WO2008020260A2 - Utilisation de sidérophores dans la prévention des maladies vasculaires essentiellement causées par un dysfonctionnement des cellules de l'endothélium, production de sidérophores et évaluation des produits carnés contenant des sidérophores - Google Patents

Utilisation de sidérophores dans la prévention des maladies vasculaires essentiellement causées par un dysfonctionnement des cellules de l'endothélium, production de sidérophores et évaluation des produits carnés contenant des sidérophores Download PDF

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WO2008020260A2
WO2008020260A2 PCT/HU2007/000076 HU2007000076W WO2008020260A2 WO 2008020260 A2 WO2008020260 A2 WO 2008020260A2 HU 2007000076 W HU2007000076 W HU 2007000076W WO 2008020260 A2 WO2008020260 A2 WO 2008020260A2
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siderophores
siderophore
heme
alkyl group
desferricoprogen
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WO2008020260A3 (fr
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József BALLA
György BALLA
István PÓCSI
László FÉSÜS
Viktória JENEY
Imre PÓCSI
Tamás EMRI
Gyöngyi GYÉMÁNT
Gyula ROMÁN
István Kovács
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R-KO-N KFT
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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
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione

Definitions

  • the invention relates to the use of fungal siderophores in the treatment of vascular diseases primarily caused by endothelial cell origin. These compounds are especially useful for oral application, which was not known in and not expected from the prior art.
  • the invention relates to a preferred route of production of fungal siderophores by using specifically isolated and selected species of fungi.
  • the invention also relates to foods, preferably meat products, especially sausage and salami, which contain siderophores in controlled and efficient amount.
  • the invention also relates to a qualifying system to determine if different foods have preventive effect on the vasculature as described above.
  • Iron is a necessary transition metal for most of living organisms in the world. Iron plays important roles in basic metabolic processes for example respiration, secretion and DNA synthesis, which are based on its ability to exist in different oxidation states and complex forming properties. Despite of the fact that iron is ubiquitous in the biosphere microorganisms have specific methods to help solubilization and uptake iron which form unsoluble iron hydroxides and iron-oxihydroxides at normal pH and aerobe conditions (Drechsler et al.: Iron chelator and Siderophores pp. 1-49, [G.Winkelmann es CJ. Carrano: Transition Metals in Microbial Metabolism, Harwood Academic Publishers,
  • Hydroxamate type siderophores ferrichromes and coprogens are produced exclusively by fungi as described by Drechsler el al. Coprogen producers are among others: Penicillium chrysogenum,
  • Penicillium roqueforti Neurospora crassa. Ferrichrome producers are among others: Penicillium chrysogenum, Penicillium roqueforti, Ustilago sphaerogena, Ustilago maydis, Neovossia indica.
  • Ferrirubin producers are among others: Aspergillus ochraceus. Ferrichrysin producers are among others: Aspergillus ochraceus,
  • coprogens are produced by Histoplasma capsulatum, Blastomyces dermatitidis, Fusarium dimerum es Cultivaria lunata (which could be human pathogenic) [Howard, D.H. (1999) Clin. Microb. Rev. 12, 394-404].
  • Ferrichrome producers are Microsporium spp, Trichophyton spp. es
  • Fig. 1 Fungal hexadentate siderophores (Fig. 1) were purified from culture fluids of Penicillium chrysogenum (coprogen, ferrichrome), Neurospora crassa
  • Neovossia indica ferrichrome
  • Aspergillus melleus ferrichrysin
  • A. ochraceus ferrichrysin, ferrirubin cultivated in defined low-iron minimal media (Charlang et al. 1981, JaIaI et al. 1984, Le ⁇ ' er et al. 2001).
  • Desferri forms of the siderophores listed above are ubiquitously used to treat iron, aluminium, or other complex-forming metal overload (Farkas et al. 1997).
  • Dionis et al described the use of desferrioxamine B (DFO, Desferal) in the treatment of acute iron toxicity and aluminium overload.
  • Enyedi et al showed the complex forming properties of desferricoprogen with metal ions with two- (Fe, Ni, Cu and Zn) or three-valences (Fe, Al, Ga, In) ( J. of Inorganic Biochemistry 98 (2004) 1957-1966).
  • Iron derived reactive oxygen species are thought to be involved in the pathogenesis of numerous vascular disorders such as atherosclerosis, microangiopathy, vasculitis and reperfusion injuries.
  • Heme is an abundant source of redox active iron and is dangerous itself if liberated from intracellular heme proteins. Heme plays crucial role in vascular endothelial cell damage, and endothelial cells have their stratagem to minimize heme mediate toxicity. Heme greatly amplifies cellular damage arising from activated oxygen produced by activated polymorphonuclear leukocytes or any other source. Free heme mediates oxidative modification of low-density lipoproteins (LDL) in which process cytotoxic lipid peroxidation products are formed. Hemoglobin is the most abundant heme protein in the vasculature; therefore it might be the potential source of heme.
  • LDL low-density lipoproteins
  • Ferritin serves as a safe storage site for the released iron. Ferritin is cytoprotective because its antioxidant, antiapoptotic and antiproliferative effects.
