News Column

Patent Issued for Adenosine Deaminase Anticancer Therapy

June 16, 2014



By a News Reporter-Staff News Editor at Cancer Gene Therapy Week -- From Alexandria, Virginia, NewsRx journalists report that a patent by the inventors Filpula, David R. (Piscataway, NJ); Sapra, Puja (Edison, NJ), filed on April 18, 2008, was published online on June 3, 2014 (see also Sigma-Tau Rare Diseases, S.A.).

The patent's assignee for patent number 8741283 is Sigma-Tau Rare Diseases, S.A. (Funchal, PT).

News editors obtained the following quote from the background information supplied by the inventors: "A tumor is an abnormal benign or malignant growth of cells or tissue that arises from uncontrolled cellular proliferation. A malignant tumor is a one that spreads from its site of origin, and is also art-known as a cancer. Thus, tumors and cancers are a family of diseases sharing the common property of uncontrolled or inappropriate cell growth. Broadly, malignant tumors are either blood derived tumors, such as leukemia, or solid tumors. Blood derived malignant tumors generally circulate in the blood, but solid malignant tumors spread throughout the body from a primary tumor. The distributed tumor cells then have the potential to develop into multiple secondary tumors, in a process of metastasis. In order for a solid tumor to undergo such a metastatic spread, solid tumor cells must escape from the primary or original tumor, enter the blood stream or lymphatic system, and from there invade the tissue of other organs, where they multiply and form new tumors. Metastasis is a complex multi-step process that involves changes in tumor cell adhesion and motility, secretion of proteolytic enzymes, chemoattractants, and proteoglycans and other factors. In addition, angiogeniesis, or the formation of new blood vessels, is also a vital step in the metastatic process (Folkman, 1995, Nature Medicine 1:27-31).

"The immune system has also been shown to inhibit the metastasis of such malignant tumor cells, and it has been reported that adenosine, in turn, may inhibit such immune protective reactions. For example, Loshkin et al., (2006, Cancer Res. 66: 7758-7765) report that adenosine inhibits activation and cytokine production in killer T cells. Adenosine negatively impacts other immune function, including both cellular elements and inflammatory functions (see, e.g., the reviews by Spychala, 2000, Pharmacology & Therapeutics 87: 161-173 and by Sitkovesky et al., 2005 Nature Reviews Immunology 5: 713-721). Sitkovesky et al., in WO 03/050241, published Jun. 19, 2003, also described methods for increasing an immune response to an antigen and for treating tumors, by administering an adenosine receptor antagonist, that can include adenosine deaminase.

"It has also been shown that adenosine promotes tumor cell migration and angiogenesis (Barcz et al., 2000, Oncol. Rep. 7(6): 1285-91; Adair, 2005 Am J Physiol Regul Integr Comp Physiol 289: R283-R296) and that adenosine stimulates the proliferation of colon cancer cells (Mujoomdar et al. 2003, Biochemical Pharmacology 66 1737-1747). It has also been reported by Asmar et al., 1966, Proc. Am. Assoc. Cancer Res. (Abstract No. 73) that the growth of certain tumor cells was inhibited, by injection of ADA, by over 50% in a mouse ascites model. These were lymphatic leukemias L1210 and L4946, lymphosarcoma 6C3HED, mammary adenocarcinoma TA3, and Ehrlich carcinoma E2. In the same Abstract, an adenocarcinoma 755 was reported to be twice as resistant to and a sarcoma 180 was completely resistant to the effect. WO03050241 A2 describes the effects of an inhibitor of adenosine receptors on B16 melanoma cells. While WO03050241A2 mentions ADA as an inhibitor of adenosine, there is no specific description for applying ADA to the treatment of specific cancers, and particularly ovarian cancer and prostate cancer.

"Thus, it seems that for some tumors, the presence of adenosine provides a 'go' signal for tumor proliferation and for tumor angiogeniesis, and a 'stop' signal for the killer T cells which would normally kill these tumors.

