Cyclic ferric (ii) citrate monohydrate, process for its production, pharmaceutical and / or nutritional and / or food compositions comprising it and its use in the therapeutic and nutritional field

Abstract

In the ferric iron citrate monohydrate defined by the chemical formula FeC6H6O7 H2O (ferrous citrate monohydrate or ferric iron citrate monohydrate) obtained according to a specific production method, the citric acid and the elemental iron are combined in a molecular complex in which the iron is gathered in the citrate moving into a ring-like form and the iron is bound to the O groups of the carboxyls 1 and 3 through ionic bonds, forming two enantiomers D and L (R and S), both of which are completely water-soluble and highly absorbable by the human body. Formula (L) and Formula (D). With a different absorption mechanism than traditional iron molecules, said ferric iron citrate monohydrate can be used in pharmaceutical compositions and / or nutritional compositions and / or food compositions, in particular for the treatment and prevention of iron deficiency anemia and all diseases and conditions associated with iron deficiency. The effectiveness is demonstrated through several examples.

Description

Technical Field

[0001] This invention relates to ferric citrate(II), particularly to ferric citrate(II) monohydrate as defined by the chemical formula FeC6H6O7·H2O (ferrous citrate monohydrate or ferric citrate(II) monohydrate), characterized by having a pH of 2.5, unlike known types, and having a non-linear, cyclic molecular structure distributed in three directions defining the so-called lattice space, wherein citric acid and elemental iron are bound in a molecular complex, wherein iron aggregates in the citrate salt that moves into a cyclic form, and iron is bound to the O groups of carboxyl groups 1 and 3 via coordinate bonds. - The group forms two enantiomers, D and L (R and S), both of which are completely water-soluble and highly absorbable by the human body.

[0002] L D The present invention also relates to a specific method for producing the ferric citrate monohydrate in the form of a ferrous molecular complex, the method giving the molecule an absorption mechanism different from that of conventional iron molecules. The method provides ferric citrate(II) obtained by the direct reaction of zero-valent iron, preferably in the form of iron filings or powder, with citric acid in water at a stoichiometric ratio of 1:1. The reaction is initially initiated in the presence of a laboratory-produced starting catalyst, such as Fe++, which forms ferrous hydroxide Fe(OH)2 in water at an amount of 5000 to 15000 ppm. For subsequent production, the amount of waste or aqueous solution containing ferric citrate, which is always used as a catalyst, is equal to 5000 ppm to 15000 ppm, preferably 10000 ppm, thereby reducing the waste content to 0.05% from a circular economy perspective.

[0003] The present invention also relates to pharmaceutical compositions and / or nutritional compositions and / or food compositions comprising the ferric citrate(II) monohydrate, and their use in the treatment and food industry (as food or food supplement), particularly in the treatment and prevention of iron deficiency anemia and all diseases and conditions related to iron deficiency.

[0004] Due to its experimentally proven solubility in water, the present invention also relates to the direct use of the said iron(II) citrate monohydrate in dry powder form as an additive dissolved in water or aqueous solutions, such as any salt or sugar. Background Technology

[0005] Iron is an essential mineral for the human body.

[0006] Iron is an essential trace element for the human body because it is a basic component of hemoglobin, myoglobin, and several iron-dependent enzymes. Iron has a variety of functions, such as transporting oxygen to tissues (hemoglobin), electron transfer in the respiratory chain (transferrin), and participating in important enzyme systems, such as the synthesis and degradation of biogenic amines (including the neurotransmitters dopamine and serotonin) and the metabolic degradation of xenobiotics (cytochrome p450 and b5).

[0007] Given the importance of iron to the human body, and because it is an essential element that is only obtained through diet, iron is continuously recycled and reused by the body due to its slow and rapid release reserves. In healthy individuals, the loss is very limited; therefore, the daily requirement necessitates a minimum exogenous intake, which can be met through diet. Furthermore, in the event of a prolonged lack of exogenous iron, the body will deplete its iron reserves stored in ferritin and hemosiderin.

[0008] In healthy subjects and under baseline conditions, the total iron content in the body is approximately 4–5 grams, of which about 65% is contained in hemoglobin molecules and 10% is contained in myoglobin. The remaining amount is primarily represented by stored iron (ferritin and hemosiderin) and a minimum amount contained in enzymes and cytochromes or associated with transferrin (transferrin).

[0009] Therefore, the human body maintains a balance among all forms of iron by controlling iron reserves, regulating absorption as needed, and recovering iron through the catabolism of red blood cells. Iron absorption depends primarily on three main factors: the amount of iron introduced into the body through diet, the release of iron from food during digestion and its bioavailability so that it can be absorbed by the small intestinal mucosa, and the intestinal lumen conditions that greatly affect its final utilization rate.

[0010] Foods of animal origin (offal, egg yolks, meat, including fishery products: oysters and clams are particularly rich in iron) and foods of plant origin (legumes, such as lentils and peas, nuts, asparagus, brewer's yeast, chicory, green chicory, and nuts) contain small amounts of iron.

[0011] Iron absorption depends largely on the food in which it is contained and its form (inorganic or organic). Generally, iron absorption from plant-based foods is low, around 10%; meat is about 30%, soybeans 20%, fish 15%, and eggs 10%; this highlights the need to know bioavailability and the element's content in food when considering iron absorption.

[0012] Currently, research on iron absorption through food remains limited and incomplete, given the various factors involved in metal absorption, and the number of study subjects is also limited. Furthermore, iron metabolism is not fully understood, as no food and / or preparation can restore proper iron metabolism in the body, and in iron-deficient subjects, supplementation only partially compensates for short-term iron deficiencies in circulation.

[0013] The reported dietary iron intake was determined based on the loss and amount necessary for growth and the formation of reserves, while also taking into account the bioavailability of this nutrient in the diet itself.

[0014] Iron deficiency leads to iron-deficiency anemia, a prevalent disease in European countries and several continents, particularly in Africa, where poor nutrition and malnutrition contribute to an increase in anemia cases. Approximately 30% of women of reproductive age and adolescents experience this dysfunction due to reduced iron reserves caused by significant blood loss during menstruation. This deficiency also negatively impacts other biological systems and mechanisms, such as thermoregulation, the immune system, and the neurotransmission system in the brain.

[0015] Clinically, iron deficiency anemia presents with fatigue, paleness, shortness of breath, and tachycardia. In such cases, some signs, such as difficulty concentrating and exhaustion, may already be detectable in the early stages of actual deficiency; simple control of blood parameters may or may not confirm iron deficiency. Complicating the diagnosis, it should be noted that determining the appropriateness of iron levels is somewhat difficult because there is no single parameter indicating a possible deficiency, which only further complicates the control of iron supplementation.

[0016] From an immune system perspective, iron deficiency is accompanied by atrophy of lymphoid tissue and reduced macrophage activity. Excessive iron intake can actually promote bacterial growth in malnourished subjects or during infection, although there are no known cases of acute iron poisoning in adults because the cumulative disease is essentially entirely hereditary (hemochromatosis).

[0017] When calculating dietary iron intake, it's important to distinguish between its two forms: heme iron (organic form) and non-heme iron (inorganic form). Their bioavailability differs significantly and greatly influences the assessment of the potential absorption of dietary iron. Heme iron is a fundamental component of hemoglobin function and is essential for red blood cell formation. Found in heme proteins in fish, meat, and eggs, heme iron is the most readily absorbed form of iron from food. Its absorption rate is approximately 25%, independent of dietary composition, as it is absorbed in the form of porphyrin complexes, unaffected by other nutrients. The oxidized state of iron in porphyrin complexes is 2+, which is the form that makes hemoglobin active in oxygen transport.

[0018] The absorption of non-heme iron is closely related to dietary composition and individual nutritional status. The latter, found in grains and vegetables, must be converted into an organic form after being absorbed in small amounts for later use. In addition, oxidized ferric iron (Fe3+) is also absorbed. 3+ It is inactive in hemoglobin and the body must first convert it into ferrous iron (Fe2+). 2+ Only iron of the non-heme form can be used for hemoglobin synthesis. Some dietary components, such as phytates (found in the bran of grains and spinach), tannins (found in tea and coffee), and polyphenols (found in olive oil), have the ability to inhibit the absorption of non-heme iron.

[0019] Experimental tests have shown that many other compounds (such as ascorbic acid (vitamin C)) and certain foods (such as meat and fish) can even enhance its absorption. Therefore, to better absorb the iron in vegetables, it is recommended to combine them with foods rich in vitamin C in the same meal: for example, lemon juice can be used as a condiment, or orange juice can be consumed with grains and vegetables. Vitamin C and heme iron found in animal-derived foods can increase the absorption of non-heme iron by 50%. The percentage of non-heme iron absorbed ranges from 2% to 13%.

[0020] Iron absorption occurs at the duodenal level of the intestine and during the first phase of fasting. The pH of the duodenum favors the absorption of iron as ferrous iron (Fe2+), which is more soluble than ferric iron (Fe3+). This provides a greater chance of chelation and higher mucosal exposure to the dissolved form before precipitation occurs.

[0021] In contrast to inorganic (non-half) iron, half (organic) iron is absorbed through a different mechanism. According to some studies, once heme separates from the globin it binds to, it is recovered from mucosal cells; according to other studies, the protein portion is removed within the mucosal epithelium. In either case, the released iron is transferred to the plasma in a form that can bind to transferrin, a protein that transports iron absorbed by the intestine from its storage site and is produced by the catabolism of hemoglobin.

