BIOAVAILABLE IRON DERIVATIVES FREE FROM GASTROLESIVITIES, PREPARATION PROCEDURE AND RELATED PHARMACEUTICAL COMPOSITIONS.

Description

TITLE OF IRON DERIVATIVES θIODISPONI0 THE E FREE FROM GASTROLESIVITY*< PR 0 CE OF ME N ÌTO OF PREPARATION AND RELATED PHARMACEUTICAL COMPOSITIONS. INV. DFS. GIANCARLO SPORTOLETTI PIER GIUSEPPE PAGELLA PIETRO CREMONESI Rome, The Register A FrofocoS lo π° l À θ < MINISTRY OF INDUSTRY, COMMERCE AND CRAFTS Provincial Office of Industry, Commerce and Crafts of Miiano COPY OF THE DEPOSIT MINUTES FOR PATENT I)' IN VENZ »ONE INO OSTRI AL E The year 1982 the day of the month oi ni ar zo fa Company ITALFA ILIACO Ci , ìWmx of Italian nationality.£La con sene m ì--, _______ m ί·.*1LAΛΐΊυ ΧΎΟύΐΰ eli-tV Via ίθ Fulvio fosti, oJO through the agent Studio Consulenza BroveUu.»ie and with domicile for legal purposes in Milan - Via Senato n. 24 at the agent's office has presented to me, the undersigned: - Stamped application for the granting of a PATENT FOR INDUSTRIAL INVENTION having as TITLE! IRON DERIVATIVES AVAILABLE FREE FROM HARMFULNESS, PREPARATION EFFECTS AND RELATED PHARMACEUTICAL COMPOSITIONS Inventor i designated τ : 1. Giéi'icarlo Sporrcletrà -- 2. Pier Giuseppe pagrila 3 0 Pietro Cremonesi KMcimàridiiXiiLKiéÀièli or Uftvi. accompanied by: - Description in duplicate of n, 37 pages of writing. π . ,.. - Drawings, plates no. 4 in duplicate. Thus CÌOmancÌa does not - Letter of appointment 4iEIóHÌarà£rbTi&W&5wnYo^ckXtti>^r'f>rooÈif^?i contents 01 & P ^'P 0^· 1 Oai't' -lM^[ó^®^hìEr5Eìa:^w^ubrìèDTaK^: jj c,!Yesìt:ìi in pari cinti, - Act of designation of the inventor. - Proof of payment on postal account no. 0066B004 in the name of the Tax and Concession Registry Office Rome of L. 288·000 - issued by the UfL Postale of Milan 57 on 2 / 3 / 1932 n. 566 - Stamp duty of L. 2OTKX 3,000=. The application, descriptions and drawings listed above have been signed by the applicant and countersigned by me and stamped with the official stamp. THE DEPOSITOR For a copy compliant with the original THE OFFICIALZE RODANTE He&ro MesckjA / / / TO (Salvatore Ravalli) on Τ,ϊί'.η ni; ιηϊι-γ,γ,,„ -------- : .j7a\ 7 ··· '·: f \ ' 7'ì Ό C : C Hon. Ministry of Industry, Trade and Craftsmanship Central Patent Office ROME The agents Messrs. Bianchetti Giuseppe, Bracco Mauro, Bianchetti Marina, Minoja Fabrizio.....dello; . - ' ' - - ........- .. . ! Patent Consulting Studio srl.......of Milan Via Rossini n.8 - ask this honorable member: Ministry to issue certified copies of the following Italian patents: y No. 1194677 granted on 22 September 1988 in the name of, ITALFARMACO SpA - Milan - titled: Pharmaceutical composition with activity: anti-inflammatory, antipyretic and analgesic - _1 certified copy of the description;............ ............. ............. ί » No. 1150213 granted on 10 December 1986 in the name of ITALFARMACO SpA - Milan - titled: Bioavailable iron derivatives free from gastro-intestinal harm, preparation process and related pharmaceutical compositions - η. 1, copy ί authenticity of the description; ì i No. 1130972 granted on 18 June 1986 in the name of DOMPE'FARMACEUTICI SpA - Milan “ entitled:; the Pharmaceutical compositions with mucolytic, bronchosecretolytic and anti-bronchospastic activity - η. 1 certified copy of the 077 j ; ii) / .description and of the verbal transfer.!-...............__ j ! i AND ! i N........ 110 4556 granted i 1.....2.1 .October . 19.8 5......in the name ..........DOMPE' FARMACEUTICI........SpA..........-.....Milan.....-.....from l..t title: j Lysine salt of 1 'acid., meta-benzoyl-hydratro-J i i .Pico, process......for_____its preparation____eJ ! pharmaceutical compositions that contain it do not _- . n. ... certified copy of the 1st.. description and of the . ver Jo al ei '1 : jdi tra se rizi on e._______ · i ! i : j We authorize Mr. .......Giorgio Gristina to collect on our behalf the certified copies mentioned above. ji THE With regards ............................,................................ .....................................................................L Mi 1 no, 10 December 1991 _ _________________________________ ι _.................................11 ..Agent Mauro Bracco SWio Corìsiìfsma Brevettale s rl 917 M GB / sm DESCRIPTION of the industrial invention entitled: BIOAVAILABLE IRON DERIVATIVES FREE FROM GASTROLESIVITIES PREPARATION PROCEDURE AND RELATED PHARMACEUTICAL COMPOSITIONS in the name of: ITALFARMACO SpA a: Viale Fulvio Testi, 330 MILAN of Italian nationality and elected domiciled for all legal purposes at the agent Studio Consulenza Patent Office - Via Senato n. £4 - Milan Designated Inventors: 1. Giancarlo Sportoletti 2. Pier Giuseppe Pagella 3. Pietro Cremonesi ' » * **» · « ·* * Filed on March 2, 1982, No. 19OO8A^M SUMMARY Succinylated proteins, with a degree of succinylation ranging from 20 to 100% and containing 0.1 to 20% iron by weight, are described. These proteins are obtained by treating succinylated proteins with iron salts at appropriate pH ranges. The resulting compounds, perfectly tolerated, are characterized by excellent iron bioavailability. Patent Consulting Studio fuaifì sr,f, - 2 DESCRIPTION pELL » INVENTION Iron, present in all tissues of the body, plays an irreplaceable physiological role. It is an essential component of hemoglobin, myoglobin, and the enzymes catalase, fumaric dehydrogenase, peroxidase, and cytochromes. DPN-cytochrome reductase and metaflavoproteins, such as ferroflavin. The tissues of an adult human contain 1.5 to 5 grams of iron, of which 60-65% is concentrated in red blood cells, while 18-30% is bound to the proteins ferritin and hemosiderin, located in the liver, spleen, bone marrow, and cells of the reticuloendothelial system. The remainder is distributed among the various enzyme systems mentioned above, in myoglobin (about 4%), and in transferrin (about 0.1%), the globulin responsible