BIOAVAILABLE IRON-ALBUMIN COMPOUNDS, A PROCESS FOR THE PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM

Abstract: Iron-albumin derivatives are described consisting of albumins acylated with dicarboxylic acid residues and containing high amounts of iron, insoluble at the stomach acid pH and highly soluble at the intestinal pH. The derivatives are characterized by a very low gastrolesivity and toxicity and by remarkable bioavailability and therapeutic effectiveness in the treatment of anaemic conditions.

Description: BIOAVAILABLE IRON-ALBUMIN COMPOUNDS, A PROCESS FOR THE PREPARATION THEREOF AND PHARMACEUTICAL COMPOSITIONS CONTAINING THEM The present invention relates to albumins acylated with dicarboxylic acid residues containing iron in a bioavailable form. The use of complexes of iron with acylated proteins for the oral martial therapy is known: see, for example, Italian Patent N. 1,150,213, which discloses an iron adducer comprising succinylated proteins, which is obtained by reacting ferric salts with carrier proteins of animal origin, such as proteins from milk,- organs or serum, or of vegetal origin. However, since said proteins have a variable composition, compounds of constant composition are hardly obtained. Moreover, even though complexes of high iron content (up to 20%) can be obtained, the high iron amount in those derivatives also involves increases in solution viscosity. As a matter of fact, the Ironproteinsuccinylate derivative (obtained according to Italian Patent N° . 1,150,213) which is used for the appropriate pharmaceutical formulation and is nowadays commercially available contains 5% by weight iron. The possibility to obtain derivatives giving low viscosity solutions, even when amounts well higher than 5% by weight iron are supported by succinylated proteins, would represent a marked improvement in the preparation of derivatives for the medicinal use. Nevertheless the most important aspect to be improved for this class of therapeutical agents undoubtedly consists in reducing or completely removing the gastrolesivity and diarrhoic effects related to the therapeutic principle. Now it has been found, and it is the object of the present invention, that using albumin as the protein to obtain said acylated compounds, bioavailable iron compounds are obtained which are highly soluble, even when they contain high amounts of iron, said compounds, moreover, being substantially devoid of toxicity and showing reduced gastrolesivity and diarrhoic side- effects. Said disadvantages, which are characteristic of these medicaments, can be related to a poor solubility of the iron derivatives or to precipitation at the intestine level or to the generation of hydroxy radicals deriving from the release of the iron in the ionic form. Different albumins can be used in the preparation of the compounds of the invention, particularly preferred being highly pure bovine serum albumin and lactalbumin. According to the invention, albumins are acylated with dicarboxylic acid residues such as, for instance, those deriving from succinic, glutaric, maleic, malic, alonic, aspartic, glutamic acids and the like. Particularly preferred is the residue from succinic acid, which proved to be the most suitable for improving some protein characteristics, such as solubility and degradability by proteases. More particularly, the present invention relates to compounds comprising iron and succinylated bovine serum albumin, supporting iron amounts from 3 to 10% by weight, as well as compounds comprising iron and succinylated lactalbumin supporting iron amounts up to 20% by weight, both these compounds being highly soluble. Said compounds can be obtained in aqueous medium by reacting the above mentioned succinylated or acylated albumins with iron ions at a pH ranging from 2 to 12, preferably from 3 to 7. The invention will be described in more detail in the following non-limiting examples. The compounds described in the Examples will be named as follows : Compounds of Examples 1-3 : Ironsuccinylalbumin. Compounds of Examples 4-5 : Ironsuccinyl- -lactalbumin. Compound of Example 6 : Ironaspartylalbumin. Compound of Example 7 : Ironmaleylalbumin. Compound of Example 8 : Ironglutarylalbumin. EXAMPLE 1 Preparation of Ironsuccinylalbumin with 6% by weight Fe content. 