[0022] In cases of confirmed iron deficiency, a diet alone, even one based on iron-rich foods, may not be sufficient to address the deficiency. Therefore, it is necessary to use targeted products with high iron content, known as dietary supplements. In the most severe cases, when the deficiency becomes dangerous, intravenous iron-based medications may be necessary to rapidly restore the body's required levels.

[0023] Among the earliest attempts by humankind to overcome this lack (dating back to the last century), the preparation of "molten iron" and "nailed apples" or "iron apples" should be mentioned.

[0024] Nowadays, when unhealthy dietary habits are introduced or the body's needs increase, supplements of natural or synthetic origin can be used as nutritional supplements. However, generally, they should be taken on an empty stomach to avoid reducing their absorption.

[0025] One of the most well-known medications used in the military treatment of iron deficiency anemia and anemia caused by acute or chronic blood loss is Ferrograd®, a sustained-release tablet of ferrous sulfate, whose daily administration of one tablet should be sufficient to meet the element's daily requirement, even in more demanding subjects. Unfortunately, there are contraindications to taking Ferrograd® as a medication. Among the side effects reported, and those common in all medications and preparations used as iron supplements, diarrhea, constipation, nausea, abdominal pain, melena, and allergic reactions should be mentioned; rare cases of gastric intolerance and acute iron poisoning have also been reported. Iron-based supplements and medications are also not recommended for people with hemochromatosis and hemolytic anemia, conditions associated with reduced numbers of red blood cells and hemoglobin, and not iron deficiency.

[0026] The most commonly used (and oldest) oral iron supplement is ferrous sulfate, although it is an inorganic compound. It is well known that metals are more readily absorbed when they exist in an organic form, that is, when they are bound to organic molecules such as citrate, fumarate, gluconate, pyruvate, malate, lactate, etc.

[0027] In inorganic salts, minerals are free and can be found alongside much smaller inorganic compounds such as potassium chloride, magnesium sulfate, and sodium chloride, which can interfere with iron absorption.

[0028] In addition to ensuring better efficacy, organic salts also reduce the likelihood of unabsorbed minerals overloading the body's elimination mechanisms. In fact, some of these inorganic salts are not absorbed and utilized, but are eliminated immediately after ingestion, creating conditions for potential physical fatigue, as excess iron that the body does not properly utilize can have pro-oxidative effects and potentially damage cells.

[0029] Despite this knowledge, as mentioned above, ferrous sulfate remains a widely used compound for iron supplementation and was the first compound developed for this purpose.

[0030] A growing awareness of the relationship between health and diet, coupled with a low propensity to change individual eating habits, has led to the development of a new class of fortified products aimed at increasing the nutritional value of food while maintaining its sensory properties. Using food as a carrier to increase the intake of certain nutrients is a method that has been tried for a decade: for example, adding iodine to table salt or iron to flour.

[0031] Other recently launched products rich in minerals and vitamins include dairy products, baby products, and juices.

[0032] A key characteristic of foods rich in minerals is their bioavailability once the product is ingested.

[0033] Existing technology As mentioned above, the use of ferric citrate (II) (also known as ferrous citrate) as a supplement is known, but to date there is no complete and accurate characterization of this compound in the literature, nor is there any information on the influence of preparation methods on the characteristics of this compound, and it does not appear in the chemical product catalogs of the most important international companies.

[0034] The compound with the chemical formula FeC6H6O7·H2O, obtained by contacting iron and citric acid, has been disclosed in Hermann Kammerer's article "Beitrrage zur kennt nise DER citonsaure", JUSTUS LIEBIGS ANNALEN DER CHEMIE, VERLAG CHEMIE GMBH, DE Vol. 170, no. 1-2, January 1, 1873, pages 176-190. However, no indication is given regarding the specific stoichiometric ratio of iron powder and citric acid used, such as the 1:1 disclosed herein, nor is it prepared using an aqueous solution rich in Fe++, nor is there any information regarding its molecular structural characteristics and its physical properties (e.g., solubility in water).

[0035] US3091626 discloses a complex method for preparing ferric citrate(II) involving the reaction of a double salt (sodium citrate and ferrous chloride) and subsequent separation from a solution of ferrous citrate, but its molecular structure is not cyclic. Furthermore, the authors of the aforementioned patent emphasize that it is impossible to prepare ferrous citrate from metallic iron and citric acid dissolved in an aqueous solution.

[0036] Even though WO 2021 / 034800 A2 does not use the same name "ferrous citrate monohydrate" to describe ferrous citrate, it discloses a product with the same empirical formula C6H8O8Fe, obtained by a method that does not allow the formation of compounds in which iron is complexed to form the innovative structure of this application.

[0037] In October 2022, the American Chemical Society published CAS number 2831338-40-8, which identified an iron salt with the chemical formula FeC6H8O7·H2O (ferrous citrate monohydrate or ferric citrate(II) monohydrate), the structural formula of which is shown below:

[0038] The presence of hydroxyl groups can be clearly seen.

[0039] Otherwise, in the iron citrate (II) of the present invention, all hydroxyl groups are deprotonated, thus having a net negative charge, which forms bonds with the positive charge present in Fe(II), thus resulting in molecular cyclization and complexation.

[0040] •H2O L D Reference: NAVIGLIO DANIELE ET AL. “Iron (II) Citrate Complex as a Food Supplement: Synthesis, Characterization and Complex Stability”. NUTRIENS, Vol.10, no.11, November 1, 2016, page 1647, XPO93174639. For brevity, we will refer to it as DN below. It describes a crystalline iron (II) citrate complex having the chemical formula FeC6H6O7·H2O and its use as a food supplement for treating iron deficiency. Furthermore, DN describes a method for preparing the iron (II) citrate complex, which involves adding citric acid monohydrate to water, heating, and adding iron filings when the temperature reaches 90°C. The resulting precipitate is filtered and freeze-dried.

[0041] This crystalline citrate complex reveals the presence of a COO- group as a whole (page 8), and iron is actually present entirely in the form of iron(II) (page 7). The FeC6H6O7·H2O structure has never been shown as cyclic. Summary of the Invention

[0042] The applicant is responsible for conducting comparative experiments with compound DN to demonstrate the different forms of the crystalline ferric citrate(II) monohydrate of this application, and the inventive activity derived from the remarkable effects clearly derived from the different characteristics that distinguish the ferric citrate monohydrate of this application from DN.

[0043] Comparative testing and demonstration The first feature to be emphasized is that the literature DN in Section 2.5 indicates the formation of ferrous citrate at pH 6.5. (Ferrous citrate (50 mg) was added to 100 mL of ultrapure water. The pH of the solution was adjusted to 6.5 using NaOH (0.1 M) or HCl (0.1 M). The pH range for evaluation was from 2 to 12. The stability of the complex was evaluated using the spectrophotometric technique described in Section 2.4).

[0044] The difference is that this patent application discloses the formation of a cyclic ferrous citrate compound with a pH of 2.5 (see page 58 of this patent application).

[0045] As is well known, in chemistry, the pH value of solution molecules significantly affects the structure and function of biomolecules: in particular, two molecules with the same crude molecular formula but existing in solutions with different pH values ​​may behave differently from a biological perspective due to the differences in the ionization of functional groups present in the molecules.

[0046] pH differences primarily affect the three-dimensional shape of molecules. For example, in proteins, the charge on ionizable groups leads to different folds or, consequently, influences interactions with other molecules, as reported in the basic textbook "Lehninger's Principles of Biochemistry." Each biomolecule has a different shape at different pH levels, and its biological function depends on pH.

[0047] We can give some practical examples, such as: Enzymes and proteins: The function of proteins, especially enzymes, depends on their three-dimensional structure, which is affected by the ionization of side groups. Therefore, the same protein can have different shapes at different pH levels, and thus different biological activities.

[0048] Enzyme activity: Many enzymes have an optimal pH value at which their activity is best. Outside of this pH range, their activity decreases or disappears completely.

[0049] In view of the above, the molecule synthesized in DN at pH 6.5 and the molecule at pH 2.5 of this patent application are two chemically different molecules.

[0050] Then a complete comparative crystallographic analysis was performed to support the claimed polymorphism and the unexpected properties of its crystallization form relative to the literature DN.

