optionally containing a second heteroatom selected from oxygen or nitrogen, wherein the second heteroatom may optionally be further substituted by hydrogen or lower alkyl; or-NRaBbIs a group represented by the following general formula (VII) or (VIII):
wherein n is an integer from 1 to 4;
-Z is hydrogen, lower alkyl or hydroxy;
-Y is hydrogen, lower alkyl, OR ', wherein R' is hydrogen, lower alkyl OR lower alkanoyl;
-a is a bond or lower alkenyl, preferably C1-C4 alkenyl; and
-X and X 'are independently hydrogen, lower alkyl, lower alkoxy, hydroxy, lower alkanoyl, nitro, haloalkyl such as trifluoromethyl, halogen, amino, mono-or di-lower alkylamino, or when X and X' are combined to form methylenedioxy;
-Rcrepresents lower alkyl;
R2represents hydrogen, halogen, substituted or unsubstituted lower alkyl or alkenyl, lower haloalkyl or haloalkenyl, cyano, acetylamino, lower alkoxy, lower alkylamino, NRdReWherein R isdAnd ReRespectively hydrogen, lower alkyl, phenyl or phenyl substituted by lower alkyl, lower alkoxy, halogen or haloalkyl such as trifluoromethyl or alkoxy, or-SRfWherein R isfIs hydrogen, lower alkyl, lower alkanoyl, benzoyl or phenyl; -W represents-OCH2-、-NHCH2-、-SCH2-or-NH (C ═ O) -;
-R3、R4and R5Each represents hydrogen, lower alkyl or lower alkenyl, branched or straight chain C1-C12Alkanoyl, benzoyl or benzoyl substituted by lower alkyl, lower alkoxy, halogen, or R4And R5Forming a five-membered ring which may be optionally substituted with a lower alkyl or alkenyl group; furthermore R3And may also represent, alone, a phosphate, monohydrogen or dihydrogen phosphate, or an alkali metal or ammonium or dialkali metal or diammonium salt thereof;
-R6represents hydrogen, halogen atom or
A portion of the R groups (i.e. R)1To R6) Is of the formula Rg-SO3-RhA sulfohydrocarbon compound of (a), wherein R isgThe group represented is selected from C1-C10Aliphatic compounds, phenyl and lower alkyl substituted by aromatic group, wherein the aromatic group as a substituent may be a substituted or unsubstituted aromatic group, RhRepresents a monovalent cation. Suitable monovalent cations include lithium, sodium, potassium, ammonium or trialkylammoniumThey dissociate under physiological conditions. The remaining R groups are hydrogen or halogen atoms, unsubstituted hydrocarbons or any other non-sulfur containing groups as defined above.
The hydrocarbon chain as used herein may be straight or branched. It is especially noted that the definition "alkyl" or "alkenyl" as used herein refers to straight or branched chain alkyl or alkenyl groups. The definition of "lower alkyl" or "lower alkenyl" refers to C respectively1-C10Alkyl or C2-C10Alkenyl, preferably C1-C6Alkyl and C2-C6An alkenyl group.
A preferred adenosine derivative of formula (VI) is N6Cyclopentyladenosine (CPA), 2-chloro-CPA (CCPA), and N6Cyclohexyl adenosine (CHA) derivatives, the preparation of these compounds being well known to the person skilled in the art. Other well known adenosine derivatives of the A1 receptor of choice are those in which R1Compounds which are acylanilino groups, wherein acylanilino group may be unsubstituted or substituted, e.g. by hydroxy, alkyl, alkoxy or-CH2C (O) R 'wherein R' is hydroxy, -NHCH3、-NHCH2CO2C2H5(ethylglycinate), tuloidide (wherein the methyl moiety may also be replaced by haloalkyl), or CH2C(O)NHC6H4CH2C (O) R * wherein R * represents a group which affords a methoxy substituent, an amide substituent (e.g. R * is-NHCH)3) Or R * is hydrazide, 1, 2-diethylamine, -NHC2H5NHC(O)CH34- (hydroxy-phenyl) propionyl, biotinylated ethylenediamine or any other suitable hydrocarbon which may represent the above compound and an A1 agonist.
