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WO2002076496A1 - Facteur letal de l'anthrax inhibant la croissance tumorale et l'angiogenese - Google Patents

Facteur letal de l'anthrax inhibant la croissance tumorale et l'angiogenese Download PDF

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Publication number
WO2002076496A1
WO2002076496A1 PCT/US2002/008656 US0208656W WO02076496A1 WO 2002076496 A1 WO2002076496 A1 WO 2002076496A1 US 0208656 W US0208656 W US 0208656W WO 02076496 A1 WO02076496 A1 WO 02076496A1
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Prior art keywords
tumor
angiogenesis
ofthe
mek
inhibitor
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PCT/US2002/008656
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English (en)
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Nicholas S. Duesbery
Craig P. Webb
George F. Vande Woude
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Van Andel Institute
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Priority to US10/472,396 priority Critical patent/US20040136975A1/en
Priority to AU2002255852A priority patent/AU2002255852B2/en
Priority to CA002442015A priority patent/CA2442015A1/fr
Priority to EP02725277A priority patent/EP1377312A4/fr
Publication of WO2002076496A1 publication Critical patent/WO2002076496A1/fr

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    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • 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
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4427—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems
    • A61K31/4439—Non condensed pyridines; Hydrogenated derivatives thereof containing further heterocyclic ring systems containing a five-membered ring with nitrogen as a ring hetero atom, e.g. omeprazole
    • 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)
    • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
    • A61K38/4886—Metalloendopeptidases (3.4.24), e.g. collagenase

Definitions

  • the present invention in the fields of molecular/cell biology and medicine is directed to the use of antlirax Lethal Factor (LF), for example, in the form of anthrax lethal toxin (LeTx), as an agent for inhibition of angiogenesis and treatment of diseases and conditions associated with undesired angiogenesis, growth of primary and metastatic tumors.
  • LF antlirax Lethal Factor
  • LeTx anthrax lethal toxin
  • Mitogen activated protein kinase kinases play pivotal roles in a variety of signal transduction pathways, aspects of which are critical for cell cycle progression and differentiation (Lewis, TS et al, (1998) in Adv. Cane. Res., eds., Vande Woude, GF et al, (Academic Press, San Diego) pp. 49-139).
  • Mitogen activated protein kinases (MAPKs), their upstream activators and their downstream effectors define multiple, conserved, eulcaryotic signal transduction pathways. Each of these pathways operates in parallel to the others, responding to particular extracellular stimuli both at a translational and post-translational level.
  • MEKs are upstream activators of members ofthe MAPK family. These members comprise extracellular-signal-regulated kinases (ERKs) also known as mitogen-activated protein kinases (MAPKs), for example, ERK 1 or ERK 2 which are the same as MAPK 1 or MAPK 2). Seven different MEK enzymes have been described. MEKs 1 and 2 phosphorylate and activate
  • MEKs 1 and 2 are stimulated by mitogens or growth factors.
  • MEKs 3, 4, and 6 have been implicated in regulation ofthe MAPK family member p38 MAPK (Derijard B et al, Science, 1995, 267:682-685; Raingeaud J et al, Mol Cell Biol, 1996, 16:1247-1255) which mediates cellular response to stimuli such as osmotic shock or cytokines (Lee JC et al, Nature, 1994, 372:739-746; Han J et al, Science, 1994, 265:808-811; Freshney NW et al, Cell, 1994, 78:1039-1049; Rouse J et al, Cell, 1994, 78 : 1027- 1037) and also may play a role in monitoring spindle assembly during mitosis (Takenaka K et al, Science, 1998,
  • MEKs 4 and 7 regulate the activity of the MAPK family member known as "stress activated protein/jun kinase” (SAPK, JNK or SAPK/JNK), which in turn regulates the transcription factor jun (Derijard et al, supra).
  • MEK 5 regulates the activity of ERK 5 (Zhou G et al, JBiol Chem, 1995, 270:12665-12669) and is activated in response to oxidative stress, hyperosmolarity and serum treatment (Kato Y et al, Nature, 1998, 395:713-716; Kato Y et al, EMBO J, 1997, 16:7054-7066).
  • MEK-ERK signalling has also been shown to play a critical role in tumor metastasis (Jeffers M et al, Proc Natl Acad Sci USA, 1998, 95:14417-22; Ward Y et al, Mol Cell Biol,
  • MEK related pathways may also play a role in tumorigenesis.
  • Activation of p38 MAPK is required for expression of and cellular response to vascular endothelial growth factor
  • NEGF neurotrophic factor
  • LF Anthrax lethal factor
  • LF is a protease that cleaves members ofthe MEK family including MEKs 1, 2 (Duesbery, ⁇ S. et al. (1998) Science 280:734-7; Nitale, G et al, (1998) Biochem Biophys Res Commun 248:706-11 and MEK3 (Pellizzari, R et al, (1999) FEBS Lett 462:199-204).
  • LF-induced proteolysis of MEK1 blocks MAPK activation (Duesbery et al, supra; Colanzi, A et al, (2000) J Cell Biol 149:331-9).
  • LF is produced by Bacillus anthracis, the Gram-positive bacterium responsible for the disease anthrax.
  • B. anthracis produces an exotoxin consisting of three proteins; protective antigen (PA), LF, and edema factor (EF) (Duesbery, ⁇ S et al. (1999) Cell Mol Life Sci 55:1599-609).
  • PA protective antigen
  • EF edema factor
  • EF is an adenylyl cyclase enzyme that dramatically elevates intracellular cAMP concentrations (Leppla, SH (1982) Proc Natl Acad Sci USA 79:3162-6).
  • Combinations of EF plus PA (termed “edema toxin" of "EdTx") cause skin edema characteristic of anthrax but are not toxic when injected intravenously into mice or rats.
  • Angiogenesis the formation of new capillaries from pre-existing ones (Folkman, J, N. Engl J. Med., 1971, 255:1182-1186; Hanahan D. et al, Cell, 1996, 56:353-364), is a normal part of embryonic development, wound healing and female reproductive function.
  • angiogenesis also plays a pathogenic role in the establishment and progression of certain diseases. Cancer, rheumatoid arthritis and diabetic retinopathy are examples of such diseases (Carmeliet P. et al, Nature, 2000, ⁇ 07:249-257). Anti-angiogenic therapy holds promise in inhibiting the progression of these diseases.
  • Angiogenesis can be triggered by several pro-angiogenic cytokines.
  • rumor cells under hypoxic conditions secrete NEGF and/or fibroblast growth factor
  • bFGF endothelial growth factor
  • ECs proliferate and migrate toward the hypoxic tumor, resulting in the generation and maturation of new blood vessels.
  • anti-angiogenic factors result from the degradation of matrix proteins - i.e., are a result ofthe action of pro-angiogenic enzymes.
  • examples include endostatin, a fragment of collagen XIII (O'Reilly, M. S. et al, Cell 1997, 88:277-285); kringle 5 of plasminogen (O'Reilly, M. S. et al, Cell, 1994, 79:315-328) and PEX, the C-terminus non- catalytic subunit of MMP-2 (Brooks P.C. et al, Cell, 1998, 92:391-400).
  • Unregulated angiogenesis contributes to the pathology of not only many neoplastic diseases but also a number of non-neoplastic diseases associated with abnormal neovascularization including arthritis, various ocular disorders, and psoriasis. See, for example,
  • ocular diseases include diabetic retinopathy, neovascular glaucoma, ocular inflammatory diseases and ocular tumors (e.g., retinoblastoma).
  • diabetic retinopathy e.g., diabetic retinopathy
  • neovascular glaucoma e.g., diabetic retinopathy
  • ocular inflammatory diseases e.g., retinoblastoma
  • ocular tumors e.g., retinoblastoma
  • neovascularization diseases which are associated with neovascularization, including retrolental fibroplasia, uveitis, retinopathy of prematurity, and macular degeneration.
  • About twenty eye diseases are associated with choroidal neovascularization and about forty with iris neovascularization (Waltman DD et al, 1978, Am. J Ophthal 55:704-710 and Gartner, S. et al, 1978, Surv. Ophthal. 22:291-312.
  • Current treatments of these diseases, especially once neovascularization has occurred, are frequently inadequate to stave off blindness. Studies have suggested that vaso-inhibitory factors which are present in normal ocular tissue (cornea and vitreous) are lost in the diseased states.
  • Anthrax lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many ofthe MEKs.
  • LeTx anthrax lethal toxin
  • LeTx effectively inhibited MAPK activation in V12 H-ras transformed NTH 3T3 cells.
  • treatment of transformed cells with LeTx caused them to revert to a non- transformed morphology, inhibited their ability to form colonies in soft agar and to invade Matrigel, without markedly affecting cell proliferation.
  • LeTx inhibited growth of ras- transformed cells implanted in athymic nude mice, in some cases causing tumor regression, at concentrations that caused no apparent systemic toxicity.
  • LeTx also greatly decreased tumor neo-vascularization.
  • the present inventors therefore conceived that LeTx, though the LF component, and other proteins or small molecules with similar modes of biochemical action, are potent inhibitors of angiogenesis, including tumor angiogenesis.
  • the present invention is directed specifically to a method for inhibiting cell migration, cell invasion, cell proliferation or angiogenesis, or for inducing apoptosis, comprising contacting cells associated with undesired cell migration, invasion, proliferation or angiogenesis with an effective amount of an inhibitor of MEK or of an enzyme that is a member ofthe MAPK family.
  • the method is for inhibiting angiogenesis.
  • the inhibitor of MEK is preferably a MEK-directed protease, such as Bacillus anthracis lethal factor or a functional derivative or homologue thereof.
  • the MEK inhibitor is an organic small molecule, preferably PD98059, U0126 or PD184352.