20 g of bovine serum albumin are dissolved in 400 ml of water. pH is adjusted to 8.0 by adding NaOH. 24 g of succinic anhydride are added in small portions, under stirring, keeping pH from 7.5 to 8 by addition of 2N NaOH and constant temperature below 25°C, preferably at 20°C. The mixture is left to react for one hour, then pH is lowered to 3 ± 0.5 by means of 2N HCl. A white precipitate forms which is recovered by centrifugation. The formed excess of succinic acid is removed by dissolving the precipitate in 400 ml of water at pH 8.0, adjusting the solution pH to 3 ± 0.5 with 2N HCl. A white precipitate forms which is recovered by centrifugation. This procedure is repeated until succinic acid is completely removed. The obtained final precipitate is dissolved in 400 ml of water by addition of 2N NaOH to pH 8.0. 7.20 g of FeCl 3 .6H 2 0 , equivalent to 1.48 g of Fe, are quickly added under stirring. A red-brown precipitate instantly forms, which is washed with 0.001N HCl, recovered by filtration (or centrifugation) and dried under vacuum. A red-brown powder (19 g) is obtained which is insoluble in water and becomes soluble adjusting pH to about neutrality by addition of 2N NaOH. The compound shows at the analysis an iron content of 6.1-6.2% by weight. By electrophoresis on cellulose acetate (as described in item 4 below) the compound moves as a unitary spot, in which iron can be determined. EXAMPLE 2 Preparation of Ironsuccinylalbumin with 3.8-4.1% by weight Fe content. The compound is prepared as described in Example 1, but adding 4.8 g of FeCl 3 .6H_0 (equivalent to 0.922 g of Iron) . The compound, moving under the electrophoretic field as a unitary spot in which iron can be determined, shows a 3.8-4.1% by weight Fe content. EXAMPLE 3 Preparation of Ironsuccinylalbumin with 9.5-10% by weight Fe content. 20 1 of demineralized water and 1 kg of bovine serum albumin are placed into a reactor, obtaining a solution of pH 6.7-6.8. A 5N NaOH solution is slowly dropped therein until pH 7.5, then 1.2 kg of succinic anhydride are added in small portions, keeping pH from 7.2 to 7.5 by addition of 5N NaOH. At the end of the addition, pH is kept to 7.5 for 30 min. then the solution is acidified to pH 3 with 6N HCl, under stirring. The obtained precipitate is centrifuged, thoroughly washed with demineralized water and redissolved in 20 1 of demineralized water with 5N NaOH to pH 7.5 , to obtain a clear solution in 30 min. The precipitation procedure at acid pH and the dissolution procedure at basic pH are repeated twice. Then the solution is slowly added with a solution of 578.5 g of FeCl,.6H„0 in 10 1 of demineralized water keeping pH from 6.5 to 6.8 by addition of 5N NaOH. Then the mixture is stirred at pH 6.6-6.8 for 30 min., acidified to pH 3 with 6N HCl and left under stirring for 30 min.. The precipitate is centrifuged, thoroughly washed with demineralized water adjusting pH to 8.5 with 5N NaOH. After having kept at the pH of 8.5 for about one hour, the obtained solution is filtered through Celite (300 g) . The precipitation procedure at acid pH and the dissolution procedure at basic pH are repeated for one time. The solution is filtered on Celite (200 g) , the product is precipitated at pH 3 with 6N HCl, stirring for 30 min. , then it is centrifuged and thoroughly washed with demineralized water. The dark brown solid is sieved and dried under vacuum at 30°C, to obtain 1 kg of Ironsuccinylalbumin. The title compound is obtained. EXAMPLE 4 Preparation of Ironsuccinyl- O^-lactalbumin with 11- 11.45% by weight Fe content. 1 kg of r -lactalbumin dissolved in 20 1 of water is placed into a reactor. A solution is obtained having pH 6.7-6.8. The protein is succinylated and purified from succinic acid as described in the previous Example. The solution of the succinylated protein is added with 1.2 kg of FeCl .δH-O in 10 1 of water, keeping pH from 6.5 to 6.9 by addition of 5N NaOH. The reaction is carried out as described in the above Example to obtain, at the end of the reaction, 1 kg of the title compound. EXAMPLE 5 Preparation of Ironsuccinyl-fX-lactalbumin with a 18.3- 18.5% by weight Fe content. 1 kg of ^.-lactalbumin dissolved in 20 1 of water is placed into a reactor, to obtain a solution with pH 6.7-6.8. The protein is succinylated and purified as described in Example 3. The solution of succinylated Q -lactalbumin is added with 2.4 kg of FeCl-..6H_0 in 10 1 of water, keeping pH from 6.5 to 6.9 by addition of 5N NaOH. The reaction is carried out as described in Example 3, to obtain 1 kg of the title compound. EXAMPLE 6 Preparation of Ironaspartylalbumin with 7.5% by weight Fe content. 