for transporting intestinal iron into the bloodstream. It is capable of binding two iron atoms, virtually inseparably at physiological pH values. Iron needs are met partly through the use of endogenous iron (derived from the breakdown of aged red blood cells) and partly through the intake of exogenous iron. Absorption of exogenous iron occurs throughout the duodenum and upper jejunum and is accumulated primarily in the liver. The first pathological manifestation of iron deficiency is hypochromic iron-deficiency anemia, the primary causes of which can be varied: chronic hemorrhages such as those occurring with gastroduodenal ulcers or neoplasms; insufficient iron. Patent Consulting Studio srl - 3 dietary intake, i.e. poor absorption as in diarrhoeal conditions; increased requirement, for example in pregnancy, breastfeeding, infectious diseases, etc.; Imperfect metabolic utilization; particular therapies such as those with ACTH and cortisone. The most appropriate therapy is iron supplementation; it is effective in reducing anemic conditions when, obviously, they result from an actual iron deficiency; however, it is generally accompanied by unwanted side effects related to the type of iron carrier used. The most widely used oral pharmaceutical preparations are based on a wide variety of organic and inorganic acid salts, gradually introduced into therapy with the hope of reducing the aforementioned side effects. Thus, oral preparations are marketed based on inorganic salts such as ferric chloride, ferrous sulfate, ferric phosphate, or organic salts such as citrate, choline, aspartate, fumarate, gluconate, glycineate, lactate, oxalate, and succinate. For deep intramuscular administration, the iron-dextran complex is recommended, while for intravenous treatments (for iron-deficiency anemia where oral therapy has failed), the iron-dextrin complex is used. For these latter types of complexes, side effects may consist of allergic reactions, temperature increases, tachycardia, leukocytosis, lymphadenopathy, for the treatment Shdio Patent Consulting srl - 4 intramuscular administration, and even anaphylactic shock, thrombophlebitis, and circulatory collapse with IV treatment; oral therapy with formulations based on the above compounds generally results in gastrointestinal damage, with necrosis and perforation of the mucosa in the most severe cases, diarrhea, and vomiting. Furthermore, poor tolerability makes it difficult to administer the appropriate amounts of iron. To minimize side effects, concomitant intake of food is recommended, but this contradicts the demonstrated variability of iron absorption depending on the composition of the food itself. It is, in fact, well-established that iron absorption from preparations is optimal when the patient is fasting. Furthermore, gastric irritation is followed by the use of antacids, which, in turn, reduce absorption, worsening the anemic state. A notable advance in martial therapy, by mouth of course, was made with the introduction onto the market of ferritin-based specialties. Ferritin is an iron globulin and is the most important iron storage protein in mammals. The commercial product, as a raw material, is extracted from horse spleen. Its iron content is 20% of the dry weight; the protein part, apoferritin, has a molecular weight of approximately StoJio Patent Consulting s,d - 5 • 445.00 and surrounds a crystalline iron oxide phosphate core. Soluble in water, it is suitable for oral administration. Ferritin therapy does not present the gastrointestinal side effects observed with the use of iron derivatives, as mentioned above. A serious limitation to its use arises both from the very high cost of the raw material and, above all, the limited availability of extraction sources. What is known so far seems to demonstrate that the optimal iron carrier for a side-effect-free intake may be a protein carrier. Many proteins have a certain affinity for iron. Therefore, attempts have been made to use certain types of proteins of animal (whey proteins, organ proteins, ovalbumin, lactoproteins) or plant (soy proteins) origin as carriers. The interaction between inorganic ferric salts and the aforementioned proteins leads to the formation of ferroprotein derivatives whose therapeutic interest is however invalidated by a series of negative characteristics, including: - the insolubility of the derivatives obtained when the percentage of iron bound to the protein reaches values ​​greater than 0.5%; - the difficulty or even impossibility of evaluating in such conditions of insolubility which fraction of the total iron is actually bound to the protein and which simply covers Brevethiaìe Consulting Studio srl - 6 • precipitated in the form of hydrated oxides, highly gastrolesive; - the lack of homogeneity and consistency in the iron composition of these derivatives. Furthermore, for some proteins, including those derived from milk, the presence of soluble iron derivatives alongside a notable quantity of insoluble derivatives has been found at various pH values, but the composition of these soluble fractions is extremely variable depending on minimal variations in the experimental parameters. It has now been surprisingly discovered that by succinylating the aforementioned proteins under appropriate conditions (a process normally used to obtain protein succinylated derivatives for food and feed use), it is possible to obtain, by reacting them with iron, ferroprotein derivatives even with high iron content. These derivatives are stable; soluble at pH values ​​greater than 5; with different but reproducible iron contents; and capable of providing iron to warm-blooded organisms, when administered orally, without inducing gastrointestinal side effects. In fact, in rats, daily oral administration for 20 consecutive days did not show any signs of gastrointestinal damage and was well