[0051] We hereby anticipate the results of the experimental section, which reports the results of the various tests conducted and provides their evidentiary value, confirming the existence of the innovative nature of the product, particularly its innovative nature relative to DN. Attached Figure Description

[0052] This specification will be better understood by referring to the accompanying drawings, in which: Figure 1a X-ray diffraction analysis of ferric citrate (II) disclosed in prior art document DN on a first sample A is shown, and repeated on a second sample B; Figure 1b Crystallographic analysis of the iron(II) citrate molecule of the present invention obtained according to the procedure of Example 1 is shown; Figure 1c Three curves, A (and curve B) and C, are shown for comparison, highlighting how the characteristic patterns of points in A (and B) and sample C are very different; Figure 2 The present invention is shown Figure 1b Microscopic images of the crystals of the ferric citrate sample; Figure 2a The percentage of atoms forming the crystal itself is shown; Figure 3 This is an EDS layered image showing how iron is distributed in the central part of the crystal; Figure 4 Thermogravimetric analysis of ferric citrate (II) obtained according to the procedure of Example 1 is shown; Figure 5a and Figure 5b The results of the solubility test of ferric citrate monohydrate according to the present invention are shown, wherein clear and transparent solutions were always obtained by dissolving samples with concentrations of 5000 ppm and 10000 ppm in distilled water at room temperature and mechanically stirring them for 1 hour and 3 hours respectively. Figure 6 This is a histogram showing the bioavailable and excreted portions of iron, expressed as a percentage (%) of the total iron measured at the end of (complete) digestion: it can be seen that gastric juice has no effect on inert molecules passing through the stomach; Figure 7 This is a histogram showing the effect of the bioavailable portion of the formulation on intestinal mucosal viability: the bioavailable portion of the formulation does not cause any reduction in intestinal epithelial cell viability; Figure 8 This is a histogram showing iron absorption in the intestinal epithelium after 1 hour and 3 hours: To assess the amount of iron absorbed by intestinal epithelial cells (expressed in μg / g per cell), iron was quantified at the apex (the portion applied to the epithelium), the lateral basal region (the portion absorbed), and the intracellular region; after 1 hour of exposure, the intracellular iron content was 70.60 ± 14.52; after 3 hours, it was 95.62 ± 6.75. Figure 9This is a histogram showing the levels of intestinal ferritin in the intestinal epithelium 1 hour and 3 hours after exposure to the formulation, with the results expressed as mean standard deviation: Ferritin was chosen as an indicator of intracellular iron content because it is involved in the micronutrient storage process and its expression depends on intracellular iron levels: For the tested molecule, a significant increase in ferritin expression was demonstrated relative to the control FD, which reached a plateau of approximately 65.15 protein μg / g 1 hour after exposure to the intestinal epithelium; Figure 10 This is a flowchart of the preparation process of ferric citrate monohydrate according to the present invention.

[0053] Comparative analysis of X-ray crystallography X-ray crystallography is a fundamental technique for determining the atomic and molecular structure of crystals. This technique utilizes the diffraction of X-rays by atoms within the crystal. The diffracted X-rays form characteristic patterns of spots (reflections) on a detector. The position and intensity of these reflections contain information about the distances between atomic planes and the orientation of atoms within the crystal.

[0054] Figure 1a The crystallographic analysis of sample A reported in DN and its replication on sample B are shown.

[0055] Figure 1b The crystallographic analysis of the molecule of this patent application, sample C, is shown, in the same proportion as DN.

[0056] exist Figure 1c In the comparison, two curves A and C (curve B is a repeat of curve A) are shown, noting how the characteristic patterns of the points are different.

[0057] For ease of comparison, we report the detection data of peaks, diffraction angles, and intensities for sample A of DN and sample C of this patent application below:

[0058] It is well known that when X-ray diffraction patterns show peaks at different distances, the sample has differences in its crystal structure.

[0059] Especially in the current context, differences in crystal structure can indicate the following: a) Different crystalline phases If the diffraction peaks of a sample are at different angular positions (2θ), this indicates that the sample belongs to a different crystalline phase defined by two different compounds or two distinct aggregates. Each crystalline phase has a unique diffraction pattern.

[0060] The varying distances between peaks indicate that the samples have different lattice parameters. This means that the unit cell sizes of the crystals in the samples are not equal.

[0061] b) Chemical composition Different peak positions can reflect changes in the chemical composition of a sample, which can alter the interplanar distance within the crystal.

[0062] c) Lattice deformation: Internal stresses or deformations within the crystal lattice can cause changes in the positions of diffraction peaks. These voltage differences can lead to peaks appearing at different distances.

[0063] It should also be noted that comparing peak values ​​and various intensities confirms the following: d) Peak intensity: ●Sample A and Sample C have peaks with different intensities. e) Peak value definition ● The peaks of samples A and C are sharp and well-defined. f) Peak position ● The position of the main peak differs from that of samples A and C, indicating that the samples have different crystal structures. g) Number of peak values ● Samples A and B show similar numbers of peaks, indicating comparable structural complexity, while sample C has more peaks in different positions.

[0064] ●Sample C has a distinct peak in the 50-70° region, while samples A and B do not.

[0065] We would like to draw attention to the fact that peaks in the 50-70° region of an X-ray diffraction pattern can provide important information about the crystal structure of the analyzed sample. According to Bragg's law, peaks at high diffraction angles (50-70°) correspond to very small interplanar distances. nλ= 2d sin(θ)n \lambda= 2d\sin(\theta)nλ= 2d sin(θ), Where d is the crystal plane distance, λ is the wavelength of the X-ray, and θ is the diffraction angle. Since sin(θ) increases with the angle, the peaks at 50-70° indicate that the corresponding crystal planes are very close to each other.

[0066] The peaks appearing in this region indicate an atomic-level crystallization sequence, revealing finer details of the sample structure.

[0067] The crystalline phase present in a sample can be determined by comparing the peaks observed in the 50-70° region with reference data from the D1 diffraction pattern. Each crystalline material has a characteristic diffraction pattern, which serves as a "fingerprint" for determining its properties.

[0068] h) Determination of impurities If the sample is a mixture of iron and other substances, the peaks in this region may differ slightly in position and intensity due to the presence of other elements and variations in crystal structure. This can help determine the composition and elemental distribution of the molecular mixture.

[0069] Peaks in the 50-70° region of an X-ray diffraction pattern represent crystal planes with small interplanar distances and provide important details about the atomic structure of the sample. These peaks are crucial for crystal phase identification, crystal structure determination, and quantitative analysis of sample composition.

[0070] This confirms that the literature DN does not define the three-dimensional mapping of atoms forming crystals, nor does it mention other elements (atoms) that may have accumulated during the experimental reaction. In contrast, the applicant's application points to an innovative production method that differs from DN, in which the use of a catalyst not only reduces the reaction time but also acts as a protection against the cyclic accumulation of ferrous citrate monohydrate, thus avoiding the presence of impurities.

[0071] From this patent application Figure 3 As can be seen, the atoms detected in the crystals belong only to carbon, hydrogen, oxygen, and iron. There is no trace of other cations or anions that could potentially interfere with the pH value and the cyclic three-dimensional molecular structure. Furthermore, it should be emphasized that the reaction rate of the reagent in the final product depends not only on the purity of the reagent but also on the intermolecular interactions.

[0072] In DN, the authors specify the following steps: "Single-crystal X-ray diffraction confirmed that the structure of the compound can be described as a pseudo-three-dimensional octahedral structure composed of coordination polyhedral iron chains."

[0073] This means that the authors of the article point out that although the compound may have a three-dimensional arrangement of atoms, the structure is dominated by units repeating along a primary dimension (usually along a specific axis or direction). In other words, the lattice exhibits a finite three-dimensionality, but the primary two-dimensional organization along the axis or plane does not form a clearly defined three-dimensional compound. Furthermore, the literature DN clearly indicates how iron is positioned at the center of the structure. In chemistry, octahedral coordination refers to the arrangement of atoms around a central atom (in this specific case, an iron atom). In this geometry, the central atom (iron) is surrounded by six atoms or groups (ligands) at approximately 90 degrees apart. Conversely, from this patent application… Figure 3 It can be confirmed that the iron atom is located on the right side of the crystal and is linked to the O atoms of various molecular carboxyl groups through strong coordination bonds. - Group bonding.

[0074] Observing polymorphs As is well known, polymorphs of a substance have the same chemical composition, but the arrangement of atoms in space is different, resulting in different physical, chemical and biological properties.

[0075] Polymorphisms of a substance may have different properties, for example: ● Melting point: The different temperatures at which each polymorph changes from a solid to a liquid state.

[0076] ●Solubility: The rate and amount at which each polymorph dissolves in a solvent.

[0077] ●Stability: Under certain temperature and pressure conditions, some polymorphs are more stable than others.

[0078] Given that the literature DN previously disclosed that the closest iron citrate monohydrate to the present invention is a linear structure with different pH and crystal differences, and given that the iron citrate monohydrate is coordinated by strong coordinate bonds, while the iron citrate of DN is coordinated by ionic attraction, it should be remembered that a dative bond (or coordination) is formed when an atom (usually a transition metal such as iron) accepts a pair of electrons from a ligand (in this case, the citrate ion).

[0079] In the case of ferric citrate complexes with coordinate bonds, regarding: ●Structure: Iron coordinates with citric acid atoms through coordinate bonds, forming a complex structure with a specific geometric shape; And about ●Solution: In solution, depending on the strength of the coordination bond and the pH of the solution, the complex may partially dissociate, but usually retains its complex configuration to some extent (as shown on pages 41-43 of this patent application).

[0080] The difference lies in the structure of two-dimensional linear ferric citrate, where iron and citrate ions are linked by ionic bonds or simpler covalent bonds, without significant complexation. Specifically, regarding... ●Structure: Iron is simply bonded to the citrate anion in a relatively uncomplicated form, potentially as a linear chain. And about ●Solution: In solution, this type of salt completely dissociates into its constituent ions (Fe2+, Fe3+, Fe ... 2+ (and citrate), compared to complexes with coordinate bonds, it has a lower tendency to form complex structures.

[0081] Regarding "equality" in solutions, some fundamental differences need to be considered: Dissociation state: Complexes with coordinate bonds: iron and citrate ions do not completely dissociate, and the salt retains part of its complex structure in solution.

[0082] Linear complexes: Salts tend to completely dissociate into Fe in solution. 2+ Ions and citrate ions.

[0083] Chemical and physical properties: Complexes with coordinate bonds exhibit significantly different chemical and physical properties from linear salts due to differences in electronic configuration and geometry (underlined in crystallographic analysis). Interactions in solution, such as solubility, reactivity, and interactions with other ions or molecules, can differ between the two.

