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WO2002002050A2 - Prevention et traitement de maladies associees aux mycoplasma - Google Patents

Prevention et traitement de maladies associees aux mycoplasma Download PDF

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WO2002002050A2
WO2002002050A2 PCT/BR2001/000083 BR0100083W WO0202050A2 WO 2002002050 A2 WO2002002050 A2 WO 2002002050A2 BR 0100083 W BR0100083 W BR 0100083W WO 0202050 A2 WO0202050 A2 WO 0202050A2
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mycoplasma
enzyme
pneumoniae
trans
infection
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WO2002002050A3 (fr
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Maria De Lourdes Higuchi
Sergio Schenkman
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Priority to CA2383850A priority patent/CA2383850C/fr
Priority to AU2001267165A priority patent/AU2001267165A1/en
Priority to EP01944764A priority patent/EP1296554B1/fr
Publication of WO2002002050A2 publication Critical patent/WO2002002050A2/fr
Priority to US10/086,913 priority patent/US7108851B2/en
Publication of WO2002002050A3 publication Critical patent/WO2002002050A3/fr
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Priority to US10/952,003 priority patent/US7335638B2/en
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    • 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)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01018—Exo-alpha-sialidase (3.2.1.18), i.e. trans-sialidase
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the prevention and treatment of diseases associated with undesirable cell proliferation, including atherosclerotic narrowing of blood vessels and malignancy, comprising preventing or treating infection by mycoplasma. It is based, at least in part, on the discovery that, in many cases, mycoplasma infection exists coincident with undesirable cell proliferation and/or proliferation of other infectious organisms.
  • Mycoplasmas are parasites of the respiratory epithelium and urogenital tract. Although mycoplasma infections are typically asymptomatic in mammals, they seem to be co-factors in diseases, such as AIDS (Acquired Immunodeficiency Syndrome), and in sequelae after mycoplasma infections having an autoimmune basis.
  • AIDS Abrered Immunodeficiency Syndrome
  • Mycoplasmas are the smallest self-replicating microorganisms and have unique properties among the prokaryotes, such as (i) their need for cholesterol to maintain their membrane envelope and (ii) the absence of an external wall.
  • Mycoplasmas are known to cause pulmonary infection in humans. See, Razin et al., "Molecular biology and pathogenicity of mycoplasmas," Microbiol. Mol. Biol. Rev.: 62(4): 1094-1156, (1998).
  • mycoplasmas can cause disease in most animals, including animals of commercial importance to the husbandry industry, such as cattle, swine, and fowl. See, Maniloff et al. Eds., Mycoplasmas, Molecular Biology and Pathogenesis, American Society for Microbiology (Washington, 1992).
  • mycoplasma may play a role in the pathogenesis of a number of human diseases, including asthma, diseases of the large intestine, rheumatoid diseases such as rheumatoid arthritis, maculopapular erythemas, stomatitis, conjunctivitis, pericarditis, Alzheimer's Disease, multiple sclerosis, the sequelae of AIDS and HIV infection, genito-urinary infections, diseases of chronic fatigue like Chronic Fatigue Syndrome, and Gulf War Syndrome.
  • the actual role of mycoplasmas in these various diseases have been difficult to determine, because most of the associations drawn to mycoplasma infection are based on serologic evidence rather than direct observation of mycoplasma organisms in disease lesions. See, Cole, "Mycoplasma interactions with the immune system: implications for disease pathology," (http://www.compkarori.com/arthritis/pi16002.htm); Cole,
  • Mycoplasma as well as chlamydia have been implicated in vascular disease, but the etiologic relationships have not been confirmed. See, Chen et al., "Carditis associated with Mycoplasma pneumoniae infection," Am. J. Pis. Child. 140:471-472 (1986); Clyde et al., "Tropism for Mycoplasma gallisepticum for arterial walls,” Proc. Natl. Acad. Sci. U.S.A.
  • prevention of or interference with the first step of mycoplasma attachment can provide an important means of controlling infection.
  • existing antibiotics have been ineffective at either preventing or breaking the adhesion of pathogenic mycoplasmas to the host cells.
  • Mycoplasma pneumoniae The attachment zone of Mycoplasma pneumoniae ("M. pneumoniae") and of other mycoplasmas is rich in glycoproteins that contain sialic acid. See, Chandler et al., "Mycoplasma pneumoniae attachment: competitive inhibition by mycoplasmal binding component and by sialic acid-containing glycoconjugates," Infect. Immun., 38(2):598-603 (1982), Glasgow and Hill, “Interactions of Mycoplasma gallisepticum with sialyl glycoproteins,” Infect. Immun.: 30:353-361 (1980), and Hansen et al., “Characterization of hemadsorption-negative mutants of Mycoplasma pneumoniae," Infect. Immun. , 32:127-136 (1981).
  • Sialic acid was initially discovered on the surface of Trypanosoma cruzi ("7. cruzT) by Pereira et al. in 1980. See, Pereira et al., "Lectin receptors as markers for Trypanosoma cruzi. Development stages and a study of the interaction of wheat germ agglutinin with sialic acid residues on epimastigotes cells," J. Exp. Med., 152:1375-92 (1980). Pereira also first demonstrated in 1983 that T cruzi has sialidase activity. See, Pereira, "A developmentally regulated neuraminidase activity in Trypanosoma cruzi," Science, 219:1444-46 (1983).
  • Trans-sialidase an enzyme expressed on the T. cruzi's surface, catalyzes the transfer of sialic acid from host glycoconjugates to glycoprotein molecules on the surface of the parasite. See, Schenkman et al., "Attachment of Trypanosoma cruzi trypomastigotes to receptors at restricted cell surface domains," Exp. Parasitol.. 72:76-86 (1991).