N used as an active ingredient according to the invention6Substituted adenosine derivatives may also be those containing an epoxide moiety, more particularly cycloalkyl epoxides containing adenosine derivatives (e.g. oxabicyclo or oxabicycloOxatricycles, the former, e.g. norbornyl, and the latter, e.g. adamantyl). Some of these compounds may be defined by the general formula (I),
wherein R is1Are groups represented by the following general formulae (IXa) and (IXb):
wherein M is lower alkyl as defined above.
With epoxidised N6Embodiments of the agonist compounds of norbornyl group include the endo and exo isomers, and more particularly may be one of the following four isomers: 2R-exo, 2R-endo, 2S-exo and 2S-endo.
Another one of N6An embodiment of the norbornyl derivative is at the purine Ring N1The-site contains one oxygen atom. This compound is referred to as N6- (5, 6-epoxynorborn-2-yl) adenosine-1-oxide.
In some cases, A1RAg may be an adenine derivative in which the β -D-ribosylyl moiety of adenosine may be replaced by hydrogen or phenyl.
A2Ran, which can be used according to the invention, is an 8-styryl derivative (X) of a1, 3, 7-substituted xanthine of the following formula (X):
wherein R is1And R3Is C1-C4Alkyl, allyl or propargyl
R7Is H, methyl or C2-C8Alkyl radical
n is 1 to 3
And X is halogen, trifluoroalkyl, alkoxy, hydroxy, nitro, amino, dialkylamino, diazo, isothiocyanate, benzyloxy, aminoalkoxy, alkoxycarbonylamino, acetoxy, acetylamino, succinylamino, 4- (4-NH)2-trans-CH2CH＝CHCH2O-3，5-(MeO)24- (4-AcNH-trans-CH)2CH＝CHCH2O)-3，5-(MeO)24- (4-tert-BOC-NH-trans-CH)2CH＝CHCH2O)-3，5-(MeO)2
A specific example of a compound of formula (X) is (3, 7-dimethyl-1-propargyl-xanthane).
A2Ran may be a compound of the formula:
or
As will be appreciated, the present invention is not limited to the above-mentioned A3RAg, A2RAg or A2Ran compounds.
The compounds on which the invention is based may be the compounds as defined above or may also be in the form of their salts or solvates, especially their physiologically acceptable salts and solvates. In addition, when containing one or more asymmetric carbon atoms, the active ingredient may include isomers and non-corresponding stereoisomers of the active ingredients described above or mixtures thereof.
The pharmaceutically acceptable salts of the above active ingredients include salts of these active ingredients with pharmaceutically acceptable organic and inorganic acids. Examples of suitable acids include nicotinic acid, hydrobromic acid, sulfonic acid, nitric acid, perchloric acid, fumaric acid, maleic acid, phosphoric acid, glycolic acid, lactic acid, salicylic acid, succinic acid, p-toluenesulfonic acid, citric acid acetate, methanesulfonic acid, formic acid, benzoic acid, malonic acid, naphthalene-2-sulfonic acid, and benzenesulfonic acid.
The active ingredient may be administered as an inactive substance (e.g. a prodrug) which is then activated by further modification at a particular site in the subject by its natural treatment. In some cases, the therapeutic function of the derivative, such as a pharmaceutical composition of the invention, is retained. These prodrugs also fall within the definition of "active ingredient" as used herein. Similarly, the definitions of "A3 RAg", "A1 RAn", "A2 RAg" and "A2 RAn" should also be understood to include prodrugs which, although they are precursors which lack antagonist or antagonistic activity (as the case may be), are active in vivo.
According to the present invention, A3RAg can be screened to select compounds with properties similar to IB-MECA activity. For example, according to the leukopenia inhibition embodiment, such compounds may be used in accordance with their ability to stimulate proliferation of bone marrow cells and leukocytes and their subsequent ability to exert efficacy in vivo. Compounds in the proliferation-inhibiting embodiments can be screened for their ability to inhibit tumor cell proliferation and subsequently exert this activity in vivo.