  • the inhibitor may be an inhibitor of a MAPK family member selected from the group consisting of ERK 1, ERK2, p38 kinase and JNK.
  • a preferred embodiment targets p38 kinase, where a preferred inhibitor is SB203580.
  • the above method may be performed so that the contacting is in vivo, such as the contacting in a mammalian subject, preferably a human.
  • the subject has a tumor and the inhibition of angiogenesis results in cessation of growth or a measurable regression of a primary or metastatic tumor.
  • This invention also provides a method for inhibiting angiogenesis in a mammalian subject, preferably a human, comprising administering to a mammalian subject in need of such inhibition an angiogenesis-inhibiting amount of a pharmaceutical composition that comprises: (a) an inhibitor of MEK or of an enzyme that is a member ofthe MAPK family; and (b) a pharmaceutically acceptable carrier or excipient, thereby inhibiting the angiogenesis.
  • the invention includes a method for treating a mammalian subject, preferably a human, having a disease or condition associated with undesired cell migration, invasion, proliferation, or angiogenesis, comprising administering to the subject an effective amount of a pharmaceutical composition that comprises :
  • the disease or condition is associated with undesired angiogenesis or neovascularization
  • the inhibitor is preferably a MEK inhibitor such as a MEK-directed protease, preferably Bacillus anthracis lethal factor or a functional derivative or homologue thereof.
  • the inhibitor is an organic small molecule; preferred MEK inhibitors of this class are PD98059, U0126 and PD184352.
  • the treatment method may employ an inhibitor of a MAPK family member selected from the group consisting of ERK 1, ERK2, p38 kinase and JNK.
  • a preferred target is p38 kinase, using, for example, the inhibitor is SB203580.
  • Diseases or conditions that are treated by the above method are tumor growth, tumor invasion or tumor metastasis wherein the angiogenesis inhibition results in reduction in size or growth rate ofthe tumor or destruction ofthe tumor.
  • Preferred a targets are solid tumors including brain tumors.
  • Figures la-lh show the effects of LeTx upon MAPK activation and cell morphology.
  • Immunoblotting of lysates from non-transformed (pDCR NTH 3T3) (Fig. la) and N12 H-ras transformed NIH 3 T3 cells (Fig. lb) show loss ofNH -terminal epitopes of MEK and phospho- epitopes of MAPK following treatment of cells with LeTx but not in control cells treated with medium alone, 100 ng/ml PA plus inactive 10 ng/ml LF(E687C), PA plus LF (100 ng/ml PA plus 10 ng/ml LF), or PD98059 (50 ⁇ M from a 50 mM stock in DMSO).
  • Non-transformed (Fig. lc) cells possessed an irregular, flattened morphology which was not substantially altered by 24 hr. exposure to LeTx (Fig. Id).
  • LeTx Fig. Id
  • the well-defined, elongated, spindle-like shape of VI 2 H-ras transformed NIH 3T3 cells Fig. le
  • Fig. If Immunostaining of N12 H-ras transformed NTH 3T3 cells incubated in the absence (Fig. lg) or presence (Fig. 1) of LeTx for 24 hr. for actin (green) showed actin stress fibers formed following LeTx treatment.
  • Figures 2a-2e show the effects of LeTx upon anchorage independent colony formation and extracellular matrix invasion.
  • VI 2 H-ras-transformed cells We evaluated the ability of VI 2 H-ras-transformed cells to form colonies in Noble agar (Figs 2a, 2b) or invade Matrigel (Figs 2c, 2d) in the presence (Figs 2b, 2d) or absence (Figs 2a, 2c) of LeTx.
  • Fig. 2e shows results of assays ofthe levels of cathepsin L by immunoblotting lysates of N12 H-ras transformed NTH 3T3 cells which had been treated with medium alone, PA plus inactive LF(E687C), or LeTx. Blots were stripped and reprobed with antibodies raised against ⁇ -tubulin to control for lane loading.
  • Figures 3a-3d show the effects of LeTx upon N12 H-ras transformed ⁇ IH 3T3 xenografts in athymic nude mice.
  • Growth of tumors derived from N12 H-ras transformed NTH 3T3 cells was measured after either sham injection or injection with Hank's buffered saline solution (HBSS) (Fig. 3a) or after injection with either HBSS or HBSS containing PA and LF (Fig. 3b).
  • Open symbols indicate the tumor on the left side, closed symbols the tumor on the right.
  • Tumor size is expressed as the product of their measured length and width.
  • Arrow heads on the x-axis indicate the times of injection.
  • the appearance of tumors from Group A (Fig. 3c) and Group B (Fig. 3d) are shown adjacent to a ruler indicating tumor size (mm).
  • Figures 4a-4h are a series of photomicrographs showing a histological analyses of tumors derived from LeTx injected mice.
  • H&E hematoxylin and eosin
  • LeTx is a potent inhibitor not only of ras-mediated oncogenic transformation and in vivo tumor growth per se but also tumor vascularization.
  • the effects on vascularization are apparent at concentrations which have no apparent side effects.
  • the inventors therefore evaluated the effect of LeTx upon (a) ras-mediated transformation in vitro and in vivo, (b) and on tumor growth in vivo, and (c) on tumor vascularization.
  • LF is intended to include close LF homologues, functional derivatives and mimetics (whether or not the latter terms are listed after an occurrence of "LF").
  • LF inhibits the MAPK pathway by proteolytically cleaving a MEK family member.
  • This discovery also provides means for identifying novel therapeutic agents, including smaller organic molecules that acting as LF mimetics or modulators, that either cleave or promote proteolytic cleavage of a MEK, thereby inhibiting the MAPK signal transduction pathway. Such agents are useful for treating cancer.
  • LF By its action on MEKs, LF can reverse or attenuate numerous cellular changes associated with oncogenic transformation, including, but not limited to, characteristic cellular morphology, intracellular patterns of actin distribution, rates of proliferation and anchorage-independent growth, hi addition, LF (or the PA component with which it is preferably administered) may be further modified to specifically target cancer cells in a more selective manner, thereby rendering it particularly useful as a cancer therapeutic.
  • Modulators that activate or promote LF proteolytic activity may be used along with LF, its homologues or mimetics to promote their anticancer activity.
  • LF mimetic is an agent, generally a polypeptide or peptide molecule, that recognizes a MEK as a substrate and cleaves the MEK at the same site cleaved by full-length, native LF.
  • LF mimetics include homologues, peptides of LF, conservative substitution variants, as well as deletion variants that retain the protease active site and proteolytic action on MEKs.
  • LF mimetics are tested using assays for LF activity, e.g., MEK mobility shift assays, MOS-induced activation of MAPK in oocytes and myelin basic protein (MBP) phosphorylation, as are known in the art.
  • assays for LF activity e.g., MEK mobility shift assays, MOS-induced activation of MAPK in oocytes and myelin basic protein (MBP) phosphorylation, as are known in the art.
  • MBP myelin basic protein
  • LF is generally the positive control for protease activity. Mimetic activity is at least about 20% ofthe activity of this control, more preferably between about 50-100% ofthe positive control.
  • “Modulators" of LF are agents that activate (promote, enhance, increase) or inhibit
  • LF proteolytic activity (suppress, block, decrease) LF proteolytic activity and are identified by assays in vitro or in vivo assays of this activity or downstream activities.
  • JNK enzyme and pathway is involved in EC motility (Shin EY et al, Exp Mol Med, 2001, 33:276-83).
  • Stable transfectant ECs expressing oncogenic H-Ras (Leu 61 showed enhanced angiogenic potential and motility compared to control EC cells.
  • JNK was constitutively activated in these transfectants.
  • Pretreatment with JNK-specific inhibitors, curcumin and trans-retinoic acid decreased the basal motility and motility stimulated by JTNtK agonists (e.g., TNF ⁇ and anisomycin) ofthe transfected EC cells in a dose-dependent manner.
  • JTNtK agonists e.g., TNF ⁇ and anisomycin
  • Angiogenesis promoted by TNF ⁇ is mediated, at least in part, by ephrin Al, a member of the ligand family for Eph receptor tyrosine kinases.
  • Cheng N et al, J Biol Chem, 2001, 276:13771-7 showed that ephrin Al induction was blocked by inhibition of p38 MAPK or S APK/JNK, but not p42/44 MAPK, using either selective chemical inhibitors or dominant- negative forms of p38 MAPK or TNF receptor-associated factor 2.
  • Heregulin stimulates NEGF secretion from breast cancer cells that can result in increased EC migration. This action is inhibited by anti-VEGF-neutralizing antibody or SB 203580 (Xiong S et al, Cancer Res, 2001, 61 : 1727-32). Heregulin activates ERK, Akt kinase, and p38 MAPK; however, only the specific inhibitor of p38 MAPK (SB 203580), but not an ERK inhibitor PD98059 nor an inhibitor of phosphatidylinositol 3-kinase-Akt pathway (Wortmannin), blocks up-regulation of NEGF by heregulin. Thus, receptor mediated activation of p38 MAPK to enhance NEGF transcription via an upstream heregulin response element, leads to increased NEGF secretion by cancer cells and a subsequent angiogenic response.
  • a functional homologue (described in more detail in a separate section below) is required to possess the characteristic of having a proteolytic action on MEK1.
  • homologous proteins to LF from other bacterial species even proteins not yet discovered, fall within the scope ofthe invention provided that these proteins have the recited biochemical and biological activity. It is within the skill in the art to obtain and express such a protein using DNA probes based on the sequence of LF or Salmonella or plant-derived homologues already characterized. Then, the protein's biochemical and biological activity can be tested readily using art-recognized methods such as those described herein, for example, a standard gel mobility shift assay for pro teo lysis of the substrate protein MEK1 , or inhibition of
  • MEK1 -mediated phosphorylation of its natural substrate, MAPK, or of a model substrate MEK1 -mediated phosphorylation of its natural substrate, MAPK, or of a model substrate.