  • Heme is absolutely required for aerobic life.
  • free heme can be quite cytotoxic, particularly in the presence of oxidants or activated phagocytes.
  • the vasculature - and in particular the endothelial lining - may be at greatest risk of exposure to free heme. This is because erythrocytes contain heme in a concentration of 20 mmol/L and are vulnerable to unexpected lysis.
  • the extracellular hemoglobin is easily oxidized, to ferrihemoglobin which, in turn, will readily release heme. Given the hydrophobic nature of heme, it is no surprise that it easily crosses the cell membranes and can synergistically enhance cellular oxidant damage.
  • Oxidative Damage caused by reactive oxygen species can be greatly amplified by 'free' redox active iron (Halliwell et al, Biochem. J.: 1984, 219, 1-14]).
  • iron-rich Staphylococcus aureus are three orders of magnitude more susceptible to killing by hydrogen peroxide than are iron-poor staphylococci (Repine, J. et al, J. Biol. Chem.: 1981, 256, 7094-7096).
  • depletion of cellular iron powerfully protects eukaryotic and prokaryotic cells against oxidant challenge (Gannon, D. et al, Lab. Invest.: 1987, 57, 37-44).
  • Heme a ubiquitous iron-containing compound, is present in large amounts in many cells (Ponka, P., Am. J. Med. ScL 1999, 318, 241-256) and is also inherently dangerous, particularly when it escapes from intracellular sites (BaIIa, G. et al, Lab. Invest: 1991, 64, 648-655; BaIIa, G. et al, Trans. Assoc. Am. Physicians.: 1990, 103, 174-917; BaIIa, J. et al, Blood: 2000, 95, 3445- 3450; Paller, M. S. et al, Proc. Natl. Acad. Sci. USA: 1994, 91 , 7002-7006). Heme greatly amplifies cellular damage arising from activated oxygen (BaIIa et al).
  • the uptake of heme is required for this synergistic toxicity and the hydrophobicity of heme is critical for entry into endothelial cells.
  • the spontaneous uptake of heme and the associated amplification of cellular oxidant sensitivity are both inhibited by hemopexin (BaIIa G et al see above).
  • the plasma heme-binding protein, hemopexin was also shown to block its catalytic activity (Gutterige et al, Biochem. J.: 1988, 256, 861-865; Eskew, J. et al, J. Biol. Chem.: 1999, 274, 638-648).
  • Hemopexin is certainly not the sole factor in plasma that protects against heme-amplified oxidant damage to endothelium. Albumin may also limit the intrusion of extracellular heme and its pro-oxidant effects. Once within the cell, heme can promote oxidative damage either directly or, perhaps more importantly, via the release of iron which can occur either through non-enzymatic oxidative degradation of heme (BaIIa G et al see above) or enzymatic, heme oxygenase catalyzed heme cleavage.
  • the iron may initially lodge within the hydrophobic interstices of the phospholipid bilayer; within this highly oxidizable matrix, iron acts as an especially active catalyst of oxidation of cell membrane constituents (BaIIa G et al see above).
  • hydrophobicity of various ferriporphyrin is critical for entry into cells and required for the synergistic oxidative toxicity. Substitution of vinyl side chains of heme with hydrogen does not alter the hydrophobicity of the resultant ferriporphyrin, iron deuteroporphyrin IX; accordingly, hypersusceptibility is similarly provoked.
  • ferrihemoglobin readily releases its heme moieties as first demonstrated by Bunn and Jandl (Bunn, H. et al, J. Biol. Chem.: 1968, 243, 465-475). Released heme from ferrihemoglobin can indeed be rapidly incorporated into hydrophobic domains of cultured endothelium and serve a source of highly damaging iron. Although ferrohemoglobin itself is not capable of sensitizing vascular endothelial cells to oxidant injury, we and others have shown it can readily be oxidized to heme- releasing methemoglobin in the presence of inflammatory-cell-derived oxidants (BaIIa, J.
  • ferrohemoglobin in the presence of activated PMNs can provide heme to endothelium which greatly enhances cellular susceptibility to oxidant-mediated cell-injury (BaIIa, J. et al, Proc. Natl. Acad. Sci. USA: 1993, 90; BaIIa J. et al, Trans. Assoc. Am. Physicians: 1992, 105, 1-6).
  • the oxidation of ferrohemoglobin to ferrihemoglobin is essential for this deleterious effect.
  • Another candidate for generating methemoglobin is nitric oxide.
  • nitric oxide Reaction of nitric oxide with free hemoglobin produces methemogobin and leads to decreased nitric oxide bioavailability, causing pulmonary hypertension, vascular damage and end-organ injury as reviewed by Gladvin et al (Free Radic. Biol. Med. 2004, 36, 707-717).
  • the initial release of heme from ferrihemoglobin can be inhibited by complexation with the hemoglobin-binding protein, haptoglobin (Bunn, H. es munkatarsai, J. Biol. Chem.: 1968, 243, 465-475).
  • ferrihemoglobin loses much of its capacity to sensitize endothelium to reactive oxygen (BaIIa, J. es munkatarsai, Proc. Natl. Acad. Sci. USA: 1993, 90).