[0084] Stability in solution: The stability of complexes with coordinate bonds is affected by factors such as pH and ion concentration, which causes them to remain intact under certain conditions (as shown in the stability studies reported on pages 43-45 of this patent application).

[0085] On the other hand, dissociated linear salts do not possess structural stability and dissociate completely in solution. (Atkins-Jones principles of chemistry).

[0086] Absorption differences Ferric citrate complexes with coordinate bonds in solution are not equivalent to linear ferric citrate salts in solution. Although they contain the same elements (iron and citrate), their structures, behavior in solution, and chemical and physical properties differ.

[0087] Furthermore, the human body's absorption of these two molecules is definitely different biochemically.

[0088] In fact, since the molecules of the present invention have a pH of 2.5 in solution and, as previously mentioned, do not dissociate into free Fe, they can resist the acidic pH of the stomach 1-3 and be transferred to the intestine in the form of a complex.

[0089] Since the pH of the DN molecule is 6.5, and given the previously established dissociation conditions, the DN molecule can dissociate from Fe in the stomach. 2+ (Fe2+) to Fe 3+ The transformation of (ferric iron) is a redox process that is influenced by chemical conditions and the acidic environment of the gastrointestinal tract.

[0090] How this happened: The stomach has a highly acidic pH, typically between 1 and 3, due to the presence of hydrochloric acid (HCl). This acidic environment favors redox reactions, particularly those involving ferrous iron (Fe²⁺). 2+ ) oxidized to trivalent iron (Fe) 3+ In the presence of an oxidizing agent, such as dissolved oxygen or other oxidizing agents present in the stomach, ferrous iron (Fe2+)... 2+ It can be oxidized to ferric iron (Fe3+). 3+ ).

[0091] The chemical composition is as follows: 4Fe 2+ +O2+4H + →4Fe 3+ +2H2O Hydrochloric acid (HCl) not only maintains the acidic pH value, but also provides the protons (H+) required for the reaction. + ).

[0092] Therefore, we can conclude that the molecule of this patent application differs from the molecule disclosed in DN because it differs in pH, its geometry, the structural stability of its coordinate bonds, the rate of absorption by the patient, and the absence of impurities, thereby allowing the resolution of a series of objective technical problems that a person skilled in the art would have required to resolve on the filing date of this patent application.

[0093] The purpose of this invention The first object of the present invention is to provide pharmaceutical compositions and / or nutritional compositions and / or food compositions comprising ferric citrate (II), which is a molecule chemically defined as a “neutral complex” in which iron 2+ is strongly bound to citrate ions and has high bioavailability, obtained by a specific and simple synthetic procedure that allows the production of ferric citrate monohydrate in crystalline form with a nonlinear but cyclic molecular structure. This allows us to definitively define the atoms and their aggregates that form the crystals by means of the microscopy techniques described below.

[0094] Another object of the present invention is to provide the use of the food and / or nutritional compositions of the present invention in treatment, particularly in the treatment and prevention of iron deficiency anemia and all diseases or conditions related to iron deficiency.

[0095] Another object of the present invention is to provide the use of the compositions of the present invention as food supplements in the field of nutrition.

[0096] Another object of the present invention is to provide the use of ferric citrate (II) prepared as described herein as an active ingredient in pharmaceutical compositions and / or nutritional compositions and / or food compositions. Detailed Implementation

[0097] Through long-term and in-depth research and development activities, the applicant has surprisingly discovered that it is possible to synthesize highly absorbable ferric citrate (II) through a specific synthetic route.

[0098] Therefore, the present invention primarily relates to iron(II) complexes having the chemical formula FeC6H6O7·H2O-ferrous citrate monohydrate or ferric(II) citrate monohydrate, the nonlinear structural formula of which is as follows: -H2O Citric acid and elemental iron are bound in a molecular complex, where iron aggregates in the citrate ion, which migrates to a cyclic form, and iron is coordinated to the O groups of carboxyl groups 1 and 3 via coordinate bonds. - On the group.

[0099] Through this reaction mechanism, we can also describe how molecules self-orient in three-dimensional space to produce a pattern of the molecule itself, which we will simply call CFC. It can take a right-handed or left-handed shape in space, forming D and L (R or S) enantiomers as previously reported.

[0100] The structural formula of FeC6H6O7·H2O

[0101] Preferably, the ferric citrate (II) is used as a treatment for iron deficiency anemia and all diseases or conditions related to iron deficiency, and is more preferably used as a therapeutic, preventive or curative treatment.

[0102] The present invention further relates to a method for preparing an iron(II) complex having the structure shown above, characterized in that the iron(II) citrate is obtained by directly reacting zero-valent iron (preferably in the form of fine iron filings or powder) and pharmaceutical-grade pure citric acid in a stoichiometric ratio of 1:1 in the presence of water, wherein the iron filings are reacted at a temperature from 40°C (1 atm) to the boiling point of the reaction mixture, preferably at a temperature below 95°C, and even more preferably below 90°C, to accelerate the oxidation and complexation process of the iron powder and citric acid, thereby producing a robust molecular structure over time.

[0103] According to a specific feature of the invention, the first reaction between zero-valent iron and citric acid is carried out in water in the presence of a laboratory-produced starting material composed of Fe(II) to form a compound Fe(OH)2 with a concentration sufficient to saturate the reaction liquid (H2O).

[0104] Then, in subsequent production steps, this presence is replaced by production waste (which is essentially an aqueous solvent containing ferrous citrate, at a concentration of approximately 1.40%).

[0105] Another advantageous feature of the present invention is that, through a circular economy mechanism, the production process ensures that chemical reaction waste is below 0.05%.

[0106] The present invention also relates to a pharmaceutical composition, or nutritional composition, or food composition, or food supplement comprising a mixture comprising, or alternatively comprising, an iron(II) complex of the chemical formula FeC6H6O7·H2O, ferrous citrate monohydrate, or ferric citrate(II) monohydrate having the structure shown in the CFC above, and optionally acceptable food or pharmaceutical grade additives, excipients, and carriers.

[0107] Preferably, the composition is characterized by containing 1 mg to 500 mg, more preferably 5 mg to 00 mg, such as 10 mg to 100 mg of ferric(II) citrate monohydrate.

[0108] Preferably, the composition is characterized in that it is for oral administration.

[0109] Preferably, the composition is used as a treatment method, and more preferably as a therapeutic, preventive, or curative treatment for iron deficiency anemia and all iron deficiency-related diseases or conditions.

[0110] According to one aspect of the invention, the present invention relates to pharmaceutical compositions and / or nutritional compositions and / or food compositions (also referred to herein only as “compositions”) comprising ferric citrate (II) having optimal bioavailability, obtained by a specific and simple synthetic method, and at least one physiologically acceptable excipient and / or carrier.

[0111] According to one embodiment, the ferric citrate (II) can be obtained and / or acquired by directly reacting zero-valent iron with citric acid (preferably in a stoichiometric ratio of 1:1).

[0112] The resulting ferric citrate (II) has high bioavailability, meaning it is highly absorbable, unlike most known and commercially available iron salts, which are not only partially absorbed but also have significant side effects in many cases.

[0113] In particular, the ferric citrate(II) contained in the composition according to the invention is synthesized by directly reacting zero-valent iron with citric acid in an aqueous solution, preferably in a stoichiometric ratio of 1:1, preferably using water saturated with iron(II), wherein all products used have undergone rigorous “food grade” testing.

[0114] Based on laboratory production studies, and then adjustments made in a pilot plant, this stoichiometric ratio proved to be the only one that could potentially maximize the final product yield to 98.32%. Variations in the stoichiometric ratio resulted in yield losses and changes in molecular stability. In fact, ratios of 1:2 and 1:3 for iron filings and citric acid, and vice versa, were tested, but the results were disappointing in terms of yield, rather than in the formation of the raw materials.

[0115] Preferably, ferric citrate (II) is synthesized by reacting very pure iron (>99.5%) scraps (or powder) with very pure anhydrous citric acid (>99.9%) in a heated reactor in the presence of water saturated with iron (II) in the form of Fe++ catalyst. This helps to maximize yield and produces no waste, thus creating a circular economy.

[0116] Preferably, and only for the first production, the starting material consisting of iron(II) that forms ferric hydroxide(II) (ferrous hydroxide) produced in the laboratory is used as the reaction catalyst in water.

[0117] refer to Figure 10 The flowchart shows that the resulting ferric citrate(II), separated from the reaction mixture by filtration (preferably by a vibrating sieve), is pearly white and is freeze-dried to remove almost all the water (residual water weight is 1.87%). The freeze-dried compound is then ground to obtain pure ferric citrate monohydrate molecules, ready for use in the target drug supplement formulation.

[0118] Advantageously, according to the features of the invention, the reaction waste, i.e., the solvent in which trace amounts (1.40%) of ferric citrate monohydrate are present, is reused as a reaction catalyst for subsequent production, without the need for starting materials. Thus, through a circular economy mechanism, chemical reaction waste in the production process is reduced to below 0.05%.

[0119] Preferably, the citric acid used in the reaction is anhydrous citric acid or citric acid monohydrate.