  • the enzyme is present both in the epimastigote form (i.e., in the invertebrate vector) and in the trypomastigote form (i.e., infectious form that circulates in the blood of the vertebrate host).
  • trans-sialidase of Trypanosoma cruzi is anchored by two different lipids
  • Glvcobioloqy. 7(6):731-5 The catalytic portion of trans-sialidase (“TSC”) has two kinds of enzymatic activity: (1) neuraminidase activity, which releases sialic acid from the complex carbohydrates; and (2) sialil- transferase activity, which catalyzes the transfer of sialic acid from glyco ⁇ jugate donors to terminal ⁇ -D galactose containing acceptors.
  • TSC trans-sialidase
  • neuraminidase activity which releases sialic acid from the complex carbohydrates
  • sialil- transferase activity which catalyzes the transfer of sialic acid from glyco ⁇ jugate donors to terminal ⁇ -D galactose containing acceptors.
  • trans-sialidase In the complete native form of trans-sialidase (“TSN"), the enzyme has a C-terminal extension having a repetitive sequence of 12 amino acids previously identified as SAPA (i.e., Shed-Acute- Phase-Antigens). Although the repetitive sequence of amino acids is not directly involved in the catalytic activity, it stabilizes the trans- sialidase activity in the blood to increase the half-life of the enzyme from about 7 to about 35 hours. See, Pollevick et al., "The complete sequence of SAPA, a shed acute-phase antigen of Trypanosoma cruzi " Mol. Biochem. Parasitol.
  • Trans-sialidase may also sialylate the host cell glycoconjugates, forming receptors that will be used by the trypomastigotes for the attachment and penetration into the target cells.
  • the trans-sialidase enzyme of T cruzi has been well characterized. See, Pollevick et al., Mol. Bioche . Parasitol. 47:247- 250 (1991); Pereira et al., J. Exp. Med.
  • the enzymatically active protein extracted from the 7 " . cruzi trypomastigotes has 4 distinct amino acid regions: (1) a N- terminal region with approximately 380 amino acids of which 30% of the sequence is similar to bacterial sialidases; (2) a region with approximately 150 residues that does not show any similarity with any known sequence; (3) a region with homology to type III fibronectin (Fnlll); and (4) a C-terminal region containing 12 repeated amino acids, which is the immuno-dominant portion and which is required for enzyme oligomerization.
  • the N-terminal and the Fnlll regions are important for trans-sialidase activity.
  • TS trans-sialidase
  • the present invention relates to methods and compositions for the prevention and treatment of disorders caused by mycoplasma infection. It is based, at least in part, on the discovery that mycoplasma infection is associated with disorders of increased cell proliferation, including arterial atheromas and various malignant neoplasic tissues. It was further noted that in many cases such atheromas and malignant tissues were also infected with chlamydia organisms.
  • the present invention provides for methods of preventing and/or treating disorders manifested by increased cell proliferation and/or co-existent proliferation of other infectious organisms whereby a subject is administered an effective amount of an agent which prevents or inhibits mycoplasma infection.
  • the agent may be an antibiotic, but, in preferred embodiments of the invention, the agent is a protein capable of removing sialic acid residues, such as a neuranimidase enzyme or, more preferably, a trans-sialidase enzyme, wherein removal of sialic acid inhibits or prevents the attachment of mycoplasma to host cells.
  • the agent is the trans-sialidase enzyme of Trypanosoma cruzi, or a portion or variant of the native enzyme which has trans-sialidase activity.
  • This aspect of the invention is based, at least in part, on the discovery that patients suffering from infection with Trypanosoma cruzi exhibited less atherosclerotic coronary artery disease and less mycoplasma in the intima.
  • the disorders to be treated according to the invention include, but are not limited to, atherosclerosis and malignancy. Without being bound to any particular theory, it is hypothesized that infection with mycoplasma may inhibit programmed cell death (apoptosis).
  • FIGURES 1 A-F Electron microscopy (A-D) and histopathological view of in situ hybridization with a M. pneumoniae bioprobe (E,F) of fatal ruptured plaque coronary artery segments.
  • FIGURE 1A Endothelial cell (End) of vasa vasorum exhibiting very small forms of Mycoplasma pneumoniae (MP) adhered to the endothelial surface. Presence of an elementary body of Chlamydia pneumoniae (CP) in the cytoplasm (original magnification: 3,300X).
  • FIGURE 1B Adventitial macrophage containing several CP bodies and MP forms (2,600X).
  • FIGURE 1C Necrotic core of atheroma plaque exhibiting many CP bodies and MP forms among abundant ruptured membrane elements (4.200X).
  • FIGURE 1P Two ellipsoid forms of MP in the interstitium (10.000X).
  • FIGURE 1E Several positive rounded brownish structures of MP inside a vulnerable plaque (1,000X).
  • FIGURE 1F Closer view of a necrotic atheromatous core exhibiting many positive brownish dots corresponding to MP (1 ,000X).
  • FIGURE 2A Illustrates a microscopic aspect of rat A (non-treated animal), showing severe chronic bronchitis and interstitial pneumonitis (H&E - x63 - original magnification).
  • FIGURE 2B - Shows rat P (after being treated for 7 days with Catalytic TS), revealing resolving interstitial pneumonitis. (H&E x100)
  • FIGURE 3A Illustrates rat A with interstitial pneumonitis seen in a high magnification view, showing vacuolated macrophages that correspond to C.pneumoniae infected cells (arrows) -H&E x 1,000.
  • FIGURE 3B - Refers to rat P, treated for 7 days, that exhibited a number of C.pneumoniae positive cells similar to rat A. However, in this case, the macrophages were detaching from the alveolar septa (arrow) - (H&E x 1,000).
  • FIGURE 4A - Refers to rat A, a non-treated animal. This rat exhibited bronchial epithelium with a proliferation of cells infected by M. pulmonis (in brown - arrows). M. pulmonis was also found in the interstitium of the alveolar septa (Immunoperoxidase - IPX - against M.