  • Hemoglobin:haptoglobin complex is eliminated from the circulation through the recently characterized CD163 receptor (Kristiansen, M., Nature: 2001, 409, 198-201), which is expressed exclusively by cells of the monocyte-macrophage lineage.
  • ferrohemoglobin or other heme proteins such as metmyoglobin and cytochrome c, all of which avidly bind heme (Smith, M. es munkatarsai, Proc.Natl. Acad. Sci. USA: 1991 , 88, 882-886), do not alter endothelial integrity.
  • the normal mechanisms for control of hemoglobin haptoglobin/hemopexin
  • oxidation of hemoglobin to ferrihemoglobin by phagocyte-mediated oxidation foster transfer of heme moieties to the vessel wall and aggravate endothelial cell damage in the short term.
  • Ferrihemoglobin present in plasma increases the level of endothelial cell associated heme in lung (BaIIa J. et al, Am. J. Phisiol.: 1995, 268, 321-327) indicating that protective effects of haptoglobin (Gutteridge, J. M., Biochim. Biophys. Acta: 1987, 917, 219-223), hemopexin (BaIIa G. et al, Lab.
  • Oxidative modification of low density lipoprotein (LDL) plays a key role in the pathogenesis of atherosclerosis (Chisolm. GM. et al. Free Radic Biol Med. 2000 Jun 15; 28(12):1815-26. Ross, R. N Engl J Med. 1999 Jan 14;340(2):115- 26.) Oxidized LDL has many damaging biological effects which contribute to the development of atherosclerosis, the leading cause of death in the developed countries. The presence of redox active transition metals is required to catalize oxidative modification of LDL.
  • oxidative modification of LDL plays a crucial role in the development of atherosclerosis: (i) clinical studies prooved that LDL undergoes oxidative modification in vivo and demonstarted its presence in atherosclerotic lesions; (ii) a lot of studies showed the damaging biologycal effects of oxidized LDL - activates and damages endothelial cells (induces apoptosis and necrosis, increases permeability of the endothelium, changes the phenotype of endothelium from anticoagulant to procoagulant, etc.) induces adhesion molecule expression, increases the secretion of chemoattractants, it causes accumulation of monocytes, proliferation of smooth muscle cells and foam cell formation, induces growth factor and collagen production and immunogenic - which contribute to the atherosclerotic lesion formation in vivo; (iii) in vivo administration of inhibitors which can block oxidative modification of LDL and subsequent pathologycal procecces can prevent
  • Oxidative modification of LDL requires the presence of redox active transition metals which iniciate and catalize oxidation of both lipid and protein moeities of LDL. Accumulation of redox active iron in the vasculature multiplies the damaging effect of reactive oxygen species.
  • Figure 1 shows chemical stucture of coprogen, ferrichrome, ferrichrysine and ferrirubin.
  • Desferricoprogen is a linear trihydroxamate; desferrichrome, desferrichrysine and desferrirubin are cyclic modified hexapeptides.
  • Figure 2 represents changes of dried cell mass, glucose and siderophore content of culture fluids during culture of Neurospora crassa.
  • Figure 3 shows the effect of L-Asp concentration and starting pH on the production of siderophore produced by Neurospora crassa.
  • Figure 4 shows the siderophore content of different mold-ripened food products.
  • Figure 5 represents correlation between intracellular HO-1 mRNA level (part A) and specific HO activity.
  • Figure 6 shows in vitro saturation of low density lipoprotein with desferri- and ferricoprogen.
  • Figure 7 demonstrates that desferri siderophores (20 ⁇ M) protect endothelial cells from oxidized LDL (200 ⁇ g/ml) mediated cytotoxicity.
  • Figure 8 represents correlation between intracellular HO-1 mRNA level (part A) and specific HO activity.
  • Figure 9 shows the levels of HO-1 mRNA in endothelial cells tretated with oxidized LDL in the presence of different desferri- or ferrisiderophores.
  • Figure 10 represents that desferricoprogen prevents heme mediated oxidation of lipid extract derived from atherosclerotic lesion.
  • Figure 11 shows that desferricoprogen delays heme mediated oxidation of atherosclerotic lesion.
  • Figure 12 demonstartes coprogen uptake of rat in case of oral administration of the drug.
  • Figure 13 demonstartes desferricoprogen uptake of rat in case of oral administration of the drug.
  • Figure 14 shows coprogen uptake of rat in case of intravenous administration of the drug.
  • Figure 15 shows desferricoprogen uptake of rat in case of intravenous administration of the drug.
  • Figure 16 represents secretion of coprogen and desferricoprogen into the urine and feces in a rat model.
  • Figure 17 demonstartes accumulation of desferricoprogen in the liver in case of oral administration of the drug in a rat model.
  • Figure 18 shows accumulation and secretion of desferricoprogen or coprogen in the liver and in intestinal epithelium in case of oral administration of the drugs in a rat model.
  • Figure 19 shows the effects of ethanol or oil on the accumulation of desferricoprogen in the liver in case of oral administration of the drug in a rat model.