10 g of bovine serum albumin are dissolved in 200 ml of water and pH is adjusted to 7.2 by addition of NaOH. 10 g of acetylaspartic anhydride are added in small portions, under stirring, keeping pH from 7.2 to 7.3 by addition of 2N NaOH. The solution is ultrafiltered, keeping the volume constant by continuous addition operating with an ultrafiltration membrane with cut-off 10,000 Daltons, for 3 hours, so as to remove acetylaspartic acid passing through the membrane. The solution containing aspartylated albumin is recovered from the ultrafilter; 7.0 g of FeCl,.6H„0 are added : a red-brown precipitate forms which is washed with water at pH 3, redissolved adjusting pH to 7.1 and freeze-dried. 9.5 g of the title compound are obtained. EXAMPLE 7 Preparation of Ironmaleylalbumin with 6.5% by weight Fe content. The iron compound is prepared as described in Example 6, but using aleic anhydride as the acylating agent. The title compound is obtained. EXAMPLE 8 Preparation of Ironglutarylalbumin with 7.0% by weight Fe content. The iron compound is prepared as described in Example 6, but using glutaric anhydride as the acylating agent. The title compound is obtained. The protein complexes prepared in the above Examples were analyzed according to the analytic methodologies described hereinbelow. 1) Determination of the iron content In all of the compounds prepared according to Examples 1-8 the iron content was determined by extracting iron from the sample with 2N HCl; the quantitative determination was carried out according to the method described in Standard Methods 14th ed. , 1975, page 208, APHA, AWWA, PCF (reaction with o- phenanthroline) . In said compound iron is present in the trivalent state, as evidenced since no Fe ions can be determined when the o-phenanthroline reaction is carried out in the absence of reducing agents. 2) Solubility as a pH function All the compounds prepared according the above Examples are insoluble at acid pH and soluble at basic pH. The solubility profile of Ironsuccinylalbumin, prepared according to the method described in Example 3, is reported by way of example. The solubility profile is taken from spectrophotometric measurements of the absorbance difference at 500 nm and 280 nm of solutions obtained treating 20 mg of Ironsuccinylalbumin with 50 ml of water, in a pH range from 2.0 to 7.0. 5 ml of the sample are diluted to 10 ml with water, centrifuged at 3,000 rpm during 10 min. and the supernatant is read at spectrophotometer. Ironsuccinylalbumin is completely soluble at pH values equal or above 6.5, whereas it is insoluble at pH below 6. The reversed precipitation curve, obtained by acidifying the Ironsuccinylalbumin solution (20 mg of the compound in water at pH 7) , shows the precipitation onset at pH 4.5. 3) Determination of the protein content The protein amount in the samples prepared according to Examples 1-8 was determined by titration of protein nitrogen (using the method described in ITALIAN OFFICIAL PHARMACOPOEIA IX ed. , vol. I, pages 191-192) . The found values range from a maximum of 85% (in the compound of Example 3) to a minimum of 65% (in the compound of Example 5). 4) Electrophoresis on cellulose acetate Electrophoresis was carried out for 20 min. at 40 V/cm, using 0.05M Tris tricine buffer, pH 8.6, which detects bands at the same electrophoretic distance for the compounds of the invention and for the respective acylated proteins. No bands which can be attributed to the starting albumins can be evidenced. The presence of iron in the bands of the compounds of the invention can be detected by means of o-phenanthroline. By way of example, in case of determinations carried out on the compounds of Examples 1-3, the distance of the spot from the origin is 37 mm. 5) UV-VIS Spectroscopic analysis (Shimadzu UV-160) UV-VIS Spectrum in the range 200-600 nm, recorded on aqueous solutions at pH 7 containing 1 mg/ml of the compounds of the invention evidences an absorbance increase from 600 to 340 nm. 6) Fluorescence emission spectrum (Apparatus: spectrofluorometer Kontron SFM25). The fluorescence emission spectra at 400 and 200 nm (excitation at 236 nm) show a poor emission, equal to about 1/10 that of the starting protein. 7) ESR Spectroscopy The spectra were recorded both at room temperature and at -160°C with a spectrometer Varian E1D, and they are characterized by a single peak of 1200 G width between the maximum and minimum slope with a value of g = 2.00. These signals can be attributed to polynuclear complexes (in this instance Fe3+ complexes) characterized by strong exchange interactions. No other signals which could be attributed to the presence of iron in the mononuclear form can be evidenced. 