IT9048540A1 - PHARMACEUTICAL COMPOSITION CONTAINING A FERRO-SCHIZOKINENE COMPLEX FOR THE TREATMENT OF FERROPENIC ANEMIA. - Google Patents

Description

DESCRIPTION of the industrial invention entitled: "PHARMACEUTICAL COMPOSITION CONTAINING AN IRON-SCHIZOKINENE COMPLEX FOR THE TREATMENT OF FERROPENIC ANEMIA"

DESCRIPTION

Object of the present invention are pharmaceutical compositions containing an iron-schizokinene complex (Fe-SK) for the treatment of iron deficiency anemia.

As is known, an essential requirement for tissue growth, both in humans and animals, is an adequate supply of iron to the body. Although there is usually a large amount of iron in the diet, the level of iron absorption from food is generally low, so that the body's iron supply can easily become critical under various circumstances. Iron deficiency anemia is commonly encountered in pregnancy and can also present problems in the newborn, particularly in certain animal species. Furthermore, in certain pathological conditions there is a poor distribution of iron in the body, which leads to a state of chronic anemia. This is observed for example in chronic diseases such as rheumatoid arthritis, and in certain haemolytic and neoplastic diseases.

Although a wide range of iron compounds are already on the market for the treatment of iron deficiency anemia, the level of iron absorption by the body of these compounds is often very low, requiring the administration of levels relatively high dosage. The administration of iron compounds in high doses, poorly absorbed, can cause siderosis of the intestinal wall and a variety of side effects such as nausea, vomiting, constipation and heavy and foul-smelling stools.

The evolution of the specific sector has also led to the definition of iron-binding complexes for the treatment of ferropenic anemia.

and in which the molar iron: complexing ratio is equal to 1: 3. In this regard, reference should be made to European patents n ° 94149, 107458 and 159917.

The object of the present invention is to provide pharmaceutical compositions for the treatment of iron deficiency anemia, which are new and involve an inventive step, since they are not obvious to those skilled in the art who are familiar with the state of the art in the field.

The present invention relates to pharmaceutical compositions containing an iron-schizokinene complex (Fe-SK), which has been shown to possess important properties for use, at relatively low dosage levels, in the treatment of iron deficiency anemia both in humans and in a veterinary context.

Schizokinene (SK) belongs to the family of siderophores, ie complexing agents capable of binding iron synthesized by bacteria (in this specific case by Bacillus megaterium) in response to the almost complete insolubility of iron in physiological conditions. The siderophores are excreted in the environment in which the bacterium lives (for example soil, aqueous environment, or human or animal host) where they meet iron, chelate it, solubilize it and transport it to bacterial cells.

In the Fe-SK complex, iron is in the ferric state, i.e. in the form of an ion with three positive charges. The molar ratio of Fe to SK overall is 1: 1.

Schizokinene is a compound formally derivable from a citric acid residue symmetrically substituted by amide bonding with the amino groups of two 1-amino-3- (N-hydroxy, N-acetyl) aminopropane residues.

Its structural formula (K.B. Mullis, J.R. Pollack, J.B. Neilands, Structure of Schizokinen an Iron - Transport Compound from Bacillus megaterium, B IOCHEMISTRY, Vol. 10, n ° 26, 1971, pp. 4894-4898) is as follows:

From the structural formula of schizokinene, it is understood that the two groups of hydroxanunic acid play an important role in the formation of the complex with iron

It has been seen that at pH greater than 3 the hydroxyl group of citric acid also participates in the complexation of the ferric ion. The formation of the complex is complete at pH 5-6.

The preparation of the Fe-SK complex is carried out in several steps, according to the following sequence:

- Bacillus megaterium culture and SK production

- extraction of SK from the culture medium of the bacterium

- chemical purification of SK

saturation of SK with Fe

- lyophilization of the Fe-SK complex.

Each of the stages involves specific operating conditions, on which the production yield of SK from the bacterium and the extraction and purification yields of this product entirely depend. In the following, a complete and detailed example of a preparative methodology will be provided, by way of non-limitation.

The preparation of the pharmaceutical compositions according to the present invention must take into account the strong hygroscopic character of the Fe (III) -SK complex (absorption of 3 moles of water per mole of complex) and of the fact that at acid pH the complex can undergo dissociation or degradation molecular.

