Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/38—Heterocyclic compounds having sulfur as a ring hetero atom
  • A61K31/382—Heterocyclic compounds having sulfur as a ring hetero atom having six-membered rings, e.g. thioxanthenes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K33/00—Medicinal preparations containing inorganic active ingredients
  • A61K33/24—Heavy metals; Compounds thereof
  • A61K33/30—Zinc; Compounds thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/55—Protease inhibitors
  • A61K38/57—Protease inhibitors from animals; from humans
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
  • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/03—Phosphoric monoester hydrolases (3.1.3)
  • C12Y301/03001—Alkaline phosphatase (3.1.3.1)

Definitions

• This invention relates to the use of human alkaline phosphatase, such as placental alkaline phosphatase or transferrin alone or alkaline phosphatase in combination with transferrin, and, optionally, ⁇ i-antitrypsin to prevent or reduce the growth of skin cancer in mammals.
• human alkaline phosphatase such as placental alkaline phosphatase or transferrin alone or alkaline phosphatase in combination with transferrin, and, optionally, ⁇ i-antitrypsin to prevent or reduce the growth of skin cancer in mammals.
• melanoma consists of local excision with a 0.5-1 cm surgical margin often associated with regional lymph node dissection.
• Other therapies often combined with surgery, include radiotherapy, chemotherapy, immunotherapy (dendritic cells and immune vaccines), biological response modifiers (such as interleukin-2) and hypothermia.
• the treatment for other skin cancers consists of mainly surgical excision; other treatments include electrocautery and curettage, chemosurgery, cryosurgery, radiotherapy and topical chemotherapy.
• Each technique has advantages and disadvantages as listed in a recent article [Martinez, J.-C. and Otley, CC.
• interferon interferon
• interleukin-2 interferon- ⁇ 2b and interleukin-2
• IFN interferon
• other cytokines were ineffective [Atkins, M.B., Elder, D.E., Essner, R., Flaherty, K.T., Gajewsky, T.F., Haluska, F.G., Hwu, P., Keilholz, U., Kirkwood, J.M., Mier, J.W., Ross, M.I., Slingluff, C.L., Sondak, V.K., Sosman, J.A., Weinstock, M.A. and King, L.
• TNF- ⁇ tumor necrosis factor- ⁇
• TNF- ⁇ tumor necrosis factor- ⁇
• TNF- ⁇ can induce hemorrhagic necrosis and regression of tumors including melanoma, and it is being utilized for the treatment of patients with locally advanced solid tumors.
• delivery of optimal doses of TNF- ⁇ is associated with severe toxicity that restricts its administration to sub- optimal doses.
• embodiments of this invention describe the use of human placental alkaline phosphatase (PALP) or transferrin alone or in combination with transferrin (TF) and (optionally) ⁇ i -antitrypsin (AT) for prevention and treatment of skin cancer.
• POP human placental alkaline phosphatase
• TF transferrin
• AT ⁇ i -antitrypsin
• the invention allows administration of compositions topically on the skin or by injection, or both.
• Embodiments of this invention demonstrate the feasibility of using human proteins for topical treatment of mammalian skin to reduce both the occurrence and growth of skin cancer. Such topical treatment may be combined with application of these proteins by an injection method as well. Also, administration of protein compositions may be part of a more complex cancer therapy.
• One of the human proteins used in an alternative embodiment of the invention is placental alkaline phosphatase (PALP) a member of the alkaline phosphatase family.
• the invention provides methods to employ PALP, and by implication other alkaline phosphatases, in humans and other mammals via topical application to prevent the occurrence or decrease the growth of already existing abnormally growing skin tumor tissues.
• alkaline phosphatase is delivered via injection directly into the dermis or skin tumor or by using other available injection methods to reduce the growth of already existing abnormally growing skin tumor tissues.
• the alkaline phosphatase may be delivered simultaneously or sequentially by an injection method and topically to reduce the growth of skin tumor.
• the other human proteins that can be used in combination with alkaline phosphatase in the embodiments for both topical and injection applications are human transferrin (TF) and human ⁇ -antitrypsin (AT).
• TF human transferrin
• AT human ⁇ -antitrypsin
• PALP + TF and PALP + TF + AT are demonstrated in alternative embodiments of the invention to exert antiskin-cancer effects upon topical application: PALP + TF and PALP + TF + AT.
• PALP + AT and TF + AT are also within the scope of the invention.
• commercial PALP preparation that contains both TF and AT in addition to PALP may be used both topically and by an injection method.
• FIG. 1 shows a digital image of a gel separation, demonstrating that the PALP used for the experiments, except when indicated otherwise, was homogeneous or near homogeneous.
• the image also shows the protein composition of the starting commercial PALP preparation, the three major bands being represented by TF alone (80 kDa), a mixture of albumin and PALP (-66-68 kDa) at a ratio of approximately 3:1, and AT (52 kDa).
• FIG. 2 shows a digital image of a gel separation, demonstrating that the Ot 1 - antitrypsin used for the experiments, except when indicated otherwise, was homogeneous, i.e. did not contain any other stained component.
• the image also shows that the protein composition of the starting commercial PALP preparation is slightly different compared to the starting material used for Fig. 1.
• TF 80 kDa
• albumin and PALP 66-68 kDa
• AT 52 kDa
• minor lower molecular weight proteins as well represented by oci-acid glucoprotein ( ⁇ 43 kDa) and degradation products of TF.
• PALP refers generally to the full-length human alkaline phosphatase protein, including isoforms and any chemically modified versions such as created, for example, by glycosylation, phosphorylation, metal-binding, or ligand-binding.
• PALP is not limited to only placental alkaline phosphatase, but also includes intestinal alkaline phosphatase, germ cell alkaline phosphatase, and tissue nonspecific alkaline phosphatase (found in bone, liver, and kidney).
• active derivative of PALP is intended to include any segment or region of PALP that alone or in conjunction with the other proteins promotes an anti skin-cancer effect.
• composition refers to one or more compounds in various combinations according to alternative embodiments of this invention.
• the combinations include a single component of either PALP or TF, or two components, either PALP and TF, or PALP and AT, or TF and AT or all three components, PALP, TF and AT.
• administered generally refers to administering the composition of PALP, TF, AT or various combinations as topical composition, injectable composition, or as a combination of a topical and injectable composition.
• the first active agent is human placental alkaline phosphatase (PALP) or an active derivative thereof.
• POP placental alkaline phosphatase
• PALP are used interchangeably to refer to placental alkaline phosphatase.
• active PALP means the human protein and its glycosylated and non-glycosylated forms as well as peptides derived from these proteins that, when administered locally or by a systemic route reduces the growth of skin cancer cells in mammalian skin tumors in a well detectable manner.
• PALP is a member of the alkaline phosphatase group of enzymes that hydrolyzes phosphate-containing compounds at alkaline pH.
• Mature PALP is a dimer of two identical glycosylated subunits. Each subunit has an approximate molecular weight of 66 kDa, as determined by gel electrophoresis.
