OA18236A - A process for producing iron (III) casein Nacetyl-aspartylated complexes and use thereof in pharmaceutical compositions. - Google Patents
A process for producing iron (III) casein Nacetyl-aspartylated complexes and use thereof in pharmaceutical compositions. Download PDFInfo
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- OA18236A OA18236A OA1201700093 OA18236A OA 18236 A OA18236 A OA 18236A OA 1201700093 OA1201700093 OA 1201700093 OA 18236 A OA18236 A OA 18236A
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- Prior art keywords
- casein
- acetyl
- iron
- aspartylated
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- 239000005018 casein Substances 0.000 title claims abstract description 110
- 235000021240 caseins Nutrition 0.000 title claims abstract description 110
- 238000000034 method Methods 0.000 title claims abstract description 31
- VTLYFUHAOXGGBS-UHFFFAOYSA-N fe3+ Chemical compound [Fe+3] VTLYFUHAOXGGBS-UHFFFAOYSA-N 0.000 title claims abstract description 16
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 6
- 238000006243 chemical reaction Methods 0.000 claims abstract description 30
- XEEYBQQBJWHFJM-UHFFFAOYSA-N iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 85
- 229910052742 iron Inorganic materials 0.000 claims description 45
- 238000007792 addition Methods 0.000 claims description 21
- FYSNRJHAOHDILO-UHFFFAOYSA-N Thionyl chloride Chemical compound ClS(Cl)=O FYSNRJHAOHDILO-UHFFFAOYSA-N 0.000 claims description 18
- VEXZGXHMUGYJMC-UHFFFAOYSA-M chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 12
- 239000012320 chlorinating reagent Substances 0.000 claims description 4
- 238000011065 in-situ storage Methods 0.000 claims description 4
- 238000004519 manufacturing process Methods 0.000 claims description 3
- 239000003085 diluting agent Substances 0.000 claims description 2
- VZGDMQKNWNREIO-UHFFFAOYSA-N Carbon tetrachloride Chemical compound ClC(Cl)(Cl)Cl VZGDMQKNWNREIO-UHFFFAOYSA-N 0.000 claims 4
- XHXFXVLFKHQFAL-UHFFFAOYSA-N Phosphoryl chloride Chemical compound ClP(Cl)(Cl)=O XHXFXVLFKHQFAL-UHFFFAOYSA-N 0.000 claims 4
- CTSLXHKWHWQRSH-UHFFFAOYSA-N Oxalyl chloride Chemical compound ClC(=O)C(Cl)=O CTSLXHKWHWQRSH-UHFFFAOYSA-N 0.000 claims 2
- MGNCLNQXLYJVJD-UHFFFAOYSA-N Cyanuric chloride Chemical compound ClC1=NC(Cl)=NC(Cl)=N1 MGNCLNQXLYJVJD-UHFFFAOYSA-N 0.000 claims 1
- JRNVZBWKYDBUCA-UHFFFAOYSA-N N-Chlorosuccinimide Substances ClN1C(=O)CCC1=O JRNVZBWKYDBUCA-UHFFFAOYSA-N 0.000 claims 1
- UHZYTMXLRWXGPK-UHFFFAOYSA-N Phosphorus pentachloride Chemical compound ClP(Cl)(Cl)(Cl)Cl UHZYTMXLRWXGPK-UHFFFAOYSA-N 0.000 claims 1
- FAIAAWCVCHQXDN-UHFFFAOYSA-N Phosphorus trichloride Chemical compound ClP(Cl)Cl FAIAAWCVCHQXDN-UHFFFAOYSA-N 0.000 claims 1
- 238000004108 freeze drying Methods 0.000 claims 1
- ZRALSGWEFCBTJO-UHFFFAOYSA-N guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 claims 1
- PCUVYBUDIWDLNI-UHFFFAOYSA-N methyl N-(N'-chloro-N-methoxycarbonylcarbamimidoyl)carbamate Chemical compound COC(=O)NC(=NCl)NC(=O)OC PCUVYBUDIWDLNI-UHFFFAOYSA-N 0.000 claims 1
- 239000000546 pharmaceutic aid Substances 0.000 claims 1
- 230000002335 preservative Effects 0.000 claims 1
- 239000003755 preservative agent Substances 0.000 claims 1
- 238000001694 spray drying Methods 0.000 claims 1
- 238000002560 therapeutic procedure Methods 0.000 abstract description 10
- 206010022971 Iron deficiency Diseases 0.000 abstract description 8
- 206010022970 Iron deficiency Diseases 0.000 abstract description 8
- RBTARNINKXHZNM-UHFFFAOYSA-K Iron(III) chloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 abstract 1
- 241001465754 Metazoa Species 0.000 abstract 1
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- 238000001914 filtration Methods 0.000 description 22
- 239000000725 suspension Substances 0.000 description 19
- OTCCIMWXFLJLIA-BYPYZUCNSA-N N-acetyl-L-aspartic acid Chemical compound CC(=O)N[C@H](C(O)=O)CC(O)=O OTCCIMWXFLJLIA-BYPYZUCNSA-N 0.000 description 15
- 239000000203 mixture Substances 0.000 description 14
- IXCSERBJSXMMFS-UHFFFAOYSA-N HCl HCl Chemical compound Cl.Cl IXCSERBJSXMMFS-UHFFFAOYSA-N 0.000 description 12
- 239000000523 sample Substances 0.000 description 12
- RAHZWNYVWXNFOC-UHFFFAOYSA-N sulphur dioxide Chemical compound O=S=O RAHZWNYVWXNFOC-UHFFFAOYSA-N 0.000 description 12
- 239000007795 chemical reaction product Substances 0.000 description 10
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 9
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 9
- 239000002244 precipitate Substances 0.000 description 9
- 235000018102 proteins Nutrition 0.000 description 9
- 108090000623 proteins and genes Proteins 0.000 description 9
- 102000004169 proteins and genes Human genes 0.000 description 9
- 229910001220 stainless steel Inorganic materials 0.000 description 9
- 239000010935 stainless steel Substances 0.000 description 9
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 8
- 238000003756 stirring Methods 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
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- 239000002253 acid Substances 0.000 description 5
- 235000013305 food Nutrition 0.000 description 5
- 230000002496 gastric Effects 0.000 description 5
- 108010084684 iron protein succinylate Proteins 0.000 description 5
