WO2008020260A2 - Use of siderophores in prevention of vascular diseases, production of siderophores and qualifying siderophore containing meat-products - Google Patents

Abstract

The invention relates to the use of fungal siderophores in the treatment of vascular diseases primarily caused by endothelial cell origin, which compounds are especially useful for oral application. Furthermore, the invention relates to a preferred route of production of fungal siderophores by using specifically isolated and selected species of fungi, 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.

Description

Use of siderophores in prevention of vascular diseases caused primarily by endothelial cell dysfunction, production of siderophores and qualifying siderophore containing meat-products

TECHNICAL FIELD

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.

Furthermore, 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.

BACKGROUND ART

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,

Amsterdam 1997.]). 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,

Aspergillus melleus.

Moreover, 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

Aspergillus spp., and Aspergillus fumigatus (Howard, 1999).

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, Leύ^' 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).

The effects of these compounds were shown in vitro systems or were administered parentally. There is no evidence in the literature about absorption of these siderophores. Moreover, many papers describe that absorption of siderophores with a molecular weight higher than 400 Da, have limited or no absorption at all. John B. Porter (Acta Haematol. 1996; 95, 13-25) described that intestinal absorption of molecules with molecular weight above 400 is unlikely, therefore clinical use of hexadentate siderophores is questionable, only a few or none can be absorbed efficiently enough to be used clinically. D.G. Maxton et al described (Clinical Science (1986) 71 ; 71-80) that intestinal permeability of polymers with a molecular weight around 500 Da is hardly detectable. R.W. Grady et al compared (The J. of Pharmacology and Experimental Therapeutics, Vol. 196 (2), 479-485) iron chelating abilities and in vivo iron secreting effects of different hydroxamates. They administered rhodotoluic acid, which is a tetradentate siderophore with a molecular weight of 344 Da or desferoxamine which has a molecular weight of about 500 parentally and measured the amount of excreted iron through the kidneys and the feces secretion system. They found that iron excreting efficiency of rhodotoluic acid is more than twice than desferoxamine.

R. Minquin et al. showed (Free Radical Biology & Medicine 38 (2005), 1206-1211) that desferrioxamine B inhibited atherosclerotic lesion formation in rabbits on cholesterol rich diet, and reduced iron content of plaques. Administration of desferrioxamine B was performed subcoutaneously, as the authors described that the drug is not absorbed through the intestine.

In contrast with the literature described above we found that fungal hexadentate siderophores are absorbed efficienty if administered orally obtaining a simple and efficient tool to treat vascular disorders which caused by elevated iron level. Particulary beneficial the oral administration of siderophores with a molecular weight of 600 Da, and even better the use of siderophores whose molecular weight is higher than 650 Da. The present investigations were carried out to test whether fungal siderophores might suppress heme-catalyzed low density lipoprotein (LDL) oxidation. We found that desferri siderophores play an important role in preventing vascular disorders particularly when administered orally. We have shown that intestinal uptake of desferri siderophores are satisfactory or in some cases excellent.

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. Plasma hemoglobin when oxidized fosters the transfer of heme moieties to endothelial cells of the vessel wall and to LDL, and sensitizes endothelial cells against oxidative damage. Endothelial cells respond to an influx of heme and oxidative stress by upregulating both heme oxygenase-1 and ferritin. Heme oxygenase-1 cleaves the porphyrin ring producing biliverdin, carbon monoxide and free redox active iron. Ferritin serves as a safe storage site for the released iron. Ferritin is cytoprotective because its antioxidant, antiapoptotic and antiproliferative effects. It has been shown that this system efficiently protects endothelial cells from the damage mediated by exogen heme and reactive oxygen species. The central importance of heme oxygenase-1 based cytoprotection was highlighted by the discovery of a child with HO-1 deficiency that had extensive endothelial cell damage.

Heme is absolutely required for aerobic life. However free heme can be quite cytotoxic, particularly in the presence of oxidants or activated phagocytes. Of all sites in the body, 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. Here, we present a brief review of the nature of heme-mediated cytotoxicity and of strategies by which normal endothelium manages to protect itself from this clear and present danger.

Damage caused by reactive oxygen species can be greatly amplified by 'free' redox active iron (Halliwell et al, Biochem. J.: 1984, 219, 1-14]). For example, 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). Conversely, depletion of cellular iron powerfully protects eukaryotic and prokaryotic cells against oxidant challenge (Gannon, D. et al, Lab. Invest.: 1987, 57, 37-44). We have shown that one critical feature of highly damaging iron to endothelium is permeation of the metal into cells. Chelation of iron by certain lipophilic chelators, such as 8-hydroxyquinoline, results in the accumulation of catalytically active lipophilic iron chelates in endothelial lipid compartments; endothelium pretreated with 8-hydroxyquinoline-iron chelate was exquisitely sensitive to both endogenous and exogenous oxidant stress (Bally, G. et al, J. Lab. Clin. Med.: 1990, 116, 546-554).

One abundant source of potentially toxic iron is heme with hydrophobic property. 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 toxicity of free heme derives from the ease with which this highly hydrophobic compound can enter and cross cell membranes, therefore readily concentrates within the hydrophobic milieu of intact cells (BaIIa G. et al see above). Both in vitro and in vivo, cells will accumulate exogenous heme and synergistically amplify the cytotoxic effects of oxidants of reagent, enzymatic, or cellular origin. Heme uptake by endothelial cells can exacerbate their damage by polymorphonuclear leukocytes (PMNs) - cells that tend to marginate along endothelial surfaces in the presence of diverse inflammatory mediators (BaIIa G.et al, Lab. Invest.: 1991 , 64, 648-655; BaIIa₁ J. et al, Blood: 2000, 95, 3445- 3450). Intriguingly, heme was shown by Graca-Souza et al to induce PMN activation as well (Blood: 2002, 99, 4160-4165). Moreover Wagner et al (Proc. Soc. Exp. Biol. Med.: 1997, 216, 456-463; Blood: 2001, 98, 1802-1811) revealed that heme can enhance endothelial cell adhesion molecule expression which regulates PMN adhesion and provokes inflammation.