[0120] Ferric citrate (II) obtained as described above was analyzed to confirm its chemical structure and purity, particularly by thermogravimetric analysis. The stability of the iron oxidation state was also repeatedly verified by colorimetric analysis with the addition of 1,10-phenanthroline to confirm that the iron oxidation state was constant (II), and a reverse test with potassium thiocyanate was performed to observe the presence of Fe+++. The stability of the oxidation state is crucial for the bioavailability of iron because, as mentioned above, the conversion of iron (II) to iron (III) leads to poor metal absorption because it cannot be directly utilized by the body.

[0121] Therefore, clinical trials were conducted to evaluate the bioavailability and intestinal absorption of iron released from the ferric citrate formulation.

[0122] As shown above, crystallographic analysis reveals that it is primarily in crystalline form, such as... Figure 1bAs shown. It can be noted that the ferric citrate(II) obtained by the method described herein (Example 1) exhibits the same crystalline form, demonstrating the reliability of the synthesis reaction. Furthermore, the resulting crystalline white compound has the molecular formula FeC6H6O7·H2O, indicating that water molecules are contained within the compound's crystal lattice, thus synthesizing ferrous citrate monohydrate. The iron content per mole of the substance is 21.0% (w / w).

[0123] The experimental section below provides a detailed description of the synthesis steps and analytical techniques.

[0124] Ferric citrate (II) prepared as described herein can be used as a food supplement and / or food additive. This supplement and this food can be used to treat and prevent iron deficiency anemia and all iron-related diseases and conditions.

[0125] Preferably, the ferric citrate (II) prepared as described herein is included in pharmaceutical compositions and / or nutritional compositions and / or food compositions.

[0126] Ferrous citrate is included in the list of permitted substances for human nutrition issued by the Ministry of Health.

[0127] The use of ferric citrate (II) prepared as described herein as an active ingredient in pharmaceutical and / or nutritional and / or food compositions is another subject of this invention.

[0128] This composition can be administered via any suitable route of administration; preferably, the composition of the present invention is suitable for oral administration.

[0129] According to a preferred embodiment, the compositions of the present invention are oral or sublingual, solid, semi-solid, gel, or liquid compositions, and may contain conventionally physiologically acceptable excipients and / or carriers, as well as food additives and nutritional products.

[0130] The compositions of the present invention may be in the form of tablets, hard capsules, soft capsules, capsules, granules, fine particles, powders, tablets, syrups, emulsions, suspensions, and solutions suitable for oral administration. In any case, other suitable food, nutritional, and / or pharmaceutical forms may be used and fall within the scope of this invention.

[0131] This composition can be taken alone or with water, or, especially when taken in powder or granule form, can be mixed with other foods, such as yogurt, cream, gel, etc.

[0132] Alternatively, the compositions of the present invention can be readily available drinkable forms, such as drinking sachets of the "strip packaging" type, in which case cream or gel form is preferred.

[0133] The types of excipients, carriers, food, nutritional products, and / or pharmaceutical additives used to prepare the compositions of the present invention, the content ratio of additives to active ingredients, and the methods for preparing food compositions, nutritional compositions, and / or pharmaceutical compositions may be appropriately selected by a skilled formulator.

[0134] For example, organic or inorganic substances, or solid or liquid substances, can be used as excipients and carriers, provided that they are edible, physiologically acceptable, and compatible with all other components of the composition.

[0135] As stated above, those skilled in the art are fully capable of selecting the most suitable carrier and excipients to prepare the composition.

[0136] Examples of excipients used in the preparation of solid food compositions, nutritional compositions and / or pharmaceutical compositions include, for example, lactose, sucrose, starch, talc, cellulose, dextrin, kaolin, calcium carbonate, stearic acid or magnesium stearate, lactose, polyethylene glycol, mannitol, sorbitol, chelating agents, anti-caking agents, sweeteners, preservatives and flavoring agents.

[0137] To prepare an orally administered liquid composition, conventional inert diluents such as water or oil, such as vegetable oil, can be used. In addition to inert diluents, the liquid composition may also contain adjuvants such as wetting agents, suspending agents, sweeteners, flavoring agents, coloring agents, and preservatives. The liquid composition can be encapsulated in an absorbable material such as gelatin.

[0138] Sweeteners can be one or more of the less common natural sugars, such as sucrose, glucose, xylitol, mannitol, or sorbitol, or synthetic products, such as sodium saccharin, aspartame, acesulfame K, or sucralose. Acidifying agents may also be added if desired, provided they are compatible with the components of the composition.

[0139] Flavorings are food-, nutritional, and / or pharmaceutically acceptable spices and flavorings of synthetic or natural oils, the latter extracted from plants, flowers, fruits, and combinations thereof, such as cinnamon, mint, fennel, and citrus leaves, bitter almonds, citrus fruits, especially orange and / or lemon, lime, vanilla, chocolate, and grapefruit oil. Chocolate, vanilla, or eucalyptus spices and fruit flavorings, especially apple, pear, peach, strawberry, apricot, orange, lemon, and grape, may also be used advantageously.

[0140] The compositions of the present invention can also be in a controlled-release form or an immediate-release / controlled-release hybrid form. The preparation of these forms is well known to those skilled in the pharmaceutical field.

[0141] Controlled-release forms, such as prolonged release, are known in the art and include, for example, the use of excipients and mediators, including blocking materials, such as polymers and copolymers of acrylic acid and methacrylic acid; cellulose derivatives, such as hydroxypropyl methylcellulose, hydroxyethylcellulose, hydroxypropyl ethylcellulose, hydroxypropyl cellulose, methylcellulose, ethylcellulose, or sodium carboxymethylcellulose; gums; waxes; glycerides or fatty alcohols or mixtures thereof.

[0142] The compositions of the present invention may be in multiple doses (e.g., when in liquid form) or in dose unit form.

[0143] Considering that only 21.0% (w / w) is absorbable iron, each dose unit may contain 1 mg to 500 mg of ferric citrate(II) monohydrate, preferably 5 mg to 200 mg, for example 10 mg to 100 mg, for example 50 mg.

[0144] The compositions of this invention can be administered once or multiple times a day, preferably once or twice a day. The dosage and daily dose can naturally vary depending on the age, sex, health status, diet and eating habits of the patient, as well as the severity of iron deficiency and the type of treatment (preventive or therapeutic). The physician will determine the correct dosage of the preparation based on the patient's physiological condition.

[0145] If desired or necessary, the compositions of the present invention may also contain other active ingredients for the prevention and treatment of the neurological diseases described herein, such as naturally derived active ingredients.

[0146] The present invention also relates to the use of the compositions described herein in treatment, particularly in the treatment and prevention of iron deficiency anemia and all diseases or conditions associated with iron deficiency.

[0147] The present invention also relates to the use of the compositions described herein as food supplements in the field of nutrition.

[0148] The present invention also relates to methods for treating and preventing iron deficiency anemia and all diseases or conditions related to iron deficiency, the methods comprising administering an effective amount of the composition described herein to a subject in need.

[0149] In clinical trials, the compositions of the present invention showed that the ferric citrate(II) contained therein was highly absorbable and no associated side effects were observed.

[0150] For purposes of pure illustration and not limitation, the invention will be described in more detail below.

[0151] Experimental Section The research began with the formation of the molecule and its preparation method, progressed to in vitro analysis of the bioavailability and intestinal absorption of iron released from the ferric citrate preparation, and culminated in an assessment of toxicity in test subjects. A clinical trial in consenting patients was then described, allowing for the analysis of representative parameters of anemia due to iron deficiency and the validation of the efficacy of treatment with ferric citrate monohydrate according to the present invention.

[0152] Experimental protocol Use high-purity reagents and solvents.

[0153] Water is produced by a milli-Q generator (Milli-Q, Bedford, MA, USA).

[0154] Analytical balance series B 204 S (Mettler-Toledo, Germany), freeze dryer model. Heto Lyolab 3000 (Analytical De Mori, Milan, Italy). Using a UV-Vis spectrophotometer, model UV-1601 (Shimadzu, Tokyo, Japan), with quartz cuvettes with a 1.00 cm optical path.

[0155] An automated powder diffractometer (Panalytical Empyrean Powder, Santa Barbara, CA, USA) and a thermogravimetric analyzer (TGA400 Perkin Elmer, Norwalk, CT, USA) were used.

[0156] The first reaction was carried out in water in the presence of a starting material consisting of iron(II) bases obtained in the laboratory.

[0157] The formation of ferrous citrate monohydrate molecules.

[0158] The idea for developing organic ferrous complexes comes from the traditional application of "iron apples" or "Nai apples" by our ancestors to individuals suffering from varying degrees of iron deficiency. Several rusty nails were inserted into the apple and kept in contact for approximately 12–24 hours. After extraction, the apple turned black and was then applied to the anemic subjects.

[0159] Because the complexed iron compounds are highly absorbed by the human body, the chemical transformation of metallic iron into ionic iron occurs in apples. Based on this popular intuition, the spontaneous process of "nailing apples" has been reproduced under stable laboratory conditions according to this invention.

[0160] In practice according to the invention, citric acid dissolved in iron(II)-rich water reacts with zero-valent iron (metallic iron, such as iron filings or powder) in a stoichiometric ratio of 1:1, and is heated at a suitable temperature (e.g., 40°C to the boiling point of the mixture, preferably about 90°C) to accelerate the oxidation and complexation process of the iron and citric acid powder, resulting in a robust molecular structure over time.

