IT8922399A1 - BIOAVAILABLE IRON-ALBUMIN DERIVATIVES, THEIR PREPARATION PROCEDURE AND RELATED PHARMACEUTICAL COMPOSITIONS. - Google Patents

Description

Description of the industrial invention entitled: BIOAVAILABLE IRON-ALBUMIN DERIVATIVES, METHODS FOR THEIR PREPARATION AND RELATED PHARMACEUTICAL COMPOSITIONS"

SUMMARY

Iron-albumin derivatives are described, consisting of albumins modified with acidic residues and containing high quantities of iron, insoluble at the acidic pH of the stomach and highly soluble at intestinal pH values.

The derivatives are characterized by very low gastrolesivity and toxicity and by valid bioavailability and therapeutic efficacy in the treatment of anemic conditions.

DESCRIPTION OF THE INVENTION

The present invention relates to albumin derivatives acylated with dicarboxylic acids containing iron in a bioavailable form.

In oral martial therapy, the use of iron complexes with acylated proteins is well known. For example, Italian patent no. 1150213, in the name of the applicant, claims an iron adductor based on succinylated proteins, obtained by reacting ferric salts with carrier proteins of animal origin, such as milk, organ, whey, or plant proteins.

The diverse composition of these non-isolated proteins, however, poses a significant challenge in consistently preparing these derivatives. Furthermore, although it is possible to obtain complexes with high iron content (up to 20%), the high amount of iron present in the derivatives also leads to increases in solution viscosity.

In fact, the ferroproteinsuccinylated derivative (obtained according to Italian patent no. 1150213) used for the appropriate pharmaceutical formulation and currently on the market contains 5% iron by weight. The possibility of obtaining derivatives that give low-viscosity solutions even when quantities well above 5% are supported by succinylated proteins would represent a clear improvement in the preparation of derivatives for pharmaceutical use.

However, for this class of therapeutic agents, the most important aspect that needs improvement is certainly the reduction or complete elimination of gastrointestinal effects related precisely to the therapeutic principle, that is, supplying iron to the body.

It has now been found, and this is the object of the present invention, that by using albumin as a protein to obtain these acylated derivatives, highly soluble bioavailable iron compounds are obtained even if they contain high quantities of iron, and the possible toxicity as well as any side effects of gastrointestinal damage of these drugs, related to poor solubility of the iron derivatives or precipitability in the intestine or the generation of hydroxyl radicals resulting from the release of iron in ionic form, are minimized and practically eliminated.

Various albumins can be used for the preparation of the derivatives of the invention, particularly preferred are high-purity bovine serum albumin and lactalbumin.

According to the invention, albumins are acylated with carboxylic groups such as, for example, succinic, glutaric, maleic, malic, malic acid, aspartic, glutamic, etc. groups. The succinic group is particularly preferred, as it has proven to be the most suitable for increasing some characteristics of proteins, in particular solubility and degradability by the action of proteases.

More particularly, the present invention relates to derivatives based on iron and highly succinylated bovine serum albumin, carrying quantities of iron between 3 and 10% by weight, and succinylated lactalbumin carrying in solution quantities of iron up to 20% by weight, both highly soluble, characterized by the fact that:

a) they are not harmful to gastroenterology, given their ability to precipitate in the acidic environment of the stomach while retaining the bound iron and to dissolve again in an alkaline environment corresponding to that of the intestine, b) they are not toxic at the cellular level given their low tendency to generate free radicals, c) they are effective in the treatment of anemia.

These derivatives can be obtained in an aqueous environment by reacting the above-mentioned succinylated or acylated proteins with iron ions at a pH between 2 and 12, preferably between 3 and 7.

Some preparation examples are reported which are not intended to be limiting, of the invention.

The invention will now be described in more detail in the following non-limiting examples.

EXAMPLE 1

Preparation of Ferrosuccinylalbumin with an iron content of 6% by weight.

20 g of serum albumin is dissolved in 400 ml of water.

The pH is adjusted to 8.0 by adding alkali (e.g., NaOH). 24 g of sucrose anhydride are added in small portions while stirring, maintaining the pH between 7.5 and 8.0 by adding alkali (e.g., 2N NaOH) and keeping the temperature constant at values below 25°C, preferably 20°C. The reaction is allowed to take place for approximately 1 hour.

The pH is lowered to values equal to 3 ? 0.5.