  • a “variant” of LF refers to a molecule substantially identical to either the full protein or to a fragment thereof in which one or more amino acid residues have been replaced (substitution variant) or which has one or several residues deleted (deletion variant) or added (addition variant).
  • substitution variant substitution variant
  • fragment residues of LF refers to any subset ofthe molecule, that is, a shorter polypeptide ofthe full length protein.
  • a preferred group of LF variants are those in which at least one amino acid residue and preferably, only one, has been substituted by different residue.
  • deletions, insertions and substitutions according to the present invention are those which do not produce radical changes in the characteristics ofthe protein in terms of its proteolytic activity.
  • substitution, deletion or insertion in advance of doing so, one skilled in the art will appreciate that the effect can be evaluated by routine screening assays such as those described here, without requiring undue experimentation.
  • the preferred longer chain variants are typically made by site-specific mutagenesis of the nucleic acid encoding the polypeptide, expression ofthe variant nucleic acid in cell culture, and, optionally, purification ofthe polypeptide from the cell culture, for example, by immunoaffinity chromatography using specific antibody immobilized to a column (to absorb the variant by binding to at least one epitope).
  • the activity of a variant present in a cell lysate or a more highly purified preparation is screened in a suitable screening assay for the desired characteristic, preferably the proteolysis of a MEK. It is also possible to follow the immunological character of the protein molecule is assayed by alterations in binding to a given antibody, and may measured by competitive immunoassay. Biochemical or biological activity is screened in an appropriate assay, as described below.
  • a “detectable moiety” or label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELIS A), biotin, digoxigenin, or haptens; and proteins for which antisera or monoclonal antibodies are available.
  • a protein is detectably labeled if it is bound, either covalently, through a linker, or through ionic, van der Waal's or hydrogen bonds, to a label such that the presence ofthe protein is detected by detecting the presence ofthe label.
  • the present invention utilizes fusion proteins comprising LF (or its homologue or functional derivative) that are fused to another peptide or polypeptide that confers useful properties on the fusion protein such as stability.
  • PA is one of three protein components ofthe "lethal” or “anthrax” toxin produced by B. anthracis.
  • the 83kDa PA binds to a cell surface receptor present on almost all vertebrate cells, and its C-terminus is necessary for this binding (Singh, Y et al, J. Biol Chem. 264:19103-19107 (1989); Novak, J. et al, J. Biol. Chem. 267:17186-17193 (1992)).
  • PA63 After binding, PA is specifically cleaved by a protease (e.g., furin, clostripain or trypsin), releasing a 20 kDa N- terminal PA fragment while a 63kDa C-terminal PA fragment (PA63) remains bound.
  • a protease e.g., furin, clostripain or trypsin
  • PA63 contains the receptor binding site at its C-terminus.
  • PA63 forms a heptameric membrane-inserted channel which mediates the entry ofthe two other protein components ofthe complex (LF and EF) into the cytosol via the endosomal pathway (Gordon et al, Infect. Immun. 56:1066-1069 (1988); Milne et al, J. Biol Chem.
  • PA refers a PA protein (or functional homologue or derivative) that has its receptor binding site intact and functional.
  • U.S. Patents 5,591,631 and 5,677,274 (incorporated by reference in their entirety) describe PA fusion proteins that target PA to particular cells, such as cancer cells and HIN-infected cells, using as fusion partners ligands for receptors on the targeted cells.
  • LF and its homologues as described herein, as antiangiogenic and/or antitumor agents, requires administration in conjunction with PA to achieve transport into cells.
  • a "chemical derivative" of LF contains additional chemical moieties not normally a part ofthe protein.
  • Covalent modifications ofthe protein are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Such chemically modified and derivatized moieties may improve the protein's solubility, absorption, biological half life, and the like. These changes may eliminate or attenuate undesirable side effects ofthe protein in vivo. Moieties capable of mediating such effects are disclosed, for example, in Remington 's Pharmaceutical Sciences, Mack Publishing Company, Easton Pennsylvania (Gennaro 18th ed. 1990).
  • Cysteinyl residues most commonly are reacted with ⁇ -haloacetates (and corresponding amines) to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, ⁇ -bromo- ⁇ -(5-imidozoyl)propionic acid, chloroacetyl phosphate, ⁇ - alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4- nitrophenol, or chloro-7-nitrobenzo-2- oxa-l,3-diazole.
  • Histidyl residues are derivatized by reaction with diethylprocarbonate (pH 5.5-7.0) which agent is relatively specific for the histidyl side chain, -bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.
  • Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents reverses the charge ofthe lysinyl residues.
  • Other suitable reagents for derivatizing ⁇ -amino-containing residues include imidoesters such as methylpicolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid; O-methylisourea, 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
  • Arginyl residues are modified by reaction with one or several conventional reagents, including phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin. Such derivatization requires that the reaction be performed in alkaline conditions because ofthe high p a ofthe guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine ⁇ -amino group. Modification of tyrosyl residues has permits introduction of spectral labels into a protein or peptide. This is accomplished by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to create
  • Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiirnide or
  • Aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Deamidation can be performed under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
  • Derivatization with bifunctional agents is useful for cross-linking the polypeptide to a water-insoluble support matrix or other macromolecular carrier.
  • Commonly used cross-linking agents include l,l-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'- dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-
  • Derivatizing agents such as methyl-3-[(p-azidophenyl)ditl io]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light.
  • reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patents 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
  • Homologues of LF (or peptide fragments or fusion proteins thereof) share sequence similarity with LF and also exhibit anti-angiogenic and anti-tumor activity.
  • a functional homologue must possess the biochemical and biological activity, preferably MEK-inhibiting, ant-angiogenic and anti-tumor activity which can be tested using in vitro or in vivo methods described herein, h view of this functional characterization, use of homologous LF proteins from other species, including proteins not yet discovered, falls within the scope ofthe invention if these proteins have sequence similarity and the recited biochemical and biological activity.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • Cys residues are aligned.
  • the length of a sequence being compared is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% ofthe length ofthe reference sequence.
  • the amino acid residues (or nucleotides from the coding sequence) at corresponding amino acid (or nucleotide) positions are then compared. When a position in the first sequence is occupied by the same amino acid residue (or nucleotide) as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology").
  • the percent identity between the two sequences is a function ofthe number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. 45:444-453 (1970) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a
  • Blossom 62 matrix or a PAM250 matrix and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W.
  • a homologue ofthe LF described herein is characterized as having (a) functional activity of native LF, and (b) sequence similarity to native LF when determined above, of at least about 30% (at the amino acid level), preferably at least about 50%>, more preferably at least about 70%o, even more preferably at least about 90%.
  • the gene and nucleotide sequence encoding LF has been described.
  • the gene encoding PA (assigned Genbank accession no. M22589) has been cloned and sequenced (Ivins, BE et al., Infect. Immun. 54:537-542 (1986); Welkos, SL et al, Gene 69:287-300 (1988); U.S. 5,591,631,
  • the protein has Swiss Prot accession number PI 3423.
  • the genes encoding MEKl and MEK-2 have been cloned and sequenced.
  • MEKl has been assigned GenBank accession no. LI 1284, and the accession no. for MEK-2 is LI 1285 (see, e.g., Zheng et al, J. Biol. Chem. 268:11435-11439 (1993)).
  • a preferred type of LF-mimetic described herein is a peptidomimetic compound which mimics the biological effects of LF or of a biologically active peptide thereof.
  • a peptidomimetic agent may be an unnatural peptide or a non-peptide agent that recreates the stereospatial properties ofthe binding elements of LF such that it has the binding activity or biological activity of LF. Similar to biologically active peptides, a peptidomimetic will have a binding face
  • a non-binding face of a peptidomimetic will contain functional groups which can be modified, for example, by various therapeutic moieties without modifying the binding face ofthe peptidomimetic.
  • a preferred embodiment of a peptidomimetic would contain an aniline on the non-binding face ofthe molecule.
  • the NH 2 -group of an aniline has a pKa ⁇ 4.5 and could therefore be modified by any NH 2 - selective reagent without modifying any NH 2 functional groups on the binding face ofthe peptidomimetic.
  • peptidomimetics may not have any NH 2 functional groups on their binding face and therefore, any NH 2 , without regard for pK a could be displayed on the non-binding face as a site for conjugation.
  • other modifiable functional groups such as -SH and -COOH could be incorporated into the non-binding face of a peptidomimetic as a site of conjugation.
  • a therapeutic moiety could also be directly incorporated during the synthesis of a peptidomimetic and preferentially be displayed on the non-binding face ofthe molecule.
  • This invention also includes compounds that retain partial peptide characteristics.
  • any proteolytically unstable bond within a peptide ofthe invention could be selectively replaced by a non-peptidic element such as an isostere (N-methylation; D-amino acid) or a reduced peptide bond while the rest ofthe molecule retains its peptide nature.
  • Peptidomimetic compounds either agonists, substrates or inhibitors, have been described for a number of bioactive peptides such as opioid peptides, NJP, thrombin, HIV protease, etc.
  • bioactive peptides such as opioid peptides, NJP, thrombin, HIV protease, etc.
  • Methods for designing and preparing peptidomimetic compounds are known in the art (Hruby, VJ, Biopolymers 53:1073-1082 (1993); Wiley, R.A. et al, Med. Res. Rev. 13:327-384 (1993);
  • such peptidomimetics may be identified by inspection ofthe cystallographically-derived three-dimensional structure of LF or an active peptide of LF either free or bound in complex with a ligand such as a MEK or an active site mimic of thr MEK protein.
  • a ligand such as a MEK or an active site mimic of thr MEK protein.