"In contrast to the above-discussed findings, Lind, et al. (U.S. Pat. No. 6,579,857) have reported that adenosine, in combination with an inhibitor of the enzyme adenosine deaminase, and/or in combination with an anticancer agent such as coformycin, is useful in a method for potentiating cell death in neoplastic cells of epithelial origin. Thus, this reference suggests that the role of adenosine in cancer is more complex and unsettled.

"As noted above, an agent for reducing endogenous adenosine levels is the enzyme adenosine deaminase. Adenosine deaminase ('ADA'), designated as EC 3.5.4.4, is an important enzyme of the purine salvage pathway. ADA converts either adenosine or deoxyadenosine, in the presence of water, into inosine or deoxyinosine and ammonia. It is known that individuals who harbor deleterious mutations in the ADA gene can develop varying degrees of an immunodeficiency disorder, from mild to severe, i.e. severe combined immunodeficiency disorder ('SCID'). SCID has been confirmed to result from the toxic accumulation of the enzyme substrates, adenosine and deoxyadenosine, in immature lymphoid cells. The onset of the disorder can range from early childhood to adults, depending on the mutations inherited. Deficiencies of ADA are one of the leading causes of SCID, in children, and is one of the leading targets for gene therapy approaches (R. Parkman et al., 2000, Gene therapy for adenosine deaminase deficiency, Ann. Rev. Med., 51:33-47).

"Previously, ADA has been commercially isolated from bovine sources and employed in treating a number of disorders, including SCID, in the form of a bovine ADA conjugated to polyethylene glycol ('PEG') polymer. PEGylated ADA for medical use is commercially available from Enzon Pharmaceuticals, Inc. as ADAGEN.RTM., PEGylated ADA. The conjugation of a PEG moiety to ADA allows the enzyme to achieve its full therapeutic effect by increasing the circulating life and rendering the ADA substantially non-antigenic, in order to minimize the potential for immunogenic reactions. It is also possible to produce recombinant human or bovine ADA enzymes for use in a conjugated form, as described by co-owned U.S. patent application Ser. No. 11/738,012, entitled 'Stabilized Proteins', and co-owned U.S. application Ser. No. 12/105,913, entitled 'Stable Recombinant Adenosine Deaminase', filed on even date herewith and claiming the benefit of priority from U.S. Patent Application Ser. No. 60/913,009, and both incorporated by reference herein in their entireties.

"Thus, there is a longstanding need in the art for new and improved methods of treating or inhibiting the growth, spread and development of cancers."

As a supplement to the background information on this patent, NewsRx correspondents also obtained the inventors' summary information for this patent: "Accordingly, the invention provides a method of treating a patient having a tumor comprising administering an effective amount of ADA to the patient in need thereof. An effective amount is that which is readily determined by one of ordinary skill in the art to reduce tissue levels of adenosine or deoxyadenosine in the patient, and wherein growth or spread of the tumor is inhibited by substantially reduced tissue levels of adenosine in the patient. The route of administration is a route such as subcutaneous, intravenous, intramuscular, intrathecal, intraperitoneal, inhalation and transurethral.

"The tumor can be malignant or nonmalignant, and is preferably a solid tumor, e.g., a tumor such as a prostate tumor, an ovarian cancer and/or a colorectal cancer.

"The adenosine deaminase is preferably conjugated to a substantially non-antigenic polymer, such as a polyalkylene oxide (PAC). The PAO preferably ranges in size from about 4,000 to about 45,000 Daltons. The PAO is preferably a polyethylene glycol ('PEG'). The molar ratio of ADA to polymer can be 1:1, or can be two or more ADA molecules per polymer, or more preferably, provides for from about 1 to about 20 polymer molecules (i.e. 11-18 PEG strands) per ADA molecule.

"The polymer-conjugated ADA is preferably administered in a dose ranging from about 10 U to about 30 U per kg or more and for a sufficient period of time to maintain inhibition of the tumor, e.g., from about 1 to about 20 days, or longer.