• the alkaline phosphatase family also includes the tissue non-specific
• PALP-like (germ cell) alkaline phosphatase Since each of these enzymes has similar phosphatase activities that may contribute to the negative control of cancer cell growth, these three enzymes may share, at least partially, the anti-cancer effects of PALP.
• placental alkaline phosphatase one of the presently known four members of the alkaline phosphatase enzyme family, can enhance both the proliferation and survival of mouse embryo fibroblasts as well as fibroblast-like cells derived from the lung of human fetus [Q.-B. She, J.J. Mukherjee, J. -S. Huang, K.S. Crilly, and Z. Kiss (2000), "Growth factor-like effects of placental alkaline phosphatase in human fetus and mouse embryo fibroblasts," FEBS Letters, 468, 163- 167 ⁇ [Q.-B. She, JJ. Mukherjee, T. Chung, and Z.
• PALP is used either alone or in combination with other human proteins to decrease the growth of melanoma in various melanoma models.
• PALP was highly purified from commercial (Sigma-Aldrich) PALP prepared by a slightly modified method described earlier [Q.-B. She, JJ. Mukherjee, J.-S. Huang, K.S. Crilly, and Z.
• PALP inhibits the growth of melanoma tumor in vivo
• PALP does not exhibit major inhibitory effects on the viability of melanoma cells in vitro. Accordingly, the effects of PALP on melanoma growth in vivo are likely to be mediated by indirect mechanisms such as, for example, activation of the anti-cancer components of the immune system or increased resistance of surrounding normal cells against tumor cells.
• modified PALP, smaller PALP- derived peptides, or modified PALP-derived peptides may be similarly effective or even more effective than the native PALP enzyme, and are each considered to be active derivatives.
• PALP isolated from placenta tissue or produced in recombinant form is considered to be similarly effective.
• Human PALP in solid form is available commercially from Sigma- Aldrich (St. Louis, MO), for example (Sigma catalog number P3895; CAS Registry Number 9001-78-9). Another commercial source of human PALP is Calbiochem (San Diego, CA; catalog number 524604).
• Human PALP and particularly an active derivative, may also be obtained by chemical synthesis using conventional methods. For example, solid-phase synthesis techniques may be used to obtain PALP or an active derivative.
• Recombinant methods to obtain quantities of PALP (and active derivative) are also suitable. Since cDNA of PALP is available, recombinant protein can be produced by one of the many existing conventional methods for recombinant protein expression. PALP has been cloned and overexpressed in a mammalian cell line as described by Millan, et al. [Kozlenkow, A., Manes, T., Hoylaerts, M.F. and Millan, J.L.
• a PALP preparation that is commercially available contains other proteins that may be removed or may be retained depending on the delivery method and the purpose of application. Commercial PALP preparations can be used as starting material to obtain homogeneous PALP, transferrin (TF) and Ot 1 -antitrypsin (AT) by successive chromatographic steps, as described in detail in Example 1.
• PALP preparations may also be used for compositions used in the practice of embodiments of the present invention, so long as the given composition comprises therapeutically effective amount of PALP, TF and AT, and the other impurities are not toxic and do not interfere with the beneficial effects of these components.
• TF and AT add to the anti-skin cancer effects of PALP.
• PALP (as well as TF and AT) may be used only in a "highly purified" form.
• a raw extract of PALP should be treated to enrich the concentration of PALP and obtain a substantially purified or highly purified preparation.
• a highly purified preparation will have a much higher concentration of the active component than found in a raw tissue extract.
• a highly purified PALP preparation does not contain detectable amounts of other proteins or contains such a minimum amount of known contaminants that the benefits of using the preparation far out-weight the accompanying potential risks.
• the term “substantially purified” is used herein to encompass compositions that are obtained from a starting material by one or more purification steps (such as solvent extraction, column separation, chromatographic separation, etc.) that enrich the concentration of PALP, relative to the starting material, to an extent that PALP is the dominating component, and the remaining components do not pose any significant health risk and do not reduce the beneficial effects of PALP.
• the term “substantially purified” should not be construed to connote absolute purity.
• the placenta-derived PALP-enriched preparation may contain TF and AT in sufficient or near sufficient amounts to support the anti-cancer effects of PALP.
• TF and AT in sufficient or near sufficient amounts to support the anti-cancer effects of PALP.
• a commercial preparation of PALP available from Sigma-Aldrich satisfies this criterion. Therefore, for the treatment of mammals either locally on the skin or by injection with the purpose of preventing or treating skin cancer the above-described commercial preparation of PALP may be applicable without further modification of the protein components.
• the commercial PALP may be supplemented with various additives or enhancers to increase its anti-cancer effects.
• a step of heat-activation of PALP preparation may be included prior to either local or systemic application.
• preparation of PALP for either local or systemic treatment may include 1-3 mM of a calcium containing compound (for example, calcium chloride) and/or 1-50 ⁇ M of a zinc containing compound (for example, zinc chloride or zinc sulfate).
• a calcium containing compound for example, calcium chloride
• a zinc containing compound for example, zinc chloride or zinc sulfate
• Substantially purified preparations of intestinal, tissue non-specific, and PALP-like (germ cell) alkaline phosphatase enzymes are all available commercially (for example, from Sigma-Aldrich). Appropriate purification methods are known for their isolation from human blood, liver, and other organs. Also, recombinant forms of each of these alkaline phosphatases have already been produced. [0036] Transferrin
• TF is the second potential component of a protein mixture developed for the prevention and treatment of skin cancer.
• TF is also a glycoprotein with an approximate molecular weight of 80 kDa. Its major function is to carry iron from the sites of intake into the systemic circulation to the cells and tissues.
• TF also serves as a growth factor for many cell types including cancer cells; for this reason, it is a standard component of several growth media used for cell culture. Whether the growth factor effects of TF are always mediated by iron or not is presently unclear.
• Melanoma is especially sensitive to oxidative stress [Baldi, A., Lombardi, D., Russo, P., Palescandolo, E., De Luca, A., Santini, D., Baldi, F., Rossiello, L., DeH'Anna, MX., Mastrofrancesco, A., Maresca, V., Flori, E., Natali, P. G., Picardo, M. and Paggi, M.G. (2005), "Ferritin contributes to melanoma progression by modulating cell growth and sensitivity to oxidative stress," Clin. Cancer Res. 11, 3175-3183].
• iron acts like a pro- oxidant.
• Chronic pro-oxidant stimuli can induce superoxide dismutase activity whereas catalase can be inactivated by H 2 O 2 , its own substrate.
• H 2 O 2 H 2 O 2
• Such imbalance between superoxide dismutase and catalase activities results in surplus H 2 O 2 formation which then, in the presence of iron, leads to generation of extremely reactive hydroxyl radicals via the Fenton reaction.
• excess intracellular iron, provided by excess transferrin may overwhelm the cellular anti-oxidant systems leading to melanoma cell death.