  • FIGURE 4B - Refers to rat P (treated for 7 days) showing a layer of M. pulmonis (arrow) detaching from the bronchial epithelial surface, and the absence of M.pulmonis from the interstitium (Immunoperoxidase - against M.pulmonis - x 1,000)
  • FIGURE 5A - Refers to rat A (non-treated animal). It shows a large quantity of M.pulmonis (in brown) on the pleural surface (arrowheads), in the interstitium, and on the alveolar surface, in a diffuse and granular shape (Immunoperoxidase - against M.pulmonis - x1 ,000).
  • FIGURE 5B - Refers to rat P, (treated for 7 days) presenting clearly defined M.pulmonis antigens on the alveolar surface; these are more compact and practically absent from the interstitium (immunoperoxidase - against M.pulmonis - x 1 ,000).
  • FIGURES 6A and 6B - Show aspects similar to FIGURES 5A and 5B, but depict a more internal region of the lung.
  • FIGURES 7A and 7B Demonstrate the same differences described in the legends to FIGURES 5A and 5B, respectively, but in a 3P view obtained using confocal laser microscopy.
  • M. pulmonis antigens were labeled with fluorescence and are shown in green.
  • the mycoplasmas in green
  • the mycoplasmas are larger, with prolongations that reach the spaces between the cells that are visible the red colored nuclei (stained with iodide propidium).
  • FIGURE 7B that represents rat P
  • the mycoplasmas are smaller and lack prolongations (acquired at 630x magnification).
  • FIGURES 8A and 8B Show ultrastructural aspects of the lungs from rats A and P, respectively.
  • FIGURE 8A shows that in rat A, the alveolar surface is completely covered by mycoplasmas
  • FIGURE 10A - Refers to rat A, showing a large number of macrophages containing C.pneumoniae antigens (in brown - arrowheads) in the lymphoid nodes at the peribronchial sites (Immunoperoxidase - against C.pneumoniae - x 1,000).
  • FIGURE 10B - Refers to rat F (a severely affected female rat treated for 9 days with Native TS), showing large numbers of plasma cells positive for C.pneumoniae antigens (in brown - arrows) at the periphery of the peribronchial lymphoid nodes. (Immunoperoxidase against C.pneumoniae, x 160). However, the alveoli are free of C.pneumoniae.
  • FIGURE 11A - Refers to rat A, seen in a higher magnification view, showing granules of C.pneumoniae in the macrophage cytoplasm, and in the extracellular space
  • FIGURE 11B - Refers to rat F (treated for 9 days with
  • FIGURES 12A and 12B - Respectively show a panoramic (x 100) and a higher magnification view (x1000) of rat G, after 12 days of treatment, revealing large areas from which M.pulmonis antigens are completely absent.
  • x100 panoramic
  • x1000 magnification view
  • FIGURES 12A and 12B - Respectively show a panoramic (x 100) and a higher magnification view (x1000) of rat G, after 12 days of treatment, revealing large areas from which M.pulmonis antigens are completely absent.
  • the reactivity of the bronchial epithelium and the interstitial inflammation still present suggest that the lung was previously severely injured. (Immunoperoxidase against M.pulmonis).
  • FIGURES 13A and 13B - Show resolving pneumonitis in rat G (12 days of treatment).
  • C.pneumoniae antigens are almost absent both at moderate magnification (x 250) and at high magnification (x 1,000), which reveals vacuolated histiocytes free of C.pneumoniae antigens (immunoperoxidase against
  • FIGURE 14 Presence of Mycoplasma pneumoniae DNA (arrows indicating brown stained regions) in neoplasic cells and in inflammatory cells from a transitional cell carcinoma from bladder, invasive, undifferenfiated form, (in situ hybridization technique - Original magnification 100x).
  • FIGURE 15 Cytological exam of ascites fluid from a patient with ovarian adenocarcinoma, exhibiting malignant neoplasic cells stained in brown due to the presence of M.pulmonis antigens, mainly on the surface, frequently forming fibrilar tufts (arrows). (Immunohistochemistry against M.pulmonis - Original magnification 100x).
  • FIGURE 16 Culture of neoplasic cells from the ascites fluid described above and doubled stained: M.pneumoniae antigens in green (fluorescein) occupy almost all the cytoplasm; nuclei in red are stained with Cy-5. Superposition of the green and red label is shown in yellow. (Laser confocal microscopy technique - original magnification 100x).
  • FIGURE 17 Cytological exam of ascites fluid from the same patient mentioned above, showing neoplasic cells forming clumps; the cells are frequently multinucleate. (Papanicolaou stain - Original magnification - 100x).
  • FIGURE 18 Electron microscopy revealing interlaced, irregular, filiform prolongations of the neoplasic cells that, together with information from other techniques, allowed identification as mycoplasmas. (Original magnification - 2,000x).
  • FIGURE 19 Electron micrograph of a neoplasic cell from ovarian adenocarcinoma. The presence of a second membrane under the plasma membrane is compatible with the idea that the external prolongations are mycoplasmas intimately adhered to the neoplasic cell. (Original magnification - 10,000x).
  • FIGURE 20 Electron microscopic view showing a neoplasic cell from an ovarian adenocarcinoma presenting many C.pneumoniae granules in the cytoplasm (arrows at the top left) and mycoplasmas in the extracellular space, adhering to the surface of the neoplasic cell (arrows at right bottom). (Original magnification 7,200x).
  • FIGURE 21 Double staining immunofluorescence technique demonstrating M.pulmonis antigens (stained in green by fluorescein, revealing the external prolongations), and nuclei in red (Cy-5) in a clump of neoplasic cells from an ovarian adenocarcinoma. The yellow regions represent the superposition of the green and red labeled areas (Original magnification - 100x).