"The amount of adenosine deaminase that is administered is effective to substantially reduce tissue levels of adenosine or deoxyadenosine in the patient, and wherein growth or spread of the tumor is inhibited by substantially reduced tissue levels of adenosine in said patient. This is, for example, a dose of adenosine deaminase ranging from about 10 U to about 30 U per kg. The dose is repeated for a sufficient period of time to maintain inhibition of the tumor, e.g., from about 1 to about 20 days, or longer. The dose is administered by any convenient route such as subcutaneous, intravenous, intramuscular, intrathecal, intraperitoneal, inhalation and transurethral.

"The adenosine deaminase is optionally purified from a bovine source or is a recombinant adenosine deaminase. The recombinant adenosine deaminase is, for example, recombinant bovine adenosine deaminase (Ser74-rbADA) comprising SEQ ID NO: 1, recombinant human adenosine deaminase (Ser74-rhADA) comprising SEQ ID NO: 3 and recombinant bovine adenosine deaminase comprising SEQ ID NO: 5 and/or variations or polymorphisms thereof. Recombinantly produced bovine adenosine deaminase, e.g., SEQ ID NO: 5, is optionally capped at Cys74 for stability in an aqueous medium.

"For purposes of the present invention, the term 'residue' shall be understood to mean that portion of a compound, to which it refers, e.g., PEG, ADA, amino acid, etc. that remains after it has undergone a substitution reaction with another compound.

"For purposes of the present invention, the term 'polymeric residue', e.g., 'PEG residue', shall each be understood to mean that portion of the polymer or PEG which remains after it has undergone a reaction with other compounds, moieties, etc.

"For purposes of the present invention, the term 'alkyl' as used herein refers to a saturated aliphatic hydrocarbon, including straight-chain, branched-chain, and cyclic alkyl groups. The term 'alkyl' also includes alkyl-thio-alkyl, alkoxyalkyl, cycloalkylalkyl, heterocycloalkyl, and C.sub.1-6 alkylcarbonylalkyl groups. Preferably, the alkyl group has 1 to 12 carbons. More preferably, it is a lower alkyl of from about 1 to 7 carbons, yet more preferably about 1 to 4 carbons. The alkyl group can be substituted or unsubstituted. When substituted, the substituted group(s) preferably include halo, oxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C.sub.1-6 hydrocarbonyl, aryl, and amino groups.

"For purposes of the present invention, the term 'substituted' as used herein refers to adding or replacing one or more atoms contained within a functional group or compound with one of the moieties from the group of halo, oxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C.sub.1-6 alkylcarbonylalkyl, aryl, and amino groups.

"For purposes of the present invention, the term 'alkenyl' refers to groups containing at least one carbon-carbon double bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkenyl group has about 2 to 12 carbons. More preferably, it is a lower alkenyl of from about 2 to 7 carbons, yet more preferably about 2 to 4 carbons. The alkenyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably include halo, oxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C.sub.1-6 hydrocarbonyl, aryl, and amino groups.

"For purposes of the present invention, the term 'alkynyl' refers to groups containing at least one carbon-carbon triple bond, including straight-chain, branched-chain, and cyclic groups. Preferably, the alkynyl group has about 2 to 12 carbons. More preferably, it is a lower alkynyl of from about 2 to 7 carbons, yet more preferably about 2 to 4 carbons. The alkynyl group can be substituted or unsubstituted. When substituted the substituted group(s) preferably include halo, oxy, azido, nitro, cyano, alkyl, alkoxy, alkyl-thio, alkyl-thio-alkyl, alkoxyalkyl, alkylamino, trihalomethyl, hydroxyl, mercapto, hydroxy, cyano, alkylsilyl, cycloalkyl, cycloalkylalkyl, heterocycloalkyl, heteroaryl, alkenyl, alkynyl, C.sub.1-6 hydrocarbonyl, aryl, and amino groups. Examples of 'alkynyl' include propargyl, propyne, and 3-hexyne.

"For purposes of the present invention, the term 'aryl' refers to an aromatic hydrocarbon ring system containing at least one aromatic ring. The aromatic ring can optionally be fused or otherwise attached to other aromatic hydrocarbon rings or non-aromatic hydrocarbon rings. Examples of aryl groups include, for example, phenyl, naphthyl, 1,2,3,4-tetrahydronaphthalene and biphenyl. Preferred examples of aryl groups include phenyl and naphthyl.