  • the structure of a LF peptide bound to its ligand can be gained by the techniques of nuclear magnetic resonance spectroscopy. The better knowledge ofthe stereochemistry ofthe interaction ofthe peptide with its ligand will permit the rational design of useful peptidomimetic agents for inactivating MEK and inhibiting angiogenesis.
  • the structure of LF or an LF in the absence of ligand could also provide a scaffold for the design of mimetic molecules.
  • small molecules are organic chemical entities that are not biological macromolecules such as proteins or peptides.
  • the small molecule inhibitors generally have a molecular mass of less than about 2000 Da, preferably less than about 1000 Da, more preferably less than about 500 Da.
  • inhibition of MEK by the small molecule inhibitor PD98059 results in the efficient antiangiogenic and antitumor effects..
  • Other small molecule inhibitors ofthe MAPK pathway or related pathways useful in the present invention include the MEK inhibitors PD184352 (Parke-Davis) (Sebolt-Leopold et al, supra) and U0126 (DuPont) (Favata, M et al, JBiol. Chem. 275:18623-18632 (1998)), the p38 MAPK inhibitor SB203580 (Schering-Plough) (Cuenda, A et al, FEBSLett. 364:229-233 (1995)), and the like.
  • Thee MEK inhibitors of this group are known to be, or are expected to be, cytotoxic to certain tumor cells.
  • Assays based on cell proliferation and tumor suppression are useful to detect MEK inhibitors such as LF functional derivatives or modulators, which are useful in inhibiting abnormal cellular proliferation and transformation, leading to antiangiogenic effects.
  • MEK inhibitors such as LF functional derivatives or modulators, which are useful in inhibiting abnormal cellular proliferation and transformation, leading to antiangiogenic effects.
  • Binding to immobilized MEK in a. 96 well plate Binding to immobilized MEK is carried out either by a competition assay with a known ligand, such as biotin-LF, or by direct binding ofthe compound being tested when the compound is labeled, e.g., biotinylated. Plates are coated at room temperature with MEK in Tris buffer- saline (TBS) (200 ng/well). After incubation for 2 hours, wells are washed with TBS, then l%BSA/TBS/Tween-20 is added to each well and incubate at 37°C for two hours. LF that had been previously biotinylated (e.g.
  • transwells are coated with type I collagen (50 ⁇ g/mL) by adding 200 ⁇ L ofthe collagen solution per transwell, then incubating overnight at 37°C.
  • the transwells are assembled in a 24-well plate and a chemoattractant (e.g., FGF-2) is added to the bottom chamber in a total volume of 0.8 mL media.
  • a chemoattractant e.g., FGF-2
  • FGF-2 chemoattractant
  • ECs such as HUVEC, which have been detached from monolayer culture using trypsin, are diluted to a final concentration of about 10 6 cells/mL with serum-free media and 0.2 mL of this cell suspension is added to the upper chamber of each transwell.
  • Inhibitors to be tested are added to both the upper and lower chambers, and the migration is allowed to proceed for 5 lirs in a humidified atmosphere at 37°C.
  • the transwells are removed from the plate stained using DiffQuik ® .
  • Cells which did not migrate are removed from the upper chamber by scraping with a cotton swab and the membranes are detached, mounted on slides, and counted under a high-power field (400x) to determine the number of cells migrated.
  • compositions ofthe invention are tested for their anti-invasive capacity.
  • cells such as ECs or tumor cells (e.g., PC-3 human prostatic carcinoma) cells to invade through a reconstituted basement membrane (Matrigel®) in an assay known as a "Matrigel® invasion assay” as described in detail by Kleinman et al, Biochemistry 25: 312-318,1986 and Parish et al, Int. J. Cancer 52:378-383,1992.
  • Matrigel® is a reconstituted basement membrane containing type TV collagen, laminin, heparan sulfate proteoglycans such as perlecan, which bind to and localize bFGF, vitronectin as well as transforming growth factor- ⁇ (TGF ⁇ ), urokinase- type plasminogen activator (uPA), tissue plasminogen activator (tPA), and the serpin known as plasminogen activator inhibitor type 1 (PALI) (Chambers et al, Cane. Res. 55:1578-1585, 1995). It is accepted in the art that results obtained in this assay for compounds which target extracellular receptors or enzymes are predictive ofthe efficacy of these compounds in vivo (Rabbani et al, Int. J. Cancer 63: 840-845, 1995).
  • Such assays employ transwell tissue culture inserts.
  • Invasive cells are defined as cells which are able to traverse through the Matrigel® and upper aspect of a polycarbonate membrane and adhere to the bottom ofthe membrane.
  • Transwells (Costar) containing polycarbonate membranes (8.0 ⁇ m pore size) are coated with Matrigel® (Collaborative Research), which has been diluted in sterile PBS to a final concentration of 75 ⁇ g/mL (60 ⁇ L of diluted Matrigel® per insert), and placed in the wells of a 24-well plate.
  • the membranes are dried overnight in a biological safety cabinet, then rehydrated by adding 100 ⁇ L of DMEM containing antibiotics for 1 hour on a shaker table.
  • the DMEM is removed from each insert by aspiration and 0.8 mL of DMEM/10 % FBS/antibiotics is added to each well ofthe 24-well plate such that it surrounds the outside ofthe transwell ("lower chamber”).
  • Fresh DMEM/ antibiotics (lOO ⁇ L), human Glu- plasminogen (5 ⁇ g/mL), and any inhibitors to be tested are added to the top, inside ofthe transwell ("upper chamber”).
  • the cells which are to be tested are trypsinized and resuspended in DMEM/antibiotics, then added to the top chamber ofthe transwell at a final concentration of 800,000 cells/mL.
  • the final volume ofthe upper chamber is adjusted to 200 ⁇ L.
  • the assembled plate is then incubated in a humid 5% CO atmosphere for 72 hours. After incubation, the cells are fixed and stained using DiffQuik® (Giemsa stain) and the upper chamber is then scraped using a cotton swab to remove the Matrigel® and any cells which did not invade through the membrane.
  • the membranes are detached from the transwell using scalpel blade, mounted on slides using Permount ® and cover-slips, then counted under a high-powered (400x) field. An average ofthe cells invaded is dete ⁇ nined from 5-10 fields counted and plotted as a function of inhibitor concentration.
  • ECs for example, HUNEC or human microvascular ECs (HMNEC) which can be prepared or obtained commercially, are mixed at a concentration of 2 x 10 5 cells/mL with fibrinogen (5mg/mL in phosphate buffered saline (PBS) in a 1 : 1 (v/v) ratio.
  • Thrombin is added (5 units/ mL final concentration) and the mixture is immediately transferred to a 24-well plate (0.5 mL per well).
  • the fibrin gel is allowed to form and then VEGF and bFGF are added to the wells (each at 5 ng/mL final concentration) along with the test compound.
  • the cells are incubated at 37°C in 5%> CO for 4 days at which time the cells in each well are counted and classified as either rounded, elongated with no branches, elongated with one branch, or elongated with 2 or more branches. Results are expressed as the average of 5 different wells for each concentration of compound. Typically, in the presence of angiogenic inhibitors, cells remain either rounded or form undifferentiated tubes (e.g. 0 or 1 branch).
  • This assay is recognized in the art to be predictive of angiogenic (or anti-angiogenic) efficacy in vivo (Min, HY et al, Cancer Res. 56: 2428-2433,1996).
  • EC tube formation is observed when ECs are cultured on Matrigel® (Schnaper et /., J Cell. Physiol. 165:107-118 1995).
  • ECs ( ⁇ 10 4 cells/well) are transferred onto Matrigel®-coated 24-well plates, and tube formation is quantitated after 48 hrs. Inhibitors are tested by adding them either at the same time as the ECs or at various time points thereafter.
  • Tube formation can also be stimulated by adding (a) angiogenic growth factors such as bFGF or VEGF, (b) differentiation stimulating agents (e.g.,. PMA) or (c) a combination of these.
  • This assay models angiogenesis by presenting to the ECs a particular type of basement membrane, namely the layer of matrix which migrating and differentiating ECs might be expected to first encounter.
  • the matrix components found in Matrigel® (and in basement membranes in situ) or proteolytic products thereof may also be stimulatory for EC tube formation which makes this model complementary to the fibrin gel angiogenesis model previously described (Blood et al, Biochim. Biophys. Ada 1032:89-118, 1990; Odedm et al., Pharmac. Ther. 49:111-124, 1991).
  • the MEK-inhibitory compounds of this invention will inhibit EC tube formation in both assays, reflective of their anti-angiogenic capability.
  • the ability ofthe compounds ofthe invention to inhibit the proliferation of EC's may be determined in a 96-well fo ⁇ nat.
  • Type I collagen (gelatin) is used to coat the wells ofthe plate (0.1-1 mg/mL in PBS, 0.1 mL per well for 30 minutes at room temperature). After washing the plate (3x w/PBS), 3-6,000 cells are plated per well and allowed to attach for 4 hrs (37 °C/5% CO 2 ) in Endothelial Growth Medium (EGM; Clonetics ) or M199 media containing 0.1-2% FBS.
  • the media and any unattached cells are removed at the end of 4 hrs and fresh media containing bFGF (1-10 ng/mL) or VEGF (1-10 ng/mL) is added to each well.
  • Compounds to be tested are added last and the plate is allowed to incubate (37 °C/5% CO 2 ) for 24-48 hrs.
  • MTS Promega
  • the absorbance at 490nm, which is proportional to the cell number, is then measured to determine the differences in proliferation between control wells and those containing test compounds.
  • a similar assay system can be set up with cultured adherent tumor cells. However, collagen may be omitted in this format.
  • Tumor cells e.g., 3,000-10,000/well
  • Serum free medium is then added to the wells, and the cells are synchronized for 24 hrs.
  • Medium containing 10% FBS is then added to each well to stimulate proliferation.
  • Compounds to be tested are included in some ofthe wells. After 24 hrs, MTS is added to the plate and the assay developed and read as described above.