  • the invention relates to the use of fungal siderophores in the treatment of vascular diseases primarily with endothelial cell origin. These compounds are applicable orally, which was not known and not expected from the prior art.
  • the invention relates to the specifically preferable production of fungal siderophores by using specifically isolated and selected species of fungi.
  • the invention also relates to foods, primarily meat products, preferably sausage and salami, which contain siderophores in controlled and efficient amount.
  • Meat products can be divided into three classes: dried products
  • the invention also provides a qualifying system to determine if different food products have preventive effect on the vasculature as described above.
  • Penicillium roquefortii Sid 4 NCAIM (P) F-001334 PenicHlium candidum ( Penicillium camemberti) Sid 5 NCAIM (P) F-001335.
  • the compounds used are the following:
  • R1 means hydrogen atom, Ci -6 alkyl group or Ci -4 hydroxy-alkyl group
  • R2 means hydrogen atom, Ci- 6 alkyl group, Ci -4 hydroxy-alkyl group or C-i- ⁇ alkanoyl group
  • R3, R4 and R5 mean Ci- ⁇ alkyl group or C 2 - 6 alkanoyl group substituted by one or two hydroxy or carboxyl.
  • R3, R4 and R5 groups are the following:
  • siderophores are produced by fermentation in which process any suitable culturing fluid and culturing conditions can be used.
  • Any suitable culturing fluid and culturing conditions can be used.
  • the presence of L-Asp and higher initial pH increase siderophore production.
  • Both ferri- and desferri-siderophores are produced during the fermentation.
  • Desferri form can be enriched by treatment of the ferri form with 8-hydroxyquinoline followed by an extraction with organic solvent e.g. dichlor- methane. n the experiments purified siderophores were used; the purification processes will be described in detail.
  • novel meat products contain one or more siderophores in a quantity which is certainly not harmful for humans. Incorporation of these compounds into the meat products can be achieved by different methods:
  • the special benefit of this invention is that it provides a method which is suitable to qualify food-, especially meat products. As it is possible to quantify siderophore content of any food products, it provides the possibility to qualify food products especially meat and dairy products from the health point of view.
  • the invention is described in details in the examples below, without limiting the protection on the processes and products shown below.
  • Fungal hexadentate siderophores (Fig. 1) were purified from culture fluids of Penicillium chrysogenum (coprogen, ferrichrome), Neurospora crassa (coprogen), Neovossia indica (ferrichrome), Aspergillus melleus (ferrichrysin) and A. ochraceus (ferrichrysin, ferrirubin) cultivated in defined low-iron minimal media (Charlang et al. 1981 , JaIaI et al. 1984, Leiter et al. 2001).
  • the purification schemes included Amberlite XAD-2, Kieselgur G and Bio-Gel P-2 liquid chromatographies and preparative HPLC on a Supelcosil-Si matrix (JaIaI & van der Helm 1991 , Leiter et al. 2001).
  • the purity of ferri-siderophores was checked by HPLC using a C-18 reversed phase column (Heymann et al. 1999, Hordt et al. 2000), and pure ferri-siderophores were deferrated using methanolic 8-hydroxyquinoline (Wong et al. 1983, Winkelmann 1993). Yields for desferricoprogen were 35 mg I "1 culture medium with P. chrysogenum and 66 mg I "1 culture medium with N. crassa.
  • the bacterial hexadentate siderophore desferrioxamine B which was used as a control in the same experiements, was purchased as Desferal® from Novartis (Basel, Switzerland).
  • Coprogen production of Neurospora crassa was optimalized, because coprogen is present in many mold-ripened food products, and it is a very promising inhibitor of in vitro LDL oxidation.
  • coprogen was produced in a 2 L flask containing 0.5 L of media which was inoculated with Neurospora crassa Sid1 NCAIM strain and cultured at 28 0 C with shaking at 250 storkes per minute for 5 days.
  • the composition of the culturing media as the following: 20 g/l glucose, 5 g/l L-Asp, 1 g/l K 2 HPO 4 3 H 2 O, 1 g/l MgSO 4 7 H 2 O, 0.5 g/l CaCI 2 2 H 2 O, 0.01 mg/l ZnSO 4 7 H 2 O and 25 ⁇ g/l biotin (pH 3.5).
  • Desferricoprogen - the iron free coprogen - was produced as described below. Coprogen dissolved in water was treated with methanolic 8- hydroxyquinoline at appropriate concentration and the mixture was stirred at 60 °C for 30 minutes. Fe 3+ - 8-hydroxyquinoline complex was then extracted completely with dichlormethane. The desferricoprogen containing aqueous phase was lyophilized, and stored in plastic vials closed hermetically at -20 0 C.
  • FIG. 2 shows changes in the dried cell mass (DCM) glucose and siderophore content of the growth media during culturing Neurospora crassa.
  • DCM dried cell mass
  • Figure 3 demonstartes the effect of L-Asp (panel A) and starting pH (panel B) on siderophore production of Neurospora crassa. Concentartion of L-Asp was changed between 2.5 g/L and 7.5 g/L, while the staring pH was kept at 3.5 (panel A). Secondly starting pH was changed between 3.5 and 6.5 while L-Asp concentration was unchanged (5 g/L). Siderophore production at pH 3.5 and L-Asp concentration of 5 g/L was considered to be 100 %.
