WO1999037312A1

WO1999037312A1 - Pharmaceutical composition comprising vanadium and/or salts thereof and its use to treat burns

Info

Publication number: WO1999037312A1
Application number: PCT/EP1999/000483
Authority: WO
Inventors: Pierre Cornelis; Daniel De Vos
Original assignee: Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
Priority date: 1998-01-27
Filing date: 1999-01-26
Publication date: 1999-07-29
Also published as: EP1051184A1; CA2318902A1; AU2719299A

Abstract

The present invention relates to the use of multivalent transition elements and/or salts thereof in a pharmaceutical composition to inhibit bacterial or yeast growth. Especially bacterial Pseudomonas growth is inhibited by using said pharmaceutical composition comprising vanadium and/or a salt thereof. Said vanadium or a vanadium salt is incorporated in a pharmaceutical composition as an essential element whereupon this pharmaceutical is applied for instance to burn-wound patients or patients having ulcers like diabetic foot ulcers.

Description

Pharmaceutical composition comprising vanadium and/or salts thereof and its use to treat burns.

Field of the invention.

The invention relates to the use of multivalent transition elements and/or salts thereof to inhibit bacterial or yeast growth. Especially bacterial Pseudomonas growth is inhibited by using vanadium and/or a salt thereof.

More specifically vanadium or a vanadium salt is incorporated in a pharmaceutical composition as an essential ingredient whereupon this pharmaceutical is applied to burns. Said pharmaceutical composition is also used to treat opportunistic infections.

Background of the invention

Bacterial infections in burn wound patients are widely known and difficult to control. Sepsis as a consequence is common and the issue often fatal. Pseudomonas aeruginosa (P.aeruginosa) emerged during the last decades, following the introduction of antibiotic therapy, as one of the most problematic gram-negative bacterium in our modern hospital settings; this organism is increasingly isolated as a nosocomial pathogen resulting in high morbidity and mortality rates. Burn wound patients and cystic fibrosis patients are very susceptible to the latter micro-organism which is inherently resistant to common antibiotics and even survives in antiseptics. Early diagnosis of P.aeruginosa infection is a key feature in the management of those patients. The source of a contamination can be endogenous, exogenous or both. It is known by several epidemiological studies that several exogenous infections are nosocomial in origin and that specific installations such as water distillation systems, hydrotherapy facilities and humidifiers can be potential reservoirs of bacteria, specifically of pseudomonads. The need actually exists for the development of alternative clinical strategies to combat and/or prevent P.aeruginosa infections. Pseudomonas aeruginosa is, as mentioned above, an opportunistic gram-negative human pathogenic bacterium which causes severe and often fatal infections particularly affecting immuno-compromised patients with severe burns. It is frequently involved in ear and eye infections and could also be considered, as mentioned above, as the main pathogen responsible of lung infections in patients developing cystic fibrosis, thus being the major cause of morbidity and mortality in these patients. Among the multiple factors recognized to be involved in the virulence of P.aeruginosa, nutritional factors of course play a primary role for the bacteria to develop in vivo. A particularly crucial factor is the bacterial iron nutrition, this element being absolutely necessary by most micro-organisms, especially for aerobic bacteria such as the pseudomonads. Iron is always tightly bound to host proteins in the organism, e.g., to transferrin, lactoferrin and haemoglobin. Therefore, pathogenic bacteria have usually developed specialized take-up systems for directly using these iron sources, as Haemophillus sp. or Neisseria sp., or have developed powerful siderophores which could successfully compete for iron with the host iron proteins, e.g., aerobactin, vibriobactin or amonabactin.

P.aeruginosa produces two types of siderophores: pyochelin and pyoverdine, the latter being the typical fluorescent peptidic siderophore produced by most of fluorescent pseudomonads. Siderophores are low molecular weight molecules (800-1500 Da) with variable chemical structure which possess specific ligands essential for the sequestration of iron. The most commonly involved ligands are the hydroxamate and cathecholate groups. Alpha-hydroxy and oxaziline groups on the other hand are rarely involved ligands. Some siderophores have three hydroxamate groups (ferrioxamine), whereas others bear only 3 catecholates (enterobactin) or are not uniform and have a mixed composition. The known P.aeruginosa strains produce three different types of pyoverdines.The complete structures of these latter types were also recently elucidated. All contain the rare amino acid δ-N-hydroxyomithine but differ in the amino acid composition of the peptidic part of the molecule which is linked to a quinolinic (chromophore) moiety. In contrast to pyoverdine, which is a high-affinity iron chelator, pyochelin is a thiazoline derivative and a low affinity iron scavenger.