  • FIGURE 22 Ovarian adenocarcinoma culture treated with TSN for 5 days. The decrease in amount of M.pulmonis antigens is remarkable (Confocal laser microscopy - Original magnification - 100x).
  • FIGURE 23 Clump of neoplasic cells in culture. The
  • TUNEL technique reveals only a single cell in apoptosis (part of the nuclei in yellow) - (Confocal laser microscopy - original magnification - 100x).
  • FIGURE 24 Ovarian adenocarcinoma cell culture to which TS was added.
  • the cells lost the adherence and entered apoptosis as detected by the TUNEL technique (positive results are nuclei in yellow), after 3 days of TS administration (Confocal laser microscopy - Original magnification - 100x).
  • FIGURE 25 Nucleotide sequence of plasmid encoding the catalytic trans-sialidase unit of trans-sialidase from T. cruzi (SEQ ID NO:1). The letters in capital represent the pET14 B and the underlined correspond to the oligonucleotideos' position.
  • FIGURE 26 Amino acid sequence of the protein encoded by the nucleic acid sequence depicted in FIGURE 25. (SEQ ID NO:2). In bold are the aminoacids not found in the original clone. DETAILED DESCRIPTION OF THE INVENTION:
  • the present invention relates to methods and compositions for preventing and/or treating conditions characterized by increased cell proliferation and/or increased proliferation of non- mycoplasma microbes and associated with mycoplasma infection.
  • the methods comprise the administration of an effective amount of an agent which prevents or decreases mycoplasma infection.
  • the level of infection is decreased by at least ten percent.
  • the level of infection may be measured by the number of mycoplasma organisms present in a tissue or fluid sample, by the immune reaction toward mycoplasma in the subject, or by any standard laboratory mycoplasma diagnostic assay.
  • the subject of the invention may be a human or a non- human subject, and the term "mycoplasma” as used herein may refer to mycoplasma capable of infecting a human and/or a non- human host.
  • the mycoplasma may be, for example but not by way of limitation, Mycoplasma (M.) buccale, M. faucium, M. fermentans, M. genitalium, M. hominis, M. lipophilum, M. oral, M. penetrans, M. pneumoniae, M. salivarium, or M. spermatophilum.
  • the agent used to prevent or decrease cell proliferation associated with mycoplasma infection may be an antibiotic or non- antibiotic agent.
  • the agent is an antibiotic
  • it may be, for example but not by way of limitation, erythromycin, azithromycin, clarithromycin, tetracycline, doxycycline, minocycline, clindamycin, ofloxacin, chloramphenicol, or any antibiotic known to have activity against mycoplasma.
  • the dose of antibiotic may be the standard dose or a lower dose.
  • the agent is not an antibiotic but rather is an agent which is able to interfere with the attachment of mycoplasma to their host cells via sialic acid residues.
  • the agent may exhibit neuraminidase and/or trans-sialidase activity.
  • the source of such activity may be, for example, a eukaryotic or prokaryotic neuraminidase and/or trans- sialidase enzyme, or an enzymatically active fragment or mutant thereof.
  • the amount of neuraminidase to be administered may be between about 1x10 "2 to 1x10 3 U per day, where a unit of enzyme activity is defined as 1 nmol of 4-MuNana hidrolyzed in one minute at 37°C in the presence of 0,5mM of 4-MuNana.
  • a unit of enzyme activity is defined as 1 nmol of 4-MuNana hidrolyzed in one minute at 37°C in the presence of 0,5mM of 4-MuNana.
  • the agent is trans-sialidase from the microorganism Trypanosoma cruzi.
  • the trans-sialidase enzyme of this microorganism is well characterized, and active fragments of the enzyme are known (for various references, please refer to the Background section, supra).
  • native trans-sialidase of T. cruzi may be utilized.
  • Such enzyme may be comprised in the supernatant of a T. criz/-infected cell culture, prepared by standard techniques and preferably sterilized ⁇ e.g., by filtration). See Umezawa et al., "Immunoblot assay using excreted/secreted antigens of Trypanosoma cruzi in serodiagnosis of congenital, acute and chronic Chagas' disease," J. Clin.
  • a portion of the native T cruzi trans-sialidase containing the catalytic portion is utilized.
  • TSC native T cruzi trans-sialidase containing the catalytic portion
  • the expression vector has the advantage of producing high yields of protein.
  • the TSC protein has the advantages of (1 ) lower immunogenicity (due to lack of the repeat-containing domain); (2) a shorter half-life and (3) a His-tag, which facilitates purification.
  • the trans-sialidase gene may be obtained from a genomic clone, isolated from a lambda Zapll library (Stratagene, http://www.stratagene.com) of T. cruzi Y strain (Silva and Nussenzweig, Folia Clin Biol 20:191-203 (1953), as described in Uemura et al., EMBO J 11:3837-3844 (1992). From the original lambda clone, which express enzymatic activity, a SK plasmid containing the trans-sialidase gene was generated (SK-154-0).
  • the preferred plasmid used is termed pTSII, and corresponds to a fragment of the original gene (clone 154-0) inserted into the sites Ndel and BamHI of the vector pET14b (Novagen - http://www.novagen.com).
  • the pTSII was constructed by the following procedure: By using SK-154-0 as template and TSPET14 (5'- GGAATTCCATATGGCACCCGGATCGAGC, SEQ IP NO:3) and RT 154 (5'-CGGATCCGGGCGTACTTCTTTCACTGGTGCCGGT, SEQ IP NO:4) a PCR product was amplified having a sequence as set forth in FIGURE 25. The corresponding amino acid sequence is depicted in FIGURE 26.
  • the final plasmid may be transformed into the bacteria Escherichia coli BLB21 OE3.
  • the construct may be made in two steps because there is an internal BamHI site in the gene.