  • Sid 5 NCAIM P
  • the main aim of this examination was to develop a reliable and easy way to use HPLC based technology which is suitable to determine the amount of siderophores in different food products.
  • the aim of the invention is to measure siderophore content of all mold-ripen meat and dairy products on the market, and to follow sideophore production in a sausage making technology.
  • Steps of the linear gradient were the followings: 0 min - water/acetinitrile
  • Coprogen content of the samples were measured by standard addition method using purified coprogen.
  • the amounts of other siderophores were measured by using relevant absorption coefficients found in the literature.
  • Figure 4 represents siderophore content of mold-ripened food products
  • B blue cheese (Roquefort-type)
  • C Camemebert cheese. It has been examined how microorganisms - introduced into the meat mixture with the starter cultures (e.g. Micrococcus, Staphylococcus, Lactobacillus, Debaryomyces hansenii) - influence siderophore production of molds in the covering culture for example through decreasing metabolizable iron. From this point of view starter yeasts are very important, because yeasts can grow quickly on additional carbon sources and survive the long making process and they are concentrated in the layer near to the surface (Encinas et al, 2000).
  • starter cultures e.g. Micrococcus, Staphylococcus, Lactobacillus, Debaryomyces hansenii
  • Sid 5 NCAIM P
  • Example 2 Purification of coprogen and other siderophores and analyze siderophore containing samples.
  • Optical density of elute was detected at 435, and 220 nm, and OD was measured at 580 nm as a reference. Siderophore peaks were identified by using relevant standards (HPLC Calibration kit - Coprogen and Fusarinines and HPLC Calibration kit - Ferrichromes; EMC Microcollections GmbH).
  • Coprogen content of the samples were measured by standard addition method using purified coprogen produced by Neurospora crassa.
  • the amounts of other siderophores were measured by using relevant absorption coefficients found in the literature.
  • Example 3 Results showing protective effects of siderophores against human low density lipoprotein oxidation and endothelial cell cytotoxicity LDL was isolated from plasma derived from EDTA (1 mg/mL)- anticoagulated venous blood taken from healthy overnight-fasted volunteers (Belcher et al. Arterioscler Thromb 13, 1779-1789 (1993), Ujhelyi et al. Clin Chem 44, 1762-1764 (1998)).
  • Density of plasma was adjusted to 1.3 g/mL with KBr, and a two-layer gradient was made in a Quick-Seal polyallomer ultracentrifuge tube (Beckman Instruments) by layering 0.9% NaCI on 10 ml of density adjusted plasma, which was then centrifuged at 302.000 x g for 3 h at 4 0 C (VTi 50.2 rotor, Beckman Instruments, Brea, CA, USA). Purity of the LDL fraction was checked by agarose gel electrophoresis. The LDL samples were kept at 4 0 C and protected from light, and the protein content was determined by the BCA protein assay (Pierce, Rockford, IL, USA).
  • FIG. 5 shows correlation between intracellular heme oxygenase-1 (HO-1) mRNA level (panel A) and specific HO activity (panel B).
  • Human umbilical vein endothelial cells (HUVECs) were treated with LDL solutions which were oxidized previously by heme and H 2 O 2 for 1 h in the standard reaction mixture supplemented with siderophores at a final concentration of 20 ⁇ M and then diluted to a final LDL concentration of 50 ⁇ g/ml.
  • Changes in HO-1 gene transcription was examined by Northern blot and HO-1 mRNA levels was quantified by videodensitometry.
  • Specific HO-1 activity was calculated from 3 independent experiments and expressed as mean ⁇ SD.
  • Figure 6 represents the saturation of LDL with desferricoprogen and coprogen in vitro. Symbols represent coprogen treatment (A), desferricoprogen treatment + FeC ⁇ added ( ⁇ ), desferricoprogen treatment without extra iron added ( ⁇ ), desferricoprogen content of LDL after desferricoprogen treatment (difference between coprogen levels with and without FeC ⁇ addition) ( ⁇ ).
  • Human umbilical vein endothelial cells were removed from human umbilical veins by exposure to dispase and cultured in medium 199 containing 15% fetal calf serum, penicillin (100 U ml '1 ), streptomycin (100 U ml " 1 ), and heparin (5 U ml *1 ) supplemented with L-glutamine, sodium pyruvate, and endothelial cell growth factor (BaIIa et al. 1993). Endothelial cells were identified by cell morphology and by the presence of von Willebrand factor.
  • HBSS Hank's balanced salt solution
  • the reduced MTT was measured spectrophotometrically at 570 nm after the formazan was dissolved in 100 ⁇ l of 10% SDS and 500 ⁇ l of hot isopropanol containing 20 mM HCl.
  • 20 ⁇ M desferoxamine B was used as a positive control (BaIIa et al. 1991).
  • LDL treated with heme and hydrogen peroxide was markedly cytotoxic whereas iron-free siderophores hindered the generation of cytotoxic LDL in a pattern quite similar to that observed in the kinetic analysis of LDL lipid peroxidation (Table 2).