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. In either case, 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).

We asked: could heme sensitize endothelial cells to oxidative challenge in the presence of plasma (BaIIa J. et al see above)? After all, plasma is enriched with binding proteins, such as albumin and hemopexin, known to inhibit heme-mediated cell damage. Exposure of endothelium to heme in the presence of whole human plasma synergizes cellular oxidant damage for added oxidants, with optimal heme-exposure duration of 60 minutes. Intriguingly, cytotoxicity studies showed little added toxicity to endothelium if water solubility of heme is conferred associatively with the arginate counterion (heme arginate). Even with efficient permeation heme arginate does not amplify oxidant-induced cytotoxicity. In support, exposure of endothelium to heme arginate in plasma free medium increases endothelial cell heme content to an extent similar to what is observed after heme treatment. Comparable heme uptake can be obtained in the presence of human plasma although at 2 orders of magnitude greater concentration for both heme arginate and heme.

The 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. On the contrary, if water solubility of heme is conferred associatively with the arginate counterion or the vinyl side chains of heme are substituted by sulfonate, propionate, or glycol leading to hydrophilic ferriporphyrins (iron deuteroporphyrin IX,2,4-bis-sulfonate, iron coproporphyrin III, and iron deuteroporphyrin IX,2,4-bis-glycol), these ferriporphyrins failed to sensitize cells to oxidants or activated polymorphonuclear leukocytes.

Although free heme is rapidly incorporated into hydrophobic domains of cells and serves as a source of highly damaging iron, the question remains as to whether intact heme liganded to proteins, as in hemoglobin, might also transfer heme to vascular endothelium. Whereas reduced (ferro- or oxy-) hemoglobin is relatively innocuous to endothelial cells, oxidized (ferri- or met-) hemoglobin greatly amplifies oxidant mediated endothelial injury (BaIIa, J. et al, Proc. Natl. Acad. Sci. USA: 1993, 90, 9285-9289; BaIIa J. et al, Trans. Assoc. Am. Physicians: 1992, 105, 1-6). This is because 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. et al, Proc. Natl. Acad. Sci. USA: 1993, 90, 9285-9289; Weiss, S. J. Biol. Chem. 1982, 257, 2947-2953; Dallegri, F. et al, Blood: 1987, 70, 1743- 1749). For instance, polymorphonuclear leukocytes, when activated with the phorbol ester PMA, markedly oxidize ferrohemoglobin to ferrihemoglobin within 30 min (BaIIa, J. et al, Proc. Natl. Acad. Sci. USA: 1993, 90). Accordingly, 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. 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). If metheme binding to globin is strengthened by haptoglobin or if released heme is religanded to hemopexin, 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.

The importance of heme release from ferrihemoglobin in such toxicity is emphasized by the fact that 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. At higher concentrations of free methemoglobin in plasma (such as might occur in certain hemolytic diseases, atherosclerosis, and malaria infections) the normal mechanisms for control of hemoglobin (haptoglobin/hemopexin) can be overwhelmed and released heme will enter the endothelial cells. These previous studies and those who revealed that hemoglobin behaves as a biologic Fenton reagent (Sadzareh, S. M. et al, J. Biol. Chem: 1984, 259, 14354-14356; Sadzareh, S.M. et al, J. Clin. Invest,: 1988, 82, 1510- 1515) made us wonder whether hemoglobin in plasma could provide heme-iron to endothelium in vivo. We demonstrated that oxyhemoglobin does not serve as a source of damaging heme-iron to endothelium. In contrast, 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. Invest: 1991 , 64, 648-655; Gutterige et al, Biochem. J.: 1988, 256, 861- 865; Eskew, J. et al, J. Biol. Chem.: 1999, 274, 638-648), and albumin can be overwhelmed and the delivery of heme-iron to the endothelium occurs in vivo (BaIIa J. et al, Am. J. Phisiol.: 1995, 268, 321-327).

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. Increasing evidence has revealed that 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 or slower the progression of vascular damage. 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.

Oxidative modification of LDL and subsequent reactions play roles in the pathomechanism of vasculitis (Lee HS.: Diabetes Res Clin Pract. 1999 Sep;45(2-3): 117-22.). Oxidation of LDL and consequent foam cell formation occurs in the case of focal segmental glomerulosclerosis (FSGS), which leads to scar formation in the vessels. Intravascular LDL oxidation leads to microangiopathic haemolytic anemia along with severe endothelial damage (Jeney V. et al, Blood. 2002 Aug 1 ;100(3):879-87). FIGURES

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 μM) protect endothelial cells from oxidized LDL (200 μg/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.

DISCLOSURE OF THE INVENTION

Based on the results demonstrated above 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.

Moreover, 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

(sausages, salami), red meat products and cold meat products. The invention also provides a qualifying system to determine if different food products have preventive effect on the vasculature as described above.

Strains of fungi with excellent siderophore producing ability have been isolated. The following fungi strains were deposited at the National Collection of

Industrial and Agricultural Microorganisms (Corvinus University, Budapest) on 2^nd of august in 2006 with the following labels:

Neurospora crassa Sid 1 NCAIM (P) F-001331

Penicillium chrysogenum Sid2 NCAIM (P) F-O01332

Penicillium nalgiovense Sid3 NCAIM (P) F-001333

Penicillium roquefortii Sid 4 NCAIM (P) F-001334 PenicHlium candidum (= Penicillium camemberti) Sid 5 NCAIM (P) F-001335.

Based on the invention we use desferri siderophores to produce oral drugs for treatment and/or prevention of vascular disorders.

The compounds used are the following:

desferricoprogen group

desferrichrome group

desferri-triacetyl fuzarinin C

In the structures above:

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-β alkanoyl group,

R3, R4 and R5 mean Ci-β alkyl group or C₂-₆ alkanoyl group substituted by one or two hydroxy or carboxyl.

