TECHNICAL FIELD

The invention relates to the use of the combination of a lyase with a selenium-containing lyase substrate to inhibit or destroy tumor cells in vitro and in vivo. More specifically, the invention concerns providing an enzyme capable of generating a toxic selenium form from a relatively nontoxic substrate in combination with said substrate. An illustrative combination is selenomethionine in combination with methioninase. The enzyme may be supplied directly or generated recombinantly in situ.

BACKGROUND ART

Selenium is an element essential for metabolism, but in the wrong form and in inappropriate amounts it is cytotoxic. This may be advantageous in the context of carcinostatic properties. Spallholz, J. E., in a review article published in Free Radical Biology and Medicine (1994) 17:45-64, proposed that the toxic selenium species is the metabolic selenide RSe.sup.- anion. The anion can participate in transthiolation reactions and generate superoxide, hydrogen peroxide and perhaps other cascading oxyradicals, according to this proposal. In a later paper from the same group, Stewart, M. J., et al., Free Radical Biology and Medicine (1999) 26:42-48, it is reported that catalytic selenite, selenocystine and selenocystamine induced apoptosis and were cytotoxic in keratinocytes, but selenomethionine was not cytotoxic and did not induce cellular apoptosis at the concentrations studied. Selenomethionine has also been shown to act as a cancer chemoprotectant by Ip, et al., Cancer Res. (1990) 50:1206-1211; El-Bayoumy, K., et al., Cancer Res. (1992) 52:2402-2407. Selenium compounds shown to inhibit carcinogenesis in rodents have been reported by Yan, Y., et aL, Biochem. Pharmocol. (1993) 45:429-437.

Apparently, diselenide forms of organic selenium compounds, such as selenocystine and selenocystamine can be converted to the toxic form RSe.sup.-. However, mammalian cells do not readily convert the monoselenide forms to toxic moieties. Thus, selenomethionine, selenohomocysteine and seleriocysteine are relatively nontoxic and noricarcinostatic.

Selenodithiols have been used to inhibit proliferation of cancer cells as described in U.S. Pat. No. 5,104,852 and selenium compounds which would otherwise be toxic have been used therapeutically in small enough concentrations as described in U.S. Pat. No. 4,512,977.

It has now been found that a particularly effective protocol for treatment of tumors comprises using the nontoxic forms of selenium as prodrugs when the targeted cells are provided with a means to convert the pro-drugs to the toxic forms. The pro-drugs themselves can be cytotoxic if at sufficiently high levels. Kajander, E. O., et al., Biochem. J (1990) 267:767-774. However, these levels are several orders of magnitude higher than those required for cytotoxicity of compounds that can readily generate RSe.sup.- in cells.

DISCLOSURE OF THE INVENTION

The invention is directed to protocols and formulations for selectively killing cancer cells through the combination of an RSe.sup.- generating enzyme, such as methioninase and its selenium-containing substrate, e.g., selenomethionine. The method can be used in chemotherapy regimen to treat a variety of cancers. The selenium-containing prodrug and/or the methioninase can also be selectively targeted to a particular cancer cell type or to a tumor site. The methioninase can be administered as a nucleic acid comprising a nucleotide sequence which encodes the enzyme that is expressible by the cancer cells or by other target host cells. The methioninase and the selenium-containing prodrug can also be directly administered to the tumor site, where possible or desired. The protocols cause cell death or retard cell growth.

The selenium-containing prodrugs include selenomethionine and related compounds. The prodrug is cleaved by a lyase at the C-Se bond to result in the release of. the active selenide moiety. This moiety is thought to be a methylselonide or a related compound of the formula RSe.sup.- where R is hydrocarbyl or H.

The enzyme administered will be one which serves as a lyase for the selenium-containing prodrug. If the selenium-containing prodrug is selenomethionine, methioninase is an appropriate choice.