• TF Another potentially important property of TF is its ability to promote migration (but not proliferation) of endothelial cells [Carlevaro, M.F., Albini, A., Ribatti, D., Gentili, C, Benelli, R., Cermelli, S., Cancedda, R. and Cancedda, F.D. (1997), "Transferrin promotes endothelial cell migration and invasion: Implication in cartilage neovascularization," J. Cell. Biol. 136, 1375-1384]. By doing so, TF was expected to promote blood vessel formation in the tumor tissue. In turn, increased vascularization should facilitate distribution of proteins and chemotherapeutic agents in the tumor thereby increasing their efficiency.
• TF has never been used alone or along with PALP and AT to control skin cancer growth.
• TF and the phrase “human TF” are used interchangeably to refer to transferrin.
• active TF means the human protein, or closely related mammalian proteins, and its/their glycosylated and non- glycosylated forms as well as peptides derived from these proteins that, when administered alone or particularly together with PALP or PALP + AT is effective to decrease the growth of melanoma and, by implication, other skin cancers.
• TF was bought from Sigma- Aldrich (T 3309; 98% pure; 300-600 ⁇ g iron per g protein). Because TF is a major component of human blood, and placenta always contains significant volume of blood, the placenta tissue is also a potential source for the isolation of this protein.
• Chromatographic separation methods are available for the purification of TF from blood or placenta. For example, it is possible to enrich TF, along with some other glycoproteins such as PALP and AT, using a so-called Concanavalin-A-Sepharose column, which separates glycoproteins based on their ability to interact with lectins such as Concanavalin-A. This step may then be followed by other column chromatography methods, such as size-exclusion chromatography, to separate glycoproteins from each other. These techniques are well known in the art.
• TF is also present (at the level of about 12% of total protein) in the commercial PALP preparation used for the purification of PALP and AT.
• TF can also be isolated in a highly purified state along with PALP and AT from commercial PALP.
• the sequence of human TF (which has approximately 10 variants) is known and the corresponding cDNA is available. This allows expression of original TF or its point and deletion mutants in any cell line of choice, for example in insect cells [Tomiya, N., Howe, D., Aumiller, J.J., Pathak, M., Park, J., Palter, K.B., Jarvis, D.L., Betenbaugh, MJ. and Lee, Y.C.
• TF preparations that are commercially available from Sigma- Aldrich contain some minor impurities (2-3% of the total protein). Such commercial TF preparations, including those that do not contain iron (apo-TF) or nearly saturated by iron (holo-TF) can be further purified by available methods to obtain homogeneous TF by successive chromatographic steps.
• TF preparations with relatively minor impurities may also be used in the practice of embodiments of the present invention, so long as the given composition comprises therapeutically effective amount of TF, and impurities are not toxic and do not interfere with the beneficial effects of the components.
• the commercial TF that was used (Sigma-Aldrich; catalog number, T 3309) was partially iron-saturated and practically did not contain endotoxin or other contaminating proteins. Such preparation may be used for both systemic and local applications without further purification.
• a raw extract or fraction should be treated to enrich the concentration of TF, with or without parallel enrichment of AT and/or PALP, and obtain a more purified preparation.
• a purified preparation will have a higher concentration of the active component than found in a raw tissue or blood extract.
• the term "purified” is used herein to encompass compositions that are obtained from a starting material by one or more purification steps (such as solvent extraction, column separation, chromatographic separation, etc.) that enrich the concentration of TF, relative to the starting material.
• purified TF should not be construed to connote absolute purity of the protein.
• a further consideration in embodiments of the invention is the degree of purity that is required for the anti-cancer effect.
• An advantage of using a preparation comprising highly purified or homogeneous TF in the methods and treatment regiments of embodiments of the present invention is that possible side effects caused by contaminating proteins will not likely be an issue.
• impure TF or TF that is purified but not homogeneous also can be used in the compositions described herein, as long as no adverse effects are observed.
• every consecutive purification step either using blood or placenta as starting material, results in some loss of the protein, using a less pure than homogeneous TF material for the compositions may be more cost-effective.
• isolation of less pure TF preparations that also contain optimal or near optimal amounts of PALP and AT may be more cost effective than assembling a preparation from highly purified TF, PALP and AT.
• the commercial PALP preparation used in embodiments of the invention contains effective amounts of TF, AT and PALP.
• the TF preparation used to treat experimental melanoma was partially saturated with iron.
• the third potential active component in the methods and compositions of the present invention is human cq -antitrypsin (AT), or an active derivative thereof.
• AT human cq -antitrypsin
• active AT means the various isoforms of the human protein, or closely related mammalian proteins, and its glycosylated and non- glycosylated forms as well as peptides derived from these proteins that can enhance the inhibitory effects of locally applied PALP and TF on the growth of skin cancer.
• AT in the literature also often called ⁇ l -proteinase inhibitor belongs to the large family of serine protease inhibitors, or serpins, that act as irreversible suicide inhibitors of proteases [Janciauskiene, S. (2001), "Conformational properties of serine proteinase inhibitors (serpins) confer multiple pathophysiological roles," Biochem. Biophys. Acta 1535, 221-235]. While AT is a particularly effective inhibitor of elastase, it also inhibits other proteases such as trypsin.
• AT was previously shown to inhibit the growth of breast cancer cells in vitro [Finlay, T.H., Tamir, S., Kadner, S.S., Cruz, M.R., Yavelow, J. and Levitz, M. (1993), " ⁇ rAntitrypsin and anchorage-independent growth of MCF-7 breast cancer cells," Endocrinology 133, 996-1002].
• locally applied AT also slightly enhanced the inhibitory effects of PALP and TF on melanoma growth.
• PALP, TF and AT in combination were about as effective as the commercial PALP preparation which contains all these 3 proteins.
• AT was shown to stimulate proliferation of normal (healthy) cells
• normal (healthy) cells Perraud, F., Besnard, F., Labourdette, G. and Sensenbrenner, M. (1988), "Proliferation of rat astrocytes, but not of oligodendrocytes, is stimulated in vitro by protease inhibitors," Int. J. Devi. Neuroscience 6, 261-266; She, Q. -B., Mukherjee, JJ., Crilly, K.S. and Kiss, Z. (2000), " ⁇ i -Antitrypsin can increase insulin- induced mitogenesis in various fibroblast and epithelial cell lines," FEBS Lett.
• AT can be proteolytically degraded by metalloproteinases and serine proteases resulting in many cases in the formation of 36-44 amino acid cleaved forms. If the concentration of the 36 amino acid fragment reaches a critical value (above 1 ⁇ M), it can stimulate the production of tumor necrosis factor- ⁇ (TNF- ⁇ ) and interleukin-6 (IL-6) by monocytes [Moraga, F., Lindgren, S. and Janciauskiene, S. (2001), "Effects of noninhibitory ⁇ -1 -antitrypsin on primary human monocyte activation in vitro," Arch. Biochem. Biophys. 386, 221- 226].