tolerated. Furthermore, always in the same type of animals, the comparison of sideremia, after oral administration, of equivalent quantities of iron in the form of ferrous sulphate, ferritin from equine spleen (sigma, Saint Louis, Missouri) and ferroprotein derivative Studio Considerai Brevellusle srI - 7 » from succinylated milk proteins, prepared as reported in example 1, shows that: a) the blood iron levels induced by the derivative, object of the present invention, compared to those from ferrous sulphate, are more prolonged over time (see table IX); b) the blood iron levels induced by the derivative, object of the present invention, compared to those of control ferritin, are higher (about 1.5:1 on average) over the six hours of the experiment (see table X). Considering the excellent tolerability of the product, its very low toxicity (greater than 4,000 mg / kg in the Swiss mouse), the high blood iron levels achievable and the ease of obtaining the raw material, the use of the derivatives, which are the object of the present invention, constitutes a clear improvement in the state of the art of martial therapy. The chemical and physical characteristics of these succinyl iron protein derivatives, as mentioned above, are reproducible and consistent, as reported in the examples, which are not intended to be exhaustive. The absence of unbound iron is demonstrated by the complete solubility in an alkaline environment (conditions in which the iron ion precipitates as a hydrated oxide) of the preparations obtained from succinylated proteins and an iron salt. The subject of this patent application is therefore the derivatives, not known until now, characterised by the fact that they contain Sfmlia Fertilizer sr1 - 8. black quantities of iron varying in percentage, animal or vegetable proteins succinylated at various levels of succinylation, and which have been shown to be suitable for the supply of bioavailable iron in warm-blooded organisms, without gastrointestinal harmful effects if administered orally, as shown in the examples below, which are indicative and not limiting. These derivatives appear to be suitable, as active ingredients, for the preparation of pharmaceutical preparations usable in the treatment of iron deficiency anemia in warm-blooded animals and, therefore, any pharmaceutical form of administration containing these derivatives as an active ingredient is a further object of this patent. The invention also provides a process for preparing the succinyl iron protein derivatives in question, a process characterized in that succinylated proteins are reacted (in a manner known per se) with iron ions at pHs between about 2 and about 10, preferably at a pH around neutrality, in aqueous solvents, and the products obtained are isolated from the reaction medium by precipitation at a pH of about 2-4, the latter pH range being achieved spontaneously or by acidification. Alternatively, the reaction product can be isolated by removing the solvent after adjusting the pH to values ​​close to neutrality. Patent Consulting Studio srì. * EXAMPLE 1 Preparation of 5% iron derivatives from succinylated whole milk powder proteins 1 kg of milk powder is suspended in 6 liters of 0.3M sodium bicarbonate and, under mechanical stirring and pH control, 500 g of succinic anhydride is added in successive portions, maintaining the pH between 7.5 and 8 by adding 4N NaOH. After the addition, the mixture is left under stirring for 2-3 hours at room temperature; during this time, the suspension dissolves completely. The opalescent solution is centrifuged or filtered until almost clear and then slowly acidified with HCl to pH 3. The precipitate that forms is separated by centrifugation or filtration and resuspended in water (approximately 8 litres); a NaOH solution is added until completely dissolved (pH approximately 7.5); it is centrifuged and the clear solution is re-acidified to pH 2.5-3 by adding HCl. The precipitate is recovered by filtration or centrifugation and washed with a pH 3 HCl solution. The residue, consisting of succinylated proteins (the degree of succinylation of which can be varied depending on the quantity of succinic anhydride used; for the example given here it is 90% with respect to the succinylatable groups present in the starting protein as reported below) is, after drying under vacuum, dispersed in distilled water; it is dissolved by adding NaOH until Patent Coiìsulertze Studio $.rt - 10, at pH 7.5, diluting the solution so that the final protein concentration is around 0.04 g / ml. To this solution is added a solution of an iron salt (example: FeClg) with a ratio of Fe^ / succinylated protein of 1:10 by weight. A suspension is obtained which is finely dispersed by mechanical stirring while the pH drops to around 2.5. It is left to stir for 3 hours at room temperature and then the suspension is filtered; the solid is suspended in water (approximately three volumes) and is dissolved by the addition of NaOH (pH 7.5) The solution which is not perfectly clear is filtered and the recovery of the product in the solid state can be carried out indifferently in two ways: a) the solution is acidified and the precipitate obtained at pH 2.5 is filtered and dried under vacuum; b) the clear solution is dialyzed against water to eliminate the sodium chloride and then freeze-dried or spray-dried. In both cases, the finished product yields are equivalent and are around 20% by weight of the starting milk powder. Product characteristics are described in Table I (example 1b). EXAMPLE 2 Preparation of 5% iron derivatives from succinylated milk proteins BreveìlosTe sf Consulting Studio! As described in example 1, with the variant that the starting product consists of 350 g of milk proteins, previously precipitated from milk powder by dispersing the powder in water (approximately 5 litres), acidifying to pR 2.5 with