Methioninase can be recombinantly produced or purified from natural sources. For example see U.S. Pat. No. 5,690,929. An expression system or cassette for production of methioninase can be used in place of the enzyme or along with it if desired. The vector permits the selective expression of the enzyme, which would localize its presence at a selected or desired site. Further, the methioninase can be complexed with a targeting molecule, e.g. an antibody that recognizes cancer cells. See, e.g. U.S. Pat. No. 5,057,313. The selenium-containing prodrug can similarly be targeted.

Thus, in one aspect, the invention is directed to a method to inhibit tumor cell growth which method comprises contacting said tumor cells with a selenium-containing prodrug and with an enzyme that liberates a toxic form of selenium from said prodrug in amounts sufficient to inhibit said tumor cell growth. In a preferred embodiment, the enzyme is an .alpha.,.gamma.-lyase or an .alpha.,.beta.-lyase and the selenium-containing prodrug is a corresponding sulfur-containing amino acid or the corresponding amine. In one preferred embodiment, the lyase is supplied by generating it internally to the targeted tumor cells or in cells adjacent to them by providing a suitable: expression system to said cells or their neighbors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the vector used for production of methioninase in situ, pQBI-Ad CMV 5MET.

FIG. 2 shows the effect of various virus titers of the methioninase-providing virus on cell survival in the presence and absence of SeMet using OVCAR-8 cells.

FIG. 3 shows the effect of various virus titers of the methioninase-providing virus on cell survival in the presence and absence of SeMet using A204 cells.

FIG. 4 shows the effect of various virus titers of the methioninase-providing virus on cell survival in the presence and absence of SeMet using PC-3 cells.

FIG. 5 shows the effect of various virus titers of the methioninase-providing virus on cell survival in the presence and absence of SeMet using A549 cells.

FIG. 6 shows the effect of SeMet concentration on OVCAR-8 cells transduced with rAd-MET.

FIG. 7 shows the combined effect of various concentrations of SeMet and various titers of control rAd on cell survival for A549 cells.

FIG. 8 shows the combined effect of concentration of SeMet and virus titers for rAd-MET on survival of A549 cells.

FIG. 9 shows the bystander effect of rAd-MET and SeMet in combination on A549 cells.

FIG. 10 shows the induction of apoptosis by the combination of rAd-MET and SeMet on A549 cells.

FIG. 11 shows the generation of oxidant by rAd-MET in combination with SeMet as detected by chemiluminescence of lucigenin.

FIG. 12 shows the effect of the combination of SeMet and rAd-MET on caspase concentrations in lysates of A549 cells.

FIG. 13 shows the effect of the combination of rAd-MET and SeMet on ascites tumor growth in vivo.

FIG. 14 shows the effect of the combination of rAd-MET and SeMet on survival of nude mice containing ascites tumors.

MODES OF CARRYING OUT THE INVENTION

It is understood that the toxicity of a selenium-based compound is highly dependent on the nature of the compound. Certain selenium compounds which are able to generate selenium anions of the general formula RSe.sup.- where R is H or a hydrocarbyl substituent are highly toxic. Forms of selenium which are known to be capable of generating this toxic moiety include the inorganic moiety selenite and organic diselenide compounds such as selenocystamine and selenocystine. However, other compounds, in particular the selenated forms of the sulfur-containing amino acids homocysteine, cysteine and methionine and their corresponding decarboxylated forms do not generate the, toxic form of selenium in mammalian cells.