  • Normal cells require a solid substrate to attach and grow. Oncogenic transformation results in loss of this phenotype so that the cells grow detached from the substrate.
  • transformed cells can grow in stirred suspension culture or suspended in semisolid medium , such as semisolid or soft agar. Techniques for soft agar growth or colony formation in suspension assays are described in Freshney, supra and Garkavtsev, I. et al, Nat Genet 14:415- 20 (1996).
  • Transformed cells successfully treated with LF or a proteolytic derivative or homologue reverts to the normal phenotype - anchorage dependence.
  • assays measuring growth or colony formation in soft agar can be used to identify LF homologues, mimetics, and modulators.
  • This type of assay will identify LF genetic constructs that, when expressed in a transformed cell, reverse the phenotype.
  • LF will cause transformed cells to revert to the normal phenotype and show contact inhibition and cessation of growth cultures at a lower cell density.
  • Assays measuring contact inhibition and density limitation of growth can be used to identify LF constructs, derivatives, etc. which are inhibit abnormal proliferation of transformed host cells.
  • Transformed cells for example of a long-term cell line are used. Delivery of LF to these cells, either as protein or via DNA expression, would reinstate contact inhibition and lower saturation densities.
  • the labeling index with [ H]-thymidine at saturation density is used to measure density limitation on growth.
  • Transformed host cells are provided with LF in a form that is expressed intracellularly, and are grown for 24 hours at saturation density in a non- limiting culture medium.
  • the percentage of cells labeling with [ 3 H]-thymidine can be is determined autoradiographically. See, Freshney, supra. Expression of LF would result in a lower labeling index compared to control transformed cells (e.g., transfected with a vector lacking an insert).
  • Transformed cells have a lower serum dependence than their normal counterparts (Temin, H, J. Natl. Cancer Inst. 37:167-175 (1966); Eagle, H. et al, J. Exp. Med. 131:863-879 (1970)); Freshney, supra). This is in part due to release of autocrine growth factors by the transformed cells.
  • an LF protein is present in a transformed cell (such as by expression of a transfected gene) the cell would release lower amounts of growth factors and would become serum dependent Therefore, measuring this parameter is useful for identifying LF constructs which could function as antiangiogenic cancer therapeutics.
  • the anti-proliferative and cyto toxic effects ofthe compositions maybe determined for various cell types including tumor cells, ECs, etc. Anti-proliferative effects would be expected against tumor cells and stimulated ECs but, under some circumstances not quiescent ECs or normal human dermal fibroblasts.
  • a typical assay would involve plating cells at a density of 5-10 x 10 3 cells/well in a 96- well plate. The compound to be tested is added at varying concentrations and allowed to incubate with the cells for 30 minutes.
  • the cells are washed 3X with media, then fresh media containing H-thymidine (e.g., 1 ⁇ Ci/mL) is added to the cells and they are allowed to incubate at 37°C in 5% CO for 24 and 48 hours.
  • Cells are lysed at the various time points using 1 M NaOH and counts per well determined using a ⁇ -counter. Proliferation may be measured non- radioactively using MTS reagent or CyQuant ® to measure total cell number.
  • MTS reagent for cytotoxicity assays (measuring cell lysis), a Promega 96-well cytotoxicity kit is used. If there is evidence of anti-proliferative activity, induction of apoptosis may be measured using TumorTACS® (Genzyme).
  • the ability ofthe compounds ofthe invention to promote apoptosis of EC's may be determined by measuring activation of caspase-3.
  • Type I collagen (gelatin) is used to coat a P100 plate and 5xl0 5 ECs are seeded in EGMF containing 10%> FBS. After 24 hours (at 37°C in5%o CO 2 ) the medium is replaced by EGM containing 2% FBS, 10 ng/ml bFGF and the desired test compound. The cells are harvested after 6 hours, cell lysates prepared in 1%> Triton and assayed using the EnzChek® Caspase-3 Assay Kit #1 (Molecular Probes) according to the manufactures' instructions.
  • Neovascularization is assessed at 5 and 7 days after implantation. On day 7, animals are anesthetized and infused with a dye such as colloidal carbon to stain the vessels. The animals are then euthanized, the corneas fixed with formalin, and the corneas flattened and photographed to assess the degree of neovascularization. Neovessels may be quantitated by imaging the total vessel area or length or simply by counting vessels.
  • Matrigel® is maintained at 4°C until use. Just prior to injection, Matrigel® is mixed with an angiogenic composition (e.g., 100 ng/mL bFGF, 100 ng/mL VEGF), then injected s.c. into mice (0.5 mL per mouse). The injected Matrigel® forms a palpable solid gel which persists for 10 days, at which time the animals are euthanized. The Matrigel® plugs are removed and angiogenesis quantitated by measuring the amount of hemoglobin in the angiogenic composition (e.g., 100 ng/mL bFGF, 100 ng/mL VEGF), then injected s.c. into mice (0.5 mL per mouse). The injected Matrigel® forms a palpable solid gel which persists for 10 days, at which time the animals are euthanized. The Matrigel® plugs are removed and angiogenesis quantitated by measuring the amount of hemoglobin in the
  • Anti-CD31 staining may be used to confirm neovessel formation and microvessel density in the plugs. (CD-31 is also known as platelet-endothelial cell adhesion molecule.)
  • Chick chorioallantoic membrane (CAM) angiogenesis assay This assay is performed essentially as described by Nguyen et al. (Microvascular Res.
  • a mesh containing either angiogenic factors (bFGF) or tumor cells plus inhibitors is placed onto the CAM of an 8-day old chick embryo and the CAM observed for 3-9 days after implantation ofthe sample.
  • Angiogenesis is quantitated by determining the percentage of squares in the mesh which contain blood vessels.
  • tumor cells for example 1-5 x 10 6 cells ofthe 3LL Lewis lung carcinoma or the rat prostate cell line MatLyLu, are mixed with Matrigel® and then injected into the flank of a mouse following the protocol described in Sec. B., above.
  • a mass of tumor cells and a powerful angiogenic response can be observed in the plugs after about 5 to 7 days.
  • the anti-tumor and anti-angiogenic action of a compound in an actual tumor environment can be evaluated by including it in the plug.
  • Measurement is then made of tumor weight, Hb levels or fluorescence levels (of a dextran-fluorophore conjugate injected prior to sacrifice).
  • the plugs are first homogenize with a tissue homogenizer.
  • Nude mice are inoculated with tumor cells and Matrigel® (1 x 10 6 cells in 0.2mL) s.c. in the right flank ofthe animals.
  • Matrigel® (1 x 10 6 cells in 0.2mL) s.c. in the right flank ofthe animals.
  • other tumors well- studied in the art may be used, e.g. , MDA-MB-231 cells (human breast carcinoma).
  • the tumors are staged to 200 mm 3 and then treatment with a test composition is initiated
  • LF or other antitumor agents of this invention are also tested for inhibition of late metastasis using an experimental metastasis model (Crowley, CW et al, Proc. Natl. Acad. Sci. USA 90 5021-5025 (1993)).
  • Late metastasis involves the steps of attachment and extravasation of tumor cells, local invasion, seeding, proliferation and angiogenesis.
  • Human prostatic carcinoma cells (PC-3) transfected with a reporter gene, preferably the green fluorescent protein
  • GFP chloramphenicol acetyl- transferase
  • CAT chloramphenicol acetyl- transferase
  • LacZ chloramphenicol acetyl- transferase
  • GFP-expressing PC-3 cells (1 x 10 6 cells per mouse) are injected iv into the tail veins of nude (nu/nu) mice. Animals are treated with a test composition at lOODg/animal/day given q.d. IP. Single metastatic cells and foci are visualized and quantitated by fluorescence microscopy or light microscopic histochemistry or by grinding the tissue and quantitative colorimetric assay ofthe detectable label.
  • the rat syngeneic breast cancer system employs Mat BUI rat breast cancer cells.
  • Tumor cells e.g., 10 in 0.1 mL PBS, are inoculated into the mammary fat pads of 10 female Fisher rats.
  • a 14-day Alza osmotic mini-pump is implanted intraperitoneally to dispense the polypeptide.
  • the polypeptide is dissolved in PBS (200 mM stock), sterile filtered and placed in the minipump to achieve a dispensing rate of about 4 mg/kg/day.
  • Control animals receive vehicle (PBS) alone or a control polypeptide in the minipump. Animals are euthanized at day 14.
  • control polypeptides In contrast, treatment with control polypeptides fail to cause a significant change in tumor size or metastasis.
  • mice are followed by weighing, palpation, and measurement of tumor size.
  • Typical tumor weight in untreated control recipients on day 12 after im inoculation is 500-2500 mg.
  • Typical median survival time is 18-28 days.
  • a positive control compound, for example cyclophosphamide at 20 mg/kg/injection per day on days 1-11 is used. Results computed include mean animal weight, tumor size, tumor weight, survival time For confinned therapeutic activity, the test composition should be tested in two multi-dose assays.
  • Test mice are male C57BL/6 mice, 2-3 months old. Following sc, im, or intra-footpad implantation, this tumor produces metastases, preferentially in the lungs. With some lines ofthe tumor, the primary tumor exerts anti-metastatic effects and must first be excised before study ofthe metastatic phase (see also U.S. 5,639,725).
  • Single-cell suspensions are prepared from solid tumors by treating minced tumor tissue with a solution of 0.3% trypsin. Cells are washed 3 times with PBS (pH 7.4) and suspended in PBS. Viability ofthe 3LL cells prepared in this way is generally about 95-99%> (by trypan blue dye exclusion). Viable tumor cells (3 x 10 4 - 5 x 10 6 ) suspended in 0.05 ml PBS are injected subcutaneously, either in the dorsal region or into one hind foot pad of C57BL/6 mice. Visible tumors appear after 3-4 days after dorsal sc injection of 10 6 cells. The day of tumor appearance and the diameters of established tumors are measured by caliper every two days. The treatment is given as one or two doses of polypeptide, per week. In another embodiment, the polypeptide is delivered by osmotic minipump.