a mineral acid and then filtering or centrifuging the protein solid and drying it. The iron content, as reported below (table 1), for the derivatives obtained in examples 1 and 2, is around 4-5% by weight. EXAMPLE 3 Preparation of iron derivatives from succinylated milk proteins with different iron contents By operating as described in examples 1 and 2 with regard to the method and the quantity of milk powder or previously precipitated milk proteins, but by modifying the weight ratio Fe / succinylated protein to the values ​​of 0.1:10, 0.2:10; 0.5:10 and 1.5:10, iron derivatives are obtained whose characteristics are reported in table I. EXAMPLE 4 Preparation of iron derivatives from succinylated egg proteins (Ovalbumin) 5 grams of ovalbumin are dissolved in 100 ml of water containing 3 grams of KHC0. To the clear solution, 2.5 g of succinic anhydride are added in successive portions, controlling the pH at values ​​between 5 and 8 by adding NaOH; $>ￎﾹￎﾺￎﾯￎﾐￎ﾿ Consulting Brevdkde ir - 12 ￢ﾀﾢit is left to react for 2 hours at room temperature; after acidifying the solution to pH 3.4, a precipitate is obtained which is isolated by centrifugation, purified by dissolving at pH 7>5, using NaOH, and subsequently reprecipitated at pH 3.4. The solid is recovered by centrifugation and dried under vacuum, the dry solid is suspended in distilled water and dissolved by adding NaOH (pH 8) to obtain a final solution of 0.04 g of protein / ml. A solution of a 3+ iron salt (e.g., ferric chloride) is added to the highly viscous solution to achieve a succinylated protein / % weight ratio of 10:1. Under these conditions, the pH drops to 2.6, resulting in the formation of a precipitate that is recovered by filtration. This precipitate is redissolved in water and NaOH is added until completely dissolved (pH 7.5). After dialysis against water to remove the sodium chloride, the solid product is recovered by freeze-drying or spray-drying. The derivative yield is equal to 33% by weight of the starting protein. Iron content and characteristics are shown in Table I. EXAMPLE 5 Preparation of iron derivatives from succinylated bovine serum proteins 50 milliliters of bovine whey protein solution (protein content approximately 4 g) are added with 1.5 g of NaHCOg and then treated with 6 g of succinic anhydride, in successive portions, Patent Consulting Studio sii * controlling the pH between 7.5 and 8 by adding NaOH. After the addition, the reaction is allowed to proceed for 2 hours at room temperature and then precipitated by acidifying to pH 2.4 with mineral acid; the precipitate is recovered by centrifugation and purified as described in the example. 4. The solid product is suspended in water and adjusted to pH 7.5 using NaOH; the resulting solution is added with an iron III salt solution (e.g., ferric chloride) until a succinylated protein / Fe weight ratio of 10:1 is achieved. The pH drops spontaneously to 2.4, resulting in a precipitate that, adjusted to pH 7.5, does not dissolve in part. The solution is separated from the residue by centrifugation, dialyzed against water to eliminate the sodium chloride, and then freeze-dried. The weight yield of iron derivative, compared to the starting protein, is approximately 30%. For iron content and characteristics, see Table I. EXAMPLE 6 Preparation of iron derivatives from succinylated porcine liver proteins 400 grams of fresh pork liver are homogenized using a Sieverson homogenizer in the presence of 800 ml of 0.2 M KHCOg solution, pH 8.3; after centrifugation at 9000 rpm at 5°C, the supernatant is collected and filtered through a 0.45 mmol cellulose acetate membrane. 50 ml of clear solution are obtained. Patent Consulting Studio s.r.l. - 14 • add 1.2 g of succinic anhydride, in successive portions, checking that the pH remains between 7.5 and & by adding NaOH· After standing for 1 hour at room temperature, the solution is acidified with mineral acid at pH 3, obtaining a precipitate which is worked as in example 4, with the variation that the final recovery is carried out by freeze-drying the solution, dialysed against water. The residue from freeze-drying is suspended in water and brought to pH 8 using soda. A not perfectly clear solution is obtained which is centrifuged; an iron solution (for example ferric chloride) is added to the clear supernatant so that the (estimated) ratio between 3+ succinylated protein and Fe is approximately 10:1. The pH of the solution drops to 2.6, resulting in a precipitate that is purified as in the previous examples. The solid obtained from freeze-drying the final solution, dialyzed against water, has the characteristics and iron content shown in Table 1. EXAMPLE 7 Preparation of iron derivatives from succinylated soy proteins 5 grams of soy protein are suspended in 100 ml of water containing 3 g of NaHCO^; it is left under stirring until completely dissolved; checking the pH between 7.5 and 8 for addition. Storio Cortsukma Brevettuale s.r.J., •ta of NaOH are added in successive portions of 2.5 g of succinic anhydride. The mixture is left to stir for 2 hours at room temperature and by adding mineral acid it is brought to pH 3.4, obtaining a precipitate that is purified as described in the previous examples. The solid residue from the final freeze-drying is suspended in 100 ml of water, the pH is corrected to 8 by adding soda and then the solution, which is not perfectly clear, is centrifuged. The clear supernatant is treated with an iron solution (e.g., ferric chloride) so that the ratio of 3+ succinylated protein to Fe is 10:1 by weight. The pH of the solution spontaneously drops to 2.6, and a precipitate forms, which is treated as in the previous examples. The solid obtained from freeze-drying the final solution has an iron content and characteristics as reported in Table I. Yield by weight with respect to the starting protein: 22%. PHYSICAL-CHEMICAL