The activity of A1RAn and A2RAn can be determined and screening can be performed using methods similar to those described for A3RAg, mutatis mutandis.
The pharmaceutical compositions of the present invention may comprise the active ingredient alone or in combination with other ingredients such as pharmaceutically acceptable carriers, diluents, excipients, additives and/or adjuvants, such as flavoring agents, coloring agents, lubricants and other pharmaceutical ingredients well known to those skilled in the art for such purpose. It will be apparent that the pharmaceutically acceptable carriers, diluents, excipients, additives used in the present invention are generally inert, non-toxic solid or liquid fillers, diluents or encapsulating materials, preferably materials which do not react with the composition of the invention.
In addition, the active ingredient may also be administered in combination with a chemotherapeutic agent, particularly in the case of the leukopenia prevention embodiment. Thus, the pharmaceutical composition of the invention may comprise a chemotherapeutic agent in addition to said active ingredient. According to some embodiments of the invention, the chemotherapeutic agent is an anti-cancer chemotherapeutic agent. It should be understood that the term means any cytotoxic drug or cocktail containing a mixture of two or more cytotoxic drugs administered to a patient for the purpose of reducing the tumor mass of the patient.
The invention finds that A3RAg is orally bioavailable and can exert dual therapeutic effects (reduction of abnormal cell proliferation and prevention or reduction of leukopenia) when administered orally. Thus, according to a preferred embodiment, the pharmaceutical composition of the invention is prepared for oral administration. Such oral compositions may further comprise pharmaceutically acceptable carriers, diluents, excipients, additives or adjuvants suitable for oral administration.
In the G-CSF-inducing embodiments of the present invention, the disclosed pharmaceutical compositions are particularly useful for increasing the level of G-CSF secreted from a cell. Such compounds can be used to promote the recovery of neutrophils or inhibit abnormal cell growth following chemotherapy and bone marrow transplantation. To date, such treatment packages have been administered with growth factors, and such treatment methods are known to have some undesirable side effects. Furthermore, it is well known that the average cost of G-CSF therapy is high.
Within the scope of the leukopenia prevention embodiments or toxicity prevention embodiments of the present invention, the disclosed pharmaceutical compositions are used to elevate the level of leukocytes in the circulatory system or counteract some other toxic side effects, such as weight loss. This aspect of the invention may be used in a variety of clinical situations. It is clear that a decrease in circulating leukocytes, particularly neutrophils, can lead to a weakening of the immune system. Examples of immune system attenuation that can be treated with this aspect of the invention are leukopenia that occurs frequently in pre-cancer or is drug-induced or neutropenia-induced by drugs.
The proliferation-suppressing embodiments are useful for treating a variety of abnormalities associated with abnormal cell growth, such as cancer, psoriasis, and some autoimmune diseases. The compositions of the invention are particularly useful for inhibiting the proliferation of tumor cells, preferably for anticancer therapy.
When A3RAg was used to treat lymphoid tumour tissue cells, the inhibition of proliferation of these cells was much more pronounced than that of adenosine or 'a 1' or 'A2' agonists, although some activity could also be found with A2RAg (see figure 5A). These results suggest that inhibition of tumor cell proliferation should be primarily due to binding of A3RAg to its receptor, but to some extent can also be mimicked by A2 RAg. The above surprising results thus provide a new therapeutic target for future anti-cancer cell inhibitor drugs.
Furthermore, it was further found that A3RAgs can also be effective in inhibiting the growth of other tumor cells besides lymphoma, such as melanoma or colon tumor (see fig. 6). It will be clear to those skilled in the art that the advantages of treating a patient with a non-specific anti-cancer agent that inhibits the growth of abnormally dividing cells while reconstituting the patient's immune system by inducing bone marrow cell proliferation are anticipated.
FIGS. 7A-7B, for example, show the different effects of A3 RAg. In this particular case, the effect of IB-MECA on tumor and normal cells was evaluated. The more pronounced effect obtained with A3RAg compared to adenosine can be more clearly shown by these results. The therapeutic effect of A3RAg was reversible when using the A3 receptor antagonist, MRS-1220.
