Methods for determining the amount of encapsulated and free iron in a sample

Abstract

Methods of increasing systemic iron availability in a human subject using plant-derived ferritin-bound iron are provided. In certain embodiments, ferritin-bound iron is orally administered in an amount effective to produce sustained systemic iron exposure characterized by measurable serum iron levels at least 24 hours after administration. In some embodiments, administration produces a delayed peak serum iron concentration and provides controlled systemic iron delivery relative to conventional iron supplements. In certain embodiments, administration of ferritin-bound iron results in a reduced hepcidin response relative to ferrous sulfate, thereby facilitating improved iron utilization. The ferritin-bound iron may be absorbed through receptor-mediated uptake mechanisms that provide controlled intracellular iron release and sustained systemic availability. The methods are useful for increasing systemic iron availability and for treating or preventing iron deficiency and iron deficiency anemia in human subjects.

Description

Attorney Docket No. 61130-3PCTMETHODS FOR DETERMINING THE AMOUNT OF ENCAPSULATED AND FREE IRON IN A SAMPLEInventor: Vincent P. HackelCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of United States Provisional Patent Application No. 63 / 619,566, filed on February 29, 2024, a continuation-in-part of United States Patent Application No. 19 / 010,844, filed on January 6, 2025, and a continuation-in-part of United States Patent Application No. 19 / 429,443, filed on December 22, 2025, the contents of which are incorporated herein by reference in their entirety.BACKGROUND

[0002] Iron is an essential micronutrient required for numerous cellular and physiological processes in humans and other animals, including oxygen transport, mitochondrial respiration, intracellular electron transport, DNA synthesis, and enzymatic reactions. Iron deficiency is the most common nutritional deficiency worldwide, affecting tens of millions of individuals in both developed and developing countries. Iron deficiency may result in iron deficiency anemia, reduced cognitive function, impaired physical performance, diminished immune function, and developmental impairments in children.

[0003] In biological systems, iron is stored primarily in ferritin, an intracellular protein complex that safely stores iron in a bioavailable form. Ferritin comprises self-assembling protein subunits that form a spherical nanocage capable of encapsulating iron in a biomineral form. This structure allows ferritin to store iron at high concentrations while preventing toxic effects associated with free iron. Ferritin proteins are widely distributed in animals, plants, and microorganisms. Plant-derived ferritin, also referred to as phytoferritin, contains iron within a protein shell that protects the iron core and facilitates controlled iron storage and release.

[0004] Ferritin is present in various tissues in animals, including liver, spleen, bone marrow, and muscle, where it functions as a reservoir of iron for physiological use. A small fraction of ferritin is present in serum and serves as a clinical indicator of systemic iron status. Ferritin structure and function allow iron to be stored in a stable, nontoxic, and bioavailable form that can be mobilized when needed.

[0005] Iron supplementation is commonly used to treat or prevent iron deficiency.Conventional iron supplements typically comprise inorganic iron salts, such as ferrousAttorney Docket No. 61130-3PCTsulfate, ferrous gluconate, or ferrous fumarate. These conventional iron salts are generally absorbed through divalent metal transporter-mediated pathways and may produce rapid increases in serum iron concentration following oral administration. Such rapid increases in circulating iron may trigger physiological regulatory mechanisms, including induction of hepcidin, a hormone that inhibits iron absorption and reduces iron bioavailability.

[0006] In contrast, ferritin-bound iron represents a structurally distinct form of iron delivery in which iron is encapsulated within a protein nanocage. Plant-derived ferritin-bound iron may be administered orally as a nutritional supplement. However, the pharmacokinetic and physiological behavior of ferritin-bound iron in human subjects has not been fully characterized, and improved methods of delivering iron that provide sustained systemic availability and improved physiological regulation remain desirable.

[0007] Recent studies have demonstrated that oral administration of plant-derived ferritin-bound iron to human subjects produces sustained systemic iron availability characterized by prolonged elevation of serum iron levels following administration. In particular, serum iron levels remain elevated relative to baseline for extended periods following administration, and peak serum iron concentrations may occur several hours after administration. This delayed and sustained systemic exposure profile differs from the rapid absorption and transient systemic exposure typically associated with conventional iron salts.

[0008] In addition, administration of plant-derived ferritin-bound iron has been observed to produce physiological responses consistent with regulated iron uptake and controlled systemic delivery. Such controlled delivery may provide improved compatibility with physiological iron regulation mechanisms and may facilitate effective treatment or prevention of iron deficiency and related conditions.

[0009] Accordingly, improved methods of increasing systemic iron availability and treating iron deficiency using plant-derived ferritin-bound iron are desirable.SUMMARY

[0010] The present invention relates to methods of increasing systemic iron availability in a human subject using plant-derived ferritin-bound iron. More particularly, the invention relates to oral administration of ferritin-bound iron compositions that provide sustained systemic iron exposure, controlled delivery, and improved physiological regulation relative to conventional iron supplements.