- 229940074442 iron protein succinylate Drugs 0.000 description 5
- 150000003839 salts Chemical class 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 102000014171 Milk Proteins Human genes 0.000 description 4
- 108010011756 Milk Proteins Proteins 0.000 description 4
- -1 aliphatic dicarboxylic acid Chemical class 0.000 description 4
- 235000005911 diet Nutrition 0.000 description 4
- 238000004090 dissolution Methods 0.000 description 4
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- 208000007502 Anemia Diseases 0.000 description 3
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- 240000004808 Saccharomyces cerevisiae Species 0.000 description 3
- 150000001263 acyl chlorides Chemical class 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
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- 150000001805 chlorine compounds Chemical class 0.000 description 3
- 238000010192 crystallographic characterization Methods 0.000 description 3
- 230000000378 dietary Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
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- BOPCNGSYUSLFFZ-WCCKRBBISA-L (2S)-2-acetamidobutanedioate;iron(2+) Chemical compound [Fe+2].CC(=O)N[C@H](C([O-])=O)CC([O-])=O BOPCNGSYUSLFFZ-WCCKRBBISA-L 0.000 description 2
- 229960005261 Aspartic Acid Drugs 0.000 description 2
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- 229940121657 Clinical Drug Drugs 0.000 description 2
- 206010018987 Haemorrhage Diseases 0.000 description 2
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 2
- 235000003704 aspartic acid Nutrition 0.000 description 2
- 230000027455 binding Effects 0.000 description 2
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- 150000001875 compounds Chemical class 0.000 description 2
- 238000011109 contamination Methods 0.000 description 2
- CWYNVVGOOAEACU-UHFFFAOYSA-N fe2+ Chemical class [Fe+2] CWYNVVGOOAEACU-UHFFFAOYSA-N 0.000 description 2
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- 231100000027 toxicology Toxicity 0.000 description 2
- 229940072107 Ascorbate Drugs 0.000 description 1
- 229940009098 Aspartate Drugs 0.000 description 1
- 241000282472 Canis lupus familiaris Species 0.000 description 1
- CKLJMWTZIZZHCS-UHFFFAOYSA-N DL-aspartic acid Chemical compound OC(=O)C(N)CC(O)=O CKLJMWTZIZZHCS-UHFFFAOYSA-N 0.000 description 1
- 206010017964 Gastrointestinal infection Diseases 0.000 description 1
- 206010020751 Hypersensitivity Diseases 0.000 description 1
- 206010050789 Hypochromasia Diseases 0.000 description 1
- 206010021972 Inflammatory bowel disease Diseases 0.000 description 1
- 206010022972 Iron deficiency anaemia Diseases 0.000 description 1
- TYQCGQRIZGCHNB-JLAZNSOCSA-N L-ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(O)=C(O)C1=O TYQCGQRIZGCHNB-JLAZNSOCSA-N 0.000 description 1
- 210000004080 Milk Anatomy 0.000 description 1
- JMQITILRHSGUCB-BYPYZUCNSA-N N-[(3S)-2,5-dioxooxolan-3-yl]acetamide Chemical compound CC(=O)N[C@H]1CC(=O)OC1=O JMQITILRHSGUCB-BYPYZUCNSA-N 0.000 description 1
- 241000700159 Rattus Species 0.000 description 1
- 230000001154 acute Effects 0.000 description 1
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- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 230000001488 breeding Effects 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 125000002843 carboxylic acid group Chemical group 0.000 description 1
- 238000002512 chemotherapy Methods 0.000 description 1
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- 230000001524 infective Effects 0.000 description 1
- 230000000968 intestinal Effects 0.000 description 1
- 230000031891 intestinal absorption Effects 0.000 description 1
- 150000004698 iron complex Chemical class 0.000 description 1
- QDUZQOIJXPPTLY-GMBKLUGCSA-N iron;(2R,3R,4S,5S)-2,3,4,5,6-pentahydroxyhexanoic acid Chemical compound [Fe].OC[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C(O)=O.OC[C@H](O)[C@H](O)[C@@H](O)[C@@H](O)C(O)=O QDUZQOIJXPPTLY-GMBKLUGCSA-N 0.000 description 1
- 230000002503 metabolic Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 150000002739 metals Chemical class 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 230000000737 periodic Effects 0.000 description 1
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- 238000005063 solubilization Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229940086735 succinate Drugs 0.000 description 1
- KDYFGRWQOYBRFD-UHFFFAOYSA-L succinate(2-) Chemical compound [O-]C(=O)CCC([O-])=O KDYFGRWQOYBRFD-UHFFFAOYSA-L 0.000 description 1
- 238000010613 succinylation reaction Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 231100000730 tolerability Toxicity 0.000 description 1
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Abstract
The present invention generally relates to new process for the preparation of iron (III) casein Nacetyl-aspartylated complexes. The product obtainable according to the method of the present invention may be safely used to the general population or animals in the therapy of Iron deficiency. The process of the invention includes the steps of: (a) reaction of casein with N-acetyl-L-aspartyl chloride, to form N-acetyl-L-aspartylated casein, (b) subsequent reaction of the N-acetyl-Laspartylated casein with ferric chloride; and (c) obtaining the Iron (III) complex with N-acetyl-Laspartylated casein.