CHARACTERIZATION OF IRON DERIVATIVES FROM PROTEINS SUCCINILATE OBTAINED ACCORDING TO EXAMPLES 1-7 The derivatives obtained according to the examples above were analyzed following the methodologies specified below: a) degree of disuccinylation This characteristic is expressed as a percentage of cynylated groups compared to the succinylatable groups of the starting protein. The method described by s. was applied. Moo.re and WH Stein, J· Biol. Chem., 211, 907, 1954, for reaction with ninhydriStudio Ceri lìÌmzì) EhtvcRuaìe srl - 16 na of free amino groups, the results are reported in Table I» b) iron content The iron content was determined both after hot extraction with 2N HCl and after complete digestion of the protein with sulfuric acid, as described in Standard Methods, 14th ed., 1975, page 208, APHA-AWWA-WPCF (reaction with o-phenanthroline). The values ​​are reported in the table. c) spectroscopic data 1) EPE spectroscopy All the derivatives described in the examples above show the same spectroscopic behavior. For example, the EPR spectrum of the succinylferroprotein derivative prepared according to Example 1 is described. The spectrum was recorded on a freeze-dried powder and shows a very broad signal, centered around g = 2.0, with a signal amplitude of approximately 2000 G. Another, very well resolved signal is present, also centered around g = 2.0. The shape of both signals is characteristic of interacting paramagnetic Fe centers (F). Reid et al. - Inorg. Chem. 2» 119, 1968). The combination of these data would indicate the presence of protein structures closed around the ligand. 2) Circular dichroism Here too the behaviour of the various derivatives described is similar; as an example, the circular dichroism spectrum of the succinylferroprotein derivative obtained according to the Studio Coirsulerai Brevettile vi - 17 example 1 · The spectrum is characterized by a negative activity in the UV region; there is an intense negative band with a maximum around 200 nm, with a shoulder, also negative, centered around 225 nm. These spectral characteristics would indicate the prevalence of a random protein structure (inter alia: F. Ciardelli and P. Salvadori - Optical Rotatory Dispersion and Circular Dichroism - Heyden & Son Ltd., London, 1973— Chapter 4.5). 3) UV-Visible absorption spectra The electronic spectral behavior of the various derivatives described is similar; Fig. 1 shows the UV-Visible spectrum for the succinylferroprotein derivative prepared as described in Example 1. The spectrum (solid line) does not show absorption maxima in the visible but only a very weak shoulder around 450 nm. In the ultraviolet there is a fairly well-defined shoulder centered around 270 nm and very intense absorption with a maximum below 220 nm. Figure 1 also shows (dotted line) the ferritin spectrum from horse spleen, for comparison (BoehringerMannheim) The absorption spectra in the visible range are reported in figure 1 and were carried out in water, pH 7. The spectrum under the same conditions of horse spleen ferritin is also reported, for comparison (Boehringer-Mannheim). Ci'r.hjl·'ίπζ» Brevcffuale srl - 18 • 4) Fluorescence emission spectrum The fluorescence emission spectra are reported in figure 2, in which the solid line corresponds to the succinylferroprotein derivatives of the invention, the dotted line to the lactoproteins as is, and the dashed line to the succinylated lactoproteins. A quenching of the protein fluorescence is observed, due to the presence of metal, d) electrophoresis The electrophoretic patterns on cellulose acetate of the various derivatives in comparison with the succinylated proteins, the original proteins and ferritin are shown in figures 3, 4, and 6. Figure 3 shows the results of electrophoresis conducted on soy proteins (path 1), succinylated soy proteins (path 2), iron derivative of succinylated soy proteins (path 3), iron derivative of non-succinylated soy proteins, soluble fraction (path 4). Figure 4 shows the results of electrophoresis conducted on ovalbumin (trace no. 1), succinylated ovalbumin (trace no. 2), iron derivative of succinylated ovalbumin (trace no. 3), iron derivative of ovalbumin (trace no. 4). Figure 5 shows the results of electrophoresis conducted on lactoproteins (trace no. 1), succinylated lactoproteins (trace no. 2), iron derivative of succinylated lactoproteins (trace no. Studio CoKdenia Brevettale sr - 19 • trace n. 3), ferritin from horse spleen (trace n. 4)· Figure 6 shows the results of electrophoresis (densitometric reading at 550 nm) conducted on iron derivative of succinylated α-toproteins (solid line), iron derivative of succinylated soy proteins (dash-dot) and iron derivative of succinylated ovalbumin (dotted line). e) gel filtration The gel filtration profile on Sephadex G 75 (Pharmacia, Uppsala) of the iron derivative of succinylated milk proteins (obtained as described in Example 1), in comparison with the succinylated proteins themselves, is shown in Figure 7, where the solid line refers to succinylated lactoproteins, the dashed line to the corresponding iron derivative; the ordinate shows the absorbance at 280 nm, the abscissa shows the progressive number of collected fractions. From the profile, it can be seen that the presence of iron gives rise to a single peak at the outlet with a retention rate similar to that of the column void. Considering that the molecular exclusion of Sephadex G 75 is approximately 75,000 Daltons, the apparent minimum molecular weight value of this aggregate is higher than this value. Furthermore, unlike it, succinylated proteins exhibit peak splitting with greater randomization. This fact is also observed at the electrophoretic level (Figure 5). Such behavior indicates that the iron present is cen. intermolecular aggregation of the protein medium. UlteStudio CofistiSenìc ButeHuale sr1 - 20 • Further