"For purposes of the present invention, the term 'cycloalkyl' refers to a C.sub.3-8 cyclic hydrocarbon. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl.

"For purposes of the present invention, the term 'cycloalkenyl' refers to a C.sub.3-8 cyclic hydrocarbon containing at least one carbon-carbon double bond. Examples of cycloalkenyl include cyclopentenyl, cyclopentadienyl, cyclohexenyl, 1,3-cyclohexadienyl, cycloheptenyl, cycloheptatrienyl, and cyclooctenyl.

"For purposes of the present invention, the term 'cycloalkylalkyl' refers to an alkyl group substituted with a C.sub.3-8 cycloalkyl group. Examples of cycloalkylalkyl groups include cyclopropylmethyl and cyclopentylethyl.

"For purposes of the present invention, the term 'alkoxy' refers to an alkyl group of indicated number of carbon atoms attached to the parent molecular moiety through an oxygen bridge. Examples of alkoxy groups include, for example, methoxy, ethoxy, propoxy and isopropoxy.

"For purposes of the present invention, an 'alkylaryl' group refers to an aryl group substituted with an alkyl group.

"For purposes of the present invention, an 'aralkyl' group refers to an alkyl croup substituted with an aryl group.

"For purposes of the present invention, the term 'alkoxyalkyl' group refers to an alkyl group substituted with an alkyloxy group.

"For purposes of the present invention, the term 'alkyl-thio-alkyl' refers to an alkyl-S-alkyl thioether, for example methylthiomethyl or methylthioethyl.

"For purposes of the present invention, the term 'amino' refers to a nitrogen containing group as is known in the art derived from ammonia by the replacement of one or more hydrogen radicals by organic radicals. For example, the terms 'acylamino' and 'alkylamino' refer to specific N-substituted organic radicals with acyl and alkyl substituent groups respectively.

"For purposes of the present invention, the term n alkylcarbonyl refers to a carbonyl group substituted with alkyl group.

"For purposes of the present invention, the terms 'halogen` or 'halo' refer to fluorine, chlorine, bromine, and iodine.

"For purposes of the present invention, the term 'heterocycloalkyl' refers to a non-aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heterocycloalkyl ring can be optionally fused to or otherwise attached to other heterocycloalkyl rings and/or non-aromatic hydrocarbon rings. Preferred heterocycloalkyl groups have from 3 to 7 members. Examples of heterocycloalkyl groups include, for example, piperazine, morpholine, piperidine, tetrahydrofuran, pyrrolidine, and pyrazole. Preferred heterocycloalkyl groups include piperidinyl, piperazinyl, morpholinyl, and pyrrolidinyl.

"For purposes of the present invention, the term 'heteroaryl' refers to an aromatic ring system containing at least one heteroatom selected from nitrogen, oxygen, and sulfur. The heteroaryl ring can be fused or otherwise attached to one or more heteroaryl rings, aromatic or non-aromatic hydrocarbon rings or heterocycloalkyl rings. Examples of heteroaryl groups include, for example, pyridine, furan, thiophene, 5,6,7,8-tetrahydroisoquinoline and pyrimidine. Preferred examples of heteroaryl groups include thienyl, benzothienyl, pyridyl, quinolyl, pyrazinyl, pyrimidyl, imidazolyl, benzimidazolyl, furanyl, benzofuranyl, thiazolyl, benzothiazolyl, isoxazolyl, oxadiazolyl, isothiazolyl, benzisothiazolyl, triazolyl, tetrazolyl, pyrrolyl, indolyl, pyrazolyl, and benzopyrazolyl.

"For purposes of the present invention, the term 'heteroatom' refers to nitrogen, oxygen, and sulfur.