Favourable are those compounds which are substituted as described below:

The meanings of R3, R4 and R5 groups are the following:

The table above shows the preferred siderophores, among them the most preferable are desferricoprogen, desferrichrome, desferrichrysin and desferrirubin. The production, purification and biological effects of siderophores used in the invention are discussed more in details.

According to the invention, siderophores are produced by fermentation in which process 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.

It has been found that siderophores can be taken up if administered orally, therefore administration of siderophores achievable in a natural and controlled way using food products. We found that meat products, especially sausages and salamis are suitable for this purpose. It is well-known that consumption of these kinds of meat products - primarily because of their high fat content - increase the risk of vascular disorders according to the conventional nutritional principles. In contrast thereto, the invention provides meat products with controlled siderophore content, whose consumption not only neutralize damaging effects of the meat product itself, but it is suitable to intake siderophores - instead of administration of pills - which can provide further protection for the vasculature.

The 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:

- mixing the isolated siderophores directly into the meat mixture during the known producing technology,

- mixing the siderophore producing fungi directly into the meat mixture during the known producing technology, or

- applying the siderophore producing microorganisms directly onto the outer surface of the semi-ready product or enrich siderophore producing microorganisms in the microflora in the last ripening step of the production. 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.

BEST MODE OF CARRYING OUT THE INVENTION

Example 1 : Production of fungal siderophores on laboratory scale

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.

Based on the literature 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 ⁰C 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 K₂HPO₄ 3 H₂O, 1 g/l MgSO₄ 7 H₂O, 0.5 g/l CaCI₂ 2 H₂O, 0.01 mg/l ZnSO₄ 7 H₂O and 25 μg/l biotin (pH 3.5). After 5 days culturing mycelium was strained through a glass filter, then filtrate was treated with FeCI₃ at the concentration of 1 g/L, pH was adjusted to 6.5 with solid NaOH, and the filtrate was centrifuged (1000 RPM, 20 min, 4 °C). Siderophore content of the supernatant was bound to a XAD-2 coloumn (250 mm * 50 mm, Supelco, Bellefonte, USA), then eluted with methanol. Coprogen was further purified from this organic solution on a silicagel coloumn (250 mm x 16 mm, Merck, Darmstadt, Germany) and was eluted with a mixture of chloroform:methanol:water=35:12:2. In the final purification step coprogen was separated from other siderophores by semi preparative HPLC using a Hewlett- Packard Series Il HPLC which is equipped with diode array detector (DAD), autosample, and ChemStation software. Separation was carried out on a Supelcosil SPLC silicagel coloumn (205 mm x 10 mm, 5 μm-es pore size). Mobil phase was a mixture of trichlormethane:methanol:water 35:12:2, and the flow rate was 3 mL/min. Optical density of the eluent was monitored at 440 nm using 580 nm as a reference wavelength. Coprogen containg samples were collected (R_f=11 ,6-12,4 min) and the solvent was evaporated.

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. Fe³⁺ - 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 ⁰C.

Because coprogen will play a crucial role in the following physiologycal and pharmacokinetic studies we further examined the effect of incubation time, L-Asp concentration and starting pH on the coprogen production.

As shown by figure 2 high yield siderophore production started after glucose is consumed in the autolysing and sporulating cultures. Based on this result coprogen fermentation requires longer culturing time (e.g. 7 days instead of 5). Figure 2 shows changes in the dried cell mass (DCM) glucose and siderophore content of the growth media during culturing Neurospora crassa. In order to measure siderophore content of the growth media mycelium was strained out through a glass filter, then filtrate was treated with FeCI₃ at the concentration of 1 g/L, pH was adjusted to 6.5 with solid NaOH, the filtrate was centrifuged (1000 RPM, 20 min, 4 °C) and the optical density was measured at 440 nm.

As shown on figure 3 increase both starting L-Asp concentration and pH favour siderophore production, therefore these parameters will be increased in the future in coprogen fermentation. 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 %.

The procedure above was used to produce siderophores by using the following fungi strains: Neurospora crassa Sid 1 NCAIM (P) F-001331 Penicillium chrysogenum Sid2 NCAIM (P) F-001332 Penicillium nalgiovense Sid3 NCAIM (P) F-001333 Penicillium roquefortii Sid 4 NCAIM (P) F-001334

Penicillium candidum (= Penicillium camemberti) Sid 5 NCAIM (P) F-001335. Siderophore production of molds used to cheese ripening is well known for decades (Ong and Neilands, 1979), but siderophore content of mold- ripened foods is not examined in detail so far.

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.

Siderophores were isolated from 6 different blue cheese (Roquefort-type) and 6 different camembert cheese. In all cases 25 g of cheese was homogenized with quartz sand in the presence of 27 ml of destilled water and 3 ml of FeCI₃ solution (6 mmol/L). Homogenate was centrifuged (10.000 RPM, 20 min, 4 °C), and the aqueous phase was separated and lyophilized. Lyophilizates were resolved in 10-20 ml of 50% methanol and centrifuged (10.000 RPM, 10 min, 4 °C). Siderophore content of the supernatant was determined by HPLC on a reversed phase coloumn (Spherisorb ODS2,

250x4.6 mm, 5 μm pore size) using gradient elution with a flow rate of 1 mi/min.

Steps of the linear gradient were the followings: 0 min - water/acetinitrile

= 6/94; 10 min - water/acetinitrile =20/80; 15 min - water/acetinitrile =15/85; and 16 min - acetonitrile 100%. 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. The amounts of other siderophores were measured by using relevant absorption coefficients found in the literature.

Results are summarized on Figure 4. We found high amount of coprogen in two products which contained 38 or 23 mg/kg coprogen. Among the 15 examined products there were 12 which had siderophore content higher than 10 mg/kg and in some cases sum of the siderophore content went up to around 100 mg/kg. Beside coprogen these products contained mainly neocoprogen I and II, trialcetylfusarinine, ferrichrome, ferrichrocine or ferrirubin. In normal conditions about 50-66% of the sum siderophore content exists in iron free form. These results suggest thet mold-ripen food products may contain significant amount of siderophore, therefore phisiologycal effects on the human body of these compounds should be examined in detail.