8) Electrophoresis on Sodium Dodecyl Sulfate (SDS) Electrophoresis was carried out on 7.5% polyaery1amide gel containing 1% by weight SDS. The electrophoretic bands were evidenced with Comassie Brilliant Blue. The following products were used as molecular weight standards: myosin (200 KD) , beta-galactosidase (116 KD) , phosphorylase b (97 KD), bovine albumin (68 KD) , ovoalbumin (42 KD) . In representative determinations, Ironsuccinylalbumin showed a single band corresponding to 100 KD molecular weight. A similar electrophoretic behaviour was shown by Ironsuccinyl-C-lactalbumin, but the molecular weight thereof is 20 KD. 9) Determination of the acylation degree and localization of the acylation The acylation degree was determined both spectrophotometrically, according to the reaction of the protein free a ino groups with ninhydrin and by fluorometric reaction with o-phthalaldehyde (G. Goodno et al. , Anal. Biochem. , 115, 203, 1981). Both these methods were used to evaluate localization of the acylation on the protein fraction constituting the prepared compounds. The obtained results prove that the end 0^-amino groups and the -amino lysin groups are acylated by more than 95%. The following additional parameters were determined on Ironsuccinylalbumin, prepared as described in Example 3. 10) Succinic acid content 7-13%, determined by GLC (Perkin Elmer 3B, 3 m x 2 mm column GP 10% SP 1200 H-PO. on Chromosorb R ) after hydrolysis of a known amount of a sample with 2.5N NaOH 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 BF,/CH_,0H. Methyl succinate is determined under the following conditions, using methyl glutarate as the internal standard: Nitrogen : 30 ml/min.- Air : 3 atm. Hydrogen : 1.5 atm. Temperature : injector 255°C column 140°C F.I.D. detector 275°C Injections : 5 μl 11) Circular dichroism (JASCO 500C apparatus) The measurements recorded in the range from 300 to 200 nm on Ironsuccinylalbumin solutions at pH 7 in comparison with the starting albumin evidence that the succinylation reaction involves the disordered structure of the protein. Figure 1 shows the dichroism spectrum of bovine serum albumin, of the corresponding acylated compound and of the compound of Example 3. 12a) H-NMR Spectrum (300 mHz; D^O; Varian Gemini 200 apparatus) The spectrum shows broad bands and a weak signal at 2.4 ppm, which signal is also present in the spectrum of the corresponding succinylated albumin, but with a more neat and defined configuration. The spectrum is reported in Figure 2. 12b) 13 C-NMR spectrum (Varian XL300 apparatus) The spectrum shows 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 (hemysuccinyl residue) due to the presence of iron, suggests for a possible implication of the succinyl groups in the bond with iron. The spectrum is reported in Figure 3. 13) X-ray spectrum (Apparatus: Siemens 500D diffractometer) The spectrum in the angular interval 2 from 9° to 51° evidences no peaks which can be attributed to the presence of crystallinity centres, thus indicating the amorphous nature of the compound. 14) Determinations of the molecular weight By means of gel-filtration on Superose 6R columns a not gaussian molecular weight profile is obtained with a main peak > 10 Daltons and a very broad 4 shoulder up to 10 D. Using the Laser Light Scattering technique with a He-Na source of 6328A, 25 mW output power, a molecular weight of 17.5 ± 2.5 Mill was determined. 15) Amino acid analysis The analysis was carried out upon decomplexation of the iron which can interfere in the analysis, by subjecting a sample to total acid hydrolysis with 6N HCl at 105°C for 24 hours and determining the amino acid composition with an automatic analyzer (Liquimat + III Kontron) using Na buffers as the eluents. The amino acid profile is reported in Table 1. Table 1 - Amino acid composition (amino acid % by mole) 16) Proteolysis The enzyme hydrolysis was carried out by treating 25 mg dissolved in 5 ml of 0.05M Tris-HCl buffer pH 7.6 with chymotrypsin (50 μg) and trypsin (50 μg) . 1 ml is withdrawn from the reaction mixture at definite times; 2 ml of a 5% trichloroacetic acid (TCA) are added, the mixture is centrifuged at 3000 rpm for 110 min. and the supernatant is spectrophotometrically read at 280 nm against a TCA blank. The absorbance change as a function of time represents the hydrolysis rate, which turns out to be comparable to that of the corresponding albumin used in the preparation, of the compound, to evidence that iron present in the compound does not inhibit the action of proteolytic enzymes. 