IT8322212A1 - Metal complexes of ovotransferrin in pharmaceutical compositions with antimicrobial activity - Google Patents

Description

Description of the Industrial Invention, entitled:

"Metal complexes of ovotransferrin in pharmaceutical compositions with antimicrobial activity"

SUMMARY

The object of the present invention? consisting of complexes of ovotransferrin with transition metals, prepared by reacting pure ovotransferrin with a salt of the transition metal in the presence of alkaline bicarbonate.

It has been found that these complexes are endowed with a good activity? antimicrobial and antifungal.

Transformed into suitable compositions, they can be administered orally, topically or parenterally in the case of bacterial and fungal infections.

The process, for their preparation and their use in pharmaceutical compositions, constitute an integral part of the present industrial invention.

DESCRIPTION

The present invention relates to the use in the pharmaceutical field of metal complexes of ovotransferrin, in particular to obtain compositions having considerable activity. antimicrobial and antifungal.

How ? known, ovotransferrin or with albumin, generally called OVT,? a gliocoprotein of molecular weight about 80.000 present in considerable quantity? in egg white.

This protein, like the other transferrins,? characterized by the ability? to bind transition metals, with a high activity? reactive.

It is also? known that ovotransferrin possesses, as in general all transferrins, the capacity? to inhibit the growth of a large number of Gram and Gram microorganisms -,

The activity? antibacterial of ovotransferrin yes? has been shown to be useful in human therapy, in controlling enteric microbial infections.

This result? especially evident in the case of bacterial enteritis of the infant fed with cow's milk.

Indeed, human milk? rich in lactotransferrin which, on the other hand, is almost missing? totally in cow's milk.

Therefore, in the case of a feeding with cow's milk, the administration of OVT can? to substitute the protection offered naturally in human milk? by lactotransferrin.

In infants fed with cow's milk, the conditions of breastfeeding can be restored, as far as this protection is concerned, by administering OVT in quantities? similar to those of lactoferrin found in human milk.

The administration of pharmaceutical preparations based on ovotransferrin therefore finds indications in the prevention and therapy of bacterial enteritis in the infant, especially if the latter? fed with cow's milk.

Purpose of the present invention,? to improve the activity? antimicrobial and anti-fungal of OVT, keeping its toxicity at low levels or practically absent.

It has now been found and forms the subject of the present invention that this and other objects are achieved by using the OVT complexes with the transition metals.

The metal complexes of conalbumin are well known (see for example Robert C. Warner and I. Weber, J.A.C.S., 75, 5094, 1953).

The use of these compounds to prepare pharmaceutical compositions having activity? antimicrobial and antifungal wasn't it for? never been described in the literature so far.

Among the complexes of OVT with such metals, the preferred ones, according to the present invention, are the complexes of zinc and copper and, of these two, the zinc complex has shown, in practice, in a more effective way. remarkable, the property? to inhibit the growth of numerous bacterial species, including those that have been shown to be resistant to the action of unsaturated OVT alone.

Furthermore, the OVT complexes with the transition metals, object of the present invention, show their inhibition effect even on non-bacterial species, of considerable importance in pathology, such as, for example, towards organisms of the genus Candida, and, moreover, they have a low toxicity? and a high tolerability? local

The remarkable properties antimicrobial and antifungal of the complexes, object of the present invention, and in particular: of those with zinc, of the OVT, were absolutely unpredictable, on the basis of current knowledge. Indeed:

1) These properties they are inexplicably greater than those presented by other OVT derivatives with other similar metals

2) are they not interpretable or deducible from the mechanism of action, generally accepted for the properties? antibacterial agents of transferrins, according to which this effect? due to the removal of iron from the culture medium: in fact, the affinity? of iron for the OVT-Zn complex? less than for the free OVT.

2

3) zinc? a metal that is part of the essential elements of organisms, and its other compounds show no activity? antibacterial.

What above I export with regard to the complex of the OV with zinc, applies in part also to the complex of copper, as well as? to other OVT complexes and, absolutely remarkable, also to the OVT complex with iron.

Activities antibacterial and antifungal presented by the complexes of the OVT with transition metals, object of the present invention, have been examined, both in vitro on numerous bacterial strains belonging to different genera, well known for their pathogenicity, and in vivo, for example, on male mice of the CD1 strain or on male rats of the Wistar strain.

All these examinations have shown clear and remarkable activities? antibacterial and antifungal, carried out by the OVT complexes, object of the present invention, and their lack of signs of toxicity? local or general or signs of hypersensitivity, even after prolonged administration.

The complexes of the OVT with the transition metals, which are the object of the present invention, can be prepared, according to known techniques, taking into account that the preparation of exactly definable complexes, from a chemical and biochemical point of view, requires a rigorous control of the mode? reaction, as regards the presence of some anions, which can become part of the complex and on which the properties depend. of the same.