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). It is also interesting that the use of cocultures of Penicillium- Penicillium and Penicillium-Geotrichum in the curing process increase siderophore production. Siderophore production of the fungi strains listed below was examined under the following conditions: 2 w/v % glucose, 2 w/v % L-Asp, 0.1 w/v % K₂HPO₄ 3H₂O, 0.1 w/v % MgSO₄ 7H₂O, 0.05 w/v % CaCI₂ 2H₂O, 0.002 w/v % ZnSO₄ 7H₂O and 25 Dg/I biotin, pH 7.0 (27 ⁰C; 250 rpm; 7 days). To culture P rockfortii and P. camembertii 2 w/v % malt-extract was used instead of glucose. Neurospora crassa Sid 1 NCAIM (P) F-001331 Penicillium chrysogenum Sid2 NCAIM (P) F-001332 Penicillium nalgiovense Sid3 NCAIM (P) F-001333 Penicillium roquefortii Sid 4 NCAIM (P) F-001334

Penicillium candidum (= Penicillium camemberti) Sid 5 NCAIM (P) F-001335 Aspergillus nidulans FGSC A116

Example 2: Purification of coprogen and other siderophores and analyze siderophore containing samples.

Siderophores were purified from culture fluids of Penicillium chrysogenum and Neurospora crassa cultivated in defined low-iron minimal media (JaIaI et al, pp. 235-269, G. Winkelmann (Ed): CRC Handbook of Microbial Iron Chelators, CRC Press, Boca Raton 1991). The purification schemes included Amberlite XAD-2, Kieselgur G and Bio-Gel P-2 liquid chromatographies and preparative HPLC on a Supelcosil-Si matrix as described previously. The purity of ferri-siderophores was checked by HPLC using a C-18 reversed phase column (Hδrdt et al. BioMetals 13, 37-46; 2000). Pure ferri-siderophores were deferrated using methanolic 8-hydroxyquinoline (Winkelmann, G., pp 219-239, Barton, B.C. Hemming (Eds): Iron Chelation in Plants and Soil Microorganisms, Academic Press, San Diego; 1993). Yields for desferricoprogen were 35 and 66 mg/L culture medium with P. chrysogenum and N. crassa respectively.

Siderophore containing samples were analized as described below: Preparation of sample: liquid samples or aqueous extract of solid samples were lyophilized and resolved in 50 v/v% methanol, then centrifuged (6000 rpm, 10 min, 4⁰C). The supernatant was analized by HPLC. HPLC: Siderophore content of the supernatant was determined by HPLC on a reversed phase coloumn (Spherisorb ODS2, 250x4.6 mm, 5 μm pore size). Twenty Dl of sample was injected directly onto the coloumnand gradient elution was performed with a floe rate of 1 ml/min. Steps of the linear gradient were the followings: 0 min: water/acetonitrile = 6/94 10 min: water/acetonitrile = 20/80

15 min: water/acetonitrile = 25/85

16 min: acetonitrile 100%.

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 4⁰C (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 ⁰C and protected from light, and the protein content was determined by the BCA protein assay (Pierce, Rockford, IL, USA).

Heme-mediated oxidation of LDL (accumulation of conjugated dienes) and of heme itself was monitored spectrophotometrically at 234 and 405 nm, respectively. Reaction mixtures contained LDL (200 mg/L protein), heme (5 μM), hydrogen peroxide (75 μM) and HEPES buffer (10 mM, pH 7.4) (BaIIa et al. 1991 , Belcher et al. 1993, Ujhelyi et al. 1998). In heme-catalyzed oxidation of LDL, heme degradation occurs in concert with formation of lipid oxidation products including conjugated dienes and lipid hydroperoxides. Thus, heme degradation reflects the progress of lipid peroxidation. The kinetics of heme disappearance was monitored at 405 nm in an automated microplate reader (model EL340, Bio-Tek Instruments, Winooski, VT, USA). LDL oxidation was monitored by the time (ΔT) required for the process to achieve maximum velocity (V_max) of heme degradation in minutes. Reaction mixtures were supplemented as indicated with desferri-siderophores at concentrations of 5, 10 and 25 μM.

As shown on figure 5 neither iron-free nor iron-saturated fungal chelators induced LDL lipid peroxidation alone. However, in the presence of heme the iron-free siderophores inhibited the formation of conjugated dienes (Fig. 5). In one representative experiment where LDL was incubated with 5 μM heme at 37⁰C, and the oxidation process was accelerated by the addition of 75 μM H₂O₂, desferricoprogen inhibited the conjugated diene generation by prolonging the initiation phase of the lipid peroxidation (Fig. 5). In contrast, under the same conditions iron-saturated coprogen had no effect on the peroxidatic reaction. Simultaneous measurements of heme degradation (followed at 405 nm) yielded similar results.

Figure 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 H₂O₂ for 1 h in the standard reaction mixture supplemented with siderophores at a final concentration of 20 μM and then diluted to a final LDL concentration of 50 μg/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. As shown in Table 1 , heme catalyzed oxidation of LDL was inhibited by desferricoprogen in a dose-dependent manner. Desferricoprogen added to the LDL-heme-H2θ2 reaction buffer at 10 μM final concentration prolonged the ΔT at Vmax value from 57+1 minutes to 109+1 minutes, resulting in a 91% increase in the resistance of LDL against heme catalyzed oxidation. The inhibition of peroxidation by desferricoprogen was compared to those of other iron-free fungal hexadentate chelators and to that of the bacterial hexadentate desferrioxamine B (Table 2). Interestingly, desferrirubin and desferrichrysin, although structurally similar to desferrichrome (Fig. 1), were significantly less effective in preventing LDL oxidation. In contrast, the ΔT at Vmax was greatly prolonged by 20 μM concentrations of desferricoprogen, desferrichrome and desferrioxamine B.