  • mice are randomized into two groups: (1) primary tumor is completely excised; or (2) sham surgery is performed and the tumor is left intact. Although tumors from 500-3000 mm 3 inhibit growth of metastases, 1500 mm 3 is the largest size primary tumor that can be safely resected with high survival and without local regrowth. After 21 days, all mice are sacrificed and autopsied.
  • Lungs are removed and weighed. Lungs are fixed in Bouin's solution and the number of visible metastases is recorded. The diameters ofthe metastases are also measured using a binocular stereoscope equipped with a micrometer-containing ocular under 8X magnification. On the basis ofthe recorded diameters, it is possible to calculate the volume of each metastasis. To determine the total volume of metastases per lung, the mean number of visible metastases is multiplied by the mean volume of metastases. To further determine metastatic growth, it is possible to measure incorporation of 125 IdUrd into lung cells (Thakur, ML et al, J. Lab. Clin. Med. 59:217-228 (1977).
  • mice Ten days following tumor amputation, 25 ⁇ g of fluorodeoxyuridine is inoculated into the peritoneums of tumor-bearing (and, if used, tumor-resected mice). After 30 min, mice are given 1 ⁇ Ci of IdUrd (iododeoxyuridme). One day later, lungs and spleens are removed and weighed, and a degree of IdUrd incorporation is measured using a gamma counter. In mice with footpad tumors, when tumors reach about 8-10 mm in diameter, mice are randomized into two groups: (1) legs with tumors are amputated after ligation above the knee joints; or (2) mice are left intact as nonamputated tumor-bearing controls.
  • the compounds that maybe employed in the pharmaceutical compositions ofthe invention include all of those compounds described above, as well as the pharmaceutically acceptable salts of these compounds.
  • Pharmaceutically acceptable acid addition salts ofthe compounds ofthe invention containing a basic group are formed where appropriate with strong or moderately strong, non-toxic, organic or inorganic acids by methods known to the art.
  • Exemplary ofthe acid addition salts that are included in this invention are maleate, fumarate, lactate, oxalate, methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate, hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts.
  • Pharmaceutically acceptable base addition salts of compounds ofthe invention containing an acidic group are prepared by known methods from organic and inorganic bases and include, for example, nontoxic alkali metal and alkaline earth bases, such as calcium, sodium, potassium and ammonium hydroxide; and nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hydroxymethyl)methylamine.
  • nontoxic alkali metal and alkaline earth bases such as calcium, sodium, potassium and ammonium hydroxide
  • nontoxic organic bases such as triethylamine, butylamine, piperazine, and tri(hydroxymethyl)methylamine.
  • the compounds ofthe invention possess the ability to inhibit the MAPK pathway and to inhibit angiogenesis, properties that are exploited in the treatment of cancer, in particular metastatic cancer.
  • a composition of this invention may be active per se, or may act as a "pro-drug" that is converted in vivo to the active form.
  • the compounds ofthe invention may be incorporated into convenient dosage forms, such as capsules, impregnated wafers, tablets or injectable preparations.
  • Solid or liquid pharmaceutically acceptable carriers may be employed.
  • the compounds ofthe invention are administered systemically, e.g., by injection or infusion.
  • injection may be by any known route, preferably intravenous, subcutaneous, intramuscular, intracranial or intraperitoneal.
  • Infusion is preferably by the intravenous route, ftrjectables or infusible preparations can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions.
  • Solid carriers include starch, lactose, calcium sulfate dihydrate, terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearate and stearic acid.
  • Liquid carriers include syrup, peanut oil, olive oil, saline, water, dextrose, glycerol and the like.
  • the carrier or diluent may include any prolonged release material, such as glyceryl monostearate or glyceryl distearate, alone or with a wax.
  • the preparation may be in the form of a syrup, elixir, emulsion, soft gelatin capsule, sterile injectable liquid (e.g., a solution), such as an ampoule, or an aqueous or nonaqueous liquid suspension.
  • sterile injectable liquid e.g., a solution
  • an ampoule e.g., an ampoule
  • aqueous or nonaqueous liquid suspension e.g., aqueous or nonaqueous liquid suspension.
  • the pharmaceutical preparations are made following conventional techniques of pharmaceutical chemistry, as appropriate, to give the desired products for oral, parenteral, topical, transdermal, intravaginal, intrapenile, intranasal, mtrabronchial, intracranial, intraocular, intraaural and rectal administration.
  • the pharmaceutical compositions may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents and so forth.
  • the present invention may be used in the treatment of any of a number of animal genera and species, and are equally applicable in the practice of human or veterinary medicine.
  • the pharmaceutical compositions can be used to treat domestic and commercial animals, including birds and more preferably mammals, as well as humans.
  • systemic administration refers to administration of a composition or agent such as the polypeptide, peptides or small organic molecule herein, in a manner that results in the introduction ofthe composition into the subject's circulatory system or otherwise permits its spread throughout the body, such as intravenous (i.v.) injection or infusion.
  • Regular administration refers to administration into a specific, and somewhat more limited, anatomical space, such as intraperitoneal, intrathecal, subdural, or to a specific organ. Examples include intravaginal, intrapenile, intranasal, mtrabronchial (or lung instillation), intracranial, intra-aural or intraocular.
  • local administration refers to administration of a composition or drug into a limited, or circumscribed, anatomic space, such as intratumoral injection into a tumor mass or peritumoral in the vicinity of a tumor, as well as subcutaneous (s.c.) and intramuscular
  • s.c. or i.m. are also routes for systemic administration
  • hrjectables or infusible preparations can be prepared in conventional forms, either as solutions or suspensions, solid forms suitable for solution or suspension in liquid prior to injection or infusion, or as emulsions.
  • the preferred routes of administration are systemic, such as i.v., the pharmaceutical composition may be administered topically or transdermally, e.g., as an ointment, cream or gel; orally; rectally; e.g., as a suppository.
  • the compound may be incorporated into topically applied vehicles such as a salve or ointment.
  • the carrier for the active ingredient may be either in sprayable or nonsprayable form.
  • Non-sprayable forms can be semi-solid or solid forms comprising a carrier indigenous to topical application and having a dynamic viscosity preferably greater than that of water.
  • Suitable formulations include, but are not limited to, solution, suspensions, emulsions, creams, ointments, powders, liniments, salves, and the like.
  • auxiliary agents e.g., preservatives, stabilizers, wetting agents, buffers, or salts for influencing osmotic pressure and the like.
  • Preferred vehicles for nonsprayable topical preparations include ointment bases, e.g., polyethylene glycol- 1000 (PEG- 1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
  • ointment bases e.g., polyethylene glycol- 1000 (PEG- 1000); conventional creams such as HEB cream; gels; as well as petroleum jelly and the like.
  • sprayable aerosol preparations wherein the compound, preferably in combination with a solid or liquid inert carrier material, is packaged in a squeeze bottle or in admixture with a pressurized volatile, normally gaseous propellant.
  • the aerosol preparations can contain solvents, buffers, surfactants, perfumes, and or antioxidants in addition to the compounds ofthe invention.
  • an effective amount ofthe compound for the preferred topical applications, especially for humans, it is preferred to administer an effective amount ofthe compound to an affected area, e.g., skin surface, mucous membrane, eyes, etc.
  • an affected area e.g., skin surface, mucous membrane, eyes, etc.
  • This amount will generally range from about 0.001 mg to about 1 g per application, depending upon the area to be treated, the severity ofthe symptoms, and the nature ofthe topical vehicle employed.
  • Antiangiogenic compositions may be administered in combination with a biodegradable, biocompatible polymeric implant which releases the troponin active agent over a controlled period of time at a selected site.
  • preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycohc acid, polylactic acid, polyethylene vinyl acetate, and copolymers and blends thereof. See, for example, Medical Applications of Controlled Release, Langer and Wise (eds.), 1974, CRC Press, Boca Raton, FL; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), 1984, Wiley, NY; Ranger et al, 1983, J. Macromol. Sci. Rev. Macromol Chem. 23:61; Levy et al, 1985,
  • a controlled release system can be placed in proximity ofthe therapeutic target, e.g., the brain, thus requiring only a fraction ofthe systemic dose (Goodson, In: Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138). Other controlled release systems are discussed in a review by Langer, R, 1990, Science 249:1527-1533)
  • compositions particularly polypeptides
  • liposomes pharmaceutical compositions in which the active protein is contained either dispersed or variously present in corpuscles consisting of aqueous concentric layers adherent to lipidic layers.
  • the active polypeptide or peptide, or the nucleic acid is preferably present in the aqueous layer and in the lipidic layer, inside or outside, or, in any event, in the non-homogeneous system generally known as a liposomic suspension.
  • the hydrophobic layer, or lipidic layer generally, but not exclusively, comprises phospholipids such as lecithin and sphingomyelin, steroids such as cholesterol, more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid, and/or other materials of a hydrophobic nature.
  • phospholipids such as lecithin and sphingomyelin
  • steroids such as cholesterol
  • more or less ionic surface active substances such as dicetylphosphate, stearylamine or phosphatidic acid
  • compositions for treating tumors and cancer may comprise, in addition to the LF or homologue, one or more additional anti-tumor agents, such as mitotic inhibitors, e.g., vinblastine; alkylating agents, e.g., cyclophosphamide; folate inhibitors, e.g., methotrexate, piritrexim or trimetrexate; antimetabolites, e.g., 5-fluorouracil and cytosine arabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin; enzymes or enzyme inhibitors, e.g., asparaginase, topoisomerase inhibitors such as etoposide; or biological response modifiers, e.g., interferons or interleukins.