A white precipitate forms and is recovered by centrifugation. To eliminate the excess succinic acid, the precipitate is redissolved in 400 ml of H2O at pH 8.0.

The reaction mixture is acidified until a pH of 3 ≤ 0.5 is reached (with N HCl2). A white precipitate is formed and recovered by centrifugation. This step is repeated until the suocinic acid is completely eliminated. The final precipitate obtained is dissolved in 400 ml of H2O by adding 2N NaOH until a pH of 8.0 is reached. 7.20 g of FeCl36 H2O equivalent to 1.48 g of Fe are added rapidly and while stirring.

A reddish-brown precipitate forms instantly which is washed with K31 at pH 3, recovered by filtration (or centrifugation) and dried under vacuum.

A reddish-brown powder (19 g) is obtained that is insoluble in water and becomes soluble by adjusting the pH to neutrality through the addition of alkalis (e.g., NaOH, NaHCO3, etc.). Upon analysis, the derivative shows an iron content of 6.1-6.2% by weight. Analyzed by cellulose acetate electrophoresis (as described in paragraph 5), it returns to the electrophoretic field in the form of a single spot in which the iron is detectable in the spot itself.

EXAMPLE 2

Obtaining Ferrosuccinylalbumin with 3.8-4.1% iron content by weight.

The derivative is prepared as described in example 1 with the only variation being that 4.8 g of FeCl 6 H2O (equivalent to 0.992 g of iron) is added.

Upon analysis, the derivative that moves under the electrophoretic plate with a single band in which iron is detectable, presents an Fe content equal to 3.8-4.1% by weight.

EXAMPLE 3

Obtaining Ferrosuccinylalbumin with 9.5-10% iron content by weight.

1.20 kg of deionized water and 1 kg of bovine albumin are loaded into a reactor, obtaining a solution with a pH of 6.7-6.8. A 5N sodium hydroxide solution is slowly dripped until the pH reaches a constant 7.5, and then 1.2 kg of succinic anhydride is added in small portions, maintaining the pH between 7.2 and 7.5 by adding 5N sodium hydroxide. After the addition, the pH is maintained at a constant 7.5 for 30 minutes, then acidified to pH 3 with 6N hydrochloric acid while stirring. The precipitate thus obtained is centrifuged, washed thoroughly with deionized water, and redissolved in 120 ml of deionized water with 5N sodium hydroxide to a constant pH of 7.5, obtaining a clear solution in 30 minutes. The precipitation procedure at acid pH and dissolution at alkaline pH is repeated twice.

The solution finally obtained is treated with a solution of 578.5 g of iron chloride hexahydrate in 1.10 g of demineralized water, maintaining the pH between 6.5 and 6.8 by adding 5N sodium hydroxide. It is then stirred at pH 6.6-6.8 for 30 minutes, acidified to pH 3 with 6N hydrochloric acid and left under stirring for 30 minutes. The precipitate is centrifuged, washed abundantly with demineralized water bringing to pH 8.5 with 5N sodium hydroxide. After maintaining a constant pH of 8.5 for 1 hour, the resulting solution is filtered through a bed of celite (300 g). The precipitation procedure at acid pH and dissolution at alkaline pH is repeated once.

The solution is filtered through a bed of celite (200 g), the product is precipitated at pH 3 with 6N hydrochloric acid, stirring for 30 minutes, centrifuged, and washed thoroughly with demineralized water. The dark brown solid is sieved and dried under vacuum at 30°C, yielding 1 kg of ferrosuccinylalbumin.

The percentage of iron contained in the derivative is between 9.5-10.0% calculated on the dry product.

EXAMPLE 4

Obtaining Ferrosuccinyl-?-lactalbumin with 11-11.45% iron content by weight.

1 kg of lactalbumin dissolved in 20 l of H2O is charged into a reactor. A solution with a pH of 6.7-6.8 is obtained. The protein is succinylated and purified from succinic acid as described in the previous example. 1.2 kg of ferrous chloride hexahydrate in 10 l of water is added to the succinylated protein solution, maintaining the pH between 6.5 and 6.9 by adding 5N sodium hydroxide. The reaction is carried out as described in the previous example, and at the end of the reaction, 1 kg of ferrosuccinyl-?-lactalbumin is obtained, which upon analysis has an iron content of 11-11.45% by weight.

EXAMPLE 5

Obtaining Ferrosuccinyl-?-lactalbumin with 18.3-18.5% iron content by weight.