17) Iron mobilization Iron mobilization is tested by reaction with desferrioxamine, which is controlled as a function of time by measuring the absorbance increase, at 487 nm, of a solution containing 7.0 ul of desferrioxamine, 500 ug of Ironsuccinylalbumin previously neutralized at pH 7.4 in 20 mM Tris-HCl buffer. The absorbance increase at 487 nm during time confirms that iron can be mobilized from Ironsuccinylalbumin. The release rate is 2.2 A for 5 min. 18) Viscosity measurements Ironsuccinylalbumin is dissolved in water at pH 7.2 at concentrations of 0.1, 1.2, 4.8. 10% w/v. The obtained solutions are viscosimetrically analyzed with a capillary viscosimeter or a Brookfield viscosimeter. Solutions prepared with Ironproteinsuccinylate (a 5% sample obtained as described in Italian Patent 1.150,213) are analyzed, by comparison and at the same concentrations. The viscosity of Ironsuccinylalbumin samples, at all the tested concentrations, is markedly lower than that of Ironproteinsuccinylate. 19 ) Hydroxy radical generation The capability of the iron compounds of the invention to generate OH radicals was investigated according to B. Halliwell, J. Gutteridge, 0. Aruoma, Anal. Biochem. 165, 215-219 (1987). FeCl, and FeSO. , which are known to be capable of generating OH radicals, are used as the controls. Representative results are reported in Table 2. Table 2 - Deoxyribose degradation at pH 4.0 In the absence of ascorbate, the formation of free radicals is significant with FeSO. and, at a lower extent, with FeCl,. Generation of free radicals deriving from Ironsuccinylalbumin, in the presence of ascorbate, is significantly lower than that of FeSO.. 20) Generation of free radicals in biologic fluids The bleomycin test /Life Chemistry report, 1987, vol. 4, pages 113-142 - J. Gutteridge, B. Halliwell/ was used for determining free radicals, due to the presence of free iron, in biologic fluids. Groups of rats are treated intraperitoneally with Ironsuccinylalbumin and Ironproteinsuccinylate (prepared according to Italian Patent 1,150,213) (in amounts corresponding to 100 mg iron/kg) , and with ferrous sulphate (in amounts corresponding to 25 mg/kg iron) . Plasma samples are collected after 2, 8 and 15 hours and the presence of iron is determined with the bleomycin test (see Table 3). Table 3 - Determination of free iron in plasma 15 h n.d. n.d. . n.d. Controls n.d. n.d. n.d. Ironsuccinylalbumin, contrary to ferrous sulphate and Ironproteinsuccinylate, induces no free iron in plasma, thus proving to be free from the injuring effects induced by free radicals deriving from iron. Toxicity study In the tests for gastrointestinal tolerance, the animals were treated orally with Ironsuccinylalbumin (a sample prepared according to Example 3, which sample being significant due the high iron content thereof, and therefore being potentially more toxic) or ferrous sulphate at dose levels of 200 mg/kg. The animals were killed at different times, up to 24 hours. The examination of stomach and intestine showed .that the intestinal and gastric injuries were significantly less marked after administration of Ironsuccinylalbumin than ferrous sulphate. The acute toxicity of the sample was compared with ferrous sulphate and the results are reported in Table 4. Ironsuccinylalbumin proves to be much less toxic than ferrous sulphate and does not induces toxicity symptoms up to 2000 mg/kg. Subacute toxicity with repeated doses by the oral route was tested with doses of 100. 250, 600 mg/kg/die of the derivative (equivalent to 1, 2.5, 6 times the treatment with 10 mg/kg/die iron). No toxicity signs are evidenced during the treatment. Tests on the dog at doses of 100, 300 and 1000 mg/kg/die (1, 3 and 10 times the therapeutic dosage) also evidenced no toxicity signs. Increase in body weight, food assumption, ophthalmic conditions, as well as electrocardiographic, hematological, biochemical and urine analysis data did not differ from the control ones. In the Ames test the derivative was tested using 5 bacterial strains (Salmonella Typhimurium Th 1535, TA 1537, TA 1538, TA 98 and TA 100) by treatment in the absence and in the presence of metabolic activation (by S9 liver fraction) . No mutagenic activity was evidenced. Toxicologic