Description
The présent invention generally relates to new process for the préparation of iron (lll) casein Nacetyl-aspartylated complexes. The product obtainable according to the method of the présent invention may be safely used to the general population or animais in the therapy of Iron deficiency. The process of the invention includes the steps of :
(a) reaction of casein with N-acetyl-L-aspartyl chloride, to form N-acetyl-L-aspartylated casein, (b) subséquent reaction of the N-acetyl-L-aspartylated casein with ferrie chloride; and (c) obtaining the Iron (lll) complex with N-acetyl-Laspartylated casein.
O.A.P.I. - B.P. 887, YAOUNDE (Cameroun) - Tel. (237) 222 20 57 00 - Site web: http:/www.oapi.int - Email: oapi@oapi.int ►
- 1 TSETI Ioulia
A process for producing iron (III) casein N-acetyl-aspartylated complexes and use thereof in pharmaceutical compositions
The présent invention generally relates to iron (III) protein complexes and to processes for the manufacture thereof. The product obtainable according to the method of the présent invention may be safely used for administration to the general population or animais in the therapy of iron deficiency.
BACKGROUND ART
Iron therapy is necessary in a wide variety of clinical situations including pregnancy, chemotherapy, acute or chronic hemorrhage, hemodialysis, inflammatory bowel disease, inadéquate diet, and gastrointestinal surgery.
Oral iron therapy is considerably less expensive than the alternative parentéral iron therapy and also poses lower risks of serious allergie reactions. On this base, in British patent application no 910321 (Olivieur Paul Gaudin), an improved process is described for the préparation of ferrous salts with aspartic acid. These salts could be used as medicines for iron deficiency.
Cremonesi P et al, in 1984 (Arzneimittel-Forschung, 34(9):948-952) reported the first Iron dérivatives of modified milk protein. Moreover, it is stated that a product containing a high amount of iron and maintaining ail the necessary characteristics of stability and solubility for a drug can be prepared by succinylating the milk proteins, before the reaction with the iron sait. For these dérivatives a particular advantage could be achieved. Iron, in the form of oligomeric complex, is tightly bonded to the protein not in chelated structures with basic residues but involving several sites of the protein chain. The solubility of the iron complex is assured by the increased availability of carboxyl groups that follows the succinylation reaction.
US Patent No. 4493829, the same period, describes succinylated protein-iron dérivatives with no gastric lésions and improved bio-availability. The same protein (vegetable and animal) modifications are described in European patent no. 0243322, by reaction of the f
-2proteins with 3-10 aliphatic dicarboxylic acid anhydrides, introducing carboxylic acid groups to the protein, thus facilitating the complexation ofthe iron.
Chemical and biological characterization of iron-protein succinylate in rats and dogs are described by Cremonesi P et al. (International Journal of Clinical Pharmacology, therapy, and toxicology, 31(1):40-51). The experiments demonstrated better gastrointestinal tolerability to an iron succinyl casein complex containing 5 % iron.
EP0939083 discloses process for the préparation of a ferro-succinylcasein complex obtained from food-grade casein used for food purposes.
US2001031748 refers to dietary of various metals, iron included, in combination to a dietary ligand administration such as ascorbate, succinate, aspartate and other ligands. The combination of such a dietary can assist to treating gastrointestinal symptoms.
Lazzari et al. reported (Clinical Drug Investigation, 2005; 25(11):679-689) an overview of 16 clinical trials in the treatment of iron deficiency with iron-acetyl-aspartylated casein (Fe-ASP), which proved to be an efficient vehicle for providing iron with high bioavailability. In open clinical trials, highly significant improvements in clinical and haematological parameters were observed after treatment with Fe-ASP in ail categories of patients with iron deficiency anaemia. In controlled clinical trials, the changes in clinical and haematological profiles observed with Fe-ASP were virtually identical to those seen with iron protein succinylate (IPS), and Fe-ASP also compared well with parentéral iron gluconate. No safety considérations were raised. Fe-ASP shows high efficacy in iron-deficient anaemia treatment, and it is an extremely well tolerated iron vehicle. Fe-ASP represents a valid alternative to IPS and shows promise as a substitute for parentéral iron therapy in selected clinical situations.
More recent, WO patent No. 2006021843 discloses a process for producing iron succinyl casein and acetyl-aspartate iron casein complexes in which the substance is little exposed to pH and température conditions. The process uses acetyl-aspartate anhydride. Moreover, this process is required no use of spécial pumps (dilacérations pumps).
t
-3SUMMARY OF THE INVENTION
The présent application describes new processes for the production of stable iron (III) casein N-acetyl-L-aspartylated complexes. The process for the préparation of complexes of iron (III) casein N-acetyl-L-aspartylated, said complexes, and pharmaceutical composi5 tions comprising said complexes ofthe invention are defined in the claims.
The complexes of the présent invention are stable and show a high stability over time.
The products may therefore be used for the therapy of iron deficiency in humans or animais.