1 kg of lactalbumin dissolved in 20 liters of H2O is charged into a reactor. A solution with pH 6.7-6.8 is obtained. The protein is succinylated and purified as described in Example No. 3.

To the succinylated lactalbumin solution, 2.4 kg of iron chloride hexahydrate in 101 ml of water are added, maintaining the pH between 6.5 and 6.9 by adding 5N sodium hydroxide. The reaction is carried out as described in Example 3, obtaining 1 kg of ferrosuccinyl-lactalbumin with an iron content of 18.3-18.5% by weight.

EXAMPLE 6

Obtaining Ferroaspartylalbumin with 7.5% iron content by weight.

10 g of breast albumin are dissolved in 200 ml of H2O and the pH is brought to 7.2 by adding NaOH.

Add 10 g of acetylaspartic anhydride in small portions while stirring, maintaining the pH between 7.2 and 7.3 by adding NaOH. The solution is ultrafiltered, maintaining the volume unchanged by continuously adding water through an ultrafiltration membrane with a molecular cutoff of 10,000 Daltons, for 3 hours to eliminate the acetylaspartic acid that passes through the membrane. The solution containing the albumin removed from the ultrafilter is recovered; 7.0 g of FeCl2 is added: a reddish-brown precipitate forms, which is washed with H2O at pH 3, redissolved, adjusting the pH to 7.1, and freeze-dried.

9.5 g of Ferroaspartylalbumin are obtained, which contains 7.5% iron by weight.

EXAMPLE 7

Obtaining Ferromaleylbumin with 6.5% iron content by weight.

The iron derivative is prepared as described in Example 6, with the only variation being that maleic anhydride is used as the acylating agent. A derivative of normal iron albumin is obtained, which contains 6.5% iron by weight.

EXAMPLE 8

Obtaining Fernoglutarylalbumin with 7.0% iron content by weight.

The iron derivative is prepared as described in Example 6, with the only modification being that glutaric anhydride is used as the acylating agent. The resulting fernoglutarylalbumin contains 7.0% iron by weight.

The complex protein derivatives prepared according to the examples described above were analyzed according to the analytical methodologies indicated below.

1) Determination of iron content

In all the derivatives prepared according to Examples 1-8, the iron content was determined by extracting the iron from the sample with 2 N HCl: the quantitative determination was carried out according to the methodology described in Standard Methods 14th ed, 1975, page 208, APH&, AWWA, WPCF (reaction with o-phenanthroline).

In the derivatives, iron is present in the trivalent state as verified by the non-determinability of Fe ions when the reaction with o-phenanthroline is carried out in the absence of reductants.

2) Solubility as a function of pH

All iron derivatives prepared according to the examples given are insoluble at acidic pH and soluble at alkaline pH.

The solubility profile of ferrosuccinylalbumin prepared according to the method described in Example 3 is illustrated as an example.

The solubility profile is obtained from spectrophotometric measurements of the difference in absorbance at 500 nm from that at 280 nm of solutions obtained by treating 20 mg of ferrosuccinylalbumin with 50 ml of H2O in the pH range of 2.0 to 7.0. 5 ml of the sample is diluted to 10 ml with H2O, centrifuged at 3000 rpm for 10 minutes and the supernatant is read spectrophotometrically.

Ferrosuccinylalbumin is completely soluble at pH values equal to or greater than 6.5 while it is insoluble at pH values lower than 6. The inverse precipitation curve, obtained by acidifying the ferrosuccinylalbumin solution (20 mg of product in H2O or pH 7) shows the onset of precipitation at a pH value equal to 4.5.

3) Succinic acid content; 7-13%, determined by GLC (Perkin-Elmer 3B, 3m x 2mm GP 10% SP 1200 H.PCt column on Chromosorb<R>) after hydrolysis of a known amount of sample with 2.5N NaCH at 100°C for 5 hours and subsequent acidification to pH 2-3 with 6N HCl. The released succinic acid is quantitatively transformed into the corresponding methyl succinate by reaction with BF3/CH3OH. Methyl succinate is determined under the following conditions using methylglutarate as an internal standard.

Nitrogen: 30 ml/minute

Air: 3 atm

Hydrogen: 1.5 atm

Injector temperature: 255?C

color it: 140?C

detector F.I.D.: 275?C

Injection: 5 ?l.