In vivo studies confirmed the results of in vitro studies, comparing the results of concurrent treatment of mice with A3RAg and a cytotoxic agent to the results of treatment of mice with cytotoxic drugs alone, A3RAg showed a chemoprotective effect on mice (see figure 8). In addition, a decrease in lesion number was found in vivo in mice treated with A3RAg, indicating the chemotherapeutic activity of A3RAg (see figure 9). FIGS. 10A-10B and 19A and 19B, for example, show that tumor-bearing mice treated with cytotoxic drugs alone exhibited a decrease in leukocytes and neutrophils in the peripheral blood, while A3RAg administration following chemotherapy resulted in a restoration of total leukocytes and an increase in the percentage neutrophils.
Therefore, it can be concluded that A3RAg has dual therapeutic effects, and can be used as a chemotherapeutic agent as well as a chemoprotectant. It is clear that the application of this dual action of the A3RAg is also within the scope of the present invention.
In any event, the administration and dosage of the pharmaceutical compositions of the present invention should be in accordance with good medical practice, taking into account the clinical condition of each patient, the site and method of administration, the timing of administration, the age, sex, weight and other factors well known to some medical practitioner.
The compositions of the present invention may be administered in a variety of ways. Administration can be oral, subcutaneous, parenteral, including intravenous, intraarterial, intramuscular, intraperitoneal, or by intranasal administration, as well as intrathecal and infusion administration as is well known to those of skill in the art.
It is well known that the course of treatment in humans is generally longer than that in animals, such as the mice exemplified herein. The duration of treatment corresponds to the course of the disease and the efficacy of the active ingredient. Treatment regimens include treatment with single or multiple doses over a period of several days or longer. The duration of treatment generally follows the course of the disease, the efficacy of the active ingredient and the type of patient being treated.
When the compositions of the present invention are administered parenterally, they are generally formulated in unit dose injectable forms (solutions, suspensions, emulsions). Pharmaceutical formulations suitable for injection include sterile aqueous solutions or dispersions and sterile powders which may be reconstituted into sterile aqueous solutions or dispersions. Carriers that can be used as solvents and dispersion media include, for example, water, ethanol, polyols (such as glycerol, propylene glycol, liquid polyethylene glycols, and the like), suitable mixtures thereof, and vegetable oils.
Nonaqueous vehicles, sometimes also used as solvent systems for the active ingredient, such as cottonseed oil, sesame oil, olive oil, soybean oil, corn oil, sunflower oil, or peanut oil and esters, such as isopropyl myristate.
In addition, various additives which enhance the stability, sterility and isotonicity of the composition may be added, including antimicrobial preservatives, antioxidants, chelating agents and buffers. The prevention of the action of microorganisms can be enhanced by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.
For oral administration, the active ingredient may be presented in the form of tablets, suspensions, solutions, emulsions, capsules, powders, syrups and the like, which are readily available to and used by the pharmacist by means of well-known techniques.
The invention, the contents of which are considered to have been disclosed in the specification, is defined by the claims and will now be described by way of example with reference to the accompanying drawings. It is to be understood that the terminology, which has been used, is intended to be in the nature of words of description rather than of limitation.
Although the foregoing specification describes in detail a few specific embodiments of the present invention, it will be understood by those skilled in the art that the present invention is not limited thereto but may be susceptible to other changes in form and detail without departing from the scope and spirit of the invention disclosed herein.
Example 1: adenosine and adenosine receptor antagonists and agonists on G-CSF production and myeloid cell proliferation
To verify the hypothesis that adenosine plays a role by stimulating G-CSF production, normal cell culture was performed in the presence of adenosine or adenosine agonists or antagonists.
For this purpose, bone marrow cells obtained from C57BL/6J or ICR mouse femurs were first dispersed by a 25G probe. Cells (3X 10' cells/well in 96 well microplates) were then cultured in RPMI medium containing 10% Fetal Bovine Serum (FBS) in the presence of adenosine (25. mu.M). Adenosine or A1 and A3 adenosine receptor agonists-CPA (an A1RAg, 0.01. mu.M), CCPA (an A1RAg, 0.01. mu.M), or IB-MECA (an A3RAg, 0.01. mu.M) were added to bone marrow cultures in the absence of adenosine; an a1 adenosine receptor antagonist, DPCPX (0.1 μ M) was added to bone marrow cultures in the presence of adenosine (25 μ M).