However, there are a number of enzymes derivable from non-mammalian sources that are able to lyse these substrates and generate toxic forms of selenium. These enzymes include L-methionine .gamma.-lyase from Aeromonas (Nakayama, T., et al., Agric. Biol. Chem. (1984) 48:2367-2369) which utilizes methionine, ethionine, homocysteine and acetyl L-homoserine as substrates; the methionine .gamma.-lyase from Pseudomonas putida described by Nakayama, T., et al., Analyt. Biochem. (1984) 138:421-424, which has a similar substrate spectrum; and the L-methionine .gamma.-lyase from Pseudomonas ovalis, Tanaka, H., et al., Biochemistry (1977) 16:100-106. A detailed description of these enzymes (EC 4.4.1.11) is found in an article by Esaki, N., et al., Methods in Enzymology (1987) 143:459-465. The anti-tumor activity of this enzyme itself has been described by Tanaka, H., et al., J. Applied Biochem. (1980) 2:439-444 and the gene encoding the P. putida enzyme as well as other corresponding enzymes have been cloned. See Inoue, H., etal., J. Biochem. (1995) 117:1120-1125. See also PCT publication WO 99/05311 and U.S. Pat. No. 5,861,154 and 5,863,788. The ability of the methionine .gamma.-lyase from P. putida to catalyze the replacement of the methylthio moiety in methionine to obtain the corresponding selenium-containing form has also been disclosed by Esaki, N., et al., Biochemistry (1979) 18:407-411. It appears, based on the results described in this paper, that amino acids other than methionine wherein,the methyl group is replaced by an alkyl substituent or even a phenyl or benzyl substituent are suitable substrates for this enzyme.

Thus, the selenium-containing prodrug may be any selenium-containing compound that can behave as a substrate for an appropriate enzyme to generate the toxic form of selenium, believed to be RSe.sup.- wherein R is H or hydrocarbyl, typically alkyl, phenyl or benzyl. The nature of this group is dependent only on the specificity of the enzyme.

The enzyme is typically a lyase of the type described above. However, any enzyme which dissociates the selenium-containing prodrug to generate the toxic selenium form is workable in the method of the invention. Such enzymes are referred to herein as "selenium prodrug lyase(s)."

The selenium prodrug lyase may be supplied as a protein or may be generated intracellularly or in situ by supplying an expression system for the enzyme. If the enzyme per se is administered, methods for administering such proteins are generally known in the art. For example, methods to administer methioninase in particular, in the context of chemotherapy are set forth in U.S. Pat. No. 5,690,929, the contents of which are incorporated herein by reference. Proteins in general can be administered by injection, typically intravenous injection or by transmembrane administration, for example, intranasally or using suppositories. Other modes of administration are also possible, including oral administration provided adequate protection from hydrolysis is included in the formulation. Such methods are generally known in the art as described in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Company, Easton Pa.

The protein may be administered per se or it may also include a targeting agent to direct the protein specifically to tumor cells. Such targeting agents may include, for example, antibodies or immunologically reactive fragments thereof, including single-chain antibodies, which are immunospecific for antigens associated with tumor cells or for antigens which appear on the organs in which the tumors reside, such as prostate-specific antigen in the case of prostate cancer. In addition, the targeting agents may include ligands for receptors that characterize the cells associated with the tumor. Coupling to such fargeting agents is also conventional and involves standard linking technologies, optionally utilizing commercially available linkers, such as those available from Pierce Chemical Company, Rockford Ill.

If the selenium prodrug lyase is to be generated intracellularly or in situ, a suitable nucleic acid molecule containing the nucleotide sequence encoding the enzyme is administered. Suitable modes of administration include injection, topical administration in formulations that include agents which enhance transmembrane or transdermal transit or any other appropriate and convenient method consistent with the situs of the tumor cells and the nature of the formulation, as will be understood by the ordinary practitioner. One particularly preferred mode of administration is local to the tumor--i.e., the nucleic acid molecule may be administered directly to the tumor tissue or to the site where the tumor resides. Because the formulations of the invention are able to provide not only a cytotoxic effect on the cells containing the prodrug and enzyme but also a bystander effect on neighboring cells, direct administration to the locus of the tumor is often satisfactory.