The three structurally different pyoverdines have been identified from several P.aeruginosa strains: from P.aeruginosa ATCC 15692 (Briskot et al., 1989, Liebigs Ann Chem, p.375-384), from P.aeruginosa ATCC 27853 (Tappe et al., 1993, J.Prakt-Chem., 335, p.83-87) and from a natural isolate, P.aeruginosa R (Gipp et al., 1991 , Z. Naturforsch, 46c, p.534-541 ). Moreover, comparative biological investigations on 88 clinical isolates and the two collection strains mentioned above revealed three different strain-specific pyoverdine-mediated iron uptake systems (Cornells et al., 1989, Infect Immun., 57, p.3491-3497; Meyer et al., 1997, Microbiology,143, p.35-43) according to the reference strains P.aeruginosa ATCC 15692 (Type I Pvd), P.aeruginosa ATCC 27853 (Type II Pvd) and the clinical isolates P.aeruginosa R and pa6 (Type III Pvd).

Pyoverdines are the main siderophores of pseudomonads such as P.aeruginosa. In vitro experiments indicated a potential role of the P.aeruginosa pyoverdine in iron release from ferritransferrin but the ability of pyoverdine to compete for iron in vivo has only recently been demonstrated (Meyer et al., 1996, Infection and Immunity, 64_> p.518-523). It was observed using a burned- mouse model that the absence of pyoverdine production in mutants raised from a virulent parental strain correlated with a loss of virulence of these mutants and that virulence was restored when the homologous pyoverdine originating from the wild-type strain was supplemented. Furthermore supplementation with a heterologous pyoverdine did not restore the virulence of the latter mutants. Thus, a precise knowledge of the pyoverdine-mediated iron uptake system used by a given P.aeruginosa isolate during infection appears a prerequisite for developing new ways of treatment of P.aeruginosa infections via bacterial iron metabolism, e.g., by blocking the pyoverdine biosynthesis or the pyoverdine- mediated iron transport.

USP 5,079,010 to S.Natterer discloses a pharmaceutical preparation which is a solution containing water and metallic trace elements to treat wounds, burns or other inflammatory changes. Such a pharmaceutical composition has among others an anti-bacterial action. As metallic trace elements can be used iron, zinc, manganese,chromium,copper,cobalt, molybdenum, tin, vanadium, nickel and selenium. An essential factor for the efficacy of the preparation is its pH which ranges between 4.0 and 1.0 and which is preferably less than 3.5. It is further disclosed that the trace elements are completely dissolved in the acid solution in order to enter into action.

Fukuda and Yamase (Biol.Pharm.Bull.20(8), 927-930,1997) discloses the in- vitro antibacterial activity of vanadate and specific vanadyl compounds against gram-positive bacteria such as Streptococcus pneumoniae. The activity of the tested compounds against pathogenic bacteria, such as the gram-negative bacteria Escherichia Coli and Pseudomonas aeruginosa, was estimated to be negligible.

Aims and Description of the invention.

Unexpectedly there has now been found that vanadium interferes with siderophores and especially with the pyoverdine-mediated iron transport. Therefore, the present invention aims to use a multivalent transition element and/or a salt thereof such as vanadium or a vanadium salt which interferes with bacterial iron uptake and inhibits bacterial growth, especially the growth of Pseudomonas aeruginosa, under iron limiting conditions.

Other multivalent transition elements which can be used for this purpose are chromium or titanium and their respective salts.

As vanadium salts can be used vanadium carbonyl, vanadium pentafluohde, vanadium pentoxide, vanadium tetrafluoride, vanadium trifluoride, vanadium trioxide, vanadium trisulfate, vanadium trisuifide, vanadyl dichloride, vanadyl sulfate or vanadyl trichloride.

The growth of P.aeruginosa in CAA medium (casaminoacids medium), a liquid medium with low iron content, is inhibited in the presence of vanadium at a concentration of 0.5 mM and completely inhibited at any concentration between 2 and 3 mM vanadium. This inhibitory effect has been observed using different P.aeruginosa strains independent of the pyoverdine-type excreted, both at 37° C and 28°C.