• TNF- ⁇ tumor necrosis factor- ⁇
• IL-6 interleukin-6
• these cytokines may exert anti-tumor effects as it has been observed, for example, in melanoma tumors [Bennloch, M., Mena, S., Ferrer, S., Obrador, E., Asensi, M., Pellicer, J.A., Carretero, J., Ortega, A. and Estrela, J.M. (2006), "Bcl-2 and Mn-SOD antisense oligodeoxynucleotides and glutamine- enriched diet facilitate elimination of highly resistant B 16 melanoma cells by tumor necrosis factor- ⁇ and chemotherapy," J. Biol. Chem. 281, 69-79].
• active fragments of AT can be chemically synthesized using solid phase chemistry or other conventional methods [Niemann, M.A., Bagott, J.E. and Miller, EJ. (1997), "Inhibition of human serine proteases by SPAAT, the C-terminal 44-residue peptide from Ci 1 -antitrypsin," Biochim. Biophys. Acta 1340, 123-130] or they can be prepared from native AT by the protease elastase as reported [Moraga, F., Lindgren, S. and Janciauskiene, S.
• Relatively pure AT is commercially available (for example, from Sigma- Aldrich; catalog number: A 9024), and it also can be highly purified from commercial PALP preparation which contains AT as a significant "contaminant.”
• essentially pure AT purified from commercial PALP preparation (Sigma-Aldrich) by a previously described method [She, Q.-B., Mukherjee, J.J., Crilly, K.S. and Kiss, Z.
• AT can be isolated in essentially pure form from human placenta. Placenta not only produces this protein [Bergman, D., Kadner, S. S., Cruz, M.R., Esterman, A.L., Tahery, M.M., Young, B.K. and Finlay, T.H. (1993), "Synthesis of cti- antichymotrypsin and cti -antitrypsin by human trophoblast," Pediatric Res.
• PALP may already contain an optimal amount of AT which effectively decreases the growth of skin cancer in the presence of PALP and TF.
• Impure commercial AT preparations that are commercially available contain impurities. Impure commercial AT preparations can be used as starting material to obtain homogeneous AT by successive chromatographic steps, as described in detail in Example 2. Impure AT preparations may also be used in formulating the compositions for use in the practice of embodiments of the present invention, so long as the given composition comprises therapeutically effective amount of AT, and impurities are not toxic and do not interfere with the beneficial effects of the components.
• a preparation containing human AT may also be obtained by extraction from placental tissue that synthesizes the protein during pregnancy.
• a preparation may be obtained by butanol extraction of homogenized placenta. Other methods of extraction from placental tissue are also suitable.
• a raw extract or fraction should be treated to enrich the concentration of AT and obtain a purified preparation.
• a purified preparation will have a higher concentration of the active component than found in a raw tissue or blood extract.
• the term "purified” is used herein to encompass compositions that are obtained from a starting material by one or more purification steps (such as solvent extraction, column separation, chromatographic separation, etc.) that enhance the concentration of AT, relative to the starting material.
• purified AT should not be construed to connote absolute purity of the protein.
• a further consideration in the practice of the invention is the degree of purity that is required for the use in the anti-cancer compositions.
• systemic application of AT will require a large degree of purification.
• the presence of other proteins (in addition to PALP and TF) and cell constituents in the AT preparation is acceptable as long as these contaminants do not cause any undesired side effects and do not decrease the effects of PALP, TF and AT.
• Embodiments of the present invention demonstrate that in animal skin cancer models both topical and subcutaneous application of highly purified PALP results in decreased skin tumor size.
• Human TF, alone or in combination with AT enhances the anti-skin cancer effects of PALP when applied topically on skin tumor.
• injected highly purified PALP adds to the inhibitory effects of topically applied commercial PALP preparation that contains significant amounts of TF and AT.
• Injected purified PALP also increases the effects of chemotherapy; this implies that topically applied PALP or mixtures of PALP + TF and PALP + TF + AT also exert similar additive effects with chemotherapy.
• compositions suitable for topical application may also include, for example, liposomal carriers made up of lipids or special detergents.
• Therapeutically effective amounts of commercial PALP or a similar composition composed of purified PALP, TF and AT may be used as the active components in the compositions described for local applications herein.
• preparations containing synthetic PALP, TF and AT or their active derivatives, or recombinant PALP, TF and AT or their active derivatives may be employed as the active components.
• active means that the given component (as specified above) alone or in combination with the two other components exerts anti-skin cancer effect measured as decreased tumor volume and tumor mass.
• therapeutically effective amount in this specification indicates a dosage of an individual component that is effective in exerting a detectable anti-skin cancer effect either alone or in the presence of the other two components.
• anti skin- cancer effect is used in relation to cancer treatment describing a detectable and quantifiable reduction in the tumor volume and tumor mass.
• preventive anti-skin cancer effect is used in relation to cancer prevention expressing a reasonable expectation that if an agent is able to decrease skin cancer growth, the same agent will also be able to reduce the occurrence of skin cancer if used regularly to treat the cancer-free skin.
• compositions suitable for topical application in the practice of embodiments of the present invention generally include commercial PALP or similar preparations assembled from purified components as minor ingredient, and the physiologically compatible carrier as a major ingredient.
• “Commercial PALP” is defined as a preparation that can be purchased from a commercial firm and that contains, as the minimum, well detectable amount of PALP.
• Commercial PALP may also contain TF, AT, albumin, and minor contaminant proteins that do not interfere with the actions of PALP, TF and AT on skin tumor growth.
• the PALP preparation available from Sigma-Aldrich is a suitable preparation because (i) it contains PALP, TF, and AT, each contributing to the anti skin cancer effect, and (ii) the other proteins that are present do not interfere with the anti skin-cancer effects of PALP, TF, and AT.
• the compositions may include one or more additives or enhancers, such as preservatives, biologically active compounds with positive effects on normal skin cells and adverse effects on skin tumors, buffers, moisture-control compounds, or antibiotics, for example.
• the composition contains the carrier and the active proteins.
• a carrier may be in any form appropriate for topical application to the skin. Any physiologically compatible carrier in which the active components are at least minimally soluble is suitable for topical compositions in embodiments of the present invention.
• a physiologically acceptable carrier for the proteins is one that is non- toxic, does not elicit an adverse physical reaction upon administration, and in which the active component is sufficiently soluble so that the composition may provide an effective amount of the active component.
• the carrier should also provide the composition an appropriate consistency for topical administration and should be capable of achieving proper distribution of the active component to the treated tissue.
• the proteins can be first dissolved in water or a suitable buffer and then mixed with the carrier.
• Suitable carriers generally include, for example, water, acetone, ethanol, ethylene glycol, propylene glycol, butane- 1, 3 -diol, isopropyl myristate, isopropyl palmitate, mineral oil, mixtures thereof, and the like. Buffered solutions and physiological saline can also serve as carriers.
• the topical composition is a gel.
• the gel may include as a carrier methylcellulose, sepharose, agar, Vaseline or petrolatum, agarose, gelatin, dextran, dextranpolyethylene, polyhydroxyethylmethacrylate, hydrophilic cellulose polymer, polyethylene glycol, polyvinylpyrrolidone, amylose, polyethyleneoxide, calcium alginate, or combination thereof.
• the proteins can be incorporated into sterile 3% by weight methyl cellulose gel, 1% by weight agarose gel, 4% by weight gelatin gel, or 1 to 3% by weight calcium alginate.