studies prove that Ironsuccinylalbumin is tolerated in the intestinal tract, has a low acute toxicity and it is not mutagenic, in comparison with ferrous sulphate which causes gastric ulcerations, is more toxic after single administration and has mutagenic properties. Table 4 Product Administration route LD50 Ironsuccinylalbumin i.p. > 2000 mg/kg Ferrous sulphate i.p. 250 mg/kg Ironsuccinylalbumin o.s. > 2000 mg/kg Ferrous sulphate o.s. 1622 mg/kg Pharmacological tests In order to pharmacologically characterize Ironsuccinylalbumin, the following parameters were evaluated: iron release at the gastric level; iron absorption and kinetic, and the antianaemic effect after a prolonged treatment using ferrous sulphate as the control (in animals with experimentally induced anaemia). The Ironsuccinylalbumin used for these tests is the one prepared in Example 3. A. Iron release in the stomach istar male rats (Charles River-Calco) fasted for 24 hours are treated orally with 2 mg Fe equivalents/kg ferrous sulphate or Ironsuccinylalbumin. 10, 20 and 30 Minutes after treatment the animals are killed, the stomach is withdrawn and the free iron in gastric juice is dosed with the Fe kit ( ako). The results reported in Table 5 (referring to a sample containing 9.5% iron, but which is representative of all the other compounds) show that Ironsuccinylalbumin causes an iron release more than 10 times lower than ferrous sulphate, which explains the lower gastrolesivity of the compound of the .invention compared with FeSO. , which is an antianaemic agent widely used in therapy. Table 5 Iron concentrations in gastric juice of rats treated with ironsuccinylalbumin or ferrous sulphate at a dose of 2 mg iron/kg (μg/ml±S.E. ) . Treatment Treatment times 10' 20' 30' Ironsuccinylalbumin 9.0+1.9 8.1±1.8 11.3±3.0 Ferrous sulphate 118.4±7.6 116.714.7 122.3±10.2 Controls 5.012.2 2.9+1.2 4.912.6 N = 7 ANOVA : Ironsuccinylalbumin vs. ferrous sulphate : p < 0.01 B. Iron absorption in the anaemic rat Sideremia was evaluated by the batophenanthroline method (Test Combination Iron - Boehringer Mannheim) in the anaemic rat (anaemia was induced by iron-free diet in the Sprague-Dawley rat and bred with iron-free diet animals born from anaemic mothers) one hour after the treatment with Ironsuccinylalbumin or ferrous sulphate at different dosages. The results obtained in different tests proved that ferrous sulphate causes a higher iron charge than Ironsuccinylalbumin samples. C. Determination of iron kinetics The sideremia values were measured in the anaemic rat at different times after a single oral treatment with ferrous sulphate and Ironsuccinylalbumin at a dose of 0.3 mg of Fe equivalents. The results show for both the compounds an iron absorption peak 1 hour after the treatment (see Table 6). Table 6 Sideremia values in the anaemic rat at different times after an oral treatment with 0.3 mg/kg of. iron as ferrous sulphate or ironsuccinylalbumin (μg/100 ml 1 E.D) Treatment Treatment times 0,3 mg Fe/kg 0 15' 30" 60' Ironsucci¬ nylalbumin / 62,519,2** 117,3+17,6** 208,6120,3** ++ ++ FeS0 4 / 197,8118,5++ 247,9115,5++ 307,7+19,2++ Anaemic 30,311,6 controls Table 6 (continued) Treatment Treatment times 0,3 mg Fe/kg 2h 3h 6h 24h Ironsuccinyl- 118,3114,3** 104,8115,0** 55,815,1 29,6+1,1 albumin ++ ++ FeS04 261,5121,5++ 219,7128,1++ 93,9122,8++ 23,810,8 ANOVA at 2 factors: ** p < 0,01 *p< 0,05: Ironsuccinylalbumin ++ p < 0,01 + p < 0,05 within the group vs. time 0 (anaemic controls) D. Antianaemic effect A prolonged treatment was carried out during 6 weeks, in the anaemic rat, by administering orally Ironsuccinylalbumin and ferrous sulphate as the control, at doses of 3, 10, 30 mg Fe/kg/die. The animals in groups of 5 animals each were killed on days 0, 7, 14, 21, 28, 35 and 42 from the treatment. The data of the hematic hemoglobin and serum iron levels (Test Combination Iron - Boehringer Mannheim) obtained with the compound prepared as described in Example 3, are reported in Table 7. These results show that the compounds are equiactive in restoring the hemoglobin levels, even though ferrous sulphate is more rapid and powerful in restoring serum iron values. Table 7 Hematological parameters during a 6 week treatment in anaemic rats with different compounds containing iron (3, 10 and 30 mg Fe/kg/die) ** (Mean 1 S.E) ANOVA: P < 0.01 P < 0.05 vs. anaemic controls Hematological Treatment Days of treatment parameters mg Fe/kg/die 0 7 14 Hanoglobin Normal controls 13 ,311, 0 13,310,5 13,710,6 (mg/dl) Anaemic controls 3 , 310 ,2 3,410,2 4,0+0,2 Ironsuccinylalbumin 3 7,9±0,6** 8,2±1,2** _Qx)nsuccinylalbumin 10 / 10,010,7 11,110,4 Ironsuccinylalbumin 30 / 11,2+0,3 12,8+0,2 FeSO. 8,010,3** 7,9+0,9** FeS0 4 10 11,210,1 11,210,8 FeS0 4 30 11,510,3 12,110,2 Seric Iron Normal controls / 128,7119,5** 111,0111,4** (μg/100 ml) Anaemic controls 19,9+6,5 38,9+3,7 Ironsuccinylalbumin 3 / 27,111,6 54,216,8* Ironsuccinylalbumin 10 / 142,7+31,7** 123,5114,4** π onsuccinylalbumin 30 / 184,619,0** 102,614,9** FeSO. 67,817,8** 81,8114,8** FeS0 4 10 n.d. 183,7142,2** FeSO. 30 189,6138,0** 147,3126,2** 4 Table 7 (continued) Hematological Treatment Days of treatment parameters mg Fe/kg/die 21 28 35 42 HEanoglcb-Ln Normal controls 13,8+0,4 13,6+2,2 15,110,5 14,8±0,8 (mg/dl) Anaemic controls 4,8±0,8 3,6±0,5 7,1+0,8 9 > 1±1 ' 4 Ircaiεuccinylalbumin 3 9,7±0,6** 10,2+1,0** Iror_stx;cinylalbumin 1012,1+0,3 13,010,113,9+0,1 H,9±0,l Ironsuccinylalbumin 30 H< 9±0 ' 4 12, 9±0, 3 14, l±O, 9 12,7 + 0,5 FeS0 4 3 12,511,0** 11,510,8** EeS0 4 10 12,6±0,4 12,510,4 13,610 15,410,7 EβS °4 3 ° 11,710,2 12,710,1 13,110,4 Seric Iron Normal controls 121,117,3** 114,5119,0** 139, 319, 8**140,5 1 16,1*" (μg/100 ml) Anaemic controls 34,213,0 30,7+2,8 40,614,9 74,7+16,9 Ironsuccinyl- 77,2111,9* 143,5138,0** albumin 3 • Ironsuccinyl- 153,518,5** 87,614,5** 113,512,3** 202,8+16,9** albumin 10 Ironsuccinyl- 133,619,1** 112,418,0** 133,514,0** 140,1125,3** albumin 30 FeSO 3 103,2123,2** 133,8110,8** 4 5^30 i Q 133,813,2** 113,819,2** 129,3123,8** 219,613,3** 4 FeS0 4 30 105,217,4** 125,7113,4**157,5137,0** The above data prove that Ironsuccinylalbumin is a protein iron complex (the iron contents can vary from 3 to 20% by weight) , the protein component of. which is albumin or lactalbumin extensively acylated, mainly at the lysine chain, as evidenced by comparative physico- chemical analysis, particularly electrophoresis, amino acid composition, succinic acid content, (NMR) , with disorganized structure (CD-circular dichroism). The protein can complex with high amounts of iron in form of a polynuclear hydrated complex (ESR) with amorphous structure, in which aquo-complexes are maintained in solution by the succinylated protein, thus forming an aggregate structure with molecular weights higher than 10 Daltons. The obtained compounds are highly insoluble in acid medium and highly soluble at neutral or basic pH values. The compounds are characterized by a very low toxicity in the animal, due to the release of iron traces, which cannot give raise to free radicals. The above mentioned effects are completely unforeseeable and they are due to the presence of albumin in the compound. The present invention also relates to the use of the novel compounds as agents effective in the treatment of anaemias, as well as to all of the industrial aspects related thereto, including the use thereof in pharmaceutical compositions, which are a further specific object of the invention. The compounds of the invention can be incorporated in pharmaceutical compositions, particularly in formulations suitable to the oral administration. For the oral administration, the compounds are formulated in form of tablets, dispersible powders, capsules, dragees, granulates, suspensions, syrups, elixirs or solutions. The preparations for the- oral use can contain one or more of the usual excipients, such as sweetening, flavouring, colouring, covering and preservative agents, in order to obtain a pleasant and palatable preparation. Tablets can contain the active ingredient in admixture with the usual pharmaceutically acceptable excipients, for example inert diluents such as calcium carbonate, sodium carbonate, lactose and talc, granulating and disintegrating agents, such as alginic acid and sodium carboxymethyl cellulose, binding agents such as starch, gelatin, gum arabic and polyvinylpyrrolidone, preservative agents such as methyl, ethyl, propyl, butyl, or alkali metals benzoates or hydroxybenzoates, and lubricants such as talc, magnesium stearate, stearic acid, glyceryl palmitostearate and the like. Syrups, elixirs, suspensions and solutions are prepared according to known methods. Together with the active ingredient, they can contain suspending agents, such as propylene glycol, methylcellulose, hydroxyethylcellulose, tragacanth gum and sodium alginate, wetting agents, such as lecithin, polyoxyethylene stearate and polyoxymethylenesorbitan monooleate and the usual