4) Determination of protein content

The quantity of proteins present in the samples prepared according to Examples 1-8 was determined by titrating the protein nitrogen (the method described according to the Official Pharmacopoeia IX ed., Vol. I, pp. 191-192 was used).

The values found are between 85% for the sample prepared according to example 3 and 65% for the cairpione prepared according to example 5.

5) Cellulose acetate electrophoresis

Electrophoresis carried out for 20 minutes at 40 V/cm using 0.05 M Tris Tricine pH 8.6 as a buffer reveals bands at the same electrophoretic distance for the derivatives of examples 1-3 and for succinylalbumin.

No bands attributable to the starting bovine albumin were detectable. The presence of iron in the bands of compounds from examples 1-3 was detectable using o-phenanthrotine.

6) UV-VIS spectroscopic analysis

The UV-VIS spectrum in the 200-600 nm region recorded on aqueous solutions at pH 7 (NaOH) containing 1 mg/ml of the capes of the invention highlights an increase in absorbance between 600 and 340 nm, presenting an evident similarity with the ferritin spectrum.

7) Fluorescence emission spectrum

The fluorescence emission spectra between 400 and 200 nm (excitation at 236 nm) show a weak emission equal to about 1/10 of that of the starting protein.

8) ESR spectroscopy

The spectra, recorded both at room temperature and at -160°C with a Varian E 1 D spectranape, are characterized by a single peak of amplitude equal to 1200 G between the positions of maximum and minimum slope with a value of g = 2.00. These signals are attributable to polynuclear complexes (in the specific case of Pe III) characterized by strong exchange interactions.

No other signs attributable to the presence of mononuclear iron were evident.

9) Circular dichroism

From the measurements recorded in the range between 300 and 200 nm on solutions of the ferrosuccinylalbumins of the invention at pH 7 compared to the starting albumin, it is evident that the succinylation reaction leads to the disorganization of the protein structure.

10a) H-NMR spectrum (300 MHz; D 0)

Broadened bands and a weak signal at 2.4 ppm, also present in the succinylalbumin spectrum but in a sharper and more defined configuration.

10b) C-NMR spectrum

Signals at 15-70 ppm (aliphatic CH), 110-140 ppm (aromatic) and 180 ppm (carboxylic). The poor resolution and intensity of the signals at 29-32 ppm (hemisuccinic residue) due to the presence of iron suggests a possible involvement of the succinyl groups in the iron binding.

11) X-ray spectrum

The spectrum in the angular interval 2 between 9° and 51° does not show peaks attributable to the presence of crystallinity centers, thus indicating the amorphous nature of the derivatives prepared according to Examples 1-8.

12) Filter gel (Superose 6)

Non-Gaussian molecular weight profile with main peak at >10<6 >Daltcns and with very broad shoulder up to 10 D.

A definite molecular weight cannot be obtained also due to possible interactions between polynuclear iron hydrate and proteins in aqueous solutions.

13) Sodium Dodecyl Sulfate (SDS) Electrophoresis was carried out in 7.5% polyacrylamide gel containing 1% SDS.

The electrophoretic bands are highlighted with Canassie Brilliant Blue. Standard molecular weights were used: myosin (200 KD), B galactosidase (116 KD), phosphorylase b (97 KD), bovine albumin (68 KD), ovalbumin (42 KD).

Ferrosuccinyl-lactalbumin exhibits the same electrophoretic behavior but has a molecular weight of 20 kD.

14) Amino acid analysis

The analysis was carried out after decomplexing the iron which can interfere with the analysis, by subjecting a sample to total acid hydrolysis with HC16N, under vacuum, at 105?C for 24 h, and determining the amino acid composition with an automatic analyzer (Liquimat III Kontron) using Na buffers as eluents.

The amino acid profile of which an example carpione is reported in table 1.

TABLE 1 - Amino acid composition (Amino acids in moles %)

15) Determination of the degree of succinylation and localization The degree of succinylation is determined both spectrophotometrically according to the reaction of the free amino groups of proteins with ninhydrin and by the fluorimetric reaction with O-phthalaldehyde (G. Goodno et al. Anal. Biochem, 115, 203 (1981). These two methods are used to evaluate the localization of succinylation on the protein fraction comprising the prepared derivatives.

From the results obtained it can be deduced that the terminal α-amino lysine groups and α-amino lysine groups are succinylated in quantities greater than 95%.