The present invention relates to pharmaceutical compositions for the treatment of iron deficiency anemia, characterized in that they contain, as at least one of the active components, a physiologically effective amount of an iron (III) -schizokinene complex in a molar ratio 1: 1, or a salt thereof containing a physiologically acceptable cation, and a vehicle suitable for the selected administration. Although the Fe (III) -SK complex is stable within a wide pH range, it tends, at pH values below 4 present in the stomach, to a situation of dissociation or even degradation of its molecular structure.

It has been found that the formulation of the Fe (III) -SK complex in the form of a pharmaceutical composition suitable for oral or nasal administration only has numerous advantages. First of all, because the oral and nasal cavities represent an environment with a pH of around 7, and a neutralized Fe (III) -SK complex is absorbed by the membranes of said cavities without any danger of dissociation or degradation of its molecular structure.

In the case of oral administration, the pharmaceutical composition according to the invention contains a solid vehicle and is suitable for chewing, or sucking (although in the process of chewing and sucking a portion of the complex can obviously pass into the stomach), or swallow. These pharmaceutical compositions can for example be in the form of chewing or bolus gums, candies, simple, chewable or effervescent tablets, particularly sugared almonds, capsules and granulates in sachets. The pharmaceutical compositions according to the present invention can be used for oral use in general. In this case they contain a solid or liquid vehicle which makes them suitable for swallowing, for example in the form of capsules or vials for extemporaneous preparation. The specific vehicles that can be used in confetti and compressed boluses are many. An example of a bolus base consists of a mixture of gelatin, glycerin, sugar, citric acid and an optional dye. The rate at which the bolus dissolves in the mouth can be varied as desired in order to obtain a good level of iron absorption, the dissolution rate being reduced, for example, by increasing the proportion of gelatin used. Boluses can conveniently be prepared by forming a melt containing an adequate amount of the iron complex and the carrier and then pouring it into a mold and allowing it to dry. A particular example of a chewable tablet base consists of a mixture of sucrose and gum arabic. Again, the rate of dissolution can be controlled by varying the ingredients in the base material. The tablets can be conveniently prepared by compression. If desired, additional flavoring agents may be incorporated into the lozenges or tablets, but this may be unnecessary, except perhaps for pediatric recipes.

When compositions for nasal administration are used, these will usually be liquid and may comprise water and / or suitable organic solvents. Such compositions can conveniently be used both in the form of drops and in the form of aerosol sprays. It is, however, possible to use solid compositions in the form of a snuff product if desired. In the case of compositions for nasal administration, the physical characteristics of the compositions may not differ so greatly from those of compositions for other modes of administration as in the case of compositions for oral administration. However, the intended manner of all forms of composition according to the present invention will generally become clear from the packaging of the composition, for example in a container for dispersing a spray or drops, and / or the instructions which are attached to it specifying the oral or nasal use.

The Fe (III) -SK complex may also be suitable for swallowing in the form of a pharmaceutical composition with delayed delivery by means of conventional protective agents.

The physiologically effective amount in the Fe (III) -SK complex can be between 0.1 and 1g.

To avoid dissociation of the Fe (III) -SK complex (even if the nature of the compositions should ensure that most of the Fe complex is released in a substantially neutral environment, partial swallowing can never be excluded) the use of free schizokinene, with the Fe (III) -SK complex, or a salt thereof containing a physiologically acceptable cation, is envisaged.

If desired, other active compounds may be present in the pharmaceutical composition according to the present invention, for example compounds with the ability to facilitate the treatment of anemia, such as folic acid. Another additional component that can be included in the composition, if desired, is a source of zinc. Iron compounds conventionally used in the treatment of iron deficiency anemia can hinder the zinc absorption mechanism in the body and this can cause severe side effects in the fetus when treating anemia in a pregnant female. It is believed, however, that iron complexes according to the present invention have the further advantage of not having this effect or of exhibiting the effect to a lesser extent than the compounds currently used in the treatment of anemia. Consequently, it can often happen that the level of the zinc providing compound added to the composition is not necessarily high or, with the preferred pharmaceutical compositions according to the present invention, it can be suppressed entirely.

The compositions according to the present invention can be formulated in the form of unit dosage, i.e. in the form of distinct portions containing a unit dose, or a multiple or subunit dose.

Up to now, a general description of the Fe (III) -SK complex for the preparation of pharmaceutical compositions for the treatment of iron deficiency anemia has been given. A more detailed description will now be provided with the help of the following examples to better clarify its purposes, characteristics, advantages and operation.