[0161] Preparation of ferric citrate(II) Pharmaceutical-grade anhydrous citric acid (400 g) was added at 10,000 ppm to 1,000 mL of Fe++-rich water, and the solution was heated in a reactor with magnetic stirring. When the solution temperature reached 90°C, 100 g of iron filings were added. The reaction was stopped when the iron filings completely disappeared and the color changed from gray to white. After the iron had completely reacted, the mixture was cooled to room temperature. Regarding the stoichiometry, the iron powder and citric acid were mixed together in a 1:1 ratio.

[0162] Advantageously, for the first production, the starting material consisting of iron(II) produced in the laboratory is used as the reaction catalyst, which is converted into iron(II) hydroxide in aqueous solution.

[0163] The precipitated pearly gray salt was washed with water and filtered under vacuum using filter paper; finally, the residue was freeze-dried after being frozen at -20°C.

[0164] Thermogravimetric analysis Thermogravimetric analysis (TGA) of ferric citrate monohydrate (~100 mg) was performed in the temperature range of 30°C to 900°C. The data showed that... Figure 4 The average of three independent replicates. Based on the obtained data, the percentage of iron in the reaction product is 21.1% ± 0.4 (w / w), because it reacts with FeC6H6O7. The calculations for H2O are consistent with the theoretical calculations, such as Figure 4 As shown in the figure.

[0165] Determination of iron oxidation state A colorimetric method based on a complex ion formed by three 1,10-phenanthroline molecules and one iron(II) ion was used to detect the oxidation state of the iron(II) ion (Fortune, WB; Mellon, MG Determination of iron withophenanthroline: A spectrophotometric study. Ind. Eng. Chem. Anal. Ed. 1938, 10, 60–64).

[0166] Iron (III) ions and thiocyanate (SCN) - The complexation of iron(III) ions can be used to detect the oxidation state of iron(III) ions (Lister, MW; Rivington, DE Some measurements on the iron(111)-thiocyanate system in aqueous solution. Can. J. Chem. 1955, 33, 1572–1590). In the first or second test mentioned in this article, any color (red) appearing in the solution indicates the presence of iron(II) or iron(III) ions, respectively.

[0167] For quantitative analysis, spectrophotometric analysis was performed at wavelengths of 520 nm and 480 nm, which are the maximum absorbance values ​​for the 1,10-phenanthroline and thiocyanate complex, respectively. For spectrophotometric quantification of iron, the required solution was prepared from analytical grade ferrous ammonium sulfate [(NH4)2Fe(SO4)2x6H2O], 1,10-phenanthroline, potassium thiocyanate (KSCN), hydroxylamine hydrochloride (NH2OHHCl), sodium acetate (NaAc), and 99.999% H2SO4 (Aldrich product number 339741). The results showed the presence of iron(II) ions and the absence of iron(III) ions.

[0168] Complexation stability Dissolve ferric citrate (50 mg) in 100 mL of water.

[0169] Adjust the pH of the solution to 2.5 using NaOH (0.1 M) or HCl (0.1 M).

[0170] The pH range for evaluation was 2 to 12. Complex stability was assessed using the aforementioned spectrophotometric technique.

[0171] Crystallographic analysis: X-ray diffraction As mentioned earlier, X-ray crystallography is a crystallographic technique that uses X-ray diffraction to obtain spectra to reveal crystalline materials. Typically, this can determine the material and molecular structure of a substance.

[0172] X-ray diffraction patterns of the synthesized samples were recorded using an automated powder diffractometer with Ni-filtered Cu K-α radiation, corresponding to a wavelength of 1.5418 Å. The patterns were recorded using continuous scans within a diffraction angle range of 5° to 60°. (Figure 1) As already mentioned, this result led to the definition of the existence of crystals.

[0173] Crystal characterization Microscopy is used to analyze crystals. These techniques allow us to verify the size of the crystal and the distribution of atoms forming the material. Using this sophisticated technique, the results allow us to precisely determine the atoms that make it up and their percentages. Figure 2 and Figure 2a ).

[0174] EDS layered image 1 Figure 3 The diagram shows that iron is distributed in the central portion of the crystal. This allows us to say that the molecules close into rings, forming molecular complexes in which iron is bound by coordinate bonds. This chemical structure is an innovative one, where iron is in a complexed form rather than a free form.

[0175] Preparation of ferric citrate(II) monohydrate capsules.

[0176] Capsules containing 50 mg of ferric citrate(II) (iron content equal to approximately 21% (w / w), thus 10 mg of highly absorbable iron) and approximately 300 mg of starch were prepared. The capsules were prepared by uniformly mixing ferric citrate(II) with starch until a homogeneous powder was obtained, and then filling the powder into capsules.

[0177] Solubility test of ferric citrate monohydrate compound At room temperature, with constant stirring for 1 hour and 3 hours, dried samples with a moisture content of 1.8% were dissolved in distilled water at concentrations of 5000 ppm and 10000 ppm.

[0178] The sample in the 5000 ppm solution dissolved completely after 1 hour, while the sample in the 10000 ppm solution dissolved completely after 3 hours. This is due to... Figure 5a and Figure 5b The photos in the images prove that the water's transparency is always the same.

[0179] Study on the content of free iron and complexed iron Two samples, in triplicate, were prepared for analysis by dissolving less than 500 mg of each of the compounds in 100 mL of distilled water.

[0180]

[0181] After magnetic stirring for 1 hour, orthophenanthroline was used for determination to verify Fe. 2+ The existence of.

[0182]

[0183] Ammonium hydroxide was then added to the sample's stock solution until the pH reached 10.67, which is the Fe...2+ The formation of ferric hydroxide creates an alkaline environment.

[0184] Fe 2+ Hydroxides are the main component in the standard solution, not in ferric citrate monohydrate.

[0185] Five hours later, the sample was filtered through filter paper. The supernatant was acidified to pH 2.45 with citric acid.

[0186] It has the following absorbance:

[0187] Comparing the absorbance before and after the reaction with 24% ammonium hydroxide, we observed:

[0188] For each sample, the percentage of free iron that can be converted into ferric hydroxide (Fe(OH)2) is as follows:

[0189] It is certain that ferric citrate monohydrate is a complex of ferrous molecules, in which the bound iron is not eroded by alkaline reagents such as ammonium hydroxide (24%). Therefore, ferric citrate monohydrate is an innovative molecule in which the fixed position of citric acid propels it toward a rapid absorption mechanism different from that of conventional iron molecules.

[0190] Product shelf life research Product shelf life studies were conducted using two methods: 1) In vitro, degradation kinetics are accelerated at elevated temperatures; 2) The samples were stored in vivo and collected monthly over a period of 18 months.

[0191] The analysis included two factors: a) microbiological analysis and b) the ferrous content based on the dosage of o-phenanthroline and the reverse test for Fe+++ using potassium thiocyanate. The test results are shown below.

[0192] In vitro analysis This analysis involved dissolving 100 mg of ferric citrate monohydrate in 100 mL of distilled water to produce a ferrous solution with a concentration of 210 ppm.

[0193] Then, at time 0, microbial parameters and ferrous iron (Fe) were analyzed. 2+ The total content of ) was determined. The results are as follows:

[0194] The first results were recorded by culturing the solution at 37°C for 4 weeks to accelerate molecular degradation kinetics. Furthermore, 37°C is the optimal temperature for the growth of Enterobacteriaceae (a family of pathogenic microorganisms).

[0195] Samples were collected every 7 days to construct molecular kinetic degradation curves.

[0196] Table 1

[0197] As observed in the experiment, the pathogenic microorganisms did not grow, and the iron content remained essentially unchanged.

[0198] In vivo analysis Fe was tested on a specific production batch. 2+ Analysis of oxidized iron content and microbiological analysis (specifically targeting the Enterobacter family). Repeat analysis was performed on the same production batch after 12 and 24 months, maintaining the same standards.

[0199] Table 2

[0200] Molecular toxicity Molecular toxicity was assessed via GLP studies (acute oral toxicity of ischemic accident in rats, assessed by the Biogem Institute in AV, Italy). In vivo analyses were performed on laboratory rats.

[0201] LD50 (lethal dose 50): Represents the amount of a substance per unit body weight that would cause death in 50% of the experimental group, expressed in mg / kg. This study involved a case series with three subjects in each group, and the dosage of the compound was gradually increased in milligrams.

[0202] The results indicate that this molecule is safe even at high doses. Throughout the experiment, no changes in the normal health and behavior of the reference IDs were observed, either on the day of administration or in the following days.

[0203] Table 3

[0204] Rk = Right kidney; LK = Left kidney; 0 = No damage; 1 = Minimal; 2 = Mild; 3 = Moderate; 4 = Marked In summary, the aim of this study was to evaluate the potential toxic effects of ferric citrate monohydrate on target organs in laboratory mice.

[0205] No adverse effects on health or mortality were observed in any of the 12 rats examined. Furthermore, no visible injuries were recorded during necropsy.

[0206] Bioavailability and bioavailability evaluation of ferric citrate monohydrate molecules The mineralized material was analyzed based on the data obtained in Section 1.3. The presence of molecular iron was also confirmed, with a measured value of 20.30% ± 0.30%. This confirms the data represented by the TGA.

[0207] The digestion process involved gastric digestion using 50 mg of material, equivalent to 10 mg of iron. This digestion process was designed to mimic human digestion and was based on the use of fluids containing typical physiological secretions that simulate digestion. Three independent replicate samples were prepared, and each sample was analyzed by ICP-MS.

[0208] Bioavailability refers to the amount of active ingredient released from a formulation into the gastrointestinal tract and absorbed by the intestines. Digestion has no effect on the iron content of the formulation.