DETAILED DESCRIPTION OF THE INVENTION
The subject of the présent invention is a new process for producing protein dérivatives of iron, namely, a ferrie complex of N-acetyl-L-aspartylated casein (hereinafter referred to as ferro-N-acetyl-L-aspartylated casein and iron (III) complex with N-acetyl-L15 aspartylated casein), preferably obtained from food-grade casein, i.e. casein used for food purposes. By the term food-grade casein is meant casein obtained from milk coming from strictly controlled breeding farms. This product présents a level of contamination lower than 1000 CFU/g for bacteria, and lower than 100 CFU/g for moulds, which contamination levels are preferably determined as described in current Eur. Pharm, mon20 ographs 2.6,12 for Total viable aérobic count, 2.6.13 for specified microorganisms and
2.6.14 for Bacteria! endotoxins. The use of food-grade casein allows obtaining particularly pure products, as compared to similar complexes obtained starting from usual milk proteins.
The présent invention refers to a process for the préparation of complexes of iron (III) casein N-acetyl-L-aspartylated comprising, preferably consisting of, the following steps:
(a) N-acetyl-L-aspartylation of casein, preferably food-grade casein, with N-acetyl-Laspartic acid chloride, prepared in situ by chlorination of the N-acetyl-L-aspartic acid with 30 a chlorinating reagent such as thionyl chloride. Although N-acetyl aspartic anhydride is generally a useful starting material for casein modification, it has several disadvantages.
For example, it is relative expensive for industrial use and also unstable against humidity on storage. Moreover there is a risk for N-acetyl aspartic anhydride of partial racemizai
-4tion (Tetrahedron: Asymmetry 18 (2007) 1625-1627). That for, the use of N-acetyl-Laspartic acid in combination with the chlorinating reagent advantages over the use of the N-acetyl aspartic anhydride in the context of the présent application, (b) Subséquent reaction of the N-acetyl-L-aspartylated casein with ferrie chloride. Step (a) preferably 5 comprises providing dissolved casein.
Preferred embodiments are described in detail as follows, wherein each of stages 1 to 9 can be used alone or in combination with any other stages for defining the présent invention, most preferably ail stages 1 to 9 are applied as described below:
Step (a):
In stage 1), dissolving of casein, preferably food-grade, is performed in alkaline pH between 8 and 9, preferably of between 8.4 and 8.6. Casein is dissolved in water.
In stage 2) N-acetyl-L-aspartyl chloride is prepared in situ by N-acetyl-L-aspartic acid and a chlorinating reagent such as thionyl chloride.
In stage 3) N-acetyl-aspartylation is performed by reaction of the dissolved casein of stage 1) with N-acetyl-L-aspartyl chloride prepared in stage 2). The reaction can be performed at a température in the range of from 15° to 40°C, such as at room température (20°C), preferably it is performed at a température in a range of from 30 to 40°C.
Preferably, the pH-value is kept within the range of pH 8-9 during reaction of casein with N-acetyl-L-aspartyl chloride.
The N-acetyl-L-aspartyl chloride is prepared in situ (without any isolation) and set for reaction with soluble casein. The N-acetyl-L-aspartic acid ratio to the casein, which is preferably used, is 1:2.
In stage 4) the N-acetyl-L-aspartylated product is purified after précipitation via acidifica30 tion at a pH value of between 2.5 and 4, preferably of between 2.5 and 3, and separated by filtration.
-5In stage 5), the product is recovered by filtration, is suspended in water and sodium hydroxide and subjected to dilacération at a pH of between 6 and 11, préférable 8 and 9, until a solution is obtained.
Step (b):
In stage 6) the reaction with ferrie chloride may be performed by adding an aqueous solution of ferrie chloride to the solution obtained at the end of stage 5), until a pH of between 2.5 and 3 is obtained. A suspension is formed, which is subjected to filtration, and crude ferro-N-acetyl-L-aspartylated casein is recovered as insoluble phase.
g of modified casein can be reacted with 6.2 g FeCI3 26.9 % w/w solution.
In stage 7), the purification is performed as follows: the crude dérivative obtained in stage 6) is suspended in water, alkalized at pH 8.5 and then agitated for 30 minutes until the product dissolution. The purified complex is clarified by subséquent filtration of the solution.
In stage 8) the ferro-N-acetyl-L-aspartylated casein is precipitated from the clarified solution of stage 7) by acidification at pH 2.5 and is then filtered and recovered.
In stage 9), the ferro-N-acetyl-L-aspartylated casein complex undergoes desiccation at a température of 60 °C and 80 °C, for a period of time of between 18 and 36 hours, preferably between 20 and 24 hours.
At the end of desiccation, the product contains a maximum moisture residue of less than 5% and a content of complexed iron of between 5% and 6% by weight, calculated on the dry substance. For a complété characterization of the product, reference is made to the data provided in Examples 1 to 3.
The process described makes it possible to obtain ferro-N-acetyl-L-aspartylated casein complex free from residue or from iron dérivatives that are insoluble or poorly soluble in water. In particular, the product is found to be completely soluble at alkaline pH values,
i.e., the ones typical of the intestinal tract, thus guaranteeing maximum iron bioavailability for the purpose of intestinal absorption.
The product according to the présent invention can be adequately compounded in phar5 maceutical formulations suitable for oral administration.
The présent invention therefore comprises pharmaceutical compositions containing ferroN-acetyl-L-aspartylated casein and appropriate excipients, such as diluents and pharmaceutical preservatives.