information can be seen from gel filtration on Sephadex G 200, in comparison with ferritin (figure 8, in which the abscissa and ordinate are as in figure 7, while the solid line corresponds to the iron derivative of succinylated β-actoproteins and the dotted line is relative to ferritin obtained from horse spleen). Also in this case the peak is univocal while the retention time is higher than that of ferritin. This seems to indicate that the apparent molecular weight is lower than that of ferritin from horse spleen. f) gastric juice stability This test is conducted on the iron derivative obtained according to Example 1 and is carried out by dissolving 25 mg of derivative in 0.5 ml of distilled water and adding this solution to 5 ml of rat gastric juice. A precipitate is obtained, and the suspension is incubated at 37°C for 60 minutes with stirring. After this period, the precipitate is separated by centrifugation, and the supernatant is analyzed for iron content. The amount of iron released into the gastric juice was 1% of the initial total. g) precipitation of the iron derivative with ammonium sulfate To a 10% (w / v) solution of iron derivative prepared as in Example 1, 30% (w / v) ammonium sulfate is added. A precipitate of iron protein derivative is obtained that has the same characteristics as the starting solution and is still soluble in water. No iron is present in the solution. The above data set demonstrates that the overall capacity Studrc Consulting Brmftuale sti - 21 The iron deficiency of succinylated proteins is not only typical of succinylated milk proteins but is characteristic of succinylated derivatives of proteins of various origins and physiological functions, whether animal or vegetable. As we have reported elsewhere, many unmodified animal or plant proteins are capable of binding iron. Indeed, when unmodified milk proteins, egg albumin, soy proteins, whey proteins, and organ (liver) extracts are reacted with iron under conditions similar to those described, to obtain the corresponding succinyl protein derivatives, products with similar iron contents are obtained, yet with completely different solubility and stability characteristics. For example, as shown in Table 1, soy protein yields a final product that, when freeze-dried, is over 75% insoluble and releases its iron upon treatment with gastric juice.In addition to the complexing capacity with respect to iron, the behavior of the succinylated iron-protein derivatives, object of the invention (i.e. solubility in an alkaline environment, stability in an acidic environment and in the presence of gastric juices even for high iron contents) is not attributable to the basic proteins, and therefore was not at all foreseeable. TOXICITY' a) The acute toxicity of iron Buccini1 protein from proteins was examined in the Sviss mouse, orally, by gastric probing. Studio Consulenza lìrwèiì uaìe srl - 22 • of milk, obtained as described in example 1, transported in distilled water. The DL^ 0 was found to be higher than 4,000 mg / kg, b) Repeated oral administration toxicity in rats, on the same derivative as in example 1 (hereinafter referred to as Ferrolat''), was investigated for 20 days of continuous treatment on male Vistai? rats with an initial weight of 1000 grams. 200, housed for 14 days before treatment and then randomized and separated into groups of 10 (I group: controls, II, III and IV group Ferrolat at 30-100-300 mg / kg / day). The preparation under examination, dissolved at the moment of use in distilled H20, was administered daily, without interruption and at the same time each day, by gavage; the controls received an equal volume (10 ml / kg) of spring water. During treatment, the rats were weighed daily, before administration, and observed for a few hours after administration of the product. Food consumption was measured at the end of treatment, 24 hours after the last administration and after approximately 20 hours of fasting. The rats under ether anesthesia were sacrificed by cutting the carotid arteries; a portion of the blood was collected in a test tube containing EDTA (2 drops of a 4% solution), the remainder was left to serum. Complete blood counts were performed on the whole blood; blood chemistry tests were performed on the serum. Study Consuhw Dif v?|hr.-:?f: $. - 23 RESULTS Weight changes and food consumption Figure 9 shows the weight growth curves obtained during the treatment up to the day before sacrifice. Table II and Table III show the values ​​relating to weight gain and food consumption of the animals undergoing treatment, respectively. The growth curve of the group of rats treated with Ferrolat at a dose of 300 mg / kg was significantly slowed, compared to the growth curve of the control group of rats, starting from the 15th day after the beginning of the treatment; the same group of rats consumed on average less food (-11.7%). Minor, statistically insignificant differences were observed in the remaining groups of rats treated with Ferrolat. Blood tests The results are reported in Table IV. Blood tests The results are reported in Table V. A modest decrease in alkaline phosphatase was observed in rats treated with Ferrolat, 300 mg / kg / day. Urine tests The results are reported in Table VI BrcVeituele s.ri Consulting Studio Organ tests Table VII shows the mean absolute weights of the main rat organs and Table VIII shows the corresponding mean weights relative to body weight. In the group of rats treated with Ferrolat 100 mg / kg / day, a significant increase in the relative weight of the spleen and lungs was observed. In the group of rats treated with Ferrolat 300 mg / kg / day, in addition to a significant decrease in body weight, a significant increase in the mean weight of the testes was observed. BLOOD LEVELS AFTER ORAL ADMINISTRATION a) Comparison of iron and ferrous sulfate Male Wistar rats weighing