• One having ordinary skill in the art will have the knowledge to vary these components to obtain sustained release of the active component.
• the carrier is Vaselinum flavum (yellow petrolatum), Vaselinum album (white petrolatum), or Vaselinum cholesterinatum.
• Vaselinum cholesterinatum consists of about 1.5 wt.-% cholesterol, about 5.0 wt.-% cerae lanae, and about 93.5 wt-% Vaselinum flavum.
• Additives or enhancers may be included in the topical protein-containing compositions. The criterion for using an additive is that it increases, or at least does not decrease, the effectiveness of the active components in achieving the desired beneficial effect.
• Additives or enhancers in compositions for topical applications may include various ingredients, for example, preservatives (such as parabens, quaternary ammonium compounds, alcohols, phenols, essential oils, and the like), buffers, antioxidants (such as vitamin E), antimicrobials, vitamins, nutrients (such as essential and non-essential amino acids, choline, inositol, minerals, trace metals, salts, nucleosides, purines, pyrimidines, monosaccharides, disaccharides, carbohydrates) and moisture-control agents (such as glycerine, propylene glycol, and the like).
• preservatives such as parabens, quaternary ammonium compounds, alcohols, phenols, essential oils, and the like
• buffers such as vitamin E
• antimicrobials such as vitamin E
• vitamins such as essential and non-essential amino acids, choline, inositol, minerals, trace metals, salts, nucleosides, pur
• PALP/TF/AT-containing compositions can also be enhanced by other locally acting anti-cancer agents.
• inhibitors of Akt kinase and extracellular signal-regulated kinase may be used for that purpose [Bedogni, B., O'Neill, M.S., Welford, S.M., Bouley, D.M., Giaccia, AJ., Denko, N.C. and Powell, M.B.
• the composition includes a penetration-enhancing additive that enhances penetration of proteins into the skin and tumor tissue.
• a penetration-enhancing additive that enhances penetration of proteins into the skin and tumor tissue.
• suitable penetration enhancers include: sulfoxides such as dimethyl sulfoxide (DMSO); alcohols such as ethanol; polyols such as propylene glycol; surfactants such as sodium lauryl sulfate, lecithin, docusate sodium, and polysorbates; fatty acids such as lauric acid, myristic acid, palmitic acid, mineral oil, and stearic acid; esters such as isopropyl palmitate and isopropyl myristate; and amides such as urea.
• DMSO dimethyl sulfoxide
• alcohols such as ethanol
• polyols such as propylene glycol
• surfactants such as sodium lauryl sulfate, lecithin, docusate sodium, and polysorbates
• fatty acids such as
• Embodiments of the present invention also provide protein-containing compositions suitable for transdermal administration. Such compositions are applied directly to the skin or incorporated into a protective carrier such as a transdermal device, i.e. a patch.
• a protective carrier such as a transdermal device, i.e. a patch.
• suitable creams, ointments, or the like can be found, for example, in the Physician's Desk Reference.
• suitable transdermal devices are described in, for example, U.S. Patent No. 4,818,540 to Chien et al. entitled "Transdermal Fertility Control System and Process".
• the protein-containing compositions can be made using a number of suitable techniques.
• the proteins, optional additives and enhancers as well as a carrier are mixed together within a commercial mixer to form a solution, a suspension, a gel, or the like. All conventional methods known in the art for mixing may be suitable.
• Various equipment are also available to manufacture liposomal preparations (which provide for controlled, sustained release of the components).
• methodologies for the formulation are well known, and can be found, for example, in Remington's Pharmaceutical Sciences, Eighteenth Edition, A.R. Gennaro, Ed., Mack Publishing Co. Easton, PA 1990.
• the compositions may be additionally processed before and after formulation.
• Sterilization for example, may be conducted individually by filter sterilization, while the whole composition can be irradiated or heat-treated or the like. Methods for conducting these steps are also conventional in the art.
• the protein-containing gel or other composition that is suitable to treat skin cancer comprises therapeutically effective amounts of PALP or an active derivative thereof, TF and AT or their active derivatives.
• Therapeutically effective amounts of active proteins may vary depending on the stage of skin cancer development, the needs or tolerance of the individual subject, and the nature of the other treatment(s).
• the concentration of the individual active protein component in a composition for topical application will be at least about 0.01 wt.-%, and more suitably, between about 0.1 and about 4 wt-% so that the total protein concentration does not exceed 6 wt.-%.
• a suitable composition for local application contains 0.4% PALP, 0.5% TF, and 1.5% AT.
• Other suitable compositions may contain 0.1% to 2% commercial PALP.
• compositions may be produced including those containing only PALP, TF, or PALP +
• TF As an example only, PALP alone may be used for prevention, while a composition (such as commercial PALP) also containing TF and AT is recommended for the treatment of skin cancer.
• a composition such as commercial PALP also containing TF and AT is recommended for the treatment of skin cancer.
• the methods described here are suitable for mammals.
• the subject can be, for example, canine, porcine, equine, bovine, or human.
• Embodiments of the invention also provides regimens for treating skin cancer comprising periodically administering by topical application to the skin specific compositions containing effective amounts of PALP, TF and AT or their active derivatives.
• therapeutically effective amounts of the active components are administered.
• the effective amounts of the individual active components that are administered need not to be identical for each separate administration. More or less of the individual active component may be administered in separate administrations, as the subject's needs dictate.
• a medical professional supervising the treatment may adjust the administered dose of the total composition and the ratio of the individual components in it to obtain the desired results.
• An important consideration is that the therapeutically effective amount of the composition also depends on the nature and frequency of other treatments. For example, if the subject is treated with both the composition and chemotherapy, the effective tolerated dose of the former may be less compared to the situation when the subject is treated with the protein composition alone.
• the composition is applied topically to an area of cancer- free skin. In another embodiment, the composition is applied topically to an area of the skin cancer. In both cases, the composition is applied periodically over a period of time.
• the term “periodically” refers to repeated administration of the same or different compositions targeted at the cancer-free skin or skin cancer tissue over the time of treatment.
• the term “periodically” includes repeated administration at fixed intervals, but also includes repeated administration over irregular intervals as is required by the subject's condition.
• the frequency of administration of the composition(s) can vary depending on the type and size of skin cancer, the nature of other treatments, and the level of success.
• the frequency of application is less if the composition is used for prevention; for example, for preventive purpose once a week application is suitable. More frequent application is needed if a skin cancer is detected.
• the composition(s) can be administered two or more times a day, or once a day, or three- times a week.
• the same composition is applied once per day for a time period determined by the level of success.
• Injection application here is defined as administration of anti-skin cancer agents by injection via any of the available routes to raise their amount in the tumor.
• Some injection methods (intravenous, intraarterial, intraportal, intraperitoneal) primarily increase the amount of injected material in the blood followed by redistribution into the tumor.
• Some other injection applications (intradermal, subcutaneous, intratissue, intracranial) lead to an increase in the amount of injected material both in the interstitial space close to the injection site and the vascular system.