  • additional anti-tumor agents such as mitotic inhibitors, e.g., vinblastine
  • alkylating agents e.g., cyclophosphamide
  • compositions comprising any known cancer therapeutic in combination with LF as disclosed herein are within the scope of this invention.
  • the pharmaceutical composition may also comprise one or more other medicaments to treat additional symptoms for which the target patients are at risk, for example, anti-infectives including antibacterial, anti-fungal, anti-parasitic, anti-viral, and anti-coccidial agents.
  • the methods of this invention may be used to inhibit tumor growth and invasion in a subject or to suppress angiogenesis induced by tumors. By inhibiting the growth or invasion of a tumor or angiogenesis, the methods result in inhibition of tumor metastasis.
  • a vertebrate subject preferably a mammal, more preferably a human, is administered an amount ofthe compound effective to inhibit tumor growth, invasion or angiogenesis.
  • the compound or pharmaceutically acceptable salt thereof is preferably administered in the form of a pharmaceutical composition as described above.
  • Doses ofthe compounds preferably include pharmaceutical dosage units comprising an effective amount ofthe polypeptide.
  • an effective amount is meant an amount sufficient to achieve a steady state concentration in vivo which results in a measurable reduction in any relevant parameter of disease and may include growth of primary or metastatic tumor or a measurable prolongation of disease-free interval or of survival. For example, a reduction in tumor growth in 20 % of patients is considered efficacious (Frei in, E., The Cancer Journal
  • an effective dose is preferably 10-fold and more preferably 100-fold higher than the 50% effective dose (ED 50 ) ofthe compound in an in vivo assay as described herein.
  • the amount of active compound to be administered depends on the precise polypeptide or derivative selected, the disease or condition, the route of administration, the health and weight ofthe recipient, the existence of other concurrent treatment, if any, the frequency of treatment, the nature ofthe effect desired, for example, inhibition of tumor metastasis, and the judgment of the skilled practitioner.
  • a preferred dose for treating a subject, preferably mammalian, more preferably human, with a tumor is an amount of up to about 100 milligrams of active compound per kilogram of body weight.
  • a typical single dosage ofthe polypeptide is between about 1 ng and about lOOmg/kg body weight.
  • dosages in the range of about 0.01-20%) concentration (by weight) ofthe compound, preferably 1-5%, are suggested.
  • a total daily dosage in the range of about 0.1 milligrams to about 10 grams is preferred for intravenous administration.
  • the foregoing ranges are, however, suggestive, as the number of variables in an individual treatment regime is large, and considerable excursions from these preferred values are expected.
  • the combination of dose and route ofthe active antiangiogenic composition is selected based on known toxicities.
  • an intratumoral or peritumoral route of administration is preferred over i.v. administration when the effective dose has toxic side effects when given systemically but less toxic or no toxic side effects when given into, or in the vicinity of, the tumor.
  • An effective amount or dose ofthe polypeptide for inhibiting cellular or enzymatic activity in vitro is in the range of about 1 picogram to about 10 nano grams per cell.
  • Effective doses and optimal dose ranges may be determined in vitro using the methods described herein.
  • the compounds ofthe invention maybe characterized as producing an inhibitory effect on endothelial cell or tumor cell proliferation, migration, invasion, or on angiogenesis or on tumor metastasis.
  • the compounds are especially useful in producing an anti-tumor effect in a mammalian host, preferably human, harboring a tumor.
  • compositions and treatment methods are useful for inhibiting cell proliferation or angiogenesis in a subject having any disease or condition associated with undesired cell proliferation, angiogenesis or metastasis.
  • Malignant and metastatic diseases and conditions include, but are not limited to, solid tumors, e.g., carcinomas, sarcomas, lymphomas and other malignant or nonmalignant tumors such as those listed in the table below (for a review of such disorders, see any textbook of clinical oncology, most recent edition, e.g., Cancer: Principles & Practice of Oncology, 5 th Ed. (DeVita, V. et al, eds), Philadelphia: Lippincott-Raven Publishers, 1997) Cancers/Tumors
  • Endotheliosarcoma myxosarcoma testicular tumor
  • angiogenesis Other classes of diseases associated with undesired or uncontrolled angiogenesis that may be treated in accordance with this invention include atherosclerosis, myocardial angiogenesis, angiofibroma, arteriovenous malformations, post-balloon angioplasty vascular restenosis, neointima formation following vascular trauma, vascular graft restenosis, coronary collateral formation, deep venous thrombosis, and ischemic limb angiogenesis.
  • Ocular neovascularization is a leading cause of blindness in the world (Lee et al, Surv. Ophthalmol 43:245-269 (1998)).
  • the most common diseases caused by this process are proliferative diabetic retinopathy, neovascular age-related macular degeneration, and retinopathy of prematurity (Neely, KA et al, Am. J. Path. 155:665-670 (1998)).
  • compositions are intended for the treatment of any ofthe above or other diseases or conditions that involve ocular neovascularization, chief among them, sickle cell retinopathy, retinal vein occlusion, neovascular glaucoma , retrolental fibroplasia, uveitis, diseases associated with choroidal or iris neovascularization, corneal graft neovascularization, as well as other eye inflammatory diseases or ocular tumors.
  • diseases or conditions that involve ocular neovascularization, chief among them, sickle cell retinopathy, retinal vein occlusion, neovascular glaucoma , retrolental fibroplasia, uveitis, diseases associated with choroidal or iris neovascularization, corneal graft neovascularization, as well as other eye inflammatory diseases or ocular tumors.
  • disorders which can be treated in accordance with the present invention include, but are not limited to, uterine disease such as endometriosis, hemangioma, arthritis, psoriasis, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
  • uterine disease such as endometriosis, hemangioma, arthritis, psoriasis, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osier- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
  • fibrosis associated with chronic inflammatory conditions This includes some ofthe conditions listed above as well as hemophilic joints, hypertrophic scars, telangiectasia, pyogenic granuloma, Von-Hippel-Landau syndrome, trachoma, vascular adhesions, lung fibrosis, chemotherapy- induced fibrosis, wound healing with scarring and fibrosis, peptic ulcers, fractures, keloids. Also included are disorders of vasculo genesis, hematopoiesis, ovulation, menstruation, pregnancy and placentation, or any other disease or condition in which invasion or angiogenesis is pathogenic or undesired.
  • Certain brain tumors are among the most highly neovascularized tumors known.
  • the present pharmaceutical compositions are therefore intended for the treatment of any of a number of brain tumors, including but not limited to glioblastoma; glioblastoma multiformae; various astrocytomas such as anaplastic astrocytoma, pilocytic astrocytoma, pleomorphic xanthoastrocytoma, subependymal giant cell astrocytoma, fibrillary astrocytoma, gemistocytic astrocytoma, protoplasmic astrocytoma; mixed oligoastrocytoma and malignant oligoastrocytoma; oligodendroglioma and anaplastic oligodendroglioma; ependymoma, including anaplastic ependymoma, myxopapillary ependymoma, and subependymo
  • Endometriosis (mentioned above) is a condition in which ectopic endometrium is present in abnormal locations, the ovary being the most common site.
  • Adenomyosis is a similar condition in which endometrial tissue has penetrated the uterine myometrium. Endometriotic tissue resembles neoplastic tissue in its ability to implant and invade. Accordingly, the present pharmaceutical compositions are intended for the treatment of endometriosis, adenomyosis, endometrial carcinoma and endometrioid tumors ofthe ovary
  • angiogenesis inhibitors may play a role in preventing inflammatory angiogenesis and gliosis following traumatic spinal cord injury, thereby promoting the reestablishment of neuronal connectivity (Wamil, AW et al, 1998, Proc. Nat 'I. Acad. Sci. USA 95:13188-13193). Therefore, the MEK-inhibitor compositions ofthe present invention are administered as soon as possible after traumatic spinal cord injury and for several days up to about two weeks thereafter to inhibit the angiogenesis and gliosis that would physically prevent reestablishment of neuronal connectivity.
  • the treatment reduces the area of damage at the site of spinal cord injury and facilitates regeneration of neuronal function and thereby prevents paralysis.
  • the compounds ofthe invention are expected also to protect axons from Wallerian degeneration, and reverse aminobutyrate-mediated depolarization (occurring in traumatized neurons).
  • Therapeutic or prophylactic utility ofthe present invention and the determination of therapeutically effective dosages can be determined or demonstrated in vivo in a suitable animal model system prior to testing in humans.
  • a suitable animal model system prior to testing in humans.
  • Such model systems may be based on the use of rats, mice, chicken, cows, monkeys, rabbits, etc.
  • any animal model system known in the art may be used.
  • NTH3T3 490 cells (mouse fibroblasts) expressing either the empty vector (pDCR) or transforming human H-ras (N12) protein
  • pDCR empty vector
  • N12 transforming human H-ras
  • PA inactive LF(E687C)
  • LF inactive LF
  • PA plus LF 100 ng/ml PA plus 10 ng/ml LF
  • MAPK phosphorylated MAPK
  • pTEpY 1 :4000; Promega, Madison, WI
  • MAPK1/2 K-23/C-14, 1 :4000; Santa Cruz Biotechnology, Santa Cruz, CA
  • PA plus LF 100 ng/ml PA plus 10 or 1 ng/ml LF (hi and lo respectively)
  • Three-dimensional Matrigel (Becton Dickinson) invasion assays were performed as described in the art (Jeffers, M et ⁇ /.(1996) Mol Cell Biol 16, 1115-25). Approximately 2.5 x 10 4 cells were mixed with growth factor-reduced Matrigel® supplemented with 1. medium alone,
  • the cell suspension was cultured for up to one week during which the cells were monitored daily.