[0209] from Figure 6 As can be seen, gastric juice has no effect on molecules, which inertly pass through the stomach.

[0210] Prior to assessing the bioavailability of iron, the effect of the bioavailable portion of the formulation on intestinal mucosal cell viability was evaluated. Figure 7 ).

[0211] Caco-2 monolayers were exposed to increased amounts of the formulation for 3 hours. Dose-response analysis showed that the bioavailable portion of the formulation did not cause any decrease in intestinal epithelial cell viability.

[0212] refer to Figure 8 To assess iron uptake, intestinal epithelium was exposed to the bioavailable portion of the formulation for 1 and 3 hours. At the end of the culture, iron in the apical, lateral basal, and intracellular portions was quantified to assess the amount of iron absorbed by the intestinal epithelial cells.

[0213] The results showed that, in addition to the portion present within the cell, the basolateral portion absorbed approximately 7 μg of iron, totaling 95.62 μg / g cell. Iron absorption was also assessed by measuring ferritin levels. Ferritin was chosen as an indicator of intracellular iron content because it is involved in the storage of micronutrients, and its expression depends on intracellular iron levels.

[0214] For the molecules tested, a significant increase in ferritin expression was observed relative to the control (FD), such as Figure 9 As shown, the protein content reached a plateau of approximately 65.15 μg / g after only 1 hour of exposure to the intestinal epithelium.

[0215] Therefore, studies have shown that the molecule has an in vitro absorption rate of 83.78%, a bioavailability of 98.45%, and a positive effect on intestinal epithelial cells.

[0216] Administer to patients who consent Capsules prepared according to the above method were used for in vivo trials on consenting subjects. Administration involved taking 1-2 capsules daily on an empty stomach. No side effects were detected in all cases, and subjects experienced less fatigue and generalized good health on the first day of treatment. Blood tests before and after ferric citrate administration showed increases in bound blood parameters.

[0217] In vivo studies of parameters representing iron deficiency anemia status A study on the in vivo absorption of ferric citrate monohydrate involved 30 patients, of whom only 11 were enrolled because their hemoglobin levels were below 12 g / dL, and their parameter values ​​were below serum iron (70-170 mg / dL) and ferritin (50-150 g / dL). The study lasted 30 days, with a total administration of 315 mg of the active ingredient, equivalent to 10.5 mg daily in Fe... 2+ Iron in its oxidized state. Patients undergo blood testing at an approved clinical site. Three samples are taken from each patient.

[0218] Recruitment Sampling 15-day controlled sampling 30-day control sampling The purpose of this test was to verify an increasing trend in hemoglobin, serum iron, and ferritin levels in the blood. In addition, parameters such as MCH (mean corpuscular hemoglobin) and the total number and size of red blood cells were also considered. The patient signed a private document and was informed of how the study would be conducted.

[0219] Subjects were recruited based on specific parameter values, namely hemoglobin deficiency (12-16 μg / dL), serum iron (70-170 μg / dL), and ferritin (50-150 ng / dL) values ​​below the reference range (12-16 μg / dL).

[0220] The patients' average parameter values ​​indicated moderate to severe anemia.

[0221] Table 4

[0222] Following treatment, we reiterate the regimen of 10.5 mg Fe daily for 30 consecutive days. 2+ Iron oxide, analyzed again on day 15, equivalent to a total administration of 157 mg of active ingredient, with the following parameter values: Table 5

[0223] And at 30 days, after a total administration of 315 mg of the active ingredient. The results are given in the table below: Table 6 Hemoglobin, ferritin, and serum iron levels, 30 days

[0224] Hemoglobin parameter analysis The iron in hemoglobin binds with oxygen, transporting it throughout the body. Therefore, oxygen transport is the most important function of hemoglobin, and also the most important function of red blood cells.

[0225] The World Health Organization (WHO) defines anemia as a decrease in hemoglobin levels to below 12 g / dL.

[0226] The subsequent classification defines its severity and subdivides it into: Mild (Hb level between 12 g / dL and 10 g / dL) Moderate (Hb range of 10 g / dL to 8 g / dL), Severe (<8 g / dL) (2.3) Analysis of hemoglobin parameters in patients treated with ferrous citrate showed that hemoglobin increased by an average of 1.4 g / dL over 15 days and by an average of 2.8 g / dL over 30 days.

[0227] Starting with a mean hemoglobin level of 11.26 g / dL, hemoglobin increased to 12.78 g / dL after 15 days. These data indicate that treatment with only 158.5 mg of ferrous iron was sufficient to restore the passive (albeit mild) state of hemoglobin. See Table 7 below for patients A04, A05, A06, A08, A10, and A12.

[0228] Table 7

[0229] On the other hand, 30 days of treatment with 315 mg of ferrous iron was sufficient to fill the significant gap in hemoglobin deficiency. Patient A03 was an exception. Patient A03's initial hemoglobin level was 8.9 g / dL. The final hemoglobin level after treatment was 13.9 g / g / dL, which means that the severe hemoglobin deficiency had been overcome, even exceeding the minimum reference value of 8%.

[0230] But what is most surprising is the small number of molecules required to obtain these results. These data contradict the scientific literature, such as the study by Schrier et al. published in Blood 2015, which demonstrated that the absorption of 1500 mg to 3000 mg of iron is necessary to correct established iron deficiency anemia.

[0231] When undergoing iron therapy, the average absorption rate is about 10% of the administered iron dose. That is, of the 100 mg of iron ingested daily, only 10 mg is absorbed. It would take 300 mg to be absorbed in one month, 1000 mg in four months, or even 8-12 months to restore hemoglobin deficiency levels to the reference values ​​for moderate anemia. Therefore, the difference between the literature data and the data demonstrated in this experimental study suggests that a dose 10 times lower is sufficient to correct established iron deficiency anemia.

[0232] Given a baseline of 12 g / dL, we can confirm that by day 15, 54.54% of patients in the study were able to correct their established anemia. More interestingly, after 30 days of treatment, 100% of patients managed to return to baseline parameters.

[0233] Ferritin parameter analysis Ferritin is the main iron storage protein in cells. Therefore, its concentration in the blood reflects the degree of iron storage in the body.

[0234] In clinical practice, measuring plasma ferritin (ferritin concentration) helps assess the amount of available iron in the whole body.

[0235] Abnormal levels of ferritin in the blood can be an indicator of underlying diseases or specific conditions, such as defects that lead to anemia.

[0236] Ferritin is a globular protein complex that represents the main iron storage protein in cells. It accumulates excess iron in a soluble and non-toxic form and transports it to where it is needed.

[0237] Excessive iron is toxic to cells because it can produce active substances that can directly damage DNA and proteins.

[0238] Therefore, ferritin plays a role in cellular detoxification by limiting the formation of reactive oxygen species and mitigating their damaging effects on cell structure. Thus, it buffers excess iron by chelating it.

[0239] A small amount of ferritin is also secreted into the blood, where it acts as an iron transporter.

[0240] Table 8 below shows the ferritin values ​​at time 0, day 15, and day 30, in ng / dL.

[0241] Table 8

[0242] Interestingly, the results showed that ferritin quantifies the amount of iron stored and ready for use in the body, increasing it by an average of 42% across all patients.

[0243] These results are also consistent with the increase in hemoglobin, because from the pathological state onwards, iron tends to bind to the parts of the body that need it most, and then as stored iron deposits, it binds to ferritin.

[0244] Serum iron parameter analysis This test measures iron levels (serum iron), which is the concentration of unbound hemoglobin iron circulating in the blood (called "transport iron"). Based on the values ​​obtained, the doctor will assess whether these levels are within the normal range. A healthy adult has approximately 3-5 grams of total iron, of which: Some of it exists in red blood cells (where iron binds to hemoglobin). A portion is stored in the body as a reserve (iron binds with ferritin and hemosiderin). One portion represents "transfer iron" (iron bound to transferrin), which is transported from the liver and intestines through the bloodstream to the tissues that need it.

[0245] In summary, since the amount of free iron in the blood is negligible, serum iron can effectively measure the iron bound to transferrin.

[0246] Low iron levels can cause anemia, usually due to deficiency (reduced intake or increased demand in the diet) or malabsorption. In addition, low iron levels may indicate severe or prolonged bleeding, pregnancy, rapid growth (in children), infectious diseases, tumors, or acute myocardial infarction.

[0247] Table 9

[0248] When the values ​​at time 0 were compared with those at day 30, the iron level, representing the circulating bound iron in the blood, showed a significant increase. This represented a 38.30% increase compared to baseline, resulting in complete recovery of baseline status in 4 of the 11 patients and near-complete recovery in 3 patients. This indicates that 63.63% of the patients ingested sufficient iron to maintain the circulation of bound iron in their blood.