Other bacterial strains in this respect susceptible for vanadium, chromium or titanium and their respective salts are Escherichia coli, Klebsiella oxytoca, Staphylococcus aureus, Stenotrophomonas maltophilia and the like.

In addition a surprising effect was obtained with respect to the growth inhibition of P.aeruginosa (Type 1,11 and III Pvd as well) if a chelator agent such as EDDHA (ethylenediaminedihydroxyphenylacetic acid) was added to the CAA- medium, optionally supplemented with vanadium or a vanadium salt. The preferred concentration and chelator used in this respect is EDDHA in a concentration of 0.2 mg/ml.

A primary aspect of the current invention is a pharmaceutical composition comprising at least one transition element and/or salt thereof which interferes with a bacterial and/or fungal/yeast iron-uptake mechanism. In more detail, the interference occurs through a high affinity siderophore mediated iron-uptake system present in bacteria and/or fungi/yeast. Preferably it occurs at the level of the pyoverdine-mediated iron transport pathway. The transition elements in the pharmaceutical composition are selected from the group comprising vanadium, chromium or titanium and/or salts thereof.

Another aspect of our invention is that vanadium and/or salts thereof can be used in a pharmaceutical composition to prevent infection in burns. It is known that in bum-wound victims opportunistic P.aeruginosa infections frequently occur; furthermore P.aeruginosa is also the main lung pathogen in cystic fibrosis patients. In patients having ulcers, like diabetic patients having foot ulcers, for instance a topical application of a pharmaceutical composition according to the invention is beneficial for said patient in order to help curing the infection as such. Optionally the pharmaceutical composition according to the invention comprising vanadium or a salt thereof may be supplemented with other metal trace elements such as zinc, silver or a salt thereof and the like.

The negative effect of vanadium on pyoverdine production, excretion or uptake, when applied in a pharmaceutical composition, is a helpful tool in the fight against said P.aeruginosa infections. Consequently application of said pharmaceutical composition to burn wound patients in order to prevent the bacterial infection is another important feature of the present invention. In addition to inhibit the bacterial growth of siderophore-excreting bacteria, vanadium or salts thereof can unexpectedly be used to inhibit the growth of yeast or fungi, especially Candida albicans. Therefore the pharmaceutical composition according to the invention, comprising at least one of the above indicated-transition elements interfering with the fungal/yeast iron-uptake mechanism, is effective against the fungi/yeasts selected from the group comprising Candida, Histoplasma, Cryptococcus or Rhodotorula, more specifically Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans or Rhodotorula piiimanae.

Thus another aspect of the present invention is a pharmaceutical composition to treat burns, Candida infection or any opportunistic infection in order to prevent a further spread of the pathogenic organisms. It is obvious that the amount/dosage of a transition element in said pharmaceutical composition varies within the limits of pharmaceutically acceptable activity (i.e. suitably prepared and approved for use in the desired application) depending on the infection/contamination to combat. Generally, the dosage needed to provide an effective amount of the composition will vary depending upon factors such as the recipient's age, condition, extent of disease, body weight of the patient and other variables which can be adjusted by one of ordinary skill in the art. To this end the dosage of the transition element can vary from about 0.01 μg/kg to about 1 mg/kg body weight of a patient, preferably from about 0.05 μg/kg to about 0.5 mg/kg body weight of a patient depending upon the type of formulation and the condition of the patient to be treated. It is well-known in the medical field that in order for a pharmaceutical composition to be effective against a certain infection/contamination, 18-24 hours after the first application an extra dosage of said pharmaceutical composition is needed.

Concerning the toxicity in mammals of multivalent transition elements such as vanadium and/or the salts thereof used according to the current invention in an appropriate pharmaceutical composition, the skilled person will apply the knowledge from, for instance, the reference Llobat.J.M. and Domingo.J.L (1984), Acute toxicity of vanadium compounds in rats and m/^'ce;Toxicol.Lett, 23: p.227-231. Alternatively the reference Domingo, J.L. et al.,(1985), Short term toxicity studies of vanadium compounds in rats; J.Appl.Toxicol, 5: p.418-421 can be used.