  • the images shown in Fig. 3 were made at day 4. Each sample was assayed in triplicate in three separate experiments.
  • V12 H-ras transformed NTH 3T3 cells (10 5 cells in a volume of 100 ⁇ l) were injected subcutaneously into 10 athymic nude mice on the left and right sides of the back behind the last rib. When tumors reached a size of 5-7 mm (approximately 2 weeks), the mice were divided into two groups as follows:
  • Group A sham-injected (insertion ofthe needle only) intra-tumorally in the left side and injected in the right side with 100 ⁇ l buffered saline.
  • Group B injected in the left side with 100 ⁇ l buffered saline and in the right side with 10 ⁇ g PA plus 2 ⁇ g LF in 100 ⁇ l buffered saline. Injections continued once daily for a total period of five days. The sizes ofthe tumors were monitored following injection. When control tumors attained a diameter of 20 mm (approximately 3-4 weeks) the mice were euthanized and the tumors dissected, trimmed, and fixed in formalin for further analyses.
  • Paraffin tissue sections (5 ⁇ m) were stained using mouse monoclonal antibodies against one of two endothelial cell markers, CD31 (BBa7, 1:100; Research Diagnostics, Flanders, NJ) and CD 34 (M7 165, 1 :25; DAKO, Carpinteria, CA), followed by FITC conjugated secondary antibodies. Slides were imaged using a Zeiss CLSM 410 confocal microscope. Images were made of representative sections that showed histologic features of a fibrosarcoma using a water immersion 40x high numerical aperture lens and were stored digitally. EXAMPLE II Cellular Consequences of LeTx Treatment
  • LeTx was active upon both non-transformed and oncogenic V12 H-ras-transformed NTH 3T3 cells by performing immunoblot analyses upon lysates of cells which had been incubated 24 h. in the presence of inactive LeTx (PA plus LF(E687C) which had a point mutation in its Zn 2+ -binding site), LeTx , or PD98059, a small organic compound that preferentially inhibits MEKl activation (Alessi, DR et al(1995) J. Biol. Chem. 270, 27489-27494; Dudley et al, supra. (Fig 1A and IB). As previously by the present inventors and colleagues, treatment of both V12 H-ras-transformed and non-transformed N H 3T3 cells with LeTx, but not inactive LeTx or PD98059, caused the loss of
  • NTH 3T3 cells transformed by oncogenic V12 H-ras undergo a change in morphology from a irregular, flattened shape to a spindle-like form with an associated loss in actin stress fibers (Dudley et al, supra) Fig 1C, IE).
  • LeTx treatment was accompanied by a reversion ofthe shape of V12 H-ras transformed NIH 3T3 cells from a spindle-like shape
  • NTH 3T3 1 1.00 ⁇ 0.04 0.81 ⁇ 0.08 0.63 ⁇ 0.07 0.70 ⁇ 0.09 0.47 ⁇ 0.03
  • Cell proliferation was assayed in non-transformed (pDCR) or V12 H-ras-transformed NIH 3T3 cells treated with either medium alone, 100 ng/ml PA plus inactive 10 ng/ml LF(E687C), PA plus LF (100 ng/ml PA plus 10 or 1 ng/ml LF Qii and lo respectively)), or PD98059 (25 ⁇ M or 50 ⁇ M from a 50 mM stock in DMSO) as described in the methods section. Data is presented relative to proliferation of control samples incubated in the presence of culture medium alone and as an average of at least three measurements plus and minus standard deviation about the mean.
  • Tumor growth and invasion is a complex multistep process that involves anchorage independent growth, motility, and proteolytic degradation ofthe extracellular matrix. Aspects of these processes may be simulated in vitro by measuring a cell's ability to (i) grow independent of substrate adhesion and form colonies in a soft agar suspension, and (ii) degrade an artificial extracellular matrix (basement membrane Matrigel®).
  • Non-transformed NTH 3T3 cells suspended in soft agar fail to proliferate and remain as single cells in suspension (Yang, JJ et al. (1998) Mol Cell Biol 18, 2586-95).
  • V12 H-ras-transformed cells continue to proliferate in the absence of substrate adhesion and form colonies of cells.
  • LeTx completely prevented colony formation of V12 H-ras- transformed cells (Fig.2a, b).
  • LeTx prevented V12 H-ras-transformed cells from invading, or branching into, the extracellular matrix (Fig. 2c, 2d).
  • V12 H-ras-transformed cells were injected subcutaneously into the left and right dorsal areas of 10 athymic nu/nu mice.
  • mice were divided the mice into two groups, A and B, when tumors had grown to a diameter of 5-7 mm.
  • Group A tumors were sham-injected on the left side and injected on the right side with buffered saline.
  • Group B tumors were injected on the left side with LeTx (10 ⁇ g PA and 2 ⁇ g
  • mice of Group A were considerably larger than those in Group B (Fig. 3).
  • mice in group B regressed in size over the course ofthe experiment.
  • MEK inhibitors such as PD98059, U0126, and PD184352 inhibit tumor cell growth in vitro or in vivo (Dudley et al, supra; Favata et al, supra; Sebolt-Leopold et al, supra. Each differs somewhat in its substrate specificity and affinity.
  • U0126 was shown to inhibit p70 s6K phosphorylation through a MEK-independent mechanism.
  • PD184352 inhibits MEKl with an IC 50 of ⁇ 0.02 ⁇ M (Sebolt-Leopold et al, supra).
  • LF has been demonstrated to inactivate MEKl but because it cleaves MEK2 (Duesbery et al, 1998, supra; Vitale, G et al. (1999) J Appl Microbiol 87, 288) and MEK3 (Pellizzari et al, supra), it is believed to inactivate these kinases as well.
  • V12 H-ras-transformed cells with LeTx caused rapid and dramatic alteration of cell morphology and the appearance of actin-stress fibers. This effect is likely to be mediated by inhibition of MEKl and/or MEK2 activity because similar effects are induced by PD98059 and U0126 (Fukuzawa et al, supra). Treatment of V12 H-ras-transformed cells with LeTx was also shown to inhibit soft agar colony formation as well as extracellular matrix invasion in vitro.
  • LeTx had only a modest effect on cell proliferation when compared to PD98059 (Dudley et al, supra) or PD 184352 (Sebolt-Leopold et al, supra) whereas LeTx was a more effective inhibitor of MAPK than was PD98059 in V12 H- ras transformed cells (see Fig. la, lb).
  • the activities of LF and the small molecule MEK inhibitors are distinct.
  • Intravenous (vs. intratumoral) injection of a similar dose of LeTx might have been expected to have undesired side effects (given that the LD 50 in mice is 12.5 ⁇ g PA and 2.5 ⁇ g LF (Ezzell, JW et al (1984) Infect.
  • each ofthe in vitro assays discussed above measures a particular aspect or aspects ofthe transformed cell phenotype. Although useful, these assays provide less valuable information than does evaluation of actual tumor growth in vivo. For example, the rate at which a primary tumor develops in vivo is not simply a reflection ofthe rate at which its cells divide but rather is a function of a complex series of cellular activities that include proliferation, vascularization, and invasion.
  • FGF fibroblast growth factor
  • LeTx has therapeutic activity that exceeds what is expected based solely upon its MEKl inhibitory activity.

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Abstract

L'invention concerne une méthode permettant d'inhiber l'angiogénèse cellulaire, consistant à mettre en contact des cellules associées à une angiogénèse non souhaitée avec une quantité efficace d'un inhibiteur de MEK ou d'une enzyme qui appartient à la famille MAPK. Les inhibiteurs MEK comprennent des protéases spécifiques de MEK, telles que le facteur létal Bacillus anthracis ou un dérivé fonctionnel de ce dernier. Des petits inhibiteurs moléculaires organiques de MEK comprennent PD98059, U0126 et PD184352. Cette mise en contact peut être réalisée in vivo, chez un patient humain ou chez un autre sujet mammifère. L'invention concerne également une méthode permettant de traiter un sujet mammifère présentant un trouble ou une affection associé à une angiogénèse non souhaitée ou à une néovascularisation, laquelle méthode consiste à administrer au sujet une quantité efficace d'une composition pharmaceutique comprenant un inhibiteur de MEK ou une enzyme appartenant à la famille MAPK susmentionnée, et un véhicule ou excipient pharmaceutiquement acceptable. La méthode thérapeutique peut être utile pour une maladie ou une affection, telle que la croissance tumorale, l'invasion tumorale ou la métastase tumorale, dans laquelle l'inhibition de l'angiogénèse se traduit par la réduction de la taille ou de la vitesse de croissance de la tumeur, ou encore, par sa destruction.
PCT/US2002/008656 2001-03-22 2002-03-22 Facteur letal de l'anthrax inhibant la croissance tumorale et l'angiogenese WO2002076496A1 (fr)

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WO2005007193A2 (fr) * 2003-07-07 2005-01-27 Vande Woude, George, F. Inhibition de l'angiogenese tumorale par combinaison de la thrombospondine-1 et des inhibiteurs du facteur de croissance endotheliale vasculaire
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WO2008019417A2 (fr) * 2006-08-16 2008-02-21 Marlyn Nutraceuticals, Inc. Traitement de maladies oculaires
WO2009132397A1 (fr) * 2008-05-01 2009-11-05 University Of South Australia Procédés et agents pour la modulation du taux et/ou de l'activité de la protéine hif-2 alpha
WO2013082511A1 (fr) * 2011-12-02 2013-06-06 Genentech, Inc. Procédés pour surmonter la résistance tumorale aux antagonistes de vegf
EP2701694A1 (fr) * 2011-04-28 2014-03-05 Duke University Procédés de traitement d'hémoglobinopathies
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US9592236B2 (en) 2011-04-28 2017-03-14 Duke University Methods of treating hemoglobinopathies
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AU2002255852B2 (en) 2006-11-09

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