• Injected commercial PALP also did not increase metastasis of melanoma cells into the lung (see TABLE 6 in the Examples), despite the presence of TF.
• AT a significant component of commercial PALP preparation, or PALP blocks the biological effect of TF required for increased metastasis.
• AT was found to interfere with the function of transferrin receptor [Graziadei, L, Gaggi, S., Kaserrbacher, R., Braunsteiner, H. and Vogel, W.
• PALP PALP + TF + AT with a ratio of about 1:1 :5, respectively.
• the injectable form of the composition is comprised of a therapeutically effective amount of PALP or an active derivative thereof as well as a physiologically acceptable carrier that does not cause an undesirable physiological effect and is capable of ensuring proper distribution of the active component in the melanoma tissue.
• the proteins are dissolved or dispersed in the physiologically acceptable carrier. Examples of carriers include physiological saline and phosphate-buffered saline.
• the protein(s) may be enclosed in liposomes such as immunoliposomes, or other delivery systems or formulations that are known to the art may be employed.
• the active protein component(s) can be readily dissolved in physiological saline (0.9% NaCl), or in any other physiologically competent carrier, to yield a solution for injection.
• One suitable composition for the practice in the method comprises PALP in a 0.9% physiological salt solution to yield a total protein concentration of 10 mg/ml.
• Another suitable composition comprises PALP in a 0.9% physiological salt solution to yield a total protein concentration of 200 mg/ml.
• a composition comprising the active protein component(s) may be administered by one of the injection methods including, intravenous, intraperitoneal, subcutaneous, intraarterial, intradermal, intratumor, intracranial or intramuscular applications.
• injection of PALP-containing composition may be complementary to the topical treatment with the compositions described above to treat skin cancer.
• Treatments with injected and topically administered protein compositions may be applied simultaneously and sequentially with either the injection procedure or topical treatment applied first.
• the injectable composition may be supplied with additives and enhancers that may be dissolved or suspended in the composition and that are expected to promote the anticancer effects of the proteins or diminish any potential side effect.
• Any chemotherapeutic agents already in clinical use such as, for example, dacacarbazine, cisplatin, vinblastin, doxorubicin, interferon alfa-2b, interleukin-12, taxol, bortezomib (PS-341) or tumor necrosis factor- ⁇ may be added as an enhancer.
• any inhibitor of melanoma cell growth that is still in clinical trial may also be used as enhancers.
• the list of such inhibitors includes docosaheaxanoic acid [Albino, A.P., Juan, G., Traganos, F., Reinhart, L., Connolly, J., Rose, D.P. and Darzynkiewitz, Z. (2000), "Cell cycle arrest and apoptosis of melanoma cells by docosahexanoic acid: Association with decreased pRb phosphorylation," Cancer Res. 60, 4139-4145], staurosporine [Zhang, X.D., Gillespie, S.K. and Hersey, P.
• the mode of injection is selected from intravenous, subcutaneous, intraperitoneal intramuscular, intraarterial, intracranial, intradermal, or intratumor.
• the preferred modes of injection are intradermal or subcutaneous near the skin tumor or directly into the tumor that provides more optimal delivery of proteins to the skin tumor.
• a common way to express a suitable dosage for systemic administration is grams of the active agent(s) per square meter of body surface area for the subject.
• Those having ordinary skill in the art are familiar with the formulas used for estimating a human subject's body surface area, based on the human's height (in cm) and mass (in kg).
• the subject may be administered a total of about 0.02 to 2.5-g PALP per m 2 body surface once daily.
• a subject may be administered by intravenous, intraarterial, intramuscular, intraperitoneal, intracranial or subcutaneous application a total of about 0,02 to 2.5-g PALP per m 2 body surface twice or three times weekly.
• the subject may be administered a total of about 0.02 to 2.5-g PALP per m 2 body surface once a week or biweekly by intravenous, intraarterial, intramuscular, intraperitoneal, intracranial or subcutaneous application.
• a preferred application is twice a week or once a week.
• One suitable treatment of skin cancer is by intradermal or intratumoral injection of the chosen composition ⁇ ). In both methods, for one injection site the subject may be administered a total of about 0.01 to 1 mg of active protein(s). Intradermal or intratumoral delivery of the composition can be performed once or twice daily, two-to-three times a week, once a week, or biweekly, as suitable. [00100] If the chosen composition is injected locally, such as when the mode of injection is intradermal or intratumoral, aliquots of about 10 to 100 ⁇ L per injection site may be administered.
• the concentration of active protein(s) in the injectable composition may be in the range of about 0.1 to 50 mg/mL. Alternatively, the concentration of the active protein may be in the range of about 0.5 to about 20 mg/mL. In one embodiment, a plurality of injection sites is treated for one administration.
• the effective tolerable dose an important consideration is whether the proteins are used alone or used as part of a more complex regimen involving other anticancer agents as well.
• Such regimens may include any other treatment (for example, chemotherapy, radiotherapy, electrochemotherapy, surgery, treatment with immune vaccines, etc.) used to control skin cancer.
• the effective tolerated amount of the injected protein(s) may be less compared to a regimen when the subject is treated with the protein(s) alone.
• Example 1 Purification and Spectrophotometry Assay of PALP.
• the purification steps described in that paper involve homogenization of human placenta in Tris, extraction with butanol, exposure to heat (55 0 C), three successive precipitations of protein with ammonium sulfate followed by re- suspension, fractionation with ethanol twice, and Sephadex-G-200-gel filtration optionally followed by continuous curtain electrophoresis to further separate PALP variants.
• FIG. 1 shows a picture of a gel separation of a preparation comprising commercial PALP without further purification, and other preparations of PALP of increasing purity. Separation of proteins was performed by conventional SDS-PAGE, and proteins were stained with coomassie blue stain. Lane 1 contains various molecular mass standards for comparison. Lane 2 represents a preparation containing commercial PALP with a strong 52 kDa band representing AT and another strong -66-68 kDa band representing a mixture of PALP and albumin.
• Lanes 3 and 4 represent preparations comprising commercial PALP material after further purification steps (described below), and lane 5 represents a preparation of homogeneous PALP obtained by the complete purification procedure described below.
• a purification procedure was performed to further purify the commercially obtained PALP to homogeneity.
• a slightly modified procedure described earlier [She, Q. -B., Mukherjee, J.J., Huang, J.-S., Crilly, K.S. and Kiss, Z. (2000), "Growth factor-like effects of placental alkaline phosphatase in human and mouse embryo fibroblasts," FEBS Lett. 469, 163-167] was used.
• the solution of commercial PALP was prepared by dissolving 350 mg of commercial PALP into 10 ml of buffer A (0.1 M sodium acetate, 0.5 M NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , adjusted to pH 6.5).
• the PALP solution was passed through a Concanavalin A- Sepharose column followed by an elution step using buffer A (50 mM ⁇ -methyl-D- mannopyranoside) as solvent.
• buffer A 50 mM ⁇ -methyl-D- mannopyranoside
• the active fractions collected from the effluent were pooled and dialyzed against buffer B (50 mM Tris-HCL at pH 7.7).