Table 1. Dose-dependence of the protective effect of desferricoprogen on LDL against oxidative modification triggered by heme-H₂O₂

Concentration of ΔT at V_max ¹ Relative ΔT a 1 max desferricoprogen (μM) (min) (% of control)

0 57 + 1 100

10 109 ± 1 191 + 2

50 206 ± 1 361 + 2

100 225 ± 6 395 ± 10

250 > 240 >420 ¹ ΔT a Vm_ax values were calculated from 3 independent experiments. The protective effects were significant (p<0.001) for all the desferricoprogen concentrations tested.

Table 2. Relative increases in the ΔT at V_maχ values due to the Fe³⁺- squevenging effect of desferri-siderophores in heme-catalysed oxidative modification of LDL by HbO₂. siderophores

Desferricoprogen 134.8 191.3 341.3

Desfθrrirubin 134.8 186.9 254.3

Desferrichrysin 104.3 119.6 139.1

Desferrichrome 152.2 191.3 302.2

Desferrioxamine B 136.9 200.0 386.9

¹ - ΔT at V_max values were normalized with ΔT at V_maχ measured in heme-H₂O₂ LDL modification systems with no siderophore supplementation. 2 - A typical set of data is shown.

The physical incorporation of both coprogen and desferricoprogen into LDL was demonstrated in vitro. Isolated LDL was incubated with coprogen and desferricoprogen within the concentration range of 10-250 μM for 2 h at 37⁰C in a volume of 2.0 ml containing 1 mg of LDL protein. The samples were dialyzed exhaustively against double-distilled water (3 x 2 h), and one series of desferricoprogen-treated LDL samples were supplemented with FeCI₃ in a molar ratio of desferricoprogeniFeCb 1 :5. All samples were freeze-dried and extracted with 1.0 ml of ice-cold methanol:double-distilled water (1 :2) by vigorous mixing for 2 min. After centrifugation at 10,000 x g for 5 min, the supernatants were analyzed using both analytical HPLC and TLC (Heymann et al. 1999, Hδrdt et al. 2000, Leiter et al. 2001).

The effectiveness of desferricoprogen in suppressing heme-catalyzed LDL oxidation (Fig. 5, Tables 1 and 2) suggested that this chelator might enter LDL. Indeed, as shown in Figure 6, incubation of LDL with coprogen or desferricoprogen led to substantial amounts of LDL-associated chelators. The incorporation of both forms into LDL was dose-dependent and saturable. Incubating 200 μg ml^"1 LDL protein and 250 μM coprogen (total coprogen content in the reaction buffer was 500 nmol) for 2 h led to the incorporation of 50.1% and 43.0% of the added coprogen and desferricoprogen (251 and 215 nmol mg^'1 LDL protein), respectively.

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 (HUVECs) 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. Confluent endothelial cells grown in 24-well tissue culture plates were washed three times with Hank's balanced salt solution (HBSS), and then exposed to a reaction mixture containing LDL (200 mg/L), heme (5 μM), H₂O₂ (75 μM) with or without the addition of iron-free or iron-saturated siderophores (20 μM). After an incubation period of 4 h, the test solutions were replaced with 500 μl of 3-[4,5- dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) (0.5 mg/L) dissolved in HBSS, and endothelial cell monolayers were incubated for another 6 h. The reduced MTT was measured spectrophotometrically at 570 nm after the formazan was dissolved in 100 μl of 10% SDS and 500 μl of hot isopropanol containing 20 mM HCl. In these experiments, 20 μM desferoxamine B was used as a positive control (BaIIa et al. 1991). As expected, 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 μM) on endothelial cells exposed to oxidatively modified LDL (200 μg/ml). LDL was oxidized with the H₂O₂ (75 μM)-heme (5 μM) 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 μM), H₂O₂ (18.75 μM) with or without the addition of iron-free or iron-saturated siderophores (5 μM) for 60 minutes followed by an 8-hour incubation with complete media alone. Endothelial microsomes were incubated with hepatic cytosol (2 mg), hemin (20 μmol/L), glucose-6-phosphate dehydrogenase (0.2 units), and NADPH (0.8 mmol/L). The formed bilirubin was extracted with chloroform and Δ 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. Cellular RNA was isolated using RNAzol (TEL-TEST, Friendswood, TX, USA), and 20 μg 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. Alternatively, 2 μg quantities of total RNA were subjected to dot blot analysis. After cross-linking to the nylon membranes, 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.

Untreated endothelial cells exhibited low levels of HO-1 mRNA expression and HO enzyme activity demonstrated by Northern blot analysis and bilirubin generation (Fig. 8, first lane and bars). Oxidized LDL caused a >40-fold increase in HO-1 mRNA expression together with a ten-fold increase in enzyme activity in human vascular endothelial cells (Fig. 8, second lane and bars). When LDL oxidation was inhibited by 20 μM desferricoprogen, increases in HO-1 mRNA levels and HO enzyme activities were almost totally prevented (third lane and bars). In contrast, coprogen did not inhibit LDL oxidation and did not prevent HO-1 induction in endothelial cells exposed to the products of the reaction (Fig. 8, fourth lane and bars). As shown in Fig. 9, among the iron-free siderophores only desferrichrysin was unable to prevent the induction of the HO-1 gene (dot B4) in agreement with its relatively weak protective effect against heme mediated LDL lipid peroxidation. The other fungal siderophores together with the positive control desferoxamine B efficiently inhibited both the oxidation of LDL and the induction of HO-1 (dots B1-3, B5). Not surprisingly, iron-complexed siderophores did not prevent the induction of HO-1 gene (dots C1-5), in agreement with their lack of effect on LDL-oxidation. Importantly, when LDL was pretreated with iron-complexed siderophores and H2O2 for 1 h in the absence of heme and then incubated with endothelial cell monolayers for 8 h at sublethal concentration neither HO-1 gene induction (Fig. 9, dots D1-5) nor increase in enzyme activity (Fig. 9, fifth lane and bars) was observed. As mentioned above, Figure 8 shows correlation between intracellular

HO-1 mRNA levels (Part A) and specific HO activities (Part B). HUVECs were treated with a series of LDL-containing reaction mixtures, which were also supplemented with 20 μM desferricoprogen or coprogen as indicated. In this case, 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 + H₂O₂- treatment of endothelial cells. B(1-5): protection of HUVECs with 20 μM desferrioxamine B, desferricoprogen, desferrirubin, desferrichrysin and desferrichrome. C(1-5): supplementation of reaction mixtures with 20 μM iron saturated ferrioxamine, coprogen, ferrirubin, ferrichrysin and ferrichrome. D(1- 5) Treatment of HUVECs with LDL + ferri-siderophore + H₂O₂ mixtures without any heme. Siderophore-iron-complexes were the same as in row C. We measured heme mediated oxidation of atherosclerotic plaques in the presence of desferricoprogen. Tissue samples were obtained at surgery of patients suffered from lumen stenosis of carotid or femoral arteries. Immediately after collection of samples vessel wall pieces were washed from contaminating blood with saline.