approximately 200 grams and fasting for 18 hours were used, randomized and separated into groups of 5; each group was sacrificed at the determined time and blood was collected from each animal by carotid resection; the iron content of the blood of each animal in the group was determined spectrophotometrically using bathophenanthrolinesulfonic acid (Fe-Test, Wako). Administration was carried out by probing in aqueous solution (5 ml / kg). The succinyl iron protein derivative used was prepared as in example 1 (Ferrolat). Dosages and results are reported in Table IX. Note that the amount of iron / kg was the same for both derivatives (Fe: 1 mg / kg). b) Comparison Ferrolat - ferritiha Consiìlenw Kr&idtaale stadium ut - 25 * • Male Wistar rats weighing approximately 200 grams and fasting for 18 hours were used, randomized and separated into groups of 9; each group was sacrificed at the determined time and blood was taken from each animal by resection of the carotid; the iron content of the blood of each animal in the group was determined spectrophotometrically using the Iron Colorimetry Method (Boehringer-Mannheim). Administration was carried out by probing in aqueous solution (5 ml / kg). Dosages and results are reported in table X. Note that the quantity of iron / kg for the succinyl iron protein derivative indicated as Ferrolat was that of Ferritin (sigma, St. Louis Missouri) and equal to 1 mg / kg. c) Comparison of succinylferroprotective derivatives obtained from different types of proteins Male Wistar rats weighing approximately 200 grams, fasted for 18 hours, were used, randomized and separated into groups of 5 (one for each dose of derivative, and a control group). Each group was sacrificed 2 hours after administration and, by resection of the carotid artery, blood was collected from each animal and iron was determined using the bacillus subtilisulfonic acid (Fe-Test, Wako). Oral administration was carried out by probing in aqueous solution (5 ml / kg). The doses of the individual derivatives used were chosen so as to always administer 1 mg / kg of Fe. Products tested: 1) succinylated soy proteins + Fe (4.42%) SìuÓjo Cmtsuìcw IWdhwIe s. - 26 * 2) succinylated ovalbumin + Fe (3.57%) 3) succinylated bovine whey proteins + Fe (5.78%) 4) succinylated milk proteins + Fe (4.5%) (Ferrolat). The results are reported in Table XI. Sbdìo Patent Consulting sd TABLE I PROTEIN SOURCE SUCCINILATION (¾ of succinylatable groups) Fe (¾ g) YIELD (1 of succinylated protein) SOLUBILITY at pH > 5 £) Milk 3) 95 6.7 30 + - „ b) It 4.6 20 + „ c) It 3.2 18 + „ d) II 2.5 18 + „ e) II 0.8 13 + Lactoproteins 95 4.5 + Ovalbumin 95 3.6 33 + Whey proteins 82 15.8 + (bovine) [5.5 30 + — (Soybean) 92 4.4 22 + (Organ) nd 8.4 nd 4· — (liver) (Milk) 35 4.3 26 + Lactoproteins 0 4.8 ί 7.7 « ​​Ovalbumin 0 10.4 + (4.5 - (Soy) 0 10.4 + - Whey proteins or 0 5 + - (bovine) (Organ) 0 ndnd (liver) 3+ a)» b), c), d), e) samples with Fe:succinylated protein ratios between 0.1:1 and 5:1 (p:p). f) complete (+); partial (+ -); insoluble (-). Patent Consulting Studio srL * TABLE II WEIGHT GAIN TREATMENT mg / kg / day Initial mean weight Final mean weight Mean weight gain+ ES Controls 224.10+2.83 343.60+8.87 119.50+7.66 Ferrolat 30 224.40+3.03 325.50+4.56 101.20+4.35 Ferrolat 100 224.40+2.30 326.10+8.06 101.70+8.08 Ferrolat 300 224.30+3.71 307*00+10.15 8^*77+8.28 X p<0.05 XX p<0.01 Significance versus controls (calculated with Student's t-test) TABLE III FOOD CONSUMPTION TREATMENT mg / kg / day Total mean intake Change Controls 32.33 - Ferrolat 30 30.42 - 5.91 Ferrolat 100 30.98 - 4.18 Ferrolat 300 28.54 -11.72 TABLE IV HEMATOLOGICAL DATA TREATMENT mg / kg / day Controls Ferrolat 30 Ferrolat 100 Ferrolat 300 Hemoglobin g £ 12.11+0.47 11.77+0.37 12.39+0.30 12.93+0.25 Hematocrit % 66.88+2.06 61.44+1.96 66.90+2.77 64.16+1.80 Leukocytes 10 3 / mm 3 16.95+2.25 16.55+1.65 23.46+2.16 19.93+2.58 Erythrocytes IOθ / mm^ 5.76+0.51 6.12+0.68 5.91+0.68 6.22+0.31 Studio Consulenza BreveBtiaìe s.rl TABLE v BLOOD CHEMICAL DATA TREATMENT . . . ntg / kg / day Controls Ferrolat $0 Ferrolat 100 Ferrolat 300 Gl ucose mg% 55.33+5.75 52.81+4.38 63.61+5.58 60.74+4.97 Azotemia mg% 32.73+1.33 34.59+2.25 28.68+2.34 32.33+2.52 GOT mll / ml 110.51+7.10 130.81+8.59 109.09+5.21 113.36+7.29 GPT mU / mf 19.58+1.52 26.86+3.43 30.14+6.02 23.15+2.57 Alkaline phosphatase mU / ml 255.65+13.82 229.53+17.03 257.53+22.35 3" 6.19+0.12 6.02+0.17 6.37+0.12 6.21+0.16 Significance X p <o,o5 xx pi.0,01Shidìo Patent Consulting sft TABLE VI URINE TESTS TREATMENT mg / kg / pH Controls Ferrolat 30 Ferrolat 100 Ferrolat 300 8.15+0.17 8.55+0.14 8.40+0.18 8.50+0.12 proteins 21.- +3.78 24.- +3.05 63.- 112.4^* 53.33+11.66 abs. glucose ass. ass. ass. abs. ketone bodies ass. ass. ass. abs. bilirubin ass. ass. ass. blood abs. ass. ass. ass. Significance R p< 0.05 xx p<0.01 Sfaiìo Patent Consulting ifl TABLE VII ABSOLUTE FRESH WEIGHT OF RAT ORGANS TREATMENT mg / kg / di Controls Ferrolat 30 Ferrolat 100 Ferrolat 300 Body weight g 310.60+7.53 300.33+3.64 296.8+6.16 X 278.44+8.89 Heart g 1.15+0.05 1.10+0.03 1.14+0.04 1.09+0.07 Lungs g 1.55+0.05 1.70+0.20 1.68+0.05 1.57+0.12 Liver g 9.14+0.35 8.89+0.35 8.78+0.28 8.35+0.44 Spleen g 1.38+0.13 1.36+0.08 1.65+0.12 1.37+0.09 Kidneys g 2.28+0.07 2.21+0.09 2.22+0.08 2.09+0.10 Adrenals mg 50.30+4.55 56.33+7.69 51.80+6.07 37»44+5.56 Brain g 1.77+0.03 1.70+0.03 1.67+0.05 1.68+0.06 Testes g 3.30+0.05 3.12+0.10 3.08+0.16 3.25+0.07 Stomach g 1.77+0.09 1.77+0.05 1.75+0.07 1.53+0.09 Significance X p<0.05 p<0.01 *** p <o,ool ρ ' ,. f λ , » 32 sudìo consulenza tirevdìuale s,r.lTABLE VIII RELATIVE FRESH WEIGHT OF RAT ORGANS TREATMENT mg / kg / day Controls Ferrolat 30 Ferrolat 100 Ferrolat 300 Body weight 310.60+7.53 300.33+3.64 296.80+6.16 A 278.44^8.89 Heart $ 0.37+0.01 0.37+0.03 0.39+0.09 0.39+0.02 Lungs $ 0.50+0.02 0.56+0.06 A 0.57+0.02 0.57+0.06 Liver 1 2.94+0.04 2.96+0.10 2.96+0.06 2.99+0.08 Spleen % 0.44+0.03 0.45+0.03 0*56+0.04 0.49+0.03 Kidneys $ 0.73+0.01 0.74+0.02 0.75+0.03 0.75+0.02 Adrenals $ 0.016+0.001 0.019+0.003 0.017+0.002 0.014+0.002 Brain ί 0.57+0.02 0.57+0.01 0.57+0.02 0.60+0.01 Testes ì 1.06+0.02 1.04+0.03 1.04+0.05 1*18+0.04 Stomach % 0.56.