  • the protective effect exerted by desferrichrysin was significant but less than those of the other chelators tested (Fig. 7).
  • Figure 7 demonstartes the protective effects of desferri-siderophores (20 ⁇ M) on endothelial cells exposed to oxidatively modified LDL (200 ⁇ g/ml). LDL was oxidized with the H 2 O 2 (75 ⁇ M)-heme (5 ⁇ M) system. Columns and bars represent means and S. E. values calculated from 3 independent experiments.
  • Heme oxygenase activity in endothelial cell microsomes was measured by bilirubin generation (BaIIa et al. 1993).
  • the induction of HO activity was determined in endothelial cells grown in 10-cm-diameter tissue culture dishes and treated with a reaction mixture containing LDL (50 mg/L), heme (1.25 ⁇ M), H 2 O 2 (18.75 ⁇ M) with or without the addition of iron-free or iron-saturated siderophores (5 ⁇ M) for 60 minutes followed by an 8-hour incubation with complete media alone.
  • Endothelial microsomes were incubated with hepatic cytosol (2 mg), hemin (20 ⁇ mol/L), glucose-6-phosphate dehydrogenase (0.2 units), and NADPH (0.8 mmol/L).
  • the formed bilirubin was extracted with chloroform and ⁇ optical density of 464 and 530 nm was measured (extinction coefficient 40 M "1 cm "1 ).
  • Heme oxygenase activity is expressed as pmol bilirubin formed/mg cell protein/60 minutes.
  • HO-1 mRNA content was analyzed in confluent HUVECs incubated with control medium or LDL test solutions as described above for the measurement of enzyme activity.
  • RNAzol TEL-TEST, Friendswood, TX, USA
  • 20 ⁇ g quantities of total RNA were run on agarose gels and transferred to nylon membrane.
  • the 28S and 18S ribosomal RNAs, and equal loading of samples were checked by ethidium bromide staining.
  • 2 ⁇ g quantities of total RNA were subjected to dot blot analysis.
  • RNAs were hybridized with biotin- labeled cDNA for HO-1 (Bioprime DNA Labeling System, Life Technologies) (Jeney et al. 2002), and the HO-1 -active bands and dots were visualized by a chemiluminescent detection system (Photogene System 2.0, Life Technologies). Autoradiographs were quantified by computer-assisted videodensitometry.
  • HO-1 mRNA levels Part A
  • Part B specific HO activities
  • HUVECs were treated with a series of LDL-containing reaction mixtures, which were also supplemented with 20 ⁇ M desferricoprogen or coprogen as indicated.
  • changes in the transcription of the HO-1 gene were analyzed by Northen blot, and mRNA concentrations were quantified by videodensitometry.
  • Specific HO activities are shown as means ⁇ S. E. calculated from 3 independent experiments.
  • Figure 9 represents dot blot analysis of the changes in the gene expression levels of HO-1 in the presence of desferri- and ferri-siderophores.
  • A(4-5) positive control, up-regulation of HO-1 after LDL + heme + H 2 O 2 - treatment of endothelial cells.
  • B(1-5) protection of HUVECs with 20 ⁇ M desferrioxamine B, desferricoprogen, desferrirubin, desferrichrysin and desferrichrome.
  • C(1-5) supplementation of reaction mixtures with 20 ⁇ M iron saturated ferrioxamine, coprogen, ferrirubin, ferrichrysin and ferrichrome.
  • oxidative modification of LDL is a key event in the development of atherosclerotic lesions. Lipid core of the atheroma is as sensitive for oxidative modification as LDL. Desferrioxamine inhibits heme mediated oxidation of lipids derived from atherosclerotic plaques. We examined whether desferricoprogen inhibits heme mediated oxidation of plaque lipids as well. Heme mediated oxidation of lipids originated from atherosclerotic soft plaques takes place about 12-18 hours, which time was increased dose dependently by desferricoprogen up to 24 hours as demonstrated on figure 10.
  • Figure 10 represents that desferricoprogen prevents heme mediated oxidation of atherosclerotic plaques.
  • Small pieces of atherosclerotic vessel wall samples were treated with heme (10 ⁇ M) in the presence or absence of desferricoprogen (25 or 50 ⁇ M) and incubated for 24 hours at 37 °C, then TBARS were determined as described.
  • Figure shows the average and S.D. of 5 independent experiments.
  • Figure 11 shows that desferricoprogen delays heme mediated oxidation of atherosclerotic plaques.
  • Neurospora crassa is a suitable organism to produce desferricoprogen in laboratory scale (up to 1 gram). Industrial scale production is achievable by using the deposited fungi strains listed in Exmple 1;
  • Figure 12-15 show that 90 % of desferricoprogen administered orally was taken up. About 5% of the administered amount is eliminated through the urine within 6 days ( Figure 13). In contrast, 75% of ferricoprogen was taken up if administered orally, and 1.5 % is excreted through the urine in 6 days ( Figure 12). Administered intravenously 10 % of desferricoprogen and 75% of coprogen is excreted through the urine within 6 days following treatment ( Figure 14,15). In the case of intravenous administration about 5% of desferricoprogen eliminated in the feces, and about 1.5% in the case of ferricoprogen.