Cultures containing RPMI media cell suspension and 5% FBS were used as controls for the above experiments.
Use 23H]Thymidine binding assays to assess the proliferation of bone marrow cells. For this, 1. mu. Ci of [3H ] per well was used after 30 hours of incubation]Thymidine is pulsed. After 48 hours of culture, cell harvest was performed and [3H ] measured with a liquid scintillation counter (LKB, Piscataway, NJ, USA)]Thymidine uptake. The test results are shown in FIG. 1, which shows that A1RAg or A3RAg has a G-CSF producing function, similar to those obtained with adenosine.
To demonstrate that adenosine and its agonists can act by stimulating G-CSF production, a further experiment was performed in which anti-G-CSF antibody (62.5ng/ml) was added to bone marrow cell cultures in the presence of adenosine (25. mu.M), CPA (0.01. mu.M) or IB-MECA (0.01. mu.M). Cell proliferation was assessed as described above. The results of this experiment are shown in FIG. 2, which shows that antibodies to G-CSF inhibit the stimulatory effect of adenosine and its agonists on myeloid cell proliferation. This result indicates that at least a part of the activity involved in the interaction with adenosine receptors is exerted by the production of G-CSF.
The cumulative effect on bone marrow cell proliferation was evaluated when using A1RAg and A3RAg (CPA and IB-MECA) conjugates. The operation of this test was performed using a similar test to the test results as indicated in fig. 1. After being dispersed, the cells were cultured in the presence of adenosine (25. mu.M), CPA (0.01. mu.M), IB-MECA (0.01. mu.M) or a combination of IB-MECA and CPA (each at a concentration of 0.01. mu.M), and then further processed by the method described above. The results are shown in FIG. 3A, which shows the enhanced effect of IB-MECA and CPA.
To compare the effect of adenosine receptor antagonists on bone marrow cell proliferation after using the above method, cell culture was performed with adenosine alone or in combination with DMPX (an A2RAn), DPCPX (an A1RAn), MRS-1220 (an A3RAn) or a combination of DPCPX and MRS-1220. The results are shown in FIG. 3B. As can be seen, blockade of the a2 receptor by DMPX can also result in increased bone marrow cell proliferation, which exceeds even adenosine alone. In contrast, the increase in proliferation with DPCPX or MRS-1220 was reduced by 50% compared to adenosine alone, whereas DPCPX in combination with MRS-1220 completely inhibited proliferation.
Cells pretreated as described above were cultured with different concentrations of IB-MECA (1. mu.M, 0.1. mu.M or 0.01. mu.M). By [ H ]3]Thymidine binding assay measures the percent stimulation. The results are shown in FIG. 3, which shows that IB-MECA stimulates bone marrow augmentation in a dose-dependent mannerAnd (4) breeding.
Example 2: modulation of tumor cell growth by adenosine and agonists thereof
Nb2-11C rat lymphoma cell (1.2X 10)4Individual cells/ml) were cultured in 96-well microplate, 1ml of RPMI medium containing 5% fetal bovine serum for 48 hours. To this was added 25 μ M adenosine, 0.01 μ M adenosine receptor agonist (CPA, an A1 RAg; DPMA, an A2RAg or IB-MECA, an A3RAg) or 0.1 μ M adenosine receptor antagonist (DPCPX, an A1 RAn; DMPX, an A2 RAn; or MRS-1220, an A3RAn) that binds adenosine (25 μ M).
Cultures of RPMI media cell suspensions containing 5% FBS were used as controls for the above experiments. The extent of cell proliferation was determined by cytometric analysis.
The results of comparison with adenosine inhibition are shown in FIGS. 5A and 5B. As can be seen, the proliferation of Nb2-11C cells was significantly inhibited after culture with IB-MECA, an A3 RAg. No growth inhibition was observed with CPA, an A1RAg, and a weaker growth inhibition was found with DPMA, an A2 Ran. CPA failed to inhibit the proliferation of both tumor cells, indicating that the adenosine a1 receptor does not possess this activity. However, the inhibitory effects of DMPA and IB-MECA suggest that the A2 and A3 adenosine receptors, respectively, play a role in the inhibitory effects.