16) Proteolysis of TTF 241 F

Enzymatic hydrolysis of samples is performed by treating 25 mg dissolved in 5 ml of 0.05 M Tris-HCl tarpane pH 7.6 with chymotrypsin (50 µg) and trypsin (50 µg). At defined times, 1 ml of the reaction is removed: 2 ml of a 5% trichloroacetic acid (TCA) solution is added. Centrifuge at 3000 rpm for 110 min, and the resulting solution is read spectrophotometrically at 280 nm against a TCA blank.

The variation in absorbency as a function of time expresses the speed of hydrolysis which is comparable to that of albumin, indicating that the iron present in the derivative does not inhibit the action of proteolytic enzymes.

Treatment of ferrous sulfate albumin with proteases produces peptides (identified by electrophoretic determinations) that are able to keep the iron still soluble in an alkaline environment.

17)Iron mobilization

The mobilization of iron from the derivatives of the invention is tested by means of the reaction with desferrioxamine, followed as a function of time by measuring the increase in absorbance at 487 nm of a solution containing, in 3 ml: 7.0 ? moles of desferrioxamine, 500 µg of ferrosuocinylalbumin previously neutralized to pH 7.4 in 20 mM Tris-HCl buffer.

The increase in absorbance at 487 nm in the tarpus confirms that iron can be mobilized from ferrosuccinylalbumin.

18) Viscosity measurements?

Ferrous sulfate albumin samples dissolved in H20 at pH 7.2 in concentrations of 0.1, 1.2, 4.8, and 10% wt. The resulting solutions are analyzed viscometrically with a capillary viscometer.

Solutions prepared with ferroprotein succinylate (a 5% carpione obtained as described in Italian patent 1150213) are analyzed for comparison and at the same concentrations.

The viscosities of the ferrosuccinylalbumin samples at all concentrations were significantly lower than those of ferroproteinsuccinylate.

19) Generation of hydroxyl radicals

The ability of iron derivatives to generate CH radicals has been investigated according to B. Hallisrell, J. Gutteridge, O. Arucma, Anal. Biochem. 165, 215-19 (1987).

As comparison samples, FeCl3 and FeSO4, known for their ability to generate OH radicals, and ferritin, which on the other hand has a poor ability to generate radicals, were used.

Example results are shown in Table 2.

TABLE 2

DEGRADATION OF DEOXYRIBOSE AT pH 4.0

In the absence of ascorbate, free radical formation is significant only with FeSO4 and to a lesser extent with FeCl3.

In the presence of ascorbate, the generation of free radicals from ferrosuccinylalbumin (the sample referred to in the table contains 9.5% iron) is lower than that of ferritin, which is inherently less reactive, and which in turn is much lower than that of FeSO4.

This experiment shows that the ferrosuccinylalbumin derivative has a very low capacity to form free radicals.

20) Generation of free radicals in biological fluids

In the determination of free radicals in biological fluids due to the presence of free iron, the bleomycin assay was used [Life Chemistry Report, 1987, vol. 4, pp. 113-142 - J. Gutteridge, B. Halliwell].

Groups of rats are treated intraperitoneally, (in quantities corresponding to 100 mg of iron/kg) with ferrosuccinylalbumin, with ferroproteinsuccinylate (prepared according to Italian patent no.

1 150213) and (in quantities corresponding to 25 mg of iron/kg) with ferrous sulphate.

Plasma samples are collected after 2, 8, 15 hours and the presence of free iron is determined by the bleomycin assay (see table 3).

TABLE 3

DETERMINATION OF FREE IRON IN PLASMA

Ferrosuccinylalbumin, unlike ferrous sulfate and the protein-succinylated iron sample, does not induce free iron in the plasma, demonstrating that it is free of the damaging effects induced by free radicals generated by the presence of iron.

Toxicity study?

In gastrointestinal tolerability studies, animals were treated orally with ferrosuccinylalbumin (sample prepared according to Example 3, significant given its high iron content and therefore potentially more toxic) or iron sulfate at dose levels of 200 mg/kg. Animals were sacrificed at various times up to 24 h.

Examination of stomach and intestinal preparations showed that intestinal and gastric damage was much less marked after the administration of ferrosuccinylalbumin than after ferrous sulfate.

The acute toxicity for the sample was compared with iron sulfate and the results are presented in Table 4.

It is noted that ferrosuccinylalbumin is much less toxic than ferrous sulfate, and does not produce signs of toxicity up to 2000 mg/kg.