[0011] In one aspect, the invention provides a method of increasing systemic iron availability in a human subject by orally administering a composition comprising plant-Attorney Docket No. 61130-3PCTderived ferritin-bound iron in an amount effective to produce sustained systemic iron exposure. In some embodiments, the sustained systemic iron exposure is characterized by measurable serum iron levels at least 24 hours after administration. This sustained exposure profile differs from conventional iron salts, which typically produce rapid increases in serum iron followed by decline.

[0012] In another aspect, the invention provides methods of treating iron deficiency and iron deficiency anemia through oral administration of plant-derived ferritin-bound iron. In some embodiments, administration produces a delayed peak serum iron concentration relative to conventional iron salts. This delayed peak concentration is associated with controlled absorption and prolonged systemic iron availability.

[0013] In a further aspect, the invention provides methods of delivering iron to a human subject while producing a reduced hepcidin response relative to conventional iron supplements, including ferrous sulfate. Reduced hepcidin induction facilitates improved iron utilization and reduces inhibition of iron absorption.

[0014] In certain embodiments, the ferritin-bound iron provides controlled systemic iron delivery characterized by sustained serum iron levels, increased systemic iron exposure, and improved physiological compatibility. In some embodiments, iron absorption is regulated based on the physiological iron status of the subject, resulting in increased uptake in iron-deficient subjects relative to iron-replete subjects.

[0015] In some embodiments, the ferritin-bound iron is absorbed through receptor-mediated uptake mechanisms that enable controlled intracellular iron release and sustained systemic availability. In certain embodiments, the ferritin-bound iron comprises an iron biomineral encapsulated within a protein shell, which facilitates controlled delivery and reduces transient increases in circulating free iron.

[0016] In some embodiments, administration of plant-derived ferritin-bound iron increases serum ferritin concentration, increases hemoglobin concentration, improves iron stores, or combinations thereof.

[0017] In some embodiments, the ferritin-bound iron is administered orally in a capsule, tablet, powder, or liquid formulation. In certain embodiments, the ferritin-bound iron is administered once daily in an amount effective to increase systemic iron availability.

[0018] These and other aspects of the invention provide improved methods of delivering iron to human subjects, particularly for treatment and prevention of iron deficiency and related conditions, while providing sustained systemic iron exposure and improved physiological regulation.Attorney Docket No. 61130-3PCTDRAWINGS

[0019] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, appended claims, and accompanying drawings where:

[0020] Figure 1 depicts a standard curve graph of horse anti- ferritin antibody (13 exposure and 12 exposure lines) and the soy anti-ferritin antibody (2 exposure and 3 exposure lines) using identical samples of soy ferritin;

[0021] Figure 2 depicts the standard curve from the data from the capillary electrophoresis from Table 4;

[0022] Figure 3 depicts the standard curve obtained from the WES system showing generation of the horse ferritin standard curve;

[0023] Figure 4 depicts a graph from the peak molecular weights for the standard curve which correspond to monomer, dimer and trimers of denatured ferritin subunits;

[0024] Figure 5 depicts WES system graph from the first extraction;

[0025] Figure 6 depicts WES system graph from the second extraction;

[0026] Figure 7 depicts the WES system data from the first and the second water extraction;

[0027] Figure 8 depicts the study schedule and menstrual cycle of the subjects;

[0028] Figure 9 depicts a flow diagram of the progress from screening to analysis;

[0029] Figure 10 depicts a graph of the changes in the serum ferritin levels for each subject before and after intake of trial supplements; and

[0030] Figure 11 depicts the graphs showing five and nine weeks of intake.DETAILED DESCRIPTION

[0031] As used herein, the following terms and variations thereof have the meanings given below, unless a different meaning is clearly intended by the context in which such term is used.

[0032] The terms “a,” “an,” and “the” and similar referents used herein are to be construed to cover both the singular and the plural unless their usage in context indicates otherwise.

[0033] The term “buffer” or “buffered solution” refers to a mixture of acid and base which, when present in a solution, reduces or modulates changes in pH that would otherwise occur in the solution when an acid or base is added.

[0034] As used herein, the term “comprise” and variations of the term, such asAttorney Docket No. 61130-3PCT“comprising” and “comprises,” are not intended to exclude other additives, components, integers or steps.

[0035] The term “ferritin” refers to the protein with caged iron oxide mineral that confers highly efficient intestinal iron absorption in living organisms and has biological and / or chemical activity and structure the same as, or substantially similar to, a natural, iron-containing ferritin. As such, ferritin includes a naturally occurring ferritin protein with iron mineral or a recombinant, iron mineral-reconstituted ferritin protein, comprising 12 or 24 ferritin subunits, wherein the subunits associate to form a spherical nanocage. Natural ferritins include animal ferritin including human ferritin, phytoferritin (plant ferritin) (derived from soybeans, and any other legume or legume process stream for example), microbial ferritins: mycoferritin (derived from fungi), or bacterial ferritin (bacterioferritin) or archaeal ferritin. Ferritin protein includes recombinant ferritin expressed by genetically-transformed microorganisms such as Escherichia coli, and other bacteria and yeasts. Ferritin expressed by genetically-transformed or recombinant microorganisms can have an amino acid sequence identical or analogous to natural ferritin. The term ferritin protein can include protein cages consisting of one or both animal isoforms, H and L or plant isoforms.