A method of preparation, preferred in the specific case of the present invention, consists in reacting the pure OVT, dissolved in an aqueous solution of an alkali metal bicarbonate, with a pH of about 8-10, with an amount? stoichiometric or slightly higher than a transition metal salt dissolved in water.

The quantity stoichiometric of salt? given by the ratio of one gram atom of metal to about 40,000 molecular weight OVT.

The reaction is carried out under stirring, preferably at a temperature lower than or equal to the room temperature, for a time ranging from a few hours to a day.

The complex formed is purified, by known dialysis techniques, ultrafiltration with membranes or molecular filtrations and then dried by lyophilization or spray-drying.

In this way, OVT complexes are obtained in the form of powder, which can be used for the preparation of pharmaceutical compositions, mainly suitable for use in warm-blooded animals, such as:

1) for oral administration: powder as it is, included, concentrated solutions, gastro-resistant granules, capsules, etc.

2) For topical use: concentrated aqueous solutions with commonly used preservatives such as benzoates, or gels, obtained with common gelling systems, etc.

3) Administration by parenteral route: aqueous solutions pi? or less concentrated, depending on the case, etc.

In order to better understand the present invention and to implement it, an exemplary embodiment is illustrated, but which must in no way constitute a limitation of the present invention.

EXAMPLE

A. Preparation of the OVT - Zn2? Complexes, OVT - Cu2 and OVT - Feg:

200 g of pure OVT in the form of lyophilized powder are added, under stirring, to one liter of a 5 mM sodium carbonate solution, keeping the pH around 9.0.

To the solution of the protein so? obtained, a solution in water of 10 M Zinc Sulphate is added, under stirring, in small volumes, until a stoichiometric Zinc concentration is reached with the protein sites, or at most 3-5% in excess.

The operation is carried out at room temperature and after the addition of the Zinc Sulphate solution, the solution is left to stand at 4? -10? C, for at least 10 hours. After this period, the OVT-Zn2 complex is further purified from low molecular weight components, possibly present by dialysis, ultrafiltration through membranes and / or molecular filtration.

At the end of these operations, the protein solution is dried by lyophilization.

By operating under the same process conditions indicated above and replacing only the zinc sulphate solution with a copper or iron sulphate solution, the 0VT-Cu and OVT-Fe complexes can be prepared.

B. Evidence of activity antimicrobial in vitro of the complexes in A.

Numerous bacterial and non-bacterial strains were examined; belonging to different genera, known for their pathogenic? t ?.

These strains came from international collections or from clinical isolates.

In the second case, identification was carried out by means of biochemical and cultural tests, in accordance with the Bergey's Manual. The data reported in table I are representative of the differences found between genera and within this genus, between species and species.

The tests were carried out in a multi-biophotometer, inoculating 10 cells / ml, coming from a pre-culture of 18 hours, in a suitable development medium - (commercial standard media) -added with 50 mM Sodium Bicarbonate. The optimal growth pH of the different microorganisms was obtained by adding ac. diluted acetic. The OVT and the lyophilized OVT-Zn2 or OVT-CU2 complexes were added to the culture medium in the quantity? of 5 mg / ml in the activity tests? antibacterial and 1 mg / ml in those of activity? antifungal. The development of the microorganisms was followed for 20 hours, with the continuous progress of multiplication. At the end of the test, the viable cells were counted by sowing the serial dilutions of the broth culture on a suitable solid medium.

For the genus Candida, the counts on solid ground were not reliable due to the phenomenon of aggregation; the cells were then counted on a Blirker blood count. From the data reported in table I, you can? note how Gram bacteria, excluding S. aureus, are completely inhibited by both unsaturated OVT, OVT-Zn and OVT-Cu. Gram bacteria - are they proven to be more? resistant to the action of OVT and OVT-Cu2, but, in almost all saci, they are very sensitive to the action of OVT-Zn. However yes? noted a considerable delay (4 to 8 hours) in the beginning of the logarithmic phase. With respect to the genus Candida, the metal derivatives of OVT prove capable of inhibiting development even at concentrations lower than those used in bacteria; only the Candida krusei yes? proved resistant. Furthermore, morphological differences have been noted in Candida, especially in the presence of OVT-Zn and 0VT-Cu2, which consist in the formation of aggregates (clumps) and in the non-formation of the germinative tube (pathogenicity criterion for some species) .

TABEL1

as could be deduced from the in vivo behavior. In evidence d? toxicity for i.p. administration there were no signs of toxicity? acute in mice up to doses of OVT-Zn2 greater than 2000 mg / kg animal weight.

With oral dosing, don't you? was it possible to highlight toxic effects up to the pi? high doses possible, either in a single administration greater than 1000 mg / kg p.a., or with the same doses for 21 consecutive days.