[0249] Total absorption of ferric citrate monohydrate molecules The absorption of ferric citrate follows this mechanism: iron is primarily absorbed in the duodenum, followed by absorption in the proximal portion of the small intestine. Intestinal cells directly absorb heme iron, while in its inorganic form, the mineral is absorbed only in its divalent form. During transport in the stomach, the acidity of the gastric environment promotes the absorption of ferric iron. 3+ Reduced to Fe 2+Iron is released along with pepsin, releasing protein-bound iron. Consequently, iron absorption is significantly reduced when gastric juice synthesis and secretion are altered (e.g., through the use of proton pump inhibitor drugs). However, at the brush border level of intestinal cells, a protein called duodenal cytochrome b (Dcytb) can reduce ferric iron to ferrous iron, allowing for its absorption. In its ferrous state, the mineral is transported across the apical membrane of intestinal epithelial cells via the ferrous transporter protein (DMT1). In our hypothetical classification of bioavailability, we prioritize heme iron, followed by the inorganic form of Fe. 2+ Further on, there is the non-heme form of Fe. 3+ Some substances in the diet, such as citric acid, help retain iron in the Fe... 2+ In contrast, dietary fiber and some plant-derived anti-nutritional substances (tannins) have the opposite effect. To empirically calculate the absorption of ferric citrate, we tested the iron levels of subjects before and after treatment with predetermined doses of the compound. Taking into account physiological losses, the difference in levels before and after intake resulted in a significant increase in the percentage of ferric citrate monohydrate absorbed, as well as in hemoglobin, serum iron, and ferritin, as described in the Brittenham GM et al. Hematology 2013 study protocol.

[0250] Table 10

[0251] "PAZIENTE" = Patient "FERRO ASSUNTO" = Iron Taken "DELTA EMOGLOBINA" = Increased Hemoglobin (DELTA HEMOGLOBIN) “DELTA SIDEREMIA” = Serum Iron Increase “DELTA FERRITINA” = Ferritin Increment "Ferro assorbito" = Iron absorbed. "% ASSORBITA" = Absorbed % “MEDIA” = Average (MEAN) The ongoing experiments have shown an incredible 91.9% iron absorption rate, an impressive figure considering that the average iron absorption rate through other types of molecules is 15-20%, as reported in the second edition of the book entitled "Nutritional Anemia," published in December 2022.

[0252] Studies evaluating side effects During the study described in Section 1.8, side effects were assessed by completing a product evaluation questionnaire. The product evaluation questionnaire was completed at the medical center during the recruitment period and the final phase.

[0253] Statistical analysis was performed using a modified intention-to-treat analysis. All randomly assigned participants received one dose of the study drug and participated in at least one post-baseline visit before being included in the analysis. A two-tailed p-value less than 0.05 was predefined as statistically significant. Side effects were assessed during the study by completing a product evaluation questionnaire. The product evaluation questionnaire was completed at the same medical center during recruitment and the final phase. The questionnaire assessed pre- and post-treatment physiological status and included six specific questions and one comparative approval question regarding previously used supplements.

[0254] Of the 11 patients selected in the study, 8 questionnaires were considered because they met statistical requirements.

[0255] Pre-intake health conditions The overall score is 1.25, with a score range of 0-10, where 0 is the lowest and 10 is the highest.

[0256] Health problems after ingestion Within the range of 0-10, the overall rating is 7.13 punN, where 0 is the minimum and 10 is the maximum, representing an increase of 470.4% compared to the initial conditions.

[0257] Self-assessment questions before symptom treatment The overall assessment score was 5.23 points, ranging from 0 to 10, where 0 is the minimum score for typical anemia symptoms and 10 is the maximum score for typical anemia symptoms.

[0258] Self-assessment questions after symptom treatment Within the range of 0-10, the overall assessment score was 1.88, where 0 is the minimum for typical anemia symptoms and 10 is the maximum for typical anemia symptoms. Typical symptoms of anemia-related diseases were reduced by 64%.

[0259] Question: Point out the side effects of currently used iron medications / supplements.

[0260] The overall assessment score was 4.99 on a scale of 0-10, where 0 represents the minimum number of symptoms caused by iron medication / supplementation and 10 represents the maximum number of symptoms caused by iron medication / supplementation.

[0261] Question: Point out the side effects of using the active ingredient ferric citrate monohydrate. The overall assessment score was 0.32 on a scale of 0-10, where 0 represents the minimum number of symptoms caused by iron medications / supplements and 10 represents the maximum number of symptoms caused by iron medications / supplements. Less than 1% of patients experienced side effects after administration of the active ingredient.

[0262] Given that the unabsorbed iron from the active ingredient ferric citrate monohydrate accounts for 10% of the total intake of 315 mg, or 31.5 mg, the assessment of side effects exceeded expectations.

[0263] The patients used a ferrous sulfate supplement for one month prior to the study, with each treatment dose being 9.6 grams (treatment lasting 4 months). Since the literature data on ferrous sulfate indicates an 85% unabsorbed amount, the unabsorbed amount is equivalent to 8.16 grams of ferrous sulfate. From this amount, it is almost obvious that those using ferrous sulfate supplements experienced both common and uncommon side effects.

[0264] Given its high bioavailability and 64-fold lower unabsorbed molecular weight in the same treatment, ferric citrate is strictly logical within the concept of a supplement with low / no side effects for patients.

[0265] In fact, when asked, "How satisfied are you with this product compared to the supplements you've used in the past?" the average response was 8.35 out of 10, with 10 being the highest level of satisfaction with the product's ferric citrate monohydrate.

[0266] Statistical analysis Statistical analyses were performed using a modified intention-to-treat analysis. All randomly assigned participants who received one dose of the study drug and attended at least one post-baseline visit were included in the analysis. We used mixed-effects linear models to compare primary and secondary efficacy endpoints between randomization groups, using complexation symmetry to interpret repeated measures for each participant. In these models, the terms randomization, time, and time-grouping were analyzed as fixed effects, and participants were analyzed as random effects. The primary outcome was the difference in the mean change from baseline for each outcome variable at the end of the treatment period (30 days). We also examined the differences in the rate of change of each analyte at all time points by testing the statistical significance of the group-time interaction terms in the models. In the latter model, when significant time effects were present, we located significant changes at post-baseline time points separately by comparing them to baseline values ​​in the mixed linear models.

[0267] Fisher's exact test was used to compare the proportion of participants who experienced adverse events at any time after the baseline visit between randomized groups. A two-tailed p-value less than 0.05 was predefined as statistically significant.

[0268] Based on the information disclosed so far, the importance of this molecular complex with cyclic iron(II) citrate monohydrate coordination bonds becomes apparent. This results in the molecule having an absorption mechanism different from that of conventional iron molecules, which allows it to achieve 91.9% water solubility, be non-toxic, have few or no side effects, and be used to treat and prevent iron deficiency anemia, as well as all diseases and conditions commonly associated with iron deficiency.

Claims

1. A ferric citrate (II), particularly a ferric citrate (II) monohydrate determined by the chemical formula FeC6H6O7·H2O (ferrous citrate monohydrate or ferric citrate (II) monohydrate), characterized in that, Its pH is 2.5, and its molecular structure is not linear but cyclic, in which citric acid and elemental iron are linked by coordinate bonds in a molecular complex. •H2O Iron aggregates in the citrate salt, which moves into a cyclic form, and iron is coordinated with the O groups of carboxyl groups 1 and 3 via coordinate bonds. - The groups combine to form two enantiomers, D and L (R and S), both of which are water-soluble: By providing the molecule itself with an enhanced absorption mechanism in the human body that differs from that of traditional iron molecules.

2. A method for preparing iron(II) crystals, wherein the iron(II) crystals have the structural formula as described in claim 1, characterized in that, The iron (II) crystals are obtained by directly reacting zero-valent iron and pure pharmaceutical-grade citric acid in a stoichiometric ratio of 1:1 in the presence of water. The zero-valent iron is preferably in the form of fine iron filings or powder. The direct reaction between the zero-valent iron and citric acid is carried out at a temperature from 40°C (1 atm) to the boiling point of the reaction mixture, preferably below 95°C, and even more preferably below 90°C, in order to accelerate the oxidation and complexation process of the iron powder and citric acid, thereby producing a well-bonded molecular structure over time.

3. The method according to the preceding claims, characterized in that, The method further includes the following steps: The obtained ferric citrate(II) was separated from the reaction mixture by filtration, preferably by a vibrating sieve, to obtain a pearly white compound; The compound was freeze-dried to remove almost all of the water (remaining water weight was 1.87%), and Then, by grinding the freeze-dried compound, pure molecules of iron citrate monohydrate in the form of dried crystalline powder are obtained, which can be used in the target drug formulation.

4. The preparation method according to claim 3, characterized in that, The reaction treats waste, namely, waste rich in Fe. 2+ The aqueous solvent is reused as a reaction catalyst for subsequent production without the need for starting materials, thereby reducing reaction waste to below 0.05%.

5. The preparation method according to claim 3, characterized in that, For each mole of the substance obtained, the iron content is equal to 21.0% (w / w).

6. The ferric citrate(II) monohydrate according to claim 1 or 2, in the form of a dry crystalline powder, as an additive that is directly soluble in water or aqueous solution, for the treatment and prevention of iron deficiency anemia and all iron deficiency-related diseases and conditions.

7. A pharmaceutical composition and / or nutritional composition and / or food composition comprising ferric citrate (II) monohydrate according to claim 1 or 2, and optionally food or pharmaceutically acceptable grade additives, excipients and carriers, for therapeutic and food industry (as food or food supplement), particularly for the treatment and prevention of iron deficiency anemia, and all iron deficiency-related diseases and conditions.

8. The composition according to claim 7, for oral or parenteral administration, characterized in that, It contains 1 mg to 500 mg, preferably 5 mg to 200 mg, such as 10 mg to 100 mg of ferric (II) citrate monohydrate.

9. The composition according to claim 7, characterized in that, Patients absorbed an average of 91.9% of the iron released from the same formulation, which is at least four times higher than the average of 15-20% absorbed by iron through other types of iron molecules.

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