  • Figure 12 Ferricoprogen, administered orally, 100 mg/kg body weight days 1 2 3 6
  • Figure 13 Desferricoprogen, administered orally, 100 mg/kg body weight days 1 2 3 6
  • Figure 14 Ferricoprogen, administered intravenously, 50 mg/kg body weight days 1 2 3 6
  • FIG. 15 Desferricoprogen, administered intravenously, 50 mg/kg body weight days 1 2 3 6
  • Figure 16 shows secretion of coprogen and desferricoprogen into the urine (U) and feces (F) in a rat model. Animals were administered with the drug either orally (100 mg/kg body weight) or intravenously (50 mg/kg body weight); data are expressed as % of administered coprogen.
  • Substantial amount (21.0 %) of orally administered coprogen is secreted in the feces within 24 hours while 0.7 % is excreted through the urine. After the first 24 hours secretion of sideophores is persisted in small amount until the third day. Evidence of the intestinal uptake of iron complexed coprogen is its presence in the urine. Substantial amount of administered coprogen (76%) is not secreted in either the feces or the urine, which suggests that coprogen is taken up and stored and/or metabolized by the intestinal flora.
  • Main part (70.1 %) of the intravenously administered coprogen is eliminated from the body within two days through the urine, but secretion of the iron-complexed form takes longer time, it is secreted on the 6 th day after administration.
  • the compound shows up in the feces on the first day (1.5 %) after administration, but it is not detectable further on. About 20 % of the compound does not appear in either the feces or the urine.
  • only small proportion (6 %) of desferricoprogen administered orally shows up in the feces on the first day, but it is still detectable on the 3 rd day.
  • the compound is continuously secreted in its iron complexed form in the urine (0.2-3.1 % of the administered amount /day).
  • Desferricoprogen administered intarvenously shows up in its iron complexed form in both urine (3.3%) and feces (4%) on the first day, and the secretion is continouing until the 6 th day through both ways.
  • About 86 % of the administered desferricoprogen is not secreted in either the feces or the urine, which suggests that it is taken up and stored and/or metabolized by the intestinal flora.
  • Importantly only negligible amount of siderophore was detected in biological samples which were chelated mostly with iron in case of desferricoprogen administration.
  • the secretion of the desferri form can not be excluded but its amount remained under the detection limit.
  • Accumulation of the coprogen in the liver is shown by the figure 17 in case of oral administration (100mg/kg body weight) of desferricoprogen in rat model in terms of the percentage of the administered coprogen.
  • the siderophore can be detected in the plasma. Absorption of the siderophore is independent from fat or oil content of the foods and alcohol has no effect on the absorption either.
  • the product of the siderophore metabolism is not known, but the tetradentate dimerumic acid which can be formed by ring-opening of the siderophore can be excluded.
  • the metabolite can be detected in feces which mean that it is secreted through bile.
  • Intravenously administered coprogen is mostly accumulated in the liver (low amount of coprogene accumulation was observed in the spleen) and form iron-coprogen complex (65-81%).
  • the siderophores are secreted in the kidney.
  • This secretion mechanism can be feasible in case of oral administration as well.
  • Microorganisms are generally used for the production of various meat products such as dried sausages, cured hams and mold coated salamis. At the beginning their spontaneous reproduction developed the sensory properties of the meat products.
  • meat products such as dried sausages, cured hams and mold coated salamis.
  • their spontaneous reproduction developed the sensory properties of the meat products.
  • the usage of microorganism in meat industry is conscious and well controlled.
  • the essence of the controlled procedure is the addition of selectively cultured molds into the cured meat products.
  • the effects of the microorganisms are well controlled by adding different kinds of carbohydrates (nutriment of the molds) or changing the conditions of the ripening room (temperature, humidity etc.).
  • Pasta making The measured amounts of typical ingredients of the meat products such as meats, bacons, salt, and spices are thoroughly mixed and ground by an industrial mincer.
  • the temperature of the used meats is typically between -2 to - 5 0 C.
  • the temperature of the bacons is typically between -5 to -7 0 C.
  • Stuffing The mixed meat-mix which contains raw materials such as meats, bacons, spices, starter culture and essential nutriments for the microorganisms - glucose, sacharose, lactose - is stuffed into casing material.
  • Casing materials can be either natural - such as gastrointestinal tracts of cattle, sheep or hog - or artificial cellulose-, collagene- or linen-based. In case of mold coated products it is practical to use collagene-based casing materials.
  • Curing has several steps.
  • the aim of the first step is to multiply microorganisms in the starter culture. Degradation of nitrites and nitrates, lactose production, decreasing pH, dehydration, blocking of harmful microorganisms, development of taste and consistency of the meat products happen in the first step.
  • the temperature is 20-24 0 C, relative humidity is 88-96 % and intensive air circulation with adding fresh air are required.
  • Variable conditions are required for controlling the development of mold coat and its thickness during the third step.