preservatives, surfactants, sweetening and buffering agents. The addition of an alkali metal hydroxide can sometimes be necessary. The most preferred forms of said pharmaceutical formulations consist or drinkable ampoules and sachets, the content of which is extemporarily dissolved or suspended in water. The active ingredient dosages can range within wide limits, depending on the nature of the used compound. Effective results are generally obtained by administering the compounds of the invention at daily dosages ranging from about 5 to about 50 mg/kg body weight. The dosage pharmaceutical forms generally contain from about 300 to about 1500 mg fo the active ingredient together with one or more of the conventional solid or liquid pharmaceutical carriers ad they can be administered one or more times a day. Some examples of pharmaceutical compositions are reported hereinbelow. EXAMPLE 9 One tablet contains: Ironsuccinylalbumin (Example 3) Glicerylpalmitostearate Sodium carboxymethylcellulose Methyl p-hydroxybenzoate Propyl p-hydroxybenzoate EXAMPLE 10 One tablet contains: Ironsuccinylalbumin (Example 1) mg 1200 Glicerylpalmitostearate Sodium carboxymethylcellulose Methyl p-hydroxybenzoate Propyl p-hydroxybenzoate EXAMPLE 11 One drinkable vial contains: Ironsuccinylalbumin (Example 3) mg 400 mg 600 mg 1200 Propyleneglycol mg 1000 mg 1500 mg 3000 Sodium benzoate mg 35 mg 52 mg 105 Methyl p-hydroxybenzoate mg 30 mg 45 mg 90 Propyl p-hydroxybenzoate mg 15 mg 22 mg 45 Sodium saccharine mg 10 mg 15 mg 30 Caramel (E 150) mg 190 mg 285 mg 570 1 N sodium hydroxide mg 1000 mg 1500 mg 3000 Depurated water q.s. to ml 10 ml 15 ml 30 EXAMPLE 12 One drinkable vial contains: Ironsuccinyl- C -lactalbumin (Example 5) mg 400 Propyleneglycol mg 1000 Methyl p-hydroxybenzoate mg 50 Propyl p-hydroxybenzoate mg 40 Sodium saccharine mg 30 Caramel mg 400 1 N sodium hydroxide mg 1000 Depurated water q.s. to ml 20 EXAMPLE 13 One drinkable vial contains: Ironaspartylalbumin mg 1200 Methylcellulose mg 800 Sodium benzoate mg 500 Propyl p-hydroxybenzoate mg 20 Sodium saccharine mg 30 1 N sodium hydroxide mg 1000 Depurated water q.s. to ml 15 EXAMPLE 14 One sachet contains: Ironsuccinylalbumin (Example 3) Sodium carboxymethylcellulose Sodium laurylsulfate Methyl p-hydroxybenzoate Propyl p-hydroxybenzoate Sodium saccharine Caramel Sorbitol q.s. to EXAMPLE 15 One sachet contains: Ironsuccinyl-C -lact lbumin (Esempio 5) Sodium carboxymethylcellulose Sodium laurylsulfate Propyl p-hydroxybenzoate Sodium saccharine Sorbitol q.s. to EXAMPLE 16 One sachet contains: Ironmaleylalbumin mg 1200 Sodium carboxymethylcellulose mg 180 Methyl p-hydroxybenzoate mg 3 Propyl p-hydroxybenzoate mg 5 Aspartame mg 30 Sorbitol q.s. to mg 2500

Claims: CLAIMS 1. Albumins acylated with dicarboxylic acid residues, containing iron in a bioavailable form. 2. Compounds as claimed in claim 1, in which acylated albumins derive from bovine albumin or lactalbumin. 3. Compounds as claimed in claims 1 or 2, in which albumins are acylated with dicarboxylic acid residues selected from succinic, glutaric, maleic, malic, malonic, aspartic and glutamic acids. 4. Compounds as claimed in claim 3, in which albumins are acylated with succinyl residues. 5. Compounds as claimed in any one of claims 1 to 4, containing 3 to 20% by weight iron. 6. Bioavailable iron containing compounds, which can be obtained from albumins acylated with dicarboxylic acid residues by interaction with iron ions in aqueous medium at pH ranging from 2 to 12, preferably from 3 to 7. 7. Pharmaceutical compositions containing as the active ingredient one compound as claimed in claims 1 to 6 in admixture with a suitable carrier or excipient. 8. Pharmaceutical compositions as claimed in claim 7, containing the active ingredient in amounts from about 300 to about 1500 mg. 9. A method for the treatment of anaemic conditions in warm blooded animals, which comprises administering one therapeutically effective dose of one of the compounds as claimed in claim 1, which dose ranges from about 10 to about 50 mg/ kg body weight. 10. The use of the compounds as claimed in claim 1 in the preparation of a medicament for the treatment of anaemic conditions in the warm blooded animals.

Inventors: BARANI ROBERTO, CARAMAZZA IDA, CREMONESI PIETRO, DEL SOLDATO PIERO, BARANI, ROBERTO, CARAMAZZA, IDA, CREMONESI, PIETRO, DEL SOLDATO, PIERO
Applicants: ITALFARMACO SPA, ITALFARMACO S.P.A.
Application year: 1990
Main classification: C07K3
IPC codes: A61K9, A61K33, C07K1