  • the PCR product may be treated with BamHI and Ndel enzymes, the resulting fragments fractionated in agarose gel electrophoresis, and then purified from the gel with Sephaglass (Amersham-Pharmacia) purification kit. So the 5' fragment Ndel- BamHI may be inserted into the pET14b vector pre-digested with BamHI and Ndel.
  • the ligation products may be transformed into E. coli K12 PH5 , selected and the expected plasmid purified.
  • this intermediate plasmid construct may be treated with BamHI, shrimp alkaline phosphatase and ligated with BamHI-BamHI-3 ' fragment purified from the gel mentioned above.
  • the ligation products again may be transformed into E. coli K12 PH5 ⁇ , selected and the expected plasmid purified.
  • the final plasmid may be confirmed by restriction analysis and used to transform the BLB21 PE3 pLys strain.
  • E. coli vectors may be used.
  • the original gene was discovered by three laboratories (Pollevick et al., Mol. Biochem. Parasitol. 47:247-250 (1991); Pereira et al., J. Exp. Med. 174:179-191 (1991) and Uemura et al., Embo J. 11:3837-3844 (1992)).
  • Other references disclose other constructs to prepare trans- sialidase, including Schenkman et al., J. Biol. Chem. 269:7970-7975 (1994); Buschiazzo et al., Cell Mol. Biol. 42:703-710 (1996) Campetella et al., Mol. Biochem. Parasitol.
  • TSC trans-sialidase
  • coli BLB 21PE3 pLys, and the plasmid pTSII may be inoculated in 50 ml LB medium containing 50 ug/ml carbenicillin and 34 ug/ml chloramphenicol. After an overnight incubation at 37°C under agitation, the 50 ml culture may be inoculated in 2 liter LB medium containing 50 ug/ml carbenicillin. The cultures may be maintained at 37C under agitation, until the absorbance at 600 nm reaches 0.5, when the cultures are shifted to 30C and the bacteria induced by addition of 0.5 mM isopropyl- ⁇ -P-thio-galactopyranoside.
  • the bacteria may be collected by centrifugation, resuspended in 50 mM sodium phosphate buffer, pH 7, containing 0.3 M NaCI, 0.1 mM dithiothreitol, 1 mM phenyl- methyl-sulfonyl-fluride, 10 ug/ml leupeptin, 1 ug/ml antipain.
  • the bacteria may be lysed by sonication (3 to 4 cycles of 5 min each) at 4°C.
  • the enzyme may be purified by a NiTA -Agarose (Qiagem) and ion exchange chromatography by using a MonoQ column as described as described by Ribeirao et al., "Temperature differences for trans-glycosylation and hydrolysis reaction reveal an acceptor binding site in the catalytic mechanism of Trypanosoma cruzi trans-sialidase". Glvcobiology, 7:1237-1246, 1997.
  • trans-sialidase activity required to confer a therapeutic benefit may vary from patient to patient and depending on the nature of the condition to be treated. Thus, the doses set forth below may require adjustment as would be apparent to the skilled artisan.
  • the present invention provides for methods of inhibiting undesirable cell proliferation comprising administering, to a subject in need of such treatment, an amount of trans-sialidase effective in inhibiting cell proliferation and/or in decreasing the level of mycoplasma infection.
  • an amount of trans-sialidase effective in inhibiting cell proliferation and/or in decreasing the level of mycoplasma infection is administered by at least ten percent.
  • the amount of enzyme administered may be between 140 and 2,000 units per day.
  • Each trans-sialidase unit is defined as follows: in a standard trans-sialidase reaction, made in 50 ul of 20 mM Hepes pH 7.0 buffer, 0.2% BSA and 0.36 nmol ( 14 C)-lactose (7.2 uM) (60 mCi/mmol), and 50 nmol (1mM) sialyl-lactose at 25C, one Unit of trans-sialidase activity is defined as the transfer of 0.36 nmol of sialic acid from unlabeled sialyllactose to the ( 14 C) lactose in 30 min under the above conditions.
  • the amount of enzyme administered may be between 10 6 and 10 13 units per day.
  • Higher TSC doses may be used because its clearance is much faster than TSN. This occurs because TSC has no 12 amino acid C-terminal repeats; See, Buscaglia et al., "Tandem amino acid repeats from Trypanosoma cruzi shed antigens increase the half-life of proteins in blood", Blood, 93:2025-2032, 1999.
  • 4mg TSC per day (preferably corresponding activity of 3.4 x 10 7 U) may be administered over a two week period, or until a desired clinical effect, or undesirable side effects occur.
  • a supernatant of a T cruzi culture, with a mean trans-sialidase activity of about 140U /day may be administered every other day for one week, or until a desired clinical effect, or undesirable side effects occur.
  • the period of treatment may be for one day or may extend for an indefinite period of time, including continuous use for years.
  • the treatment period is between 1 week and 8 weeks.
  • the route of administration may be intravenous, intraperitoneal, intrathecal, oral, by inhalation, subcutaneous, intramuscular, or any other appropriate route.
  • the agent of the invention may be comprised in a suitable pharmaceutical vehicle. It may be used together with other agents directed toward treating either the mycoplasma infection or the undesirable cell proliferation.
  • an agent having neuraminidase and/or trans-sialidase activity may be used in conjunction with, for the treatment of atherosclerotic vascular disease, an anti-platelet or anti-thrombotic agent or, for the treatment of a malignancy, a standard chemotherapeutic agent or radiation therapy.
  • Pisorders characterized by undesirable cell proliferation include atherosclerotic vascular disease (for example of the coronary arteries, carotid arteries, cerebral vasculature, aorta, etc.), and malignancies including but not limited to ovarian carcinoma, breast cancer, prostate cancer, colon cancer, lung cancer (small cell and non-small cell varieties, mesothelioma, etc.), pancreatic cancer, gastric cancer, thyroid cancer, melanoma, leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, testicular carcinoma, etc.