  • the aim of the fourth step is to decrease water content of the salamis (water activity should below 0.91) by drying. In this step 15-17 0 C, 80-88% relative humidity and variable air circulation are required. Quality control:
  • the quality control of the salamis is an obligatory step prior to sale. Every batch is tested for pathogenic microbes and chemical tests are performed to determine water, fat, salt, and nitrate contents and their ratios are also calculated. The outlook, color, odor and taste properties of the products are also tested by senses. The correct parameters of these tests are controlled by the Hungarian Food Law and the Hungarian Food Book. Only such products can be packed and saled which meet all testing parameters.
  • Raw materials are: cattle meat, hog meat, industrial bacon, fat-back.
  • Additives are: sodium nitrite/nitrate mixture, spices, sodium-ascorbate, mono- and complex carbohydrates.

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Abstract

L'invention se rapporte à l'utilisation de sidérophores fongiques dans le traitement des maladies vasculaires essentiellement causées par les cellules de l'endothélium, les composants selon ladite invention convenant particulièrement à une administration par voie orale. L'invention concerne également une voie de production privilégiée de sidérophores fongiques par l'utilisation d'espèces fongiques spécifiquement isolées et sélectionnées, ainsi que des aliments, et de préférence des produits carnés, en particulier la saucisse et le salami, qui contiennent des sidérophores en quantité efficace et contrôlée. L'invention se rapporte de plus à un système d'évaluation permettant de déterminer si différents aliments ont un effet préventif sur le système vasculaire, comme décrit ci-dessus.
PCT/HU2007/000076 2006-08-16 2007-08-16 Utilisation de sidérophores dans la prévention des maladies vasculaires essentiellement causées par un dysfonctionnement des cellules de l'endothélium, production de sidérophores et évaluation des produits carnés contenant des sidérophores Ceased WO2008020260A2 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
HU0600653A HU0600653D0 (en) 2006-08-16 2006-08-16 Process for producing siderofor and siderofor-laden meat-product and use thereof
HUP0600653 2006-08-16
HU0700535A HUP0700535A2 (en) 2007-08-14 2007-08-14 Siderofor use for treatment vascular system, process for producing siderofor, and meat product that contain siderofor and qualification thereof
HUP0700535 2007-08-14

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8241872B2 (en) 2008-03-14 2012-08-14 Astellas Pharma Inc. Microorganism producing cyclic compound
WO2013034911A1 (fr) * 2011-09-06 2013-03-14 Ip Science Limited Produits et méthodes
US8598115B2 (en) 2008-03-14 2013-12-03 Astellas Pharma Inc. Cyclic compound and salt thereof
CN105018352A (zh) * 2015-07-23 2015-11-04 云南大学 一种产曲酸真菌菌株及制备方法
CN114958946A (zh) * 2022-03-09 2022-08-30 山西勰成生物科技有限公司 一种发酵型红枣螯合肽铁的制备方法

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BE628495A (fr) * 1962-02-16
US3342795A (en) * 1962-03-02 1967-09-19 Ciba Geigy Corp Ferrichrysin, desferrichrysin, and derivatives thereof
CH437639A (de) * 1962-04-05 1967-06-15 Ciba Geigy Verfahren zur Herstellung von Coprogen und Desferricoprogen
US5573800A (en) * 1989-02-21 1996-11-12 Viskase Corporation Antimicrobial composition for surface treatment of foodstuffs
US7595073B2 (en) * 2003-02-28 2009-09-29 Kraft Foods Global Brands Llc Use of siderophores and organic acids to retard lipid oxidation
WO2005067970A1 (fr) * 2004-01-14 2005-07-28 Gekkeikan Sake Co., Ltd. Complement de fer et son utilisation

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8241872B2 (en) 2008-03-14 2012-08-14 Astellas Pharma Inc. Microorganism producing cyclic compound
US8598115B2 (en) 2008-03-14 2013-12-03 Astellas Pharma Inc. Cyclic compound and salt thereof
WO2013034911A1 (fr) * 2011-09-06 2013-03-14 Ip Science Limited Produits et méthodes
CN103929981A (zh) * 2011-09-06 2014-07-16 Ip科技有限公司 产品和方法
JP2014531418A (ja) * 2011-09-06 2014-11-27 アイピー サイエンス リミテッド 生成物および方法
US20140370047A1 (en) * 2011-09-06 2014-12-18 Ip Science Limited Products and methods
AU2012306078B2 (en) * 2011-09-06 2016-03-31 Ip Science Limited Products and methods
US10052355B2 (en) 2011-09-06 2018-08-21 Ip Science Limited Products and methods
JP2018154638A (ja) * 2011-09-06 2018-10-04 アイピー サイエンス リミテッド 生成物および方法
CN105018352A (zh) * 2015-07-23 2015-11-04 云南大学 一种产曲酸真菌菌株及制备方法
CN114958946A (zh) * 2022-03-09 2022-08-30 山西勰成生物科技有限公司 一种发酵型红枣螯合肽铁的制备方法
CN114958946B (zh) * 2022-03-09 2024-03-26 山西勰成生物科技有限公司 一种发酵型红枣螯合肽铁的制备方法

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