A further subject of the invention refers to the use of the ferrie complexes described above for the préparation of médicaments for the treatment of pathological conditions resulting from iron deficiency due, for example, to physiological alteration, periodic hemorrhage, infective diseases, pregnancy, breast-feeding, and imperfect metabolic utiliza15 tion. One example of these pathological conditions is anaemia, in particular hypochromie sidéropénie anaemia.
In order to provide an illustration of the présent invention, which in no way limits the scope thereof, the following experimental examples are given.
EXAMPLES
Example 1 - Préparation of Iron Casein N-acetyl-L-aspartylated Complex (A) Casein dissolution.
In a stainless steel reactor with sufficient shaker and pH detector, are added 5 g of casein used for food or pharmaceutical purposes on 70 g of de-ionized, microbiologically pure water. The mixture is kept under stirring until a homogeneous suspension is obtained. Approximately 1.5 g of a 15 % w/w sodium hydroxide (NaOH) solution is added, at a température of 20 °C, in a period of 10 minutes, until a pH value of 8.5 ± 0.1 is 30 obtained. The solution is shaken for another 15 minutes.
(B) N-Acetyl-L-Aspartyl Chloride préparation.
In a stainless steel reactor with sufficient shaker are added, 15.6 mL (15 équivalents) of thionyl chloride (SOCI2), 2.5 g of N-acetyl-L-aspartic acid and the mixture is kept under stirring at 75 °C. The mixture is kept under stirring until the reaction completion (1-1,5 h) by the formation of the acyl chloride product. In this reaction, the sulfur dioxide (SO2) 5 and hydrogen chloride (HCl) generated are both gases that can leave the reaction vessel, driving the reaction forward. The excess of thionyl chloride is evaporated by heating the reaction mixture at 75 °C, close to the boiling point (74.6 °C) ofthe SOCI2.
(C) Casein acetylaspartylation.
îo The solution obtained from stage (A), of the dissolved casein, is added gradually to the, the N-acetyl-L-aspartic chloride (B), with simultaneously addition about 3.1 g of 15 % w/w NaOH, maintaining pH at 8.5 ± 0.1, at room température. The solution obtained is left under strong agitation for 2h.
is (D) Transfer and purification of casein acetyl aspartate via précipitation.
The solution obtained from stage (C) is transferred into an enameled reactor provided with a mixer and a suitable probe for reading pH values. The solution, kept at a température of 20 °C, is then acidified with approximately 2.28 g of HCl 6 N solution in a period of 40 minutes, until a pH of 3 is obtained. On reaching the target pH, addition of acid is stopped and precipitate is obtained. The decrease in the pH value causes the formation of a precipitate of N-acetyl-L-aspartylated casein depurated from other salts, other reaction products, and other soluble impurities.
(E) Filtration and dilacérations.
The N-acetyl-L-aspartylated casein obtained in stage (D) is recovered by filtration. The moist product is removed from the filter, and then put into a stainless-steel reactor equipped with an anchor mixer and with a probe for reading pH values which has been charged with 70 ml of depurated water, and then subjected to agitation. The suspension is kept under agitation for 10 min, at a température of 20 °C, and then subjected to dilacération. After 20 minutes of dilacération, approximately 3.1 g of NaOH 15 % w/w solution are added to the suspension, until a stable pH value of between 8.1 and 8.4 is obtained. The aspartylated casein suspension is filtered, the reactor and filter are washed
-8with 50 ml of depurated water, and the washing water is then added again to the clarified solution.
(F) . Reaction with ferrie chloride and filtration of crude ferro-N-acetyl-L-aspartylated casein.
The solution from stage (E) is sent on to an enamelled reactor equipped with impeller mixer and with a probe for reading pH values. To this homogeneous solution is added
6.2 g in total of FeCI3.6H2O 26.9% w/w (Iron solution), accordingly:
i) Addition of 1.7 g of iron solution with simultaneous incrémental addition of 0.6 g 15 % w/w NaOH in order to set the pH value between 8.3-8.5. The solution is left for agitation for 15 minutes.
ii) Addition of the rest 4.5 g of iron solution until the pH of the solution to maintain between 2.8-3.0. The suspension is kept under agitation for 1 h. The pH is adjusted with HCl 6 N solution or 15% w/w NaOH solution, respectively.
A suspension of ferro-N-acetyl-L-aspartylated casein is obtained. The reaction product is kept under agitation for 20 minutes, and the crude ferro-N-acetyl-L-aspartylated casein dérivative is recovered by filtration.
(G) Complex dissolving and filtration.
The solid complex is dispersed in 70 g of purified water and then 3.85 g of NaOH 15 % w/w solution are added until a pH of 8.5 ± 0.1. The solution is kept under agitation for 30 minutes. The remained solution is filtered in order the undissolved impurities to be separated. The insoluble residue is less than 0.5 wt%.
(H) Précipitation and isolation of pure moist ferro-N-acetyl-L-aspartylated casein.
To the clear solution, which is put into an enamelled reactor equipped with an impeller mixer and with a probe for reading the pH value, are added by pouring 2.28 g of 6 N HCl aqueous solution, at a température of between 20 °C and 25 °C, in approximately 20-30 minutes, until a pH value of 2.8-3.0 is obtained. At the end of the addition, the reaction product is kept stirred for 20 minutes, and the precipitate of pure ferro-N-acetyl-L
-9aspartylated casein that has formed is recovered by filtration and subséquent washing with purified water.
(I) Desiccation of ferro-N-acetyl-L-aspartylated casein.