These studies show that rodents accept vanadium compounds at a moderate toxicity at a level of 0.2-1.1 mg/ml.

In Rehder.D, (1992), BioMetals, 5: p.3-12 is disclosed that for human tissues the c(V) is estimated at 0.1-1 micromolar. On the other hand measurements of Versieck et al, show in normal human serum and plasma a level of 0.03-0.7 ng/ml. (Versieck, J. et al., 1987, CRC,p.71 , Trace elements in human serum and plasma.)

Concerning the dosage of the multivalent transition element to be used in an appropriate pharmaceutical composition according to the invention, reference is made to Natesampillae et al., in Critical Reviews in Biochemistry and Molecular Biology, 31 , (5): p.339-359 (1996). Herein is disclosed that the LD50 of vanadyl sulfate for rats is 450mg/kg (9mM).

The pharmaceutical composition comprising vanadium or salts thereof according to the invention can be formulated in any acceptable manner. For general knowledge concerning pharmaceutical compositions as such, reference is herewith made to "Dermatological Formulations", Percutaneous Absorption, by Brian W.Barry, (1983), Ed. Marcel Dekker.lnc. and to " Encyclopedia of pharmaceutical technology", by J.Swarbrick and J.C.Boylan,(1996/1997),Vol.14, Ed. Marcel Dekker, Inc. Some non-limitative examples are formulations as hydrogel, (dry)-sprays, tablets, injection preparations, suntan composition such as suntan lotion, cream, balm or salve, as impregnating agent in (sticking) plasters and the like.

Another possibility for a pharmaceutical composition according to the invention is to impregnate, or an uptake of, vanadium or salts thereof into preferably proliferating skin cells of a burn-wound victim in order to prevent that a Pseudomonas infection occurs. Said proliferating skin cells, preferably from the patient concerned, can be applied for instance as a layer of cells to the area of the burn in order to cure the burned area by a skin-grafting technique known to a skilled person.

In order to clarify what is meant in this description by some terms a further explanation is hereunder given.

"Transition element" refers to the position of the chemical elements concerned as mentioned in the "Periodic Chart of Elements" known to a skilled person.

With "interfering with an iron uptake mechanism" is meant that the presence and/or activity of a transition element (e.g. vanadium or a salt thereof) has a direct or indirect impact on the iron uptake mechanism of the bacteria or yeast concerned.

The term "to treat burns" also means the prevention of infection upon a contamination by, for instance, P.aeruginosa in the burns.

"Opportunistic infection" means an infection caused by a microorganism, e.g. bacteria, yeast or fungi, which will develop to a disease or infectious pathogenic status in a patient, if in said patient already a predetermined situation has been developed like a trauma or immune deficiency. Brief description of the Figures-

Figure 1 a, b and c:

Bacterial growth in CAA-medium at 37°C of three different P.aeruginosa strains in the presence/absence of 1 mM Vanadium as determined at OD of 600nm. Figure 1 d. e and f:

Pyoverdine excretion/production of the three different P.aeruginosa strains in the presence/absence of 1 mM Vanadium as measured in the culture supernatant at OD of 400nm. Figure 2 a and 2 b:

Effect of vanadium on the growth of P.aeruginosa (a) and production of pyoverdine (b) in the presence of vanadium.

The invention is hereunder further explained by way of example without being restrictive in the scope of the current invention.

EXAMPLES

Example 1.

Bacterial strains and general growth conditions

The following P.aeruginosa strains are used : P.aeruginosa ATCC 15692 (Briskot et al., 1989, Liebigs Ann Chem, p.375-384), P.aeruginosa ATCC 27853 (Tappe et al., 1993, J.Prakt-Chem., 335, p.83-87) and from a natural isolate, P.aeruginosa R (Gipp et al., 1991 , Z. Naturforsch, 46c, p.534-541 ). Three different strain-specific pyoverdine-mediated iron uptake systems have been described (Comeiis et al., 1989, Infect Immun., 57, p.3491-3497; Meyer et al., 1997, Microbiology, 143, p.35-43) viz. a system for P.aeruginosa ATCC 15692 (Type I Pvd), P.aeruginosa ATCC 27853 (Type II Pvd) and the clinical isolates P.aeruginosa R and pa6 (Type III Pvd) respectively. Bacterial stocks, kept at -80° C, are grown overnight in LB-medium (Maniatis et al., 1982, Molecular Cloning, CSH, NY) to revive the bacteria.