[0036] A “ferritin protein subunit” is defined as one of the 12 or 24 polypeptide subunits that make up a ferritin protein. The numbering system used herein for the identification of amino acids within ferritin subunits is based on the original sequence of horse spleen L ferritin (Swiss Protein Database Accession Number P02791). The horse spleen numbering system can be easily converted to a numbering system based on the human H sequence (Swiss Protein Database accession number P02794; the human L sequence accession number is P02792), which has four additional amino acids at the N-terminus. The human H sequence numbering therefore adds 4 to the corresponding amino acid number in horse spleen ferritin. For example, L134 by horse spleen numbering corresponds to L138 by human H sequence numbering.

[0037] “Apoferritin” refers to the iron-free form of the protein, i.e., the protein in the unmineralized state.

[0038] A “ferritin pore” is one of the external or internal ferritin cage pores that lead to the eight Fe2+exit / entry ion channels in an assembled ferritin protein cage; the channels and pores are formed by trimers of ferritin subunits. In an intact, 24 subunit ferritin protein cage, there are eight three-fold axes of symmetry, each at a junction of three ferritin subunits. Each ferritin pore and ion channel is formed by these three-way junctions of ferritin subunits. The pores can be visualized in crystals of ferritin proteins by X-ray crystallography and analyzedAttorney Docket No. 61130-3PCTin solutions by changes in the rate of Fe2+exit.

[0039] An “immunoblot” or “immunodetection” is a specific type of biochemical test that measures the presence or concentration of a protein (referred to as the “analyte”) in solutions that frequently contain a complex mixture of substances including other proteins. The methods and techniques involved in immunoassays are well known by those in the art.

[0040] “Isolation” or “isolation of ferritin” as used herein means separation of ferritin from other components in the plant or animal material, which provides a substantially pure target compound, such as a substantially pure ferritin.

[0041] Substantially pure ferritin contains ferritin in an amount of from about 5% to about 100%, from about 15% to about 80%, from about 50% to about 85%, from about 65% to about 95% by weight of the total protein in the material processed by the method of the invention.

[0042] The terms “individual,” “subject” and “patient” are used interchangeably herein, and generally refer to a mammal. The term “mammal” is defined as an individual belonging to the class Mammalia and includes, without limitation, humans, experimental animals such as, for example, rodents, domestic and farm animals, zoo, sports, and pet animals, such as for example, cows, sheep, dogs, horses, cats and cows.

[0043] A “legume” can be one or more soybeans, yellow peas, green peas, lentils, chickpeas (also called garbanzos), peanuts, trefoil, pinto beans, Great Northern beans, navy beans, red beans, black beans, dark or light red kidney beans, fava beans, baby lima beans, pink beans, mayocoba beans, small red beans, black-eyed peas (also called cow peas), cranberry beans, white beans, rice beans, butter beans, and combinations of any of the foregoing. The legume can be any of a variety of species, including, e.g., a Phaseolus species (e.g., Phaseolus vulgaris), a Pisum species (e.g., Pisum sativum), a Lens species (e.g., Lens vulgaris, Lens culinaris), a Cicera species (e.g., Cicera arietenum), a Vigna species (e.g., Vigna unguiculata), a Glycine species (e.g., Glycine max), and combinations of any thereof.

[0044] The term “nutraceutical formulation” refers to a food or part of a food that offers medical and / or health benefits including prevention or treatment of disease. Nutraceutical products range from isolated nutrients, dietary supplements, genetically engineered designer foods, functional foods, herbal products and processed foods such as cereal, soup and beverages. The term “functional foods,” refers to foods that include “any modified food or food ingredients that may provide a health benefit beyond the traditional nutrients it contains.” Nutraceutical formulations of interest include foods for veterinary or human use, including food bars (e.g. cereal bars, breakfast bars, energy bars, nutritional bars); chewingAttorney Docket No. 61130-3PCTgums; drinks; fortified drinks; drink supplements (e.g., powders to be added to a drink); tablets; lozenges; candies; and the like.

[0045] The term “reconstituted,” “mineralized” or “remineralized” refers to the addition of iron atoms to ferritin protein.

[0046] The term “solution” refers to a composition comprising a solvent and a solute, and includes true solutions and suspensions. Examples of solutions include a solid, liquid or gas dissolved in a liquid and particulates or micelles suspended in a liquid.

[0047] A “supplement” or “dietary supplement” as used herein is useful for supplementing, replenishing, and increasing the iron supply to humans, animals and plants, and for treating various disorders and conditions. A dietary supplement can be formulated for oral administration. As contemplated in the present invention, a dietary supplement includes ferritin in an amount of from about 10% to about 90% by weight of the total protein in the supplement. For example, subject dietary supplement includes ferritin in an amount of from about 0.1% - 9%, 10% to about 15%, from about 15% to about 20%, from about 20% to about 25%, from about 25% to about 30%, from about 30% to about 35%, from about 35% to about 40%, from about 40% to about 45%, from about 45% to about 50%, or from about 55% to about 90%, and from about 90% to about 100% by weight of the total protein in the supplement. For oral preparations, a subject dietary supplement can be formulated with appropriate additives