• FIG. 1 in lane 3 SDS-PAGE separation of the collected and dialyzed fraction is shown in FIG. 1 in lane 3.
• the collected and dialyzed fraction from the previous step was then passed through a Q-Sepharose column.
• the fraction of interest was eluted with buffer B using a linear gradient of 0-250 mM potassium phosphate at a pH of 7.5.
• the active fractions from the Q-Sepharose column were pooled and dialyzed against phosphate- buffered saline and concentrated by Amicon ultrafiltration.
• SDS-PAGE separation of the collected and dialyzed fraction is shown in FIG. 1 in lane 4, which demonstrates that at least two major proteins are still present in the fraction after dialysis.
• the collected and dialyzed fraction from the previous step was purified to homogeneity by t-butyl hydrophobic interaction chromatography (HIC).
• HIC t-butyl hydrophobic interaction chromatography
• the fraction Prior to adding the fraction to the t-butyl HIC column, the fraction was made 2 M in ammonium sulfate, and the pH was adjusted to 6.8.
• the 5-ml bed volume t-butyl HIC cartridge (BIO-RAD, Hercules, Calif.) was connected to a fast performance liquid chromatography (FPLC) system from PHARMACIA (Peapack, NJ.).
• FPLC fast performance liquid chromatography
• the fraction was introduced to the HIC column, and the column was eluted with buffer C (100 mM sodium phosphate buffer, 2 M ammonium sulfate at pH 6.8).
• the column was eluted with buffer C until a first protein-containing fraction completely eluted, and then a negative gradient of 2 M-to-0 M ammonium sulfate in 100 mM sodium phosphate at pH 6.8 was passed over the column.
• the negative linear gradient was used to elute a second protein-containing fraction, which contained the enzymatically active PALP protein.
• the enzymatically active PALP fraction from the HIC separation was dialyzed against phosphate buffered saline and concentrated by Amicon ultrafiltration. The presence and purity of the PALP enzyme in the fraction was confirmed by SDS- PAGE. After electrophoretic separation, the gel was stained using coomassie blue or silver stain for visual observation of protein bands. When a single protein band with an approximate molecular weight of 66 kDa was not observed, the last chromatographic step was repeated. The pure PALP was further identified by sequence analysis performed by the Mayo Clinic Protein Core Facility (Rochester, MN, US).
• PALP enzyme activity was assayed using a spectroscopic method by monitoring the hydrolysis of 4-nitrophenylphosphate (as an increase in absorbance at 410 nm) at room temperature (22 0 C) as described in Chang, G.-G., Shiao, M.-S., Lee, K.-R. and Wu, J. -J. (1990), "Modification of human placental alkaline phosphatase by periodate-oxidized monophosphate," Biochem. J. 272, 683-690.
• a partially purified human placental alkaline phosphatase preparation was acquired from Sigma- Aldrich, Inc. AT is a major component of the commercially obtained PALP. AT was first further purified by successive Concanavalin A- Sepharose and Q-Sepharose chromatography as described by Chang et al. for the isolation of PALP [Chang, T.-C, Huang, S.-M., Huang, T.-M. and Chang, G.-G. (1992), "Human placenta alkaline phosphatase: An improved purification procedure and kinetic studies," Eur. J. Biochem.
• the Q-Sepharose fraction which still contained placental alkaline phosphatase in addition to AT, was further purified to homogeneity by r-butyl HIC chromatography [She, Q. -B., Mukherjee, J.J., Crilly, K.S. and Kiss, Z. (2000), "cti -Antitrypsin can increase insulin-induced mitogenesis in various fibroblast and epithelial cell lines," FEBS Lett. 473, 33-36].
• the 5 ml bed volume f-butyl HIC cartridge was connected to a PHARMACIA FPLC system and the fractions containing AT were pooled.
• the purity was confirmed by SDS-PAGE (polyacrylamide gel electrophoresis) using coomassie blue stain.
• the purified protein was identified as AT by sequence analysis. The sequence analysis was performed by the Mayo Clinic Protein Core Facility (Rochester, MN, USA). The protein concentration was determined by the Lowry assay, using bovine serum albumin as standard, with a protein assay kit from Sigma-Aldrich, Inc. according to the instructions. This purification procedure has been previously published [She, Q.- B., Mukherjee, J.J., Crilly, K.S. and Kiss, Z. (2000), " ⁇ -Antitrypsin can increase insulin-induced mitogenesis in various fibroblast and epithelial cell lines," FEBS Lett. 473, 33-36].
• FIG. 2 is an image of a stained gel.
• the gel includes the commercially obtained partially purified placental alkaline phosphatase preparation (shown in lane 2) further purified by successive Concanavalin A-Sepharose (lane 3), Q-Sepharose (lane 4), and t-butyl HIC chromatography using 2 M-to-0 M ammonium sulfate gradient (lane 5).
• Lane 1 contains molecular mass standards of 97 kDa (top), 66 kDa, 45 kDa, 31 kDa, and 22 kDa (bottom) in that order.
• FIG. 1 contains molecular mass standards of 97 kDa (top), 66 kDa, 45 kDa, 31 kDa, and 22 kDa (bottom) in that order.
• the B16 mouse melanoma tumors were developed in first generation hybrid BDFl (C57 Bl female x DBA/2 male) adult female mice kept at specified pathogen free (SPF) hygienic level. These mice have the complete immune system.
• the human H-168 melanoma tumors were developed in the homozygous line of CB. - 171 cr scid/scid mice (with severely compromised immune deficiency) which is an inbred mutant strain.
• tumor tissue fragments of about 0.1 -cm 3 were surgically implanted subcutaneously into the intrascapular region to develop the tumors. Each tumor fragment contained l-1.5xl ⁇ 6 cells.
• the animals were kept in macrolon cages on ventilated rack at 22-24 0 C (50-60% humidity) with lighting regimen of 12/12h light/dark.
• the animals had free access to tap water and were fed with sterilized standard diet (Charles River VRFl, Germany) ad libitum.
• the animals were taken care of according to the "Guiding Principle for the care and use of Animals" based upon the Helsinki declaration and they were approved by the local ethical committee.
• mice bearing the rapidly growing B16 melanoma tumors were used after 8-11 days of tumor transplantation, while mice bearing the slower growing H- 168 melanoma were used after 17 days of transplantation.
• the size of the tumors was in the 0.3 -0.6 cm 3 range; animals were selected so that in the same treatment group the difference in tumor size was no greater than 0.1 cm 3 .
• PALP, TF, or AT were used topically in a cream alone or in combination; in each case, 150-mg cream was applied on the tumor and the vicinity of tumor area (with about 0.3-0.5 cm margin).
• Example 4 PALP preparations inhibit the growth ofB16 melanoma.