Furthermore, it can be seen that the effect of adenosine on the proliferation of Nb2-11C cells is essentially abolished in the presence of DPCPX, an A1Ran, which is essentially ineffective, whereas in the presence of MRS-1220, an A3Ran, the effect of adenosine is essentially abolished. DMPA, an A2Ran, may play a secondary but still important role. From these findings, it can be concluded that the growth of tumor cells can be effectively inhibited by A3RAg or A2 Ran.
Inhibition of growth of B-16 melanoma, HCT-116 colon tumor and Nb2-11C lymphoma by A3RAg, IB-MECA was evaluated in the same manner as described above. The results are shown in FIG. 6, expressed as a percentage of inhibition or proliferation.
Example 3: differential effects of adenosine A3 receptor agonists on tumor cells and normal cells
The effect of adenosine, A3RAns and A3RAgs on tumor cell growth was examined using the assay described above.
Briefly, Nb2-11C lymphoma or bone marrow cell cultures were performed in the presence of adenosine, or IB-MECA. The dual effect of A3RAg, as shown in fig. 7A and 7B, inhibited tumor cell growth while stimulating bone marrow cell proliferation.
Example 4: in vivo studies
The 40C 57BL6/J mice were divided into four groups, each group being treated with one of the following protocols:
1. control group: each mouse was injected intraperitoneally (i.p.) daily with 1ml of physiological saline from the first day of tumor inoculation until the mice were sacrificed;
2. chemotherapy groups: on the one hand cyclophosphamide is injected i.p. 24 hours after tumor cell inoculation, and simultaneously 1ml physiological saline is injected i.p. per mouse every day from the first day of tumor inoculation to the time when the mouse is sacrificed;
3. adenosine A3 receptor agonist (A3RAg) group: IB-MECA was orally administered daily from the day of tumor inoculation until mice were sacrificed
A3RAg + chemotherapy group: on the one hand, cyclophosphamide is injected i.p. 24 hours after tumor cell inoculation, and IB-MECA is orally administered at 3 mug/kg body weight every day
Blood was taken from the tail vein of the mice on days 5 and 9 and blood samples were obtained for counting White Blood Cell (WBC) numbers. The results are shown in FIG. 8.
In addition, mice were sacrificed 18 days later and melanoma tumor lesions in their lungs were calculated. The results are shown in FIG. 9.
To evaluate the chemoprotective effect of A3RAg, further experiments were performed. Mice were treated with cyclophosphamide (50mg/kg body weight in 0.3ml PBS). At 48 and 72 hours after cytotoxic drug administration, mice were i.p. injected with adenosine (25. mu.g/kg body weight) or IB-MECA (3 or 6. mu.g/kg in 0.2ml PBS). White Blood Cell (WBC) number and neutrophil phagocytic cell number were examined. The results are shown in FIGS. 10A (WBCs) and 10B (neutrophils), respectively.
As can be seen, the number of white blood cells and neutrophils in the peripheral blood of mice treated with cyclophosphamide only was reduced compared to mice treated with IB-MECA only. When adenosine or IB-MECA was administered, the total white blood cell count was restored, which had a very significant effect and was completely restored after 168 hours (7 days).
Example 5: adenosine A3 receptor agonists prevent weight loss in mice treated with chemotherapeutic drugs
Four groups of nude mice (BALB/C lineage), 10 per group, were treated as follows:
group 1: mice not treated [ request confirmation ].
Group 2: mice were injected intraperitoneally (i.p.) with 5-fluoro-uracil (5-FU, 30mg/kg body weight in PBS) for 5 consecutive days
Group 3: mice were injected i.p. with 5-FU as in the second group, but from the next day onwards, mice were given Cl-IB-MECA (6ug/kg body weight in 0.2ml PBS) orally every other day.
Group 4: mice received Cl-IB-MECA administration as above.