After drying and weighing samples were frozen in liquid nitrogen and stored at - 70⁰C until assay.

Small pieces of vessel wall samples were dispersed in phosphate buffer containing heme (10 μM) and desferricoprogen (25 es 50 μM) and incubated for 24 or 48 hours at 37 °C. For the measurement of TBARs 250 μl of reaction mixture was combined with 250 μl thiobarbituric acid reagent (0.375 g 2- thiobarbituric acid, 2.08 ml cc HCI, 15 ml 10 % trichloroacetic acid and distilled water to a final volume of 100 ml). After heating at 9O⁰C for 20 minutes the samples were cooled and extracted with 250 μl n-buthanol. The upper phase was measured spectrophotometrically at 532 nm. Results were calculated using a molar extinction coefficient of 1.56*10⁵ M^'1 cm^"1 and are presented as nmol thiobarbituric acid-reactive substances/ mg tissue.

As prevoiously described (BaIIa et al 1991 , 1993) 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 wondered 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. On the other hand desferricoprogen has no effect if the oxidation takes longer (48 hours) as shown in figure 11. We concluded that chelate forming ability of desferricoprogen with iron retards lipid oxidation of the atherosclerotic plaques.

Figure 10 represents that desferricoprogen prevents heme mediated oxidation of atherosclerotic plaques. Small pieces of atherosclerotic vessel wall samples were treated with heme (10 μM) in the presence or absence of desferricoprogen (25 or 50 μM) 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. Small pieces of atherosclerotic vessel wall samples were treated with heme alone (10 μM, black bars) or in the presence of desferricoprogen (50 μM, grey bars) and incubated for 24 or 48 hours at 37 °C, and then TBARS were measured as described. Figure shows the average and S. D. of 5 independent experiments.

Main conclusions of Example 1, 2 and 3 are:

- 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;

- Based on our results siderophores - especially desferricoprogen and desferrichrome - significantly increase the resistance of LDL against heme mediated oxidation. In similar dose-response fashion, these siderophores also inhibited the generation of LDL products cytotoxic to human vascular endothelium. A surrogate marker for the non-cytocidal effects of oxidized LDL, induction of heme oxygenase-1 (HO-1), showed similar patterns. At sublethal concentrations oxidized LDL caused a >40-fold increase in HO-1 mRNA and a ten-fold increase in HO-1 enzyme activity in human vascular endothelial cells. However, when iron-free fungal siderophores were added to LDL/heme oxidation reactions, the product failed to induce HO-1 (not in the case of desferrichrysin). We conclude that fungal hexadentate siderophores effectively prevent LDL oxidation caused by redox-active, heme-derived iron as well as subsequent injury of endothelial cells caused by the LDL oxidation products.

- Chelate forming ability of desferricoprogen with iron retards lipid oxidation of the atherosclerotic plaques.

Example 4

Animal studies were performed to determine whether siderophores can be taken up if they were administered orally, therefore they are suitable for oral treatment or food additives. FLFi hybrid rats weighing 150-350 g were fed a normal chow diet were used in the experiments. After weighing rats were treated with either desferricoprogen or ferricoprogen at a concentration of 50 mg/kg (20 mg/ml in saline, sterile filtered) intravenously through femoral vein, or orally at a concentration of 100 mg/kg, through a orogastric tube. Urine and feces were collected for 24 hours, while rats were kept in separated cages with free access to water and food.

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. Based on the experiments excretion of administered desferricoprogen is quite slow both through urine and feces; within 6 days only 15% of the administered amount is eliminated. Excretion of ferricoprogen is much faster, within 6 days up to 75% of the administered amount is eliminated. Retarded excretion of desferricoprogen suggests that the compound interacts with particles of blood plasma, for example low density lipoprotein, which we have demonstrated in an in vitro experiment (Figure 6). Table 3 contains experimental data which were used to generate figure 12-15 as well. Table 3

Figure 12: Ferricoprogen, administered orally, 100 mg/kg body weight days 1 2 3 6

Urine (U) 0.7 0 0.6 0

Feces (F) 21 0.4 0.25 0

Sum (S) 21.7 0.4 0.85 0

Figure 13: Desferricoprogen, administered orally, 100 mg/kg body weight days 1 2 3 6

Urine (U) 0.2 3.1 0 1.2 Feces (F) 6 0.05 0.15 0

Sum (S) 6.2 3.15 0.15 1.2

Figure 14: Ferricoprogen, administered intravenously, 50 mg/kg body weight days 1 2 3 6

Urine (U) 33 35.6 5 1.2

Feces (F) 1.5 0 0 0

Sum (S) 34.5 35.6 5 1.2

Figure 15: Desferricoprogen, administered intravenously, 50 mg/kg body weight days 1 2 3 6

Urine (U) 3.3 2 3.4 0.65

Feces (F) 4 0.25 0.3 0.15

Sum (S) 7.3 2.25 3.7 0.8

Further animal studies were performed to determine whether siderophores can be taken up if they are administered orally, therefore they are suitable for oral treatment or food additive. FLFi hybrid rats weighing 150-350 g were fed a normal chow diet were used in the experiments. After weighing rats were treated with either desferricoprogen or ferricoprogen at a concentration of 50 mg/kg (20 mg/ml in saline, sterile filtered) intravenously through femoral vein, or orally at a concentration of 100 mg/kg, through an orogastric tube. Some animals were given 0.5 ml 20 % ethanol or 0.5 ml olive oil after the oral administration of the siderophore. Urine and feces were collected for 24 hours, while rats were kept in separated cages with free access to water and food.