• the aggressive B 16 mouse melanoma model was developed as described under Example 3. On day 11 after transplantation of tumor cells, the animals were either remained untreated (Group 1) or were treated with 15 mg/kg highly purified PALP via subcutaneous administration (Group 2). In Group 3, the tumors were treated locally with 150-mg of cream containing 4 mg commercial PALP in 1 g of Vaselinum cholesteratum. In Group 4, the animals were treated simultaneously locally with commercial PALP containing cream and via subcutaneous injection with highly purified PALP. Each treatment was performed once daily for seven consecutive days (i.e., treatments were terminated on day 18). Each group included 7 animals; the mean values ⁇ std. dev. for tumor volumes are shown in TABLE 1.
• Example 5 Effects of topical combined treatments of B16 melanoma with PALP, TF and AT.
• PALP is composed of about 10% PALP, 12% TF, 30%
• Example 3 On day 8 after transplantation of tumor cells, the animals either remained untreated (Group 1) or in other animal groups the tumors were treated locally with creams (150 mg per application) containing active components as follows: Group 2, 0.4 mg of purified PALP per 1-g Vaseline; Group 3, 0.5 mg of purified partially iron- saturated TF per 1-g Vaseline; Group 4, 0.4 mg of PALP + 0.5 mg TF per 1-g Vaseline; Group 5, 1.2 mg of purified AT per 1-g Vaseline; Group 6, 0.4 mg of PALP + 0.5 mg TF + 1.2 mg of purified AT per 1-g Vaseline; and Group 7, Vaselinum cholesteratum alone. Each treatment was performed once daily on days 8, 11, 13, 15, 18 and 20.
• PALP preparation such as the presently used commercial preparation
• Ghosh and Fishman Ghosh, N.K. and Fishman, W.H. (1968), "Purification and properties of molecular- weight variants of human placental alkaline phosphatase," Biochem. J. 108, 779-792] and containing at least PALP and preferably PALP, TF and AT, can be used for the local treatment of skin cancer as the active component of anti-skin cancer products.
• Example 6 Synthesis of N,N-diethyl-N-methyl-2-[(9-oxo-9H- thioxanthen-2-yl)methoxy]-ethanaminium iodide (coded as CCDTHT).
• Example 7 Combined effects of CCDTHT and purified PALP on the growth of Bl 6 melanoma.
• CCDTHT was synthesized as referred to in Example 6.
• the Bl 6 mouse melanoma model was developed as described under Example 3; in each of the three experimental groups five mice were used. In Group 1, from day 11 (11 days after tumor implantation) until the end of experiment the animals remained untreated.
• mice received 4.6 mg/kg CCDTHT once daily from day 11 until day
• mice received 4.6 mg/kg CCDTHT once daily from day 11 until day 22 plus 14 mg/kg highly purified PALP on days 11, 13, 15, 17 and 21. Both
• CCDTHT and PALP were administered by subcutaneous injection.
• Example 8 Effects of commercial PALP and chemotherapy on tumor growth in human melanoma bearing mice.
• Example 3 When treatments were applied, they always started on day 17 following tumor transplantation. In each experimental group 5 animals were included. Animals in Group 1 remained untreated over the whole experimental period. Animals in the second group were treated with 4.5 mg/kg CCDTHT + 0.25 mg/kg of pyrrolidinedithiocarbamate + 0.12 mg/kg of zinc chloride 3 x 5 days for 3 weeks (with 2 days rest after each 5-day period) followed by treatments with only 4.5 mg/kg of CCDTHT for 2 x 5 days (again with 2 days rest after each 5-day period). For simplicity, in TABLE 4 where the results are presented, this complex treatment is named "chemotherapy”.
• Animals in the third group were treated with 1.5 mg of commercial PALP 5 x 5 days for 5 weeks (with 2 days rest after each 5-day period).
• Animals in the fourth group were treated with 4.5 mg/kg CCDTHT + 0.25 mg/kg of pyrrolidinedithiocarbamate + 0.12 mg/kg of zinc chloride + 1.5 mg of commercial PALP 3 x 5 days for 3 weeks (with 2 days rest after each 5-day period) followed by treatments with 4.5 mg/kg of CCDTHT + 1.5 mg of commercial PALP for 2 x 5 days (again with 2 days rest after each 5-day period).
• chemotherapy the complex treatment with agents other than PALP is named "chemotherapy”.
• Example 9 Effects of commercial PALP on the tissue infiltration of lymphocytes and formation of blood vessels in B16 melanoma.
• the excised tumor samples were fixed in 4% paraformaldehyde in phosphate-buffered saline and embedded in paraffin so that several consecutive cross- sections could be made. Sections (5-6 ⁇ m) were stained with hematoxylin/eosin ("H&E") with a standard procedure well known in the art.
• H&E hematoxylin/eosin
• Nine sections derived from 3 animals were evaluated for each treatment for estimating lymphocyte infiltration and blood vessel formation.
• the size of lymphocytes is about double the size of melanoma cells and the two cell types can be easily distinguished. Blood-containing blood vessels are stained red, which ensures their straightforward recognition.
• tumor samples also contained about 2.8-times more red-stained blood vessels per mm 2 than untreated tumor samples (data are not shown separately).
• PALP acted by enhancing the number of blood vessels in the tumor thereby facilitating distribution of chemotherapeutic agents in the tumor tissue.
• increased distribution of chemotherapeutic agents in the tumor tissue will proportionally enhance their anti-tumor effects. Since stimulation of blood vessel formation is likely to take a relatively long time period, this may explain why the effect of PALP and chemotherapy in combination was particularly strong only after about 40 days of starting the treatment.
• Example 10 Comparison of the effects of injected commercial PALP, purified PALP and transferrin as well as locally applied commercial PALP on lung metastasis ofB16 melanoma.
• B16 melanoma cells are highly metastatic that are known to preferably form metastatic colonies in the lung.
• the goal of this experiment was to determine if any of the components present in commercial PALP or the mode of application affect the metastatic process.
• the B 16 melanoma model described under Example 3 was used.
• the treatments of mice bearing the rapidly growing B 16 melanoma were started 11 days after tumor transplantation; additional treatments were performed on each day for six consecutive days.
• seven animals were used.
• animal tumors were treated locally with 200 mg Vaseline (control group).
• animal tumors were locally treated with 200 mg Vaseline cream containing 4 mg commercial PALP per 1-g Vaseline.
• animals were subcutaneously injected commercial PALP (1.5 mg per animal or 60 mg per kg).
• animals were treated with commercial PALP both locally and subcutaneously.
• mice were subcutaneously injected highly purified PALP (0.35 mg per animal or 14 mg per 1 kg).
• animals were subcutaneously injected partially iron-saturated human TF (0.4 mg per animal or 16 mg per 1 kg).
• animal tumors were locally treated with 200 mg Vaseline cream containing 1.0 mg human TF per 1 g Vaseline.
• the tumors were untreated during the entire period of experiment.
• the macroscopic colonies were counted in the lung (7 animals in each group) 21 days after starting the treatments.
• the data are presented in TABLE 5; numbers in the parentheses indicate the smallest and highest numbers of metastatic lung colonies in the same group. The results show that subcutaneous administration of TF leads to the doubling of lung metastatic colonies, while other treatments have practically no effects on the metastatic process.

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