The nucleic acid molecule is typically a vector, most commonly a viral vector, although naked DNA can, in some instances, be used. The viral vectors may be retroviral vectors, which preferentially replicate in rapidly proliferating cells, thus conferring specificity for tumor cells on the vector, or may include adenoviral vectors or other conventional vector-based molecules. Specificity in this case may be conferred by localized administration and/or by placing the expression of the nucleotide sequence encoding the enzyme under control of a promoter which is operable selectively in tumor tissue. Table 1 below sets forth a list of tumor-associated viruses and oncogenes which comprise promoters specific for the tumors with which they are associated. Promoters associated with these genes and viruses may be used to direct expression selectively in the appropriate tumor.

                                    TABLE 1
                          Oncogenes and tumor viruses
    Acronym     Virus              Species Tumor origin      Comments
    abl         Abelson leukemia   Mouse   Chronic           TyrPK(src)
                                           myelogenous
                                           leukemia
    erbA        Erythroblastosis   Chicken                   Homology to human
                                                             glucocorticoid
                                                             receptor
    erbB        Erythroblastosis   Chicken                   TryPK EGF/TGFc
                                                             receptor
    ets         E26 myeloblastosis Chicken                   Nuclear
    fes (fps).sup.a  Snyder-Thellen     Cat                       TryPK(src)
                sarcoma
                Gardner-Arnstein
                sarcoma
    fgr         Gardner-Rasheed    Cat                       TyrPK(src)
                sarcoma
    fms         McDonough sarcoma  Cat                       TyrPK CSF-1
                                                             receptor
    fps (fes).sup.a  Fujinami sarcoma   Chicken                   TyrPK(src)
    fos         FBJ osteosarcoma   Mouse                     Nuclear, TR
    hst         NVT                Human   Stomach tumor     FGF homologue
    intl        NVT                Mouse   MMTV-induced      Nuclear, TR
                                           carcinoma
    int2        NVT                Mouse   MMTV-induced      FGF homologue
                                           carcinoma
    jun         ASV17 sarcoma      Chicken                   Nuclear, TR
    hit         Hardy-Zuckerman 4  Cat                       TyrPK GFR L
                sarcoma
    B-lym       NVT                Chicken Bursal lymphoma
    mas         NVT                Human   Epidermoiod       Potentiates
     response
                                           carcinoma         to angiotensin II
    met         NVT                Mouse   Osteosarcoma      TyrPK GFR L
    mil (raf).sup.b  Mill Hill 2 acute  Chicken                   Ser/ThrPK
                leukemia
    mos         Moloney sarcoma    Mouse                     Ser/ThrPK
    myb         Myeloblastosis     Chicken Leukemia          Nuclear, TR
    myc         MC29               Chicken Lymphomas         Nuclear TR
                myelocytomatosis
    N-myc       NVT                Human   Neuroblastomas    Nuclear
    neu (ErbB2) NVT                Rat     Neuroblastoma     TryPK GFR L
    ral (mil).sup.b  3611 sarcoma       Mouse                     Ser/ThrPK
    Ha-ras      Harvey murine      Rat     Bladder, mammary  GTP-binding
                sarcoma                    and skin carcinomas
    Ki-ras      Kirsten murine     Rat     Lung, colon       GTP-binding
                sarcoma                    carcinomas
    N-ras       NVT                Human   Neuroblastomas    GTP-binding
                                           leukaemias
    rel         Reticuloendothe-liosis Turkey
    ros         UR2                Chicken                   TyrPK GFR L
    sis         Simian sarcoma     Monkey                    One chain of PDGF
    src         Rous sarcoma       Chicken                   TyrPK
    ski         SKV770             Chicken                   Nuclear
    trk         NVT                Human   Colon carcinoma   TyrPK GFR L
    yes         Y73, Esh sarcoma   Chicken                   TyrPK(src)

Suitable viral vector constructs are those known in the art. For example, vectors derived from a parvovirus (U.S. Pat. Nos. 5,252,479 and 5,624,820), a paramyxovirus such as simian virus 5 (SV5) (U.S. Pat. No. 5,962,274), a retrovirus such as HIV (U.S. Pat. Nos. 5,753,499 and 5,888,767), and a baculovirus such as a nuclear polyhedrosis virus (U.S. Pat. No. 5,674,747) can be used. Vectors derived from adenovirus have been extensively studied (U.S. Pat. Nos. 5,670,488, 5,817,492, 5,820,868, 5,856,152 and 5,981,225 all incorporated herein by reference).