Casaminoacids medium (CAA), a liquid medium with low iron content, was used for the experiments (Hofte et al., 1993). Its composition is 5 g.l^"1 CAA, 1.3 g.l^"1 K₂HP0₄.3H₂0 and 0.25 g.l^"1 MgS0₄.7H₂0. The pH value of this medium is above 4, preferably between 4.5 and 10 and has more preferably a pH value of about 7.

The V0S0₄.5H₂0 was purchased from Merck and a 100 mM stock solution was prepared.

The metal solution of vanadate was filter sterilized using a bacterial filter (0.2 μm pore size) and added after autoclaving to avoid metal precipitation.

Bacteria were grown in a New Brunswick Innova shaker at 200 rpm at 28° C.

Example 2.

Effect of vanadium on the growth and pyoverdine production in P.aeruginosa.

a) Effect of vanadium on growth of P.aeruginosa in iron-deficient (CAA) medium

The growth of P.aeruginosa in iron-deficient CAA-medium (liquid culture) was strongly inhibited in the presence of vanadium at a concentration of 0.5 mM, while a complete inhibition occurred at 2 mM (see figure 1a, b, c and 2a). This inhibitory effect was observed for the different P.aeruginosa strains, independent of the producing pyoverdine-type at both 37°C and 28°C. Vanadium has less effect on the bacterial growth in the presence of iron. After about 1 day the growth of P.aeruginosa restarts in 1 mM vanadate medium, indicating that the effect is not irreversible. The same experiment was performed using solid medium (agar plates) and also on this medium a concentration of 1 mM vanadate completely inhibits the P.aeruginosa growth, while a concentration of 0.5 mM allows some growth.

b) Production of pyoverdine by P.aeruginosa in the presence of vanadium

The production of pyoverdine was determined by 400 nm absorption of culture supernatant (figures 1 d, e, f and 2 b) as well as by visual inspection using UV- fluorescence (table 1). The growth inhibition by vanadium clearly corresponds to an inhibition or shortage of pyoverdine production.

Furthermore, instead of green fluorescent pyoverdine, a non-fluorescent but yellow-brown colour was produced. In the presence of iron no pyoverdine was produced (no fluorescence in supernatant).

Table 1 : Colour and fluorescence of P.aeruginosa culture (supernatant) after 24 hours of growth.

uM vanadium colour fluorescence

0 green XXX

10 green XX

25 green-brown X

50 gold-brown -

75 gold-brown -

100 gold-brown -

250 gold-brown -

500 gold-brown -

600 brown -

700 brown -

750 brown -

800 brown -

Production of a colour by P.aeruginosa in the presence of vanadium

When P.aeruginosa restarts growing in the presence of vanadium, the production and excretion of yellow-brown colour was observed. The production of this yellow-brown colour is specific for pyoverdine-producing strains, since a pyoverdine-deficient mutant does not produce this colour. This yellow-brown is not simply a vanadium-pyoverdine complex. Following IEF (iso-electric focusing) of the supernatant from P.aeruginosa type I cultures in CAA-medium, two pyoverdine isoforms can be detected under UV as distinct bands. When vanadium is added directly to this supernatant prior to IEF, the 2 pyoverdine bands can still be detected, although at a slightly different level than in the absence of vanadium. In contrast, when supernatant from P.aeruginosa cells which were grown in vanadium-containing CAA-medium was analysed through IEF, no bands at all could be detected under UV. Thus, vanadium binds to pyoverdine (resulting in a band-shift) but this complex is not the same as the yellow-brown colour that appears when P.aeruginosa cells are grown in the presence of vanadium.

Example 3

Susceptibility of other bacteria for vanadium

Other bacterial species have been tested for their capacity to grow in the presence of increasing amounts of vanadium. These include Escherichia coli, Serratia marcessens as members of the gram-negative Enterobacteriaceae and Streptococcus spp. and Staphylococcus aureus respectively as representatives for gram-positive bacteria. All strains were clinical isolates, taken from routine medical microbiology bench.