Then we examined the accumulation of desferricoprogen administered either intravenously or orally in the body. Blood samples (centrifuged to separate plasma and blood cells), liver, spleen and intestinal epithelium of the experimental animals were taken. Feces samples were shaken in 40 ml of water for 30 minutes, then centrifuged (1000 rpm, 15 min), frozen and lyophilized. Urine, plasma, blood cells, liver, spleen and intestinal epithelium samples were frozen and lyophilized. Liophilized samples were resolved in 20 ml of 50% methanol, shaken and divided into two 10 ml aliquots. One of them was treated with FeCb (6-fold of the administered coprogen), and the precipitate was spined down by centrifugation, and the supernatant was analyzed by HPLC.

We have examined secretion of ferri- or desferricoprogen administered orally or intravenously into the urine and feces. 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. Surprisingly, 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. Importantly the compound is continuously secreted in its iron complexed form in the urine (0.2-3.1 % of the administered amount /day). This observation proves the uptake of desferri siderophores. About 93% of the administered desferricoprogen is not secreted in either the feces or the urine, which suggests that desferricoprogen is taken up and stored and/or metabolized by the intestinal flora similarly to that of ferricoprogen.

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. However, the secretion of the desferri form can not be excluded but its amount remained under the detection limit.

Based on these experiments absorption of both coprogene and desferricoprogene from intestine and their secretion to the urine and feces (via bile) were proven. 76-93 percentages of the administrated siderophores appeared neither in the feces nor in urine with the exception of the intravenous administration of ferricoprogen. There are more possible explanations of this observation; for example the accumulation of the compounds or their metabolic products in the liver, whose hypothesis was tested in the following study.

We established that desferricoprogen administered either intravenously or orally likely accumulated or partially metabolized in the liver.

As shown by Figure 19; 14-19% of the intravenously administrated coprogen accumulated in the liver within 5 minutes after administration and this amount increased further to 14-34 % 0.5-2.0 hours post administration. During this time an unknown siderophore metabolite appears in increasing amount which has shorter retention time. As the chemical composition and molar extinction coefficient of the siderophore metabolite is unknown its concentration can not be evaluated. Important to know, that this metabolite is not the dimerumic acid which is a tetra-dentate ligand and formed from the hydrolysis of the ester bond in the coprogen. This conversion product was appeared in feces samples with various concentrations.

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. Accumulation of coprogen in the liver and in intestinal epithelial cells and its secretion (feces and urine) are shown in figures 18 and 19 in case of oral administration of desferricoprogen and coprogen (100mg/ kg body weight) respectively in a rat model. Results are expressed as percentage of the administered coprogen and were calculated from three independent experiments (t= 24 hrs).

The absorbed coprogen enriched in the liver showing linear time dependence (fig.17) and coprogen content of the liver reached 9-10 % of total administered coprogen one day after oral dosage. The siderophore metabolite having shorter retention time was detected again in the liver. Approximately 0.1 percent of the orally administered desferricoprogen remained in the intestinal epithelium after 24 hrs while there was no detectable siderophore in the intenstinal epithelial cells if coprogen was dosaged (fig. 18). Although low amount of the intravenously administered siderophore was detected in the spleen (0.05-0.071 %) 2 hrs postinjection there was no detectable siderophore in the spleen after oral administration (datas are not shown). Addition of ethanol or oil into the drugs before oral administration did not influence the accumulation of desferricoprogen in the liver 24 hours post-administration (fig. 19). Both coprogen secretion of the animals (feces and urine) and low siderophore content of the intestinal epithelium remained unaffected by ethanol or oil pretreatment of the samples as well (datas are not shown).

Table 4 Localization of desferricoprogen in the body in cases of intraven

Coprogen Coprogen in Coprogen Treatment of sampling in plasma blood cells In liver mg Sample Treatment mg/ml mg/ml

5 min 1.610 Without FeCI₃ Intravenous desferricoprogen

< 0.01 mg/ml 0 1.830 With FeCI₃ 50 mg/kg, total: 13.0 mg

0.5 h 2.56 Without FeCI₃ Intravenous desferricoprogen

< 0.01 mg/ml 0 3.50 With FeCI₃ 50 mg/kg, total: 10.3 mg

1 h 1.25 Without FeCI₃ Intravenous desferricoprogen

≤ 0.01 mg/ml 0 1.89 With FeCI₃ 50 mg/kg, total: 8.9 mg

2 h 2.60 Without FeCI₃ Intravenous desferricoprogen

"1

≤ 0.01 mg/ml 0 2.60 With FeCI₃ 50 mg/kg, total: 12.3 mg

1 h 0.000 Without FeCI₃ Oral desferricoprogen

Non detectable 0 0.000 With FeCI₃ 100 mg/kg, total: 15.2 mg

2 h 0.068 Without FeCI₃ Oral desferricoprogen

Non detectable 0 0.077 With FeCI₃ 100 mg/kg, total: 19.6 mg

4 h 0.128 Without FeCI₃ Oral desferricoprogen

≤ 0.01 mg/ml 0 0.66 With FeCI₃ 100 mg/kg, total: 18.8 mg

24 h 0.70 Without FeCI₃ Oral desferricoprogen

≤ 0.01 mg/ml 0 1.90 With FeCI₃ 100 mg/kg, total: 22.0 mg

accumulation and conversion rate of the siderophore in the liver can not be determined while there is no precise assay to measure the concentration of the metabolite (coprogene derivative).