The nucleic acid molecule can be delivered directly to a tissue of the host animal by injection, by gene gun technology or by lipid mediated delivery technology. The injection can be conducted via a needle or other injection devices. The gene gun technology is disclosed in U.S. Pat. No. 5,302,509 and the lipid mediated delivery technology is disclosed in U.S. Pat. No. 5,703,055.

In still another specific embodiment, the nucleic acid molecule is delivered to a cell of host animal ex vivo and the cell is then delivered to a suitable tissue of the host animal, preferably through injection or intravenous drip.

General methods for ex vivo transduction and whole animal administration include Ca.sub.3 (PO.sub.4).sub.2 -DNA transfection (Sambrook, et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press, 1989), DEAE dextran-DNA transfection (Sambrook, et al., Molecular Cloning, 2nd Edition, Plainview, N.Y. Cold Spring Harbor Press, 1989), electroporation (e.g., protocols from Bio-Rad), transfection using "LIPOFECTIN".TM. reagent (e.g., protocols from BRL-Life Science), gene gun technology (U.S. Pat. No. 5,302,509), or viral gene delivery system (Kaplitt, et al., Viral Vectors, Academic Press, Inc., 1995).

Although the prodrug and the selenium prodrug lyase may be delivered concomitantly, it is preferred that the enzyme be provided first, followed by administration of the selenium-containing prodrug. This is in order that the cells will be preconditioned to generate the toxic form of selenium. In any event, the tumor to be treated must be proximal both to the lyase and the prodrug to elicit the toxic response.

In addition to the selenium-containing prodrug and the prodrug lyase, chemotherapeutic agents can be employed in suitable therapeutic protocols. Such agents as 5-FU, cyclophosphamide, doxorubicin, BCNU, methotrexate and other drugs may be employed along with the protocols of the invention. The efficacy of these drugs is enhanced by the lyase and the prodrug.

The tumors to be treated may include solid tumors such as those of the breast, prostate, colon, lung, brain, pancreas, liver, and the like or may be lymphomas or other leukemias or metastases of solid tumors. The selection of the appropriate expression system both from the standpoint of the vector and the control sequences for expression will depend on the nature of the tumor targeted. The nucleotide sequence encoding the enzyme may also be provided with a fusion amino acid sequence which confers target specificity, as was the case for direct administration of the enzyme.

The following examples are intended to illustrate but not to limit the invention.

EXAMPLE 1

Construction of a Methioninase-Producing Vector

FIG. 1 shows a diagram of a vector constructed to provide viral infection of tumor cells and expression the methioninase derived from P. putida. The nucleotide sequence encoding the enzyme is placed under control of the CMV-5 promoter and enhancer. The backbone vector is commercially available. The resulting vector is propagated according to standard techniques in adenovirus.

To prepare the vector, the methioninase gene cloned from P. putida as described in Inoue, H.,et al, J. Biochem (1995) 117:1120-1125 was amplified by PCR and the 1.2 kb gene was ligated into the transfer vector pQBI-Ad CMV 5GFP (obtained from Quantum, Montreal, Quebec, Canada) downstream of the CMV-5 promoter at the BglII/PmeI site. The resulting vector is shown in FIG. 1. The vector was cotransfected into 293A cells (Quantum) using the calcium phosphate method along with Ad CMV Lac Z.DELTA.E1/.DELTA.3 viral DNA cut with ClaI (Quantum). Transfected cells were overlain with 1.25% Sea Plaque agarose (FMC Bioproducts, Rockland, Me) and incubated at 37.degree. C. for 14-21 days. Primary plaques were isolated and used to infect 293A cells to generate primary crude viral lysate. Methioninase gene expression in the viral lysate was confirmed by an enzymatic assay. After a second plaque purification, a single plaque was amplified in 293A cells. Ad CMV 5GFP .DELTA.E1/.DELTA.E3 was used as a control vector (-rAd) and purchased from Quantum. The control rAd was expanded in 293A cells and purified by cesium chloride centrifugation and subsequent fractionation on Sephadex G25. The above-described construction of rAd-MET is reported by Miki, K., et al., Cancer Res. (in press) incorporated herein by reference.