EXAMPLE 1

Bacillus mecraterium culture and schizokinene production

The strain of Bacillus megaterium that produces SK must be strictly maintained on a mineral medium containing glucose or iron-free sucrose. It must also be prepared, conditioned and used in glass bottles or Petri dishes that are equally iron-free. All the glassware used to prepare the product must be washed with 6N HC1, then rinsed with distilled, demineralized and bidistilled water.

1. Composition and method of preparation of the culture medium to maintain the strain (SK agar-agar):

SK agar-agar can be prepared in carefully washed pyrex bottles as indicated below, with high quantity chemicals free from mineral impurities. The composition of SK agar-agar is as follows (quantity in grams per 1 liter of SK broth):

The pH is adjusted to 7.0 with concentrate. 15 g of agar (Difco Lab Detroit, Mich. USA) are added per liter of culture medium. This gelling agent is melted by heating the medium to 100 ° C in a bain-marie for 10 minutes. It is brought in an autoclave at 115 ° C for 20 minutes.

This SK culture medium is divided, at the exit of the autoclave, in tubes or in sterilized Petri dishes, or it is stored in bottles at laboratory temperature.

The Bacillus megaterium strain is spread on this culture medium and divided into Petri dishes; these are incubated at 37 ° C for 24-48 hours, then stored at 4 ° C in the dark. Prolonged storage of the strain can be obtained by storing in liquid nitrogen a suspension of cells in sterile physiological solution added with glycerol treated in an autoclave (10%, V / V).

2. Production of SK from the bacterium

Culture medium

The production is carried out in SK broth, the composition of which is identical to that of the SK agar-agar without the addition of agar. This broth is prepared in 10 liter pyrex flasks, which allow the preparation of 6 liters of culture medium. After treatment in an autoclave (115 ° C, 20 minutes) and cooling, the SK broth shows an important precipitate. It is decanted and the supernatant is transported aseptically, with the help of a sterile peristaltic pump, in a sterile 10 liter flask. 5 liters of sterile clear medium (SK-base broth) can also be recovered.

1.5 g of arginine (hydrochloride) per liter are added to this broth-base. This amino acid must not be autoclaved. At the moment, a concentrated solution is prepared which is sterilized by membrane filtration (Millex Millipore, pores of 0.22 μπι), which is then added to the broth in an adequate quantity to obtain a final concentration of 1.5 g / liter.

Inoculation and culture

A fresh culture of the strain is prepared on SK agar. A dense suspension of these cells is prepared in 10 ml of sterile physiological solution (or in 10 ml of SK-base broth); the 5 liters of SK broth are inoculated with this suspension. It may be interesting to add a small amount of pure SK to this medium: the start of the growth process in this case is faster.

The flask is incubated at 37 ° C for 6 days, under violent stirring (thermostated water bath with stirrer, or stove under magnetic stirring). Soil aeration is very important for SK production.

At the end of the incubation period, the culture medium is freed from the cells (centrifugation, membrane filtration). This phase no longer requires aseptic conditions if the SK extraction is done quickly. In any case, the filtrate must be stored at 4 ° C.

The pH of the concentrate is brought to 2 with concentrated HC1 (36 °) and it is poured into a decantation ampoule. Two volumes of phenol (crystallized) -chloroform mixture (1: 1, weight and weight) are added and the mixture is stirred. It is left to decant and the lower aqueous phase is discarded. 4 volumes of ethyl ether are added to the organic (upper) phase. He shakes violently and lets himself be decanted.

It is extracted with a small amount of distilled water (1/20 of the amount of ethyl ether used). He shakes violently several times. Schizokinene passes into the aqueous fraction. This is recovered in an evaporation flask. The extraction is carried out three times with water and the aqueous fractions are gathered in the same flask. The aqueous extract is concentrated under vacuum at 40 ° C (rotary evaporator) up to 1/10 of its volume.

3. Purification:

The purification is carried out on Dowex resin (AG-2-X10), in a column with a diameter of 2.5 cm and a length of 25 cm. The concentrated aqueous extract is applied to the column. It is then washed with 100 ml of distilled water, then eluted with a 0.2 M NH4Cl solution. The collected fractions (5 ml each) are analyzed with the iron perchlorate reagent: all those which give a red color are collected in a flask and concentrated under vacuum up to 1/10 of volume. The schizokinene of this concentrate is pure.

4. Iron saturation of SK

The pH of the SK concentrate is brought to 7.0 with KOH.