  • atherosclerotic vascular disease for example of the coronary arteries, carotid arteries, cerebral vasculature, aorta, etc.
  • malignancies including but not limited to ovarian carcinoma, breast cancer, prostate cancer, colon cancer, lung cancer (small cell and non-small cell varieties, mesothelioma, etc.), pancreatic cancer, gastric cancer, thyroid cancer
  • the agent of the invention may also be used to treat disorders characterized by co-infection with mycoplasma and at least one other microbe, where the microbe may be a virus (e.g., Human Immunodeficiency Virus) or bacterium (e.g. a chlamydia).
  • the amount of agent administered is an amount which inhibits or prevents mycoplasma adhesion and/or infection.
  • EXAMPLE 1 ASSOCIATION OF MYCOPLASMA INFECTION WITH ATHEROSCLEROSIS OF CORONARY ARTERIES
  • Mycoplasma pneumoniae is related to the presence of atherosclerosis in coronary arteries. It was also demonstrated that large numbers of Chlamydia pneumoniae are present in the atheromatous plaques possibly leading to plaque rupture and thrombosis. The proliferation of chlamydia appears to be a consequence of the close association between this bacterium and mycoplasmas, which results in inflammation and rupture of the atheromatous plaque in the coronary arteries of patients who had died due to acute myocardial infarction. These data were obtained by analyzing autopsy material.
  • mycoplasmas to be absent or minimal in the intimal layer of the coronary artery segments of non- atherosclerotic patients, which contrasts with the high concentration in the intimal layer of the coronary arteries of atherosclerotic patients.
  • Chlamydia pneumoniae was present in the majority of the segments of the 4 groups of coronary arteries, although to a much greater degree in the group exhibiting ruptured and thrombosed plaque segments.
  • the electron microscopic characteristics that allowed the identification of these microorganisms as Mycoplasma pneumoniae included their rounded structures which contain a granulous chromatin-like material enveloped by a cytoplasmic membrane, in the absence of an external cell wall.
  • M. pneumoniae were adhered to the endothelial surface of the vasa vasorum (FIGURE 1 A) or were in the cytoplasm of cells also infected with C. pneumoniae.
  • the mycoplasmas were present in blood monocytes and macrophages (FIGURE 1B) or in the interstitium. Large numbers of these microorganisms were present inside the atheroma together with C. pneumoniae and were associated with several membrane components possibly corresponding to degenerated bacteria (FIGURE 1C).
  • Mycoplasma were also found in large cylindrical or elliptical forms in the extracellular matrix (FIGURE 1P). These results were confirmed by in situ hybridization with a M. probe from Enzo Piagnostics (New York, NY USA). This technique, described in (Sambiase et al., "CMV and transplant-related coronary atherosclerosis: an immunohistochemical, in situ hybridization and polymerase chain reaction in situ study," Modern Pathology 13:173-179 (2000)), revealed a larger number of mycoplasmas mainly in unstable plaque segments throughout the fatty material or in the necrotic core (FIGURES 1 E and 1F).
  • TSC catalytic portion - recombinant form
  • TSN complete native TS form
  • Group A - Two control animals were killed without injecting any substance; another rat was killed on the seventh day, after receiving inactivated TSC substance for 5 consecutive days. There was no difference between this latter rat and the first two. These three animals were used as control animals exhibiting the usual pattern of the disease.
  • Group B Five animals each received TSN (complete active native TS substance) every two days, employing a dose of 0.5mL/animal, and were killed after seven, nine and twelve days of treatment. Another rat, rat 0, received TSC (active TS substance catalytic portion, produced from a cloned bacteria) at a dose of 140 ⁇ g/day for five consecutive days, and was killed after seven days. Results: Group A - Non-treated rats and rats treated with the inactivated TS substance.
  • M.pulmonis and C.pneumoniae were performed by transmission electron microscopy, confocal laser microscopy and immunohistochemistry.
  • Lung was the most frequently injured organ. All rats presented histopathological signs of chronic tracheobronchitis, with different intensities of multi-focal, interstitial pneumonitis (FIGURE 2A). A lympho-histiocytic infiltrate was present in the alveolar septa with many vacuolated macrophages in the interstitium and in the alveolar lumen (FIGURE 3A). The tracheo-bronchial epithelium showed proliferation of the epithelial lining cells which were highly infected by M.pulmonis on their surface.
  • the mycoplasmas were present both strongly attached to the epithelial cells, forming an irregular surface that stretched between adjacent cells, and in the cytoplasm of the lining cells (FIGURE 4A).
  • a large amount of M.pulmonis was present in the form of thin, delicate granules covering large areas of the pleural or subpleural areas (FIGURE 5A), and also lining the alveolar surfaces, in macrophages or in the interstitial spaces (FIGURE 6A).
  • Both confocal (FIGURE 7A) and electron microscopy (FIGURE 8A) emphasized the irregular shape of the mycoplasmas, which extensively covered the alveolar surface and expanded into the alveolar septum.
  • Electron microscopy and confocal laser microscopy provided qualitative data, while the immunohistochemistry performed using the previously described antibodies, anti- pulmonis and anti-C. pneumoniae antibodies, provided quantitative data.
  • the average number of cells positive for C.pneumoniae/ ⁇ OO field of lung tissue was obtained, and the percent area positive for M.pulmonis was obtained using an image analysis system (Leica Quantimet 500). The lack of a typical morphology for M.pulmonis did not permit quantification of their number.