The weight of the mice was measured on days 7, 10 and 14, and the results are shown in FIG. 11.
As can be seen, 5-FU had a significant effect on mouse weight compared to the control, and when Cl-IB-MECA was administered in combination with 5-FU, the weight reduction was partially prevented. Cl-IB-MECA by itself does not cause weight loss per se. This experiment demonstrates that a3 adenosine receptor agonist has a full protective effect against some of the toxic effects of chemotherapy.
Example 6: Cl-IB-MECA protected mice from myelotoxic effects of chemotherapeutic drug doxorubicin ICR mice were treated with doxorubicin (i.p. injection, 10mg/kg in 0.5ml PBS). Cl-IB-MECA (6. mu.g/kg body weight) mice were dosed orally 24, 48 and 72 hours after cytotoxic drug dosing. At 72 hours, 96 hours, 120 hours and 144 hours, the mice were sacrificed and blood samples were taken. In addition, bone marrow was aspirated from the femur of the mouse, and after staining the preparation with coumasille blue, the aspirated preparation was subjected to cell counting of nucleated cells.
Three groups of mice were used for the experiments:
group 1: (control) mice were dosed with PBS only.
Group 2: mice were treated with doxorubicin alone.
Group 3: doxorubicin was administered as above in combination with Cl-IB-MECA.
The white blood cell count results are shown in FIG. 12A, and the bone marrow nucleated cell count results are shown in FIG. 12B. These results clearly show that there was a significant increase in both peripheral leukocyte and bone marrow nucleated cell numbers when Cl-IB-MECA was administered. This is a clear protective effect of A3RAg against the myelotoxic effects of doxorubicin
Example 7: anti-G-CSF antibodies neutralize Cl-IB-MECA myeloprotective effects
ICR mice were divided into six groups as follows:
group 1: control group-vehicle only administration.
Group 2: the modulation was performed with anti-G-CSF antibody (5. mu.g/mouse).
Group 3: chemotherapy group-cyclophosphamide (CYP-50mg/kg body weight) was administered.
Group 4: chemotherapy (50mg/kg body weight CYP) + anti-G-CSF antibody (5. mu.g/mouse).
Group 5: chemotherapy (50mg/kg body weight CYP) + Cl-IB-MECA (6. mu.g/kg body weight) + anti-G-CSF antibody (5. mu.g/mouse)
Group 6: chemotherapy (50mg/kg body weight CYP) + Cl-IB-MECA (6. mu.g/kg body weight) + anti-G-CSF antibody (5. mu.g/mouse)
Each group had 10 mice and the experiment was repeated twice.
CYP was injected intraperitoneally, with 0.2ml PBS as vehicle.
Cl-IB-MECA was administered orally (with 0.2ml PBS) 48 and 72 hours after cyclophosphamide administration.
anti-G-CSF antibody was administered intravenously (with 0.2ml PBS) 72 hours after the administration of the chemotherapeutic agent.
Blood samples were collected 124 hours after chemotherapy. White Blood Cell (WBC) counts were determined using a Coulter counter and different cell counts were performed using smear preparations stained with May-Grundvald-Giemsa solution.
The WBC count results are shown in fig. 13. It can be found that mice treated with cyclophosphamide alone show a decrease in the number of WBCs in the peripheral blood. The WBC number and the percentage of neutrophils were significantly higher in the group treated with Cl-IB-MECA than in the group treated with chemotherapy (no metastasis results of neutrophils were indicated). When the anti-G-CSF antibody was administered to the control group or the chemotherapy group, the expected decrease in the number of WBCs was observed. The administration of anti-G-CSF antibodies to mice treated with chemotherapeutic drugs and Cl-IB-MECA abrogated the protective effects of Cl-IB-MECA, as clearly seen in FIG. 13. From these results, it was concluded that the protective effect of Cl-IB-MECA on the immune system was exerted by the activity of Cl-IB-MECA in promoting the production and secretion of G-CSF.