The moist pure product obtained in the previous stage is dried under vacuum, at a température increasing from 60 °C to 70 °C, for an overall period of 24 hours. After the desiccation process, 6.5 g of pure iron (III) casein N-acetyl-L-aspartylated complex are obtained, with a water content < 5 wt % (Karl Fisher), having the following chemical and physicochemical characteristics:
| Product characteristics | Value |
| Colour | Brown-red |
| Solubility | Soluble in NaOH pH = 8 |
| PH | 2.8 |
| Free iron (not complexed) | <0.05% w/w |
| Total iron | 5.5 % w/w |
| Proteins | 75.2 % w/w |
| Complexed N-acetyl-L-aspartic acid | 7.1 % w/w |
| Free N-acetyl-L-aspartic acid | 0.1 % w/w |
| Chlorides | 1.8 % w/w |
| Bacterial content | <103 CFU/g |
| Mold and yeast content | <102 CFU/g |
Example 2 - Préparation of Iron Casein N-acetyl-L-aspartylated Complex (A) Casein dissolution.
In a stainless steel reactor with sufficient shaker and pH detector, are added 5 g of casein used for food or pharmaceutical purposes on 70 g of de-ionized, microbiologically pure water. The mixture is kept under stirring until a homogeneous suspension is obtained. Approximately 1.5 g of a 15 % w/w sodium hydroxide (NaOH) solution is added, at a température of 20 °C, in a period of 10 minutes, until a pH value of 8.5 ± 0.1 is obtained. The solution is shaken for another 15 minutes.
-ίο- (B) N-Acetyl-L-Aspartyl Chloride préparation.
In a stainless steel reactor with sufficient shaker are added, 15.2 mL (15 équivalents) of thionyl chloride (SOCI2), a catalytic amount of 40 pL of dimethyl formamide (DMF), 2.5 g of N-acetyl-L-aspartic acid and the mixture is heated to 75 °C under reflux conditions. The mixture is kept under stirring until the reaction completion by the formation of the acyl chloride product. In this reaction, the sulfur dioxide (SO2) and hydrogen chloride (HCl) generated are both gases that can leave the reaction vessel, driving the reaction forward. The excess of thionyl chloride is evaporated by heating the reaction mixture gradually up to 75 °C, close to the boiling point (74.6 °C) ofthe SOCI2.
(C) Casein acetylaspartylation.
The solution obtained from stage (A), ofthe dissolved casein, is added to the N-acetyl-Laspartic chloride of stage (B), with simultaneously addition about 3.1 g of 15 % w/w NaOH, maintaining pH at 8.5 ± 0.1, at room température. The solution obtained is left under strong agitation for 2h.
(D) Transfer and purification of casein acetyl aspartate via précipitation.
The solution obtained from stage (C) is transferred into an enameled reactor provided with a mixer and a suitable probe for reading pH values. The solution, kept at a température of 20 °C, is then acidified with approximately 2.28 g of HCl 6 N solution in a period of 40 minutes, until a pH of 3 is obtained. On reaching the target pH, addition of acid is stopped and precipitate is obtained. The decrease in the pH value causes the formation of a precipitate of N-acetyl-L-aspartylated casein depurated from other salts, other reaction products, and other soluble impurities.
(E) Filtration and dilacérations.
The N-acetyl-L-aspartylated casein obtained in stage (D) is recovered by filtration. The moist product is removed from the filter, and then put into a stainless-steel reactor equipped with an anchor mixer and with a probe for reading pH values which has been charged with 70 ml of depurated water, and then subjected to agitation. The suspension is kept under agitation for 1 hour, at a température of 20 °C, and then subjected to dilacération. After 20 minutes of dilacération, approximately 3.1 g of NaOH 15 % w/w solution are added to the suspension, until a stable pH value of between 8.1 and 8.4 is
- u obtained. At the end of the addition, the reaction product is once more treated in the same reactor, for a period of approximately 2 hours. This operation facilitâtes the disgregation of the solid particles of N-acetyl-L-aspartylated casein. The aspartylated casein suspension is filtered, the reactor and filter are washed with 50 ml of depurated water, and the washing water is then added again to the clarified solution.
(F) . Reaction with ferrie chloride and filtration of crude ferro-N-acetyl-L-aspartylated casein.
The solution from stage (E) is sent on to an enamelled reactor equipped with impeller mixer and with a probe for reading pH values. To this homogeneous solution is added
6.2 g in total of FeCI3-6H2O 26.9% w/w (iron solution), accordingly:
i) Addition of 1.7 g of Iron solution with simultaneous incrémental addition of 0.6 g 15 % w/w NaOH in order to maintain pH between 8.3-8.5. The solution is left for agitation for 15 minutes.
ii) Addition of the rest 4.5 g of Iron solution until the pH of the solution is between 2.83.0. The suspension is kept under agitation for 1 h. The pH is adjusted with HCl 6 N solution or 15% w/w NaOH solution, respectively.
A suspension of ferro-N-acetyl-L-aspartylated casein is obtained. The reaction product is kept under agitation for 20 minutes, and the crude ferro-N-acetyl-L-aspartylated casein dérivative is recovered by filtration.
(G) Complex dissolving and filtration.
The solid complex is dispersed in 70 g of purified water and then 3.85 g of NaOH 15 % w/w solution are added until a pH of 8.5 ± 0.1. The solution is kept under agitation for 30 minutes. The remained solution is filtered in order the undissolved impurities to be separated. The insoluble residue is less than 0.5 wt%.