EXAMPLE 2

Effect of Varying Methioninase in the Presence of Selenomethionine on Various Tumor Cell Lines

Cells (4.times.10.sup.3) of various tumor lines were seeded onto 96-well plates. After 24 hours, serial dilutions of the methioninase expressing vector (rAd-MET) or a control vector containing no methioninase encoding nucleotide sequence (rAd) were added to the wells with and without 20 .mu.M of selenomethionine (SeMet) (20 .mu.M). After incubating the plates for three (3) days, the medium was replaced with 0.5 mg/ml MTT for two (2) hours and analyzed using a microplate reader (BioRad), in a standard MTT assay. Results were recorded as percentage of surviving cells.

The cell lines tested were human cancer cell lines OVCAR-8 (ovarian cancer), A549 and EKVX (lung cancer), Hep-2 (head and neck cancer), MIA PaCa-2 and BxPC-3 (pancreatic cancer), human rhabdomyosarcoma cell line A204 and human prostate cell line PC-3. The results with respect to the OVCAR-8 cell line and cell lines A204, and PC-3 are shown in FIGS. 2, 3 and 4 in X/Y graphs; FIG. 5 is a bar graph of the results with respect to the A549 cell line. In each case, the presence of SeMet drastically lowered the IC.sub.50 for the rAd-MET vector. The results are further summarized in Table 1.

                             TABLE 1
    Comparison of IC.sub.50 of rAd-MET and Control-rAd in human tumor
                 cell lines with or without SeMet
                  IC.sub.50 rAd-MET (pfu) IC.sub.50 Control-rAd (pfu)
    cell lines     SeMet (+)   SeMet (-)   SeMet (+)   SeMet (-)
    OVCAR-8       2.7 .times. 10.sup.3  3.0 .times. 10.sup.5  3.8 .times.
     10.sup.6  4.6 .times. 10.sup.6
    A549          7.0 .times. 10.sup.3  3.3 .times. 10.sup.5  6.7 .times.
     10.sup.5  8.0 .times. 10.sup.5
    Hep-2         5.9 .times. 10.sup.3  3.3 .times. 10.sup.5  7.9 .times.
     10.sup.5  5.9 .times. 10.sup.5
    MIA PaCa-2    1.7 .times. 10.sup.4  3.6 .times. 10.sup.5  9.3 .times.
     10.sup.5  1.1 .times. 10.sup.6
    BxPC-3        3.5 .times. 10.sup.4  3.1 .times. 10.sup.6  2.7 .times.
     10.sup.6  3.0 .times. 10.sup.6
    EKVX          3.7 .times. 10.sup.3  4.8 .times. 10.sup.5  1.2 .times.
     10.sup.6  1.7 .times. 10.sup.6

                             TABLE 2
        Comparison of SeMet IC.sub.50 in human tumor cell
                   lines transdued with rAd-MET
                                    SeMet IC.sub.50 (.mu.M)
        cell lines      rAd-MET       Control-rAd     Wild Type
        OVCAR-8            0.59          170.3          223.6
        A549               0.5           327.1          485.9
        Hep-2              1.66           387            411
        MIA PaCa-2         3.86           338            422
        BxPC-3             3.38          222.6          311.8
        EKVX               1.66          686.4          1360