  • FIGURE 2B Upon examination of the lung, histological sections showed that after 7 days of treatment the treated animals presented resolving pneumonitis (FIGURE 2B). There was a great reduction of M.pulmonis in the alveolar lumen. In the respiratory epithelium, the mycoplasma were detaching from the septum to the lumen, with a clear decrease in the number of adhesion points (FIGURE 3B). At the respiratory epithelium, M. pulmonis was only found on the surface of epithelial cell, but not in their cytoplasm or between them, as shown by immunohistochemistry (FIGURE 4B). The mycoplasmas on the alveolar and pleural surfaces had better defined limits (FIGURE 5B).
  • FIGURE 6B shows in closer view that the mycoplasma are still not into the cytoplasm of the cells.
  • the morphology of M.pulmonis also changed, as visualized by confocal laser microscopy in 3P view; they were seen without the dendritic extensions that were visually seen in the non treated animals, the change seen by confocal microscopy: smaller micoorganisms and in lower amount (FIGURE 8B).
  • the C. pneumoniae by immunohistochemistry were present in macrophages with stronger staining and more homogeneous, than in the non-treated group, possibly suggesting peribronchial lymphoid nodes (FIGURE 10B).
  • FIGURE 11B There was a change in the morphological aspect of positive cells for C. pneumoniae in the alveoli.
  • degenerated C. pneumoniae were detected by electron microscopy (FIGURE 9B).
  • the sialidases irreversibly catalyse the transfer of sialic acid from glycoconjugates to water, in a reaction recognized as hydrolysis.
  • the sialidases may also transfer sialic acid between galactose molecules, and can catalyze an reversible reaction denominated trans-sialation or more generically, trans-glycosylation.
  • the efficiency of the transferase activity versus hydrolysis depends on the concentration of acceptors containing free ⁇ -galactose.
  • the T.cruzi trans-sialidase "TS" differs from the other sialidases because the acceptor concentration necessary for trans-glycosylation is much lower. Also, TS has a much lower catalytic efficiency in promoting hydrolysis, not depending on the acceptor concentration.
  • This patent thus aims to investigate whether the removal of sialic acid or trans-sialation might provide a protective effect against mycoplasmas. Using mycoplasma-infected rats, we tested the effect of bacterial sialidases that exhibit neuraminidase activity and very little trans-sialidase activity.
  • Literature data show that mycoplasmas are sensitive to neuraminidase treatment and to pronase and other chemical agents, in vitro.
  • the bacterial sialidases exhibit a lower specificity than the T.cruzi TS which acts only on sialic acid linked terminally by ⁇ 2,3 linkages.
  • the bacterial sialidases hydrolyze bonds with terminal linkages ⁇ 2,3; ⁇ 2,6; ⁇ 2,8, branched linkages, and glycoconjugates containing substitutions in the de ⁇ - galactosyl and adjacent residues, such as those found in the Lewis antigens, which are important factors in the linkage of adhesion molecules of the immune system.
  • Rat # 1 was treated for 5 consecutive days with Vibrio cholerae neuraminidase**.
  • Rat # 2 was treated for 5 consecutive days with Clostridium perfringes neuraminidase * **.
  • Rat # 3 was treated for 5 consecutive days with the catalytic form of recombinant T.cruzi TS of (TSC).
  • TSC recombinant T.cruzi TS of
  • Rat # 1 received Vibrio cholerae from Roche Diagnostics, via daily intraperitoneal injection of 68 ul of the substance diluted in 432 ⁇ l of physiological saline.
  • rat # 2 1.6ul of Clostridium perfringes from Biolabs (catalog: #728S), diluted in 498.4 ⁇ l of physiological saline, was injected.
  • Rat # 4 received no infection.
  • the amount of injected enzyme in each animal was estimated using an enzymatic assay employing a fluorescent substrate (4-methyl-umbelliferil-N-acetyl-neuramic acid), provided in the table below: Comparative data on Enzymatic activity:
  • Enzymatic activity corresponds to the amount of picomoles of methyl-1-umbeliferil-n-acetyl-neuraminic acid hydrolyzed in 1 minute at 37°C.
  • mycoplasmas are present in cancer cells and may affect the natural biological process of cell death (apoptosis), transforming cells into permanently differentiated cells, thus playing a fundamental role in the pathogenesis of malignant neoplasia.
  • apoptosis cell death
  • transforming cells into permanently differentiated cells thus playing a fundamental role in the pathogenesis of malignant neoplasia.
  • malignant neoplasias such as adenocarcinomas of the bladder, lung, stomach and large intestine, as well as mesotheliomas, are severely infected with mycoplasmas in association with Chlamydia pneumoniae.
  • TSN native trans-sialidase
  • the cell culture in each well was stained using a double immunofluorescent staining technique, employing the following combinations: nuclear stain (Cy-5) + M.pulmonis antigens (fluorescein); nuclear (Cy-5) + M. pneumoniae antigens (fluorescein); and nuclear stain (Cy-5) + apoptotic nuclei detected by the TUNEL method (fluorescein).
  • FIGURES 21, 22, 23 and 24 The results are depicted in FIGURES 21, 22, 23 and 24.
  • the culture cells that did not receive TSN grew, maintaining initial cohesion, forming a clump of neoplasic cells (FIGURES 21 and 23).
  • the cultures receiving TSN exhibited cells that lost adherence each other, taking on the appearance of a cell monolayer (FIGURES 22 and 24).
  • the TUNEL technique demonstrated that the samples receiving TSN contained a large number of apoptotic cells already after 3 days of treatment, that increased after 5 days (FIGURE 24). In contrast, the cultures that did not receive TSN showed very few cells in apoptosis (FIGURE 23).
  • M. pneumoniae antigen stain was found to occur simultaneously with an increase in the number of neoplasic cells that had entered apoptosis, in the wells receiving TSN. This is consistent with the conclusion that the removal of mycoplasmas from neoplasic cells induces apoptosis in these cells.
  • EXAMPLE 5 EFFECT OF TRANS-SIALIDASE ON HUMAN CANCERS IN VIVO.