A concentrated solution of FeC ^ (10%, W / V) is added until the absorption is saturated at 500 nm, measured with the spectrophotometer. In practice, it is preferred to define the saturation curve with the help of an aliquot of diluted concentrate, then calculate the amount of iron needed to saturate all of the remaining schizokinene concentrate and carry out this operation at once. The dosages carried out on different samples show that, when saturated, the complex contains about 6 to 10% of iron (weight-weight).

The Fe (III) -SK complex is then divided into freeze-drying ampoules, frozen and lyophilized. The product is hygroscopic and can absorb 3 moles of water per mole of complex. It is therefore necessary to keep it in corked or sealed bottles (or ampoules).

5. Production yield

The yield is about 0.9 g of complex per liter of culture medium.

EXAMPLE 3

Plasma iron levels in rats after single administration of the Fe (III) -SK complex. Comparison: microencapsulated ferritin.

35 Sprague-Dawley rats, weighing about 200-220g, fasted for 24 hours, were divided into the following groups:

1st group (5 specimens): control (no treatment);

2nd group (15 specimens): treated with microencapsulated ferritin, suspended in water, administered orally at a dose corresponding to 6 mg Fe / kg;

3rd group (15 specimens): treated with Fe (III) -SK, dissolved in water, administered orally at a dose corresponding to 6 mg Fe / kg.

The rats belonging to the second and third groups were divided into three subgroups from which blood was taken, carefully avoiding haemolysis, respectively 1 hour, 2 hours and 6 hours after administration.

The piasmatic concentration of iron was

determined by atomic absorption with lamp

specific .

Tables 1 and 2 show respectively the

piasmatic concentration of iron after

oral administration of the Fe (III) -SK complex e

of ferritin-based comparator ad

a dose corresponding to 6 mg Fe / kg (the values are

expressed in μg / 100 ml of plasma).

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Claims (17)

CLAIMS 1 Pharmaceutical composition for the treatment of iron deficiency anemia, essentially characterized by the fact of containing, as at least one of the active components, a physiologically effective quantity of an iron (III) -schizokinene complex, or of a salt thereof containing a cation physiologically acceptable, and physiologically acceptable vehicles suitable for administration. 2 Pharmaceutical composition for the treatment of iron deficiency according to claim 1, in which the physiologically effective amount of the iron (III) -schizokinene complex is comprised between 0.1 and 1g. 3 Pharmaceutical composition for the treatment of iron deficiency anemia as per claim 2, which contains a solid vehicle and is suitable for chewing or sucking. Pharmaceutical composition for the treatment of iron deficiency anemia as per claim 3, in the form of chewable tablets. 5 Pharmaceutical composition for the treatment of iron deficiency anemia according to claim 3, in the form of granules. Pharmaceutical composition for the treatment of iron deficiency anemia according to claim 3, in the form of chewing gum, 7 'slices or boluses. Pharmaceutical composition for the treatment of iron deficiency anemia according to claims 1 and 2, which contains a solid or liquid vehicle which makes it suitable for swallowing. 8 Pharmaceutical composition for the treatment of iron deficiency anemia as per claim 7, in the form of tablets, effervescent tablets, capsules or dragees. Pharmaceutical composition for the treatment of iron deficiency anemia according to claim 7, in the form of vials for extemporaneous preparation. 10 Pharmaceutical composition for the treatment of iron deficiency anemia according to claims 1 and 2, which contains a liquid or solid vehicle and is suitable for nasal administration. 11th Pharmaceutical composition for the treatment of iron deficiency anemia according to claims 10, in the form of drops. 12. Pharmaceutical composition for the treatment of iron deficiency anemia according to claim 10, in the form of an aerosol spray. 13 Pharmaceutical composition for the treatment of iron deficiency anemia according to claim 10, in the form of a snuff product. Pharmaceutical composition for the treatment of iron deficiency anemia as per the preceding claims, containing compounds capable of facilitating the treatment of anemia. Pharmaceutical composition for the treatment of iron deficiency anemia as per claim 14, wherein the compound capable of facilitating the treatment of anemia is folic acid. 16. Pharmaceutical composition for the treatment of iron deficiency anemia as per the preceding claims, containing free schizokinene, or a salt thereof containing physiologically acceptable cations. 17 Pharmaceutical composition for the treatment of iron deficiency anemia as per the preceding claims, containing a source of zinc. Formulations of pharmaceutical compositions for the treatment of iron deficiency anemia according to the preceding claims, in the form of distinct portions containing a physiologically effective unit dose, or a multiple or submultiple dose thereof.