+0.02 0.59+0.02 0.59+0.02 0.54+0.02 Significance * p<0.05 aa p<0.01 aaa ρζΟ,ΟΟΙ - 33 - Sfatilo Patent Consultancy tei TABLE IX BLOOD LEVELS: FERROLAT VS FERROUS SULFATE COMPARISON TREATMENT Serum iron ^tg% hours after treatment(3 30' 1 2 4 6 Controls HO S ml / kg 99.22 59.91 98.25 80.17 74.- +24.92 + 6.85 +13.28 + 6.37 + 10.10 (5) (5) (5) (5) (5) Ferrolat; 22.37 mg / kg 227^5 27^8 2O?il6 14§*18 (Fe equal to 1 mg / kg) +16.40 +18.91 +22.51 +27.74 +16.73 (5) (5) (5) (5) (5) xxx XXX xxx xxx FeSO.TH 0: 4.98 mg / kg 336.27 272.14 273.56 129.50 80.52 4 Z (Fe equal to 1 mg / kg p.os) +27.79 (5) +24.09 (5) +19.32 (5) +6.21 (S) +5.65 (5) Significance towards controls: xp <o,o5XX p<0.01 xxx p<0.001 Significance between the two iron preparations: 30': p <0.01; 4h: p <0.05» 6h: p <0.01 TABLE X BLOOD LEVELS: FERROLAT-FERRÌTIN COMPARISON TREATMENT Serum iron ug / 100 ml hours given treatment 1 2 4 6 Ferritin (F-4503 Sigma 168.79 170.66 148.08 89.22 dilution 1.08¾ v / v, +15.35 +14.70 +18.69 +12.75 5 ml / kg equal to 1 mg / kg of Fe) xxx XX Ferrolat 21.46 mg / kg 302.07 269.81 223.72 106.24 (equal to 1 mg / kg of Fe) +15.59 +29.94 +18.51 +11.30 in 5 ml / kg Controls, distilled water 5 ml / kg XXX 80.40 +6.39 xp<0.05 xx p<0.01 xxx p<0.001 Significance is calculated with Student's t-test versus Ferritin Studio CcnsuSma Patent s,f1 TABLE XI BLOOD LEVELS: COMPARISON BETWEEN BUCCINILFERROPROTEIN DERIVATIVES OBTAINED FROM DIFFERENT TYPES OF PROTEINS TREATMENT Serum iron in ug / 100 ml mean values ​​+ ES Controls 83.99+7.56 Soy succ. ¥¥¥ 260.68+29.52 Ovalbumin succ. 235.82+46.06 * Whey protein succ. ** 277.63+49.35 Ferrolat 305.81+23.20 *** Significance versus controls xp<0.05 XX p<0.01 xxx p<0.001 The present invention also relates to all industrially applicable aspects related to the use of the succinyl iron protein derivatives described and claimed herein as agents suitable for the treatment of iron deficiency anemia. Therefore, an essential aspect of the invention consists of pharmaceutical formulations containing predetermined quantities of such derivatives. The latter can be administered orally, for example in the form of tablets, capsules, vials, sachets containing granules, syrups, etc. The following formulations are cited purely by way of example: Brevelbal Consulting Studio « - 35 - vials containing 5-10-40 mg of iron in the form of ferrosuccinyl proteins, in addition to aqueous solvent, stabilizers, flavorings, etc. commonly used in pharmaceutical technology; - 5-10-40 mg iron tablets in the form of ferrosuccinyl proteins, in addition to excipients, disintegrants, etc. commonly used in pharmaceutical technology; - capsules containing 5-10-40 mg of iron in the form of ferrosuccinyl proteins; - single-dose sachets containing an effervescent granule equal to 5„io_4o mg of iron in the form of ferrosuccinyl proteins, in addition to sugar, flavourings, stabilisers etc. commonly used in pharmaceutical technology.

Claims

1. CLAIMS 1. Succinylated iron-containing proteins.

2. succinylated proteins containing 0.1 to 20% iron by weight.

3. Succinylated proteins according to claims 1-2, characterised in that the degree of succinylation is between 20 and 100%.

4. succinylated proteins according to claims 1-3, characterised in that the starting protein is a protein of vegetal or animal origin.

5. Milk protein with a degree of succinylation greater than 70%, containing more than 4% iron by weight. Studio Consulenza Brevetkiale srl - 36 6. Ovalbumin with a degree of succinylation greater than 70%, containing more than 4% by weight of iron.

7. Soy protein with a degree of succinylation greater than 70%, containing more than 4% iron by weight.

8. A process for the preparation of succinylated proteins containing iron according to the preceding claims, characterized in that a succinylated protein is reacted with iron salts in an aqueous medium, at a pH between approximately 2 and approximately 10, and the resulting product is isolated by precipitation at a pH of the order of 2-4, the latter pH range being achieved spontaneously or by acidification.

9. Process according to claim 8, characterized in that the reaction between succinylated protein and iron salts occurs at a pH around neutrality.

10. A process for the preparation of succinylated proteins containing iron according to claims 1-7, characterized in that a succinylated protein is reacted with iron salts in an aqueous environment at a pH between about 2 and about 10, and the reaction product is isolated by removing the solvent after adjusting the pH to values ​​close to neutrality.

11. Pharmaceutical compositions for the treatment of side-rodent anemia, characterized in that they contain as an active ingredient - 37 succinylated proteins containing iron according to claims 1-7. Milan, 2 March 198 2 Staio Constflenja Breveffuale srì. 1 9908A / 82 day of treatment 360 199 0 8 M 82 Figure 7 Figure 8 Stage Patent Consultancy sii ] 1 99 08 A / 82 «> II ? ; ; Figure 3 Figure 4 Figure 5 1 9 η Π 8 M 82 250 500 Shirìio Consulenza BreveRuaìf? srl. ACT OF......DESIGNATION! OF INVENTIONS^ A, 3 A / 82 The undersigned STUDIO CONSULTENZA PATENTTUALE, agent of the company ITALFARMACO SpA, Viale Fulvio Testi, 330, MILAN, hereby declares that the inventors of the patent application for industrial invention entitled: BIOAVAILABLE IRON DERIVATIVES FREE FROM GASTROLESIVITIES, PREPARATION PROCEDURE AND RELATED PHARMACEUTICAL COMPOSITIONS filed on 2 March 1982 are:

1. Giancarlo Sportoletti 2. Pier Giuseppe Pagella 3.Pietro Cremonesi. Staio Patent Consulting, Via Senato, 24, 20121 Milan.