Example 8: Cl-IB-MECA for inhibiting the development of HCT-116 human colon cancer in nude mice
By subcutaneous injection of 1X 10 to nude mice (BALB/C lineage)6HCT-116 human colon cancer cells form tumors (Harlan, Jerusalem, Israel). Mice were treated every other day with 6. mu.g/kg body weight of Cl-IB-MECA (in 0.2ml PBS) orally. Mice treated with vehicle (PBS) only. Each group had 10 mice. Tumor was determined by measuring the perpendicular diameter of each tumor twice a weekGrowth rate of tumor according to the formula pi/6 [ D ]1D2The size of the tumor is estimated. The results are shown in FIG. 14. It can be seen that the treatment groups had significant inhibition of tumor growth.
A separate set of independent experiments was performed on Cl-IB-MECA and 5-fluorouracil (5-FU) combination therapy. 1X1O6HCT-116 cells were injected subcutaneously into nude mice. One day later, 5-FU (30mg/kg body weight in 0.2ml PBS) was injected intraperitoneally, followed by 4 consecutive days. Every other day, mice were orally administered Cl-IB-MECA (with 0.2ml PBS) at 5. mu.g/kg body weight. Mice treated with vehicle (PBS) or 5-FU alone were used as controls. Each group contained 10 mice. The growth rate of the tumors was determined by measuring the diameter of each tumor perpendicular to each other twice a week according to the formula pi/6 [ D ]1D2]The size of the tumor is estimated.
The results are shown in FIGS. 15 and 16. It was observed that the growth of tumors treated with 5-FU, Cl-IB-MECA and a combination of Cl-IB-MECA and 5-FU was significantly inhibited. After 20 days, the synergy of Cl-IB-MECA and 5-FU was clearly seen by noting the tumor mass, as shown in FIG. 16 (FIG. 16 represents the results of day 30).
Example 9: Cl-IB-MECA for stimulating bone marrow cell proliferation by inducing G-CSF production
Bone marrow cells (3X 10) in 96-well microplates6Cells/ml) were cultured. To this was added Cl-IB-MECA at a final concentration of 10nM, with or without anti-G-CSF-antibody at final concentrations of 0.05 and 0.5. mu.g/ml. Use 23H]Thymidine binding assays measure cell proliferation. The results are shown in FIG. 17.
It can be seen that anti-G-CSF antibodies inhibit bone marrow cell proliferation in a dose-dependent manner. This experiment also shows that Cl-IB-MECA activity is exerted by the G-CSF pathway, which includes secretion of G-CSF from cells.
Example 10: Cl-IB-MECA inhibits tumor cell growth and stimulates myeloid cell proliferation and differentiation
B-16 melanoma cells (5X 10) in 96-well microplates5Cells/ml) and bone marrow cells (3X 10)6Cells/ml). The culture was RPMI medium supplemented with 10% FTS. To this was added Cl-IB-MECA at a final concentration of 0.01. mu.M or 0.1. mu.M, with or without the adenosine A3 receptor antagonist, MRS-1523. With the aforementioned [2 ], [3H]Thymidine binding assays measure cell proliferation. The results are shown in FIG. 18. It can be seen that the proliferation of B-16 melanoma cells and bone marrow cells was unchanged compared to the control in the presence of MRS-1523. In contrast, Cl-IB-MECA exerted the effect of inhibiting B-16 melanoma cell proliferation and stimulating bone marrow cells.
These results indicate a dual role for the a3 adenosine receptor agonist.
Example 11: CI-IB-MECA as chemoprotectants
Similar examples to example 4 were performed with Cl-IB-MECA and the results are shown in FIGS. 19A and 19B, which demonstrate the chemoprotective activity of Cl-IB-MECA.
Example 12: effect of IB-MECA and Cl-IB-MECA on bone marrow cell proliferation
Murine bone marrow cells were cultured as described above. IB-MECA or Cl-IB-MECA at concentrations of 1 or 10nM were added in the presence or absence of A3RAn, MRS-1523. The antagonist was added at a concentration of 10 nM. The results are shown in FIG. 20.
As can be seen from FIG. 20, the effects of IB-MECA and Cl-IB-MECA are both dose dependent. Furthermore, it can be seen that this effect can be largely inhibited by A3 Ran.