Growth at 37°C in CAA-agar plates containing 0-1-2-5-7-10 mM vanadate was monitored at 24 h and 72 h. Growth could be observed for all strains at 1 mM of vanadium, but for concentrations of 2mM and higher, bacterial growth was fully inhibited.

Example 4

Effect of vanadium on the growth of Pvd type I. II and III P.aeruginosa strains. From P.aeruginosa strains three structurally different pyoverdines have been identified: Pvd type I, II and III respectively (see text above). These three types have been tested for growth / inhibition under several conditions. Clinical isolates comprising the three types (I, II and III), have been tested as well and demonstrate the same growth / inhibition characteristics as given hereunder. Growth / inhibition was measured at OD 600nm and is depicted in tables A and B.

Table A

CAA medium only CAA medium+ CAA medium+ 2mM Vanadium 3 mM Vanadium

Type 1 (1) (2) (3)

Type II (1) (2) (3)

Type III (1 ) (2) (3)

(1 ): growth starts after 5 hours to the same extent for the three types

(2): growth inhibition for the three types, however after 20 hours type III restarts growing

(3): total growth inhibition for the three types to the same extent as of the onset

Table B

CAA+10μM Fe3+ CAA+10μM Fe3+ CAA+10μM Fe3+ +2mM Vanadium +3mM Vanadium

Type I (4) (5) (6)

Type II (4) (5) (6)

Type III (4) (5) (6)

(4): growth starts after 5 hours for the three types (5): type I starts growing after 24 hours type II starts growing after 48 hours type III starts growing after 12 hours (minimum inhibition period) (6): total growth inhibition for the three types to the same extent as of the onset Example 5.

Growth of different bacteria in CAA, CAA + V (1 mM), CAA + V (2 mM), and CAA + V (3 mM) without and with FeCl₃ (10 μM) expressed as duration of lag phase (in hours)

* Cultures done m LB (iron-rich medium)

Each culture was done in triplicate and values for PAOl, ATCC 27853, and PA6 are the average of at least three independent expeπments Example 6.

Growth of different bacteria in CAA, CAA + V (1 mM), CAA + V (2 mM), and CAA + V (3 mM) without and with FeCI₃ (10 μM) expressed as time to reach an OD 600 nm of 0.2

* Cultures done in LB (iron-rich medium)

Each culture was done in triplicate and values for PAOl, ATCC 27853, and PA6 are the average of at least three independent experiments. Example 7.

Effect of EDDHA on growth of Pseudomonas aeruginosa without and in combination with vanadium expressed as duration of lag phase (in hours)

Each culture was done in triplicate

Example 8.

Effect of EDDHA on growth of Pseudomonas aeruginosa without and in combination with vanadium expressed as as time to reach an OD 600 nm of 0.2

Claims

1. Pharmaceutical composition comprising at least one transition element and/or salt thereof which interferes with a bacterial and/or fungal/yeast iron- uptake mechanism.

2. Pharmaceutical composition according to claim 1 wherein the interference occurs through a high affinity siderophore mediated iron-uptake system present in bacteria and/or fungi/yeast.

3. Pharmaceutical composition according to claim 2 wherein the interference occurs at the level of the pyoverdine-mediated iron transport pathway.

4. Pharmaceutical composition according to claim 1 , 2 or 3 wherein the bacteria are gram-negative bacteria.

5. Pharmaceutical composition according to claim 4 wherein the gram-negative bacteria are selected from the group comprising Pseudomonas, Klebsiella, Stenotrophomonas, Escherichia, more specifically E.coli.

6. Pharmaceutical composition according to claim 1 , 2 or 3 wherein the fungus/yeast is selected from the group comprising Candida, Histoplasma, Cryptococcus or Rhodotorula, more specifically Candida albicans, Histoplasma capsulatum, Cryptococcus neoformans or Rhodotorula pilimanae.

7. Pharmaceutical composition according to any of the preceeding claims wherein the transition elements are selected from the group comprising vanadium, chromium or titanium and/or salts thereof.

8. Use of a pharmaceutical composition according to any of the claims 1-7 to treat burns.

9. Use of a pharmaceutical composition according to any of the claims 1-7 to treat yeast infection.

10. Use of a pharmaceutical composition according to claim 9 wherein said yeast is Candida sp.

11. Use of a pharmaceutical composition according to any of the claims 1-7 to treat opportunistic infections.