TABLE II

C. Evidence of activity antibacterial in vivo of the complexes prepared in A,

These analyzes demonstrate the potential therapeutic applications of the complexes of the invention in the field of the therapy of bacterial and fungal infections in warm-blooded animals. This example, reported in table II, is not, therefore, limiting the use of the substance, nor is it limited. much less for the route of administration and the dosages, which, moreover, can be extended, from time to time, according to the current knowledge of the state of the art available to connoisseurs of the subject. Male mice of COI strain of about 27 g were used for the test, roughly divided into three groups of about 50 animals each. The animals were infected with cavity inoculation. peritoneal of 0.5 ml of a broth culture of Salmonella SA 12 (5X10 total cells) in gastric mucin at 4%. After 15 minutes 0.5 ml of physiological solution is inoculated in the controls, and 0.5 ml of a 100 mg / ml solution of 0VT or of the OVT-Zn complex (pH 7) in the treated ones. The observation of infected animals is carried out every 12 hours; lethality? maximum? 72 hours after inoculation; after this period, in fact, there were no more? deaths. In the untreated group, the percentage of surviving mice was 10%, in the group treated with 0VT it was 9.8%, in the group treated with the OVT-Zn complex it was 28%. In vitro tests conducted at the same time have shown that the activity? antibacterial of 0VT-Zn against Salmonella SA 12 was much greater than 0VT, The observation of infected animals is performed every 12h; the data reported refer to 72 h from inoculation as the number of surviving animals is subsequently maintained constant,

Parallel to each in vivo test, an in vitro test is performed: 10 cells / ml of Salmonella SA12 in BHI

How can you? note the greater in vitro inhibition of Salmonella SA 12 growth by OVT-Zn? in accordance with the greater protection operated in vivo by the same.

D. Evidence of activity? in vivo antifungal of the complexes prepared in A.

Male rats of the Wistar strain of about 300 g were used, immunosuppressed by administering high doses of cortisones for 15 days, randomly divided into 5 groups of 10 animals each. A small portion of the abdomen skin of all rats was shaved, thoroughly disinfected and scarified. Subsequently, a drop of a 12 hour broth culture of Candida albicans was placed on the lesion. The lesion was then sprinkled, in the animals of one group, with OVT.

Subsequently, the animals of the second group were treated with the OVT-Zn complex, those of the third group with OVT-Cu2, those of the fourth group with OVT-Fe2, while those of the fifth group were not treated. The lesion was covered with an occlusive dressing.

Every 24 hours, the lesion was discovered, serosity was taken with a slide. present, a new bandage was proceeded, preceded in the animals treated by a new dressing with OVT or OVT-Metal complex. The slide was washed with 1 ml of physiological solution and the cell count was carried out on the latter on a Burker blood count. Gi? from the first day of treatment, in animals treated with an OVT-Metal complex, a decrease in the number of cells is noted, which disappear from the lesion after a few days; rei group treated with unsaturated OVT there is a decrease more? contained in the number of Candide at the site of the lesion, and also the disappearance occurs with a slight delay, compared to the previous groups.

Finally, in the untreated group, the decrease in the number of cells? much more? slow and still, after several days, you will rir considerable quantities? by Candide.

During the whole treatment, there were no signs of toxicity. local or general, due to saturated or unsaturated OVT, n? signs of hypersensitivity attributable to the protein itself.

Claims (10)

1. Complexes of ovotransferrin with transition metals to activity? antimicrobial and antifungal. 2. Ovotransferrin complexes, according to claim 1, in which the transition metal? zinc. 3. Ovotransferrin complexes, according to claim 1, wherein the transition metal? copper. 4. Ovotransferrin complexes, according to claim 1, wherein the transition metal? iron. 5. Process for the preparation of the ovotransferrin complexes with a transition metal, according to any one of the preceding claims, in which the pure ovotransferrin, dissolved in an aqueous solution of an alkali metal bicarbonate with a pH of about 8- is reacted. 10, with a quantity? stoichiometric or slightly higher than a salt of the transition metal, dissolved in water. 6. Process according to claim 5, in which the reaction is carried out at a temperature lower than or equal to the ambient one, for a time ranging from a few hours to a day. 7. Process according to claims 5 and 6, wherein the complex of ovotransferrin with a transition metal is purified by dialysis, ultrafiltration with membranes or filtrations and then dried by lyophilization or spray-drying. 8. Use of complexes of ovotransfemin with a transition metal, according to any one of the preceding claims from 1 to 4, for the preparation of pharmaceutical compositions, having the same activity. anti-microbial and anti-fungal. 9. Pharmaceutical compositions containing an ovotransferrin complex with a transition metal, according to any one of the preceding claims from 1 to 4, suitable for OS administration, for topical use, or for relative administration, having activities? anti-microbial and anti-fungal. 10. Pharmaceutical compositions, according to claim 9, prepared in the form of powders, aqueous solutions or gels, suitable for topical administration in the therapy of skin infections in warm-blooded animals.