(H) Précipitation and isolation of pure moist ferro-N-acetyl-L-aspartylated casein.
To the clear solution, which is put into an enamelled reactor equipped with an impeller mixer and with a probe for reading the pH value, are added by pouring 2.28 g of 6 N HCl
- 12aqueous solution, at a température of between 20 °C and 25 °C, in approximately 20-30 minutes, until a pH value of 2.8-3.0 is obtained. At the end of the addition, the réaction product is kept stirred for 20 minutes, and the precipitate of pure ferro-N-acetyl-Laspartylated casein that has formed is recovered by filtration and subséquent washing 5 with purified water.
(I) Desiccation of ferro-N-acetyl-L-aspartylated casein.
The moist pure product obtained in the previous stage is dried under vacuum, at a temîo perature increasing from 60 °C to 70 °C, for an overall period of 24 hours. After the desiccation process, 6.6 g of pure iron (III) casein N-acetyl-L-aspartylated complex are obtained, with a water content < 5 wt% (Karl Fisher), having the following chemical and physicochemical characteristics:
| Product characteristics | Value |
| Colour | Brown-red |
| Solubility | Soluble in NaOH pH = 8 |
| PH | 2.7 |
| Free Iron (not complexed) | <0.05% w/w |
| Total iron | 5.7 % w/w |
| Proteins | 84.1 % w/w |
| Complexed N-acetyl-L-aspartic acid | 7.3 % w/w |
| Free N-acetyl-L-aspartic acid | 0.2 % w/w |
| Chlorides | 1.6 % w/w |
| Bacterial content | <103 CFU/g |
| Mold and yeast content | <102 CFU/g |
Example 3 - Préparation of Iron Casein N-acetyl-L-aspartylated Complex (A) Casein dissolution.
In a stainless steel reactor with sufficient shaker and pH detector, are added 5 g of casein used for food or pharmaceutical purposes on 70 g of de-ionized, microbiologically pure water. The mixture is kept under stirring until a homogeneous suspension is ob5 tained. Approximately 1.5 g of a 15 % w/w sodium hydroxide (NaOH) solution is added, at a température of 20 °C, in a period of 10 minutes, until a pH value of 8.5 ± 0.1 is obtained. The solution is shaken for another 15 minutes.
(B) N-Acetyl-L-Aspartyl Chloride préparation.
In a stainless steel reactor with sufficient shaker are added, 15.6 ml (15 équivalents) of thionyl chloride (SOCI2), a catalytic amount of 100 pl_ of dimethylformamide (DMF) and 2.5 g of N-acetyl-L-aspartic acid. The mixture is kept under stirring at 75 °C under reflux conditions until the reaction completion by the formation of the acyl chloride product. In this reaction, the sulfur dioxide (SO2) and hydrogen chloride (HCl) generated are both gases that can leave the reaction vessel, driving the reaction forward. The excess of thionyl chloride is evaporated by heating the reaction mixture gradually up to 75 °C, without the cooling condenser, close to the boiling point (74.6 °C) ofthe SOCI2.
(C) Casein acetylaspartylation.
The solution obtained from stage (A), ofthe dissolved casein, is added to the N-acetyl-Laspartic chloride of stage (B), with simultaneously addition about 3.1 g of 15 % w/w NaOH, maintaining pH at 8.5 ± 0.1, at room température. The solution obtained is left under strong agitation for 2h.
(D) Transfer and purification of casein acetyl aspartate via précipitation.
The solution obtained from stage (C) is transferred into an enameled reactor provided with a mixer and a suitable probe for reading pH values. The solution, kept at a température of 20 °C, is then acidïfied with approximately 2.28 g of HCl 6 N solution in a period of 40 minutes, until a pH of 3 is obtained. On reaching the target pH, addition of acid is stopped and precipitate is obtained. The decrease in the pH value causes the formation of a precipitate of N-acetyl-L-aspartylated casein depurated from other salts, other reaction products, and other soluble impurities.
(E) Filtration and dilacérations.
The N-acetyl-L-aspartylated casein obtained in stage (D) is recovered by filtration. The moist product is removed from the filter, and then put into a stainless-steel reactor equipped with an anchor mixer and with a probe for reading pH values which has been charged with 70 ml of depurated water, and then subjected to agitation. The suspension is kept under agitation for 1 hour, at a température of 20 °C, and then subjected to dilacération. After 20 minutes of dilacération, approximately 3.1 g of NaOH 15 % w/w
-15solution are added to the suspension, until a stable pH value of between 8.1 and 8.4 is obtained. At the end of the addition, the reaction product is once more treated in the same reactor, for a period of approximately 2 hours. This operation facilitâtes the dilacération and then solubilization of the solid particles of N-acetyl-L-aspartylated casein. The aspartylated casein suspension is filtered, the reactor and filter are washed with 50 ml of depurated water, and the washing water is then added again to the clarified solution.
(F) . Reaction with ferrie chloride and filtration of crude ferro-N-acetyl-L-aspartylated casein.
The solution from stage (E) is sent on to an enamelled reactor equipped with impeller mixer and with a probe for reading pH values. To this homogeneous solution is added
6.2 g in total of FeCI3-6H2O 26.9% w/w (Iran solution), accordingly:
i) Addition of 1.7 g of iron solution with simultaneous incrémental addition of 0.6 g 15 % w/w NaOH in order to set the pH at between 8.3-8.5. The solution is left for agitation for 15 minutes.
ii) Addition of the rest 4.5 g of Iron solution until the pH of the solution is between 2.83.0. The suspension is kept under agitation for 1 h. The pH is adjusted with HCl 6 N solution or 15% w/w NaOH solution, respectively.