"In some embodiments, substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls and mercaptoalkyls; substituted alkenyls include carboxyalkenyls, aminoalkenyls, dialkenylaminos, hydroxyalkenyls and mercaptoalkenyls; substituted alkynyls include carboxyalkynyls, aminoalkynyls, dialkynylaminos, hydroxyalkynyls and mercaptoalkynyls; substituted cycloalkyls include moieties such as 4-chlorocyclohexyl; aryls include moieties such as napthyl; substituted aryls include moieties such as 3-bromo phenyl; aralkyls include moieties such as tolyl; heteroalkyls include moieties such as ethylthiophene; substituted heteroalkyls include moieties such as 3-methoxy-thiophene; alkoxy includes moieties such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halo shall be understood to include fluoro, chloro, iodo and bromo.

"For purposes of the present invention, 'positive integer' shall be understood to include an integer equal to or greater than 1 and as will be understood by those of ordinary skill to be within the realm of reasonableness by the artisan of ordinary skill.

"For purposes of the present invention, the term 'linked' shall be understood to include covalent (preferably) or noncovalent attachment of one group to another, i.e., as a result of a chemical reaction.

"The terms 'effective amounts' and 'sufficient amounts' for purposes of the present invention shall mean an amount which achieves a desired effect or therapeutic effect as such effect is understood by those of ordinary skill in the art.

"For purposes of the present invention, the term 'adenosine' shall be understood to include the nucleosides adenosine and deoxyadenosine. Adenosine also includes adenosine and deoxyadenosine present in the form of AMP, ADP, ATP, dAMP, dADP or dATP.

"For purposes of the present invention, 'adenosine-mediated tumor' or 'adenosine deaminase-responsive tumor' shall be understood as broadly including any types of tumors which benefit from the administration of ADA, or active fraction thereof, etc., regardless of the route of administration.

"For purposes of the present invention, 'treatment of adenosine-mediated tumor' or 'treatment of adenosine deaminase-responsive tumor' or 'inhibition of adenosine-mediated tumor growth' or 'inhibition of adenosine deaminase-responsive tumor growth' shall be understood to mean that symptoms or conditions are minimized or attenuated when compared to that observed in the absence of the ADA treatment. The treated conditions can be confirmed by, for example, tumor growth inhibition and/or decrease in adenosine levels in cancer cells or tissues.

"Broadly speaking, successful treatment shall be deemed to occur when the desired clinical response is obtained. For example, successful treatment can be defined by obtaining e.g., 10% or higher (i.e. 20% 30%, 40%) tumor growth inhibition. Alternatively, successful treatment can be defined by obtaining at least 20% or preferably 30%, more preferably 40% or higher (i.e., 50% or 80%) decrease in adenosine levels in cancer cells or tissues, including other clinical markers contemplated by the artisan in the field, when compared to that observed in the absence of the ADA treatment described herein.

"Further, the use of singular terms for convenience in description is in no way intended to be so limiting. Thus, for example, reference to a composition comprising an enzyme refers to one or more molecules of that enzyme. It is also to be understood that this invention is not limited to the particular configurations, process steps, and materials disclosed herein as such configurations, process steps, and materials may vary somewhat.

"It is also to be understood that the terminology employed herein is used for the purpose of describing particular embodiments only and is not intended to be limiting, since the scope of the present invention will be limited by the appended claims and equivalents thereof."

For additional information on this patent, see: Filpula, David R.; Sapra, Puja. Adenosine Deaminase Anticancer Therapy. U.S. Patent Number 8741283, filed April 18, 2008, and published online on June 3, 2014. Patent URL: http://patft.uspto.gov/netacgi/nph-Parser?Sect1=PTO1&Sect2=HITOFF&d=PALL&p=1&u=%2Fnetahtml%2FPTO%2Fsrchnum.htm&r=1&f=G&l=50&s1=8741283.PN.&OS=PN/8741283RS=PN/8741283

Keywords for this news article include: Antiarrhythmic Agents, Biotechnology, Pharmaceuticals, Drugs, Sulfur, Genetics, Nitrogen, Oncology, Proteins, Chalcogens, Hydrolases, Proteomics, Therapeutics, Solid Cancers, Bioengineering, Organic Chemicals, Radiologic Agents, Adenosine Deaminase, Cancer Gene Therapy, Cyclic Hydrocarbons, Radiologic Adjuncts.

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Source: Cancer Gene Therapy Week


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