  • the first patient a 64 year old, female, had been diagnosed with ovarian adenocarcinoma in 1990, when the tumor was resected. She received chemotherapy (2 cycles) interrupted as a result of toxicity. In July 1997, she presented a recurrence of the cancer, and was treated by chemotherapy with Carboplatine. In February 1998, a second recurrence was found, and she was treated with radiotherapy. In March 1999, after a further recurrence of the tumor, chemotherapy with 3 cycles of Taxol was performed. This treatment was also interrupted by cytotoxicity. Intestinal hemorrhage by tumoral rectal infiltration appeared. Subsequently, laparotomy revealed a recurrence of the tumor that was considered inoperable and a colostomy was performed.
  • the protocol used on this patient is as follows.
  • the patient was administered 50 ml of native trans-sialidase (TSN"), intraperitoneally, corresponding to 140 U activity, on alternate days, during a period of 14 days.
  • Enzyme activity - 1 U corresponds to 30,000 cpm at 37 °C, during 30 minutes.
  • the patient soon began the protocol. She received recombinant catalytic fragment trans-sialidase (TSC) 4.0 mg/day, during 14 consecutive days. The corresponding activity was 3.4 x 10 7 U/ day. She presented with fever at the end of the second week of treatment that was controlled with Cypro 1.0 g/day. The number of blood leucocytes was unaltered. Tomography showed a reduction in the tumor, and the patient showed improvement in the clinical state.
  • TSC catalytic fragment trans-sialidase

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Abstract

L'invention concerne la prévention et le traitement de maladies associées à une prolifération cellulaire indésirable, y compris le rétrécissement athérosclérotique de vaisseaux sanguins et le cancer, comprenant la prévention et le traitement d'infections par des mycoplasma. L'invention est basée, en partie, sur la mise en évidence que, dans de nombreux cas, une infection aux mycoplasma coexiste avec une prolifération cellulaire indésirable et/ou avec une prolifération d'autres organismes infectieux.
PCT/BR2001/000083 2000-07-03 2001-07-03 Prevention et traitement de maladies associees aux mycoplasma Ceased WO2002002050A2 (fr)

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DE60135843T DE60135843D1 (de) 2000-07-03 2001-07-03 Prävention und behandlung von mycoplasma-assoziierten krankheiten mit trans-sialidase und/oder neuraminidase
CA2383850A CA2383850C (fr) 2000-07-03 2001-07-03 Prevention et traitement de maladies associees aux mycoplasma
AU2001267165A AU2001267165A1 (en) 2000-07-03 2001-07-03 Prevention and treatment of mycoplasma-associated diseases
EP01944764A EP1296554B1 (fr) 2000-07-03 2001-07-03 Prevention et traitement de maladies associees aux mycoplasma par l'utilisation de trans-sialidase et/ou de neuraminidase
US10/086,913 US7108851B2 (en) 2000-07-03 2002-03-01 Prevention and treatment of mycoplasma-associated diseases
US10/952,003 US7335638B2 (en) 2000-07-03 2004-09-28 Prevention and treatment of mycoplasma-associated diseases

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003082324A1 (fr) * 2002-03-28 2003-10-09 Maria De Lourdes Higuchi Prevention et traitement de maladies associees au mycoplasme
US7674832B2 (en) 2003-03-28 2010-03-09 Higuchi Maria De Lourdes Compositions for promoting wound healing and treating psoriasis
US7732410B2 (en) 2003-03-28 2010-06-08 Higuchi Maria De Lourdes Compositions for inhibiting atherosclerosis
US8551940B2 (en) 2003-03-28 2013-10-08 Maria de Lourdes Higuchi Diagnosis, prevention and treatment of disorders characterized by undesirable cell proliferation
US8822232B2 (en) 2003-03-28 2014-09-02 Maria De Lourdes Higuchi Diagnosis, prevention and treatment of disorders characterized by undesirable cell proliferation

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
BASEMAN ET AL.: 'Sialic acid residues mediate mycoplasma pneumoniae attachment to human and sheep erythrocytes' INFECTION AND IMMUNITY vol. 38, no. 1, October 1982, pages 389 - 391, XP002950818 *
NEYROLLES ET AL.: 'Identification of two glycosylated components of mycoplasma penetrans: a surface-exposed capsular polysaccharide and a glycolipid fraction' MICROBIOLOGY vol. 144, May 1998, pages 1247 - 1255, XP002950817 *
ROBERTS ET AL.: 'Sialic acid-dependent adhesion of mycoplasma pneumoniae to purified glycoproteins' J. BIOL. CHEM. vol. 264, no. 16, 05 June 1989, pages 9289 - 9293, XP002950819 *
See also references of EP1296554A2 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003082324A1 (fr) * 2002-03-28 2003-10-09 Maria De Lourdes Higuchi Prevention et traitement de maladies associees au mycoplasme
US7674832B2 (en) 2003-03-28 2010-03-09 Higuchi Maria De Lourdes Compositions for promoting wound healing and treating psoriasis
US7732410B2 (en) 2003-03-28 2010-06-08 Higuchi Maria De Lourdes Compositions for inhibiting atherosclerosis
US7906114B2 (en) 2003-03-28 2011-03-15 Maria De Lourdes Higuchi Compositions for promoting wound healing and treating psoriasis
US7914781B2 (en) 2003-03-28 2011-03-29 Higuchi Maria De Lourdes Compositions for inhibiting atherosclerosis
US8551940B2 (en) 2003-03-28 2013-10-08 Maria de Lourdes Higuchi Diagnosis, prevention and treatment of disorders characterized by undesirable cell proliferation
US8822232B2 (en) 2003-03-28 2014-09-02 Maria De Lourdes Higuchi Diagnosis, prevention and treatment of disorders characterized by undesirable cell proliferation

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