Desferricoprogen administered orally appears in the liver after 2 hrs (0.4 %) then its amount almost doubled after 4 hrs (0.7%) and it can be increased to

9-10 % after 24 hrs (Table 4 and Fig 17). At least 37-88% of the siderophore is complexed with iron in the liver which is independent evidence that the orally administrated siderophore is absorbed via intestine. The orally administered desferricoprogen can be detected in the serum. An extra HPLC peak - which suggests metabolization of coprogen - can be detected in the plasma. In contrast desferricoprogen can not be detected either in blood cells or in the spleen.

Based on the experiments above we can state that siderophore administered orally is absorbed via intestine and transported to the liver. Major proportion of the siderophore forms complex with iron then this iron-siderophore complex is stored and metabolized in the liver and finally secreted in the urine.

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.

Example 5: Technological description of salami making by using mold

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. Nowadays, 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 ⁰C. The temperature of the bacons is typically between -5 to -7⁰C. 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:

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. In this step the temperature is 20-24 ⁰C, relative humidity is 88-96 % and intensive air circulation with adding fresh air are required.

In the second phase water solubilized selectively cultured molds are introduced on the surface of the salami by dipping or spraying techniques. For the propagation of mold 17-21 ⁰C temperature, 85-90% relative humidity and low air circulation 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 ⁰C, 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.

Ingredients used for salami making:

Raw materials are: cattle meat, hog meat, industrial bacon, fat-back. Additives are: sodium nitrite/nitrate mixture, spices, sodium-ascorbate, mono- and complex carbohydrates.

The following siderophore producing fungi strains were used during the procedures:

Neurospora crassa Sid 1 NCAIM (P) F-001331 Penicillium chrysogenum Sid2 NCAIM (P) F-001332 Penicillium nalgiovense Sid3 NCAIM (P) F-001333 Penicillium roquefortii Sid 4 NCAIM (P) F-O01334 Penicillium candidum (= Penicillium camemberti) Sid 5 NCAIM (P) F-O01335

In tune with the literature (Ong es Neilands 1979) we found that high amount of fungal siderophores - e.g. coprogen - can be present in certain foods like cheese which were ripen by Penicillium roqueforti or Penicillium camemberti. The amount of coprogen can reach 40 mg/kg in Roquefort-type cheese and approximately 50-70% of coprogen is not complexed with iron. Importantly we have found that Penicillium nalgiovense - which is primarily used for salami making - is able to produce high amount of siderophors e.g. coprogen (160-225 mg/L coprogen in liquid culture lacking iron). Important to note that siderophores spectra produced by P. Roqueforti and P. Nalgiovense is very similar; besides coprogen, both produce dimerumic acid, neocoprogen and triacetyl-fuzarinine in the absence of iron. This microbiological aspect is very important in enrichment of salamis with siderophores (mostly with coprogen) at a similar or higher concentration than described in cheese.

Claims

Use of fungal desferri-siderophores in the preparation of pharmaceutical compositions for treatment of vascular diseases primarily with endothelial cell origin.

Use of fungal desferri-siderophores in the preparation of pharmaceutical compositions which can be administered orally for the treatment of vascular diseases primarily with endothelial cell origin.

The use according to claims 1 or 2 wherein the siderophore is a member of the desferricoprogen group:

wherein

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 Ci_-6 alkanoyl group,

R3, R4 and R5 mean Ci_-6 alkyl group or C_2-6 alkanoyl group substituted by one or two hydroxy or carboxy.

4. The use according to claim 3 wherein the structures of the siderophores are listed below:

5. The use according to claims 1 and 2 wherein the structure of the siderophore is a member of the desferrichrome group below:

In the structure above

R1 means hydrogen atom, C_1-6 alkyl group or Ci_-4 hydroxy-alkyl group,

R2 means hydrogen atom, C_1-6 alkyl group, Ci_-4 hydroxy-alkyl group or Ci_-6 alkanoyl group,

R3, R4 and R5 mean C_1-6 alkyl group or C_2-6 alkanoyl group substituted by one or two hydroxy or carboxy.

6. The use according to claim 5 wherein the structures of the siderophores are listed below:

7. The use according to any of the claims 3 to 6 wherein A, B, C and D have the following meanings:

8. The use according to claims 1 or 2 wherein the siderophore is desferri- triacetyl fuzarine C:

9. The use according to claims 1 or 2 wherein the siderophore is desferricoprogen.

10. Process for preparing the members of

- desferricoprogen family

- desferricrome family

- and for the production of the desferri-triacyl fuzarinine C

wherein

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 Ci-6 alkanoyl group, R3, R4 and R5 mean Ci_₆ alkyl group or C₂..₆ alkanoyl group substituted by one or two hydroxy or carboxy, wherein the fungi strains such as Penicillium chrysogenum, Penicillium roqueforti, Neurospora crassa, Penicillium roqueforti, Ustilago sphaerogena, Ustilago maydis, Neovossia indica, Aspergillus ochraceus or Aspergillus melleus are cultured on culture medium containing mineral salts, carbon- and nitrogen source and siderophores are purified from the liquid medium. 11. The method according to claim 10 wherein the following siderophore producing fungi strains are used:

Neurospora crassa Sid 1 NCAIM (P) F-001331

Penicillium chrysogenum Sid2 NCAIM (P) F-001332

Penicillium nalgiovense Sid3 NCAIM (P) F-O01333

Penicillium roquefortii Sid 4 NCAIM (P) F-001334

Penicillium candidum (Penicillium camemberti) Sid 5 NCAIM (P) F- 001335

12. Food, mostly meat products contain siderophores in efficient amounts. 13. The foods according to claim 12 which contain members of

- desferricoprogen siderophore family

- desferrichrome siderophore family

- and desferri-triacetyl fuzarinine siderophore family.

14. Method for introduction of siderophores into the siderophore containing meat-products selected from any of the processes:

- Isolated siderophores are mixed directly into the pasta,

- Siderophore producing fungi strains (see above) are mixed directly into the meat pasta,

- Water solubilized siderophore producing fungi strains are introduced on the surface of the meat-products during the curing step.

15. Method for qualification and quantification of siderophore content of meat- products wherein the quantity is compared to the required siderophore amount of a healthy individual.