A suspension of ferro-N-acetyl-L-aspartylated casein is obtained. The reaction product is kept under agitation for 20 minutes, and the crude ferro-N-acetyl-L-aspartylated casein dérivative is recovered by filtration.
(G) Complex dissolving and filtration.
The solid complex is dispersed in 70 g of purified water and then 3.85 g of NaOH 15 % w/w solution are added until a pH of 8.5 ± 0.1. The solution is kept under agitation for 30 minutes. The remained solution is filtered in order the undissolved impurities to be separated. The insoluble residue is less than 0.5 wt%.
(H) Précipitation and isolation of pure moistferro-N-acetyl-L-aspartylated casein.
To the clear solution, which is put into an enamelled reactor equipped with an impeller mixer and with a probe for reading the pH value, are added by pouring 2.28 g of 6 N HCl 5 aqueous solution, at a température of between 20 °C and 25 °C, in approximately 20-30 minutes, until a pH value of 2.8-3.0 is obtained. At the end of the addition, the reaction product is kept stirred for 20 minutes, and the precipitate of pure ferro-N-acetyl-Laspartylated casein that has formed is recovered by filtration and subséquent washing with purified water.
(I) Desiccation of ferro-N-acetyl-L-aspartylated casein.
The moist pure product obtained in the previous stage is dried under vacuum, at a température increasing from 60 °C to 70 °C, for an overall period of 24 hours. After the desiccation process, 6.4 g of pure iron (III) casein N-acetyl-L-aspartylated complex are is obtained, with a water content < 5 wt% (Karl Fisher), having the following chemical and physicochemical characteristics:
| Product characteristics | Value |
| Colour | Brown-red |
| Solubility | Soluble in NaOH pH = 8 |
| PH | 2.8 |
| Free iron (not complexed) | <0.05% w/w |
| Total iron | 5.3 % w/w |
| Proteins | 88.0 % w/w |
| Complexed N-acetyl-L-aspartic acid | 7.0 % w/w |
| Free N-acetyl-L-aspartic acid | 0.6 % w/w |
| Chlorides | 1.8 % w/w |
| Bacterial content | <103 CFU/g |
| Mold and yeast content | <102 CFU/g |
I
- 17Cited literature
1. GB910321 Improvements in and relating to a process of preparing Ferrous Salts of Aspartic Acid and Medicines containing these Salts.
2. Arzneimittel-Forschung, 1984; 34(9):948-952 Iron dérivatives of modified milk protein.
3. US4493829, 1985, Bio-available succinylated protein-iron dérivatives which do not cause gastric lésions, method of préparation and related pharmaceutical compounds.
4. EP0243322, 1987, Compounds containing bioavailable iron, process for their préparation and pharmaceutical compositions containing them.
5. International Journal of Clinical Pharmacology, therapy, and toxicology, 1993; 31(1):40-51 Chemical and biological characterization of iron-protein succinylate (ITF 282)
6. EP0939083 Ferro-succinylcasein complex, process for its préparation and pharmaceutical compositions containing it.
7. US2001031748 Use of métal complexes to treat gastrointestinal infections.
8. Clinical Drug Investigation, 2005; 25(11):679-689 Overview of clinical trials in the treatment of iron deficiency with iron-acetyl-aspartylated casein.
9. W02006021843 A process for producing iron succinyl casein and acetyl-aspartate iron casein complexes and use thereof in pharmaceutical mixtures.
10. Tetrahedron: Asymmetry 18 (2007) 1625-1627.
Claims (6)
1. Process for the préparation of iron (ΙΠ) complex with N-acetyl-L-aspartylated casein, comprising the steps of s (a) reaction of casein with N-acetyl-L-aspartyl chloride, ta form N-acetyl-Laspartylated casein, (b) subséquent reaction of the N-acetyl-L-aspartylated casein with ferrie chloride; and (c) obtaining the iron (III) complex with N-acetyl-L-aspartylated casein.
2. The process of daim 1, wherein the casein utilized is food-grade casein.
3. The process of daim 1, wherein step (a) comprises providing a solution of casein, and wherein addition of N-acetyl-L-aspartyl chloride to the casein solution is péris formed at a pH of between 6 and 9.
4. The process of claim 1, wherein the N-acetyl-L-aspartyl chloride is prepared in situ, preferably by using chlorinating reagents selected from the group consisting of thionyl chloride (SOCI2), phosphorus pentachloride (PCI5)f phosphorus trichloride zo (PCI3), carbon tetrachloride (CCI4), oxalyl chloride (COCI)2, phosphoryl chloride (POCI3), 2-chloro-l,3-bis(methoxycarbonyl)guanidine (Palau'Chlor), cyanuric chloride and /V-chlorosuccinimide.
5. The process of daim 1, wherein the iron (III) complex with N-acetyl-L-
25 aspartylated casein obtained in step (c) is darified before being sent on for desiccation or spray-drying or freeze-drying.
6. A process for preparing a pharmaceutical composition comprising an iron (III)
30 complex with N-acetyl-L-aspartylated food-grade casein, said process comprising the process according to anyone of claims 1-5 and combining said iron (III) complex with N-acetyl-L-aspartylated food-grade casein with pharmaceutical excipients, in particular selected from diluents and preservatives.
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP14386034.4 | 2014-12-19 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA18236A true OA18236A (en) | 2018-09-04 |
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