Subacute oral toxicity with repeated doses was conducted with doses of 100, 250, and 600 mg/kg/day of the derivative (equivalent to 2.5 and 6 times the 10 mg/kg/day iron treatment). No signs of toxicity were observed during treatment.

Even in studies at doses of 100, 300, and 1000 mg/kg/day (1, 3, and 10 times the therapeutic dose), no signs of toxicity were observed. Body weight gain, food consumption, ophthalmic status, electrocardiographic, hematological, biochemical, and urinalysis data did not differ from controls.

In the Ames test, the derivative was tested using five bacterial strains (Salmonella Tiphimurium TH 1535, TA 1537, TA 1538, TA 98 and TA 100) with treatment in the absence and presence of metabolic activation (from liver fraction S 9). No mutagenic activity was observed.

Toxicological studies demonstrate that the ferrous derivative succinylalbumin is tolerated in the gastrointestinal tract, has low acute toxicity and is not mutagenic, compared to ferrous sulfate which causes gastric ulceration, is more toxic after a single administration and has mutagenic properties.

TABLE 4

Pharmacological trials

To pharmacologically characterize the ferrosuccinylalbumin derivative, the following were evaluated: gastric iron release; iron absorption and kinetics; and the antianemic effect of prolonged treatment using ferrous sulfate as a comparison (in animals with experimental anemia).

Iron release into the stomach

Male Wistar rats (Charles River-Calco) fasted for 24 hours are treated orally with 2 mg Fe equivalents/kg of ferrous sulfate or ferrosuccinylalbumin.

After 10, 20, 30 minutes from treatment the animals are sacrificed, the stomach is collected and the free iron in the gastric juice is measured using the Fe Kit (Wako).

The results, in table 5, (referring to a sample containing 9.5% iron but representative of all other derivatives) show that ferrosuccinylalbumin causes an iron release more than 10 times lower than ferrous sulphate.

In a similar experiment, ferroproteinsuccinylate caused an iron release only 8 times lower than ferrous sulfate.

TABLE 5

Iron concentrations in gastric juice of rats treated with ferrosuccinylalbumin or ferrous sulfate at a dose of 2 mg iron/kg (?g/ml ? S.E.)

Iron absorption in anemic rats

Serum iron levels were assessed using the bathophenanthroline method (Combination Iron Test - Boehringer Mannheim) in anemic rats (anemia was induced by an iron-deprived diet in Sprague-Dawley rats and by rearing animals born to anemic mothers on an iron-deprived diet) one hour after treatment with ferrosuccinylalbumin or ferrous sulfate at various doses. Results from various experiments suggest that ferrous sulfate causes a greater iron load than ferrosuccinylalbumin samples.

Determination of iron kinetics

Serum iron levels were measured in anemic rats at various temperatures after a single oral treatment with ferrous sulfate and ferrosuccinylalbumin at a dose of 0.3 mg Fe equivalents. The results show a peak iron absorption for both derivatives 1 hour after treatment (Table 6).

TABLE 6

Iron values in anemic rats at different times after oral treatment with

0.3 mg/kg iron as ferrous sulfate or ferrosuccinylalbumin (pg/100 ml ? E.D)

Antianemic effect

A prolonged treatment of 6 weeks was performed in anemic rats, administering orally ferrosuccinylalbumin and ferrous sulphate as a comparison at doses of 3, 10, 30 mg Fe/kg/day.

On days 0, 7, 14, 21, 28, 35 and 42 after treatment, the animals in groups of 5 are sacrificed.

The results of the levels of blood hemoglobin and serum iron (Test Combination Iron - Boehringer Mannheim), in table 7 (the data obtained with the derivative prepared according to the method described in example 3 are reported) show the equiactivity of the compounds in restoring the levels of hemoglobin, even though ferrous sulphate is faster and more powerful in restoring the values of serum iron.

TABLE 7

Hematological parameters during a 6-week treatment in anemic rats with different

iron-based compounds (3, 10 and 30 mg Fe/kg/day)

(Mean ? S.E.) ANOVA: ** P < 0.01 * P < 0.05 Vs anemic controls

TABLE 7: Second part

* * * The set of data obtained demonstrates that ferrosuccinylalbumin is a protein-iron complex (iron contents can vary from 3 to 20% by weight) in which the protein component is albumin or lactalbumin extensively acylated mainly at lysine as shown by comparative chemical-physical analyses, in particular electrophoresis, amino acid composition, succinic acid content, (NMR) with a disorganized structure (CD).

The protein is capable of absorbing high quantities of iron in the form of a polynuclear hydrated complex (ESR) similar to that of ferritin (UV-VIS) with an amorphous structure, in which aqueous iron complexes are maintained in solution by the succinylated protein forming an aggregate structure with molecular weights greater than 106 Daltons.

Derivatives are obtained that are highly insoluble in acidic environments and highly soluble at neutral and alkaline pH values.

The derivatives are characterized by very low toxicity in animals, characterized by the fact that they release trace amounts of iron, unable to generate free radicals. These effects mentioned, completely unpredictable, are caused by the presence of albumin in the derivative.

The present invention also relates to the use of the new compounds as effective agents in the treatment of anemia, with regard to all industrially applicable aspects of said use, including their incorporation into pharmaceutical compositions, which are a further specific object of the invention. The compounds according to the invention can therefore be incorporated into pharmaceutical formulations, particularly those suitable for oral administration. For oral administration, the substances are formulated as tablets, dispersible powders, capsules, tablets, granules, suspensions, syrups, elixirs, or solutions. Preparations for oral use may contain one or more of the usual additives, such as sweeteners, flavorings, colorings, coatings, and preservatives, in order to obtain a preparation that is pleasant in appearance and taste. The tablets may contain the active substance mixed with the usual pharmaceutically acceptable excipients, such as inert diluents such as calcium carbonate, sodium carbonate, lactose and talc; granulating and disintegrating agents such as starch, alginic acid and sodium carboxymethylcellulose; binding agents such as starch, gelatin, arabic gum and polyvinylpyrrolidone; preservatives such as methyl, ethyl, propyl or bufolic acid benzoates or hydroxybenzoates, or alkali metal benzoates; and lubricating agents such as talc, magnesium stearate, stearic acid, glyceryl palmitostearate and analogues.

Syrups, elixirs, suspensions, and solutions are prepared in known ways. Along with the active ingredient, they may contain suspending agents such as propylene glycol, methylcellulose, hydroxyethylcellulose, gum tragacanth, and sodium alginate; wetting agents such as lecithin, polyoxyethylene stearate, and polyoxymethylene sorbitan monooleate; and the usual preservatives, surfactants, sweeteners, and buffering agents. Sometimes, the addition of an alkali metal hydroxide may also be necessary. For these pharmaceutical formulations, the most preferred presentations are drinkable vials and sachets; the contents of these are dissolved or suspended in water just before use. Finally, capsules or tablets may contain the active ingredient alone or mixed with an inert solid diluent such as calcium carbonate, calcium phosphate, or kaolin. The active ingredient dosages used can vary within wide limits, depending on the nature of the compound employed. Generally, good results are obtained by administering the compounds according to the invention at daily dosages ranging from approximately 5 to approximately 50 mg per kg of body weight. The pharmaceutical dosage forms generally contain approximately 300 to approximately 1500 mg of active ingredient in combination with one or more common solid or liquid pharmaceutical vehicles and are suitable for administration once or more times a day.

Some illustrative examples of the pharmaceutical compositions which are the object of the present invention are reported below.

EXAMPLE 9

EXAMPLE 10

EXAMPLE 11

EXAMPLE 12

EXAMPLE 13

EXAMPLE 14

EXAMPLE 15

EXAMPLE 16

Claims (8)

1. Albumins acylated with dicarboxylic acid residues, containing iron in a bioavailable form. 2. Compounds according to claim 1 wherein the acylated albumins are derived from bovine albumin or lactalbumin. 3. Compounds according to claim 1 or 2 wherein the albumins are acylated with dicarboxylic acid residues selected from succinic, glutaric, maleic, malic, malic, aspartic, glutamic acid. 4. Compounds according to claim 3 wherein the albumins are acylated with succinyl residues. 5. Compounds according to any of claims 1-4 containing from 3 to 20% by weight of iron. 6. Bioavailable iron compounds obtainable from albumins acylated with dicarboxylic acid residues by interaction in an aqueous environment with iron ions at a pH between 2 and 12, preferably between 3 and 7. 7. Pharmaceutical compositions containing as active ingredient a derivative of claims 1-6 in mixture with a suitable vehicle or excipient. 8. Pharmaceutical compositions as defined in claim 7, containing the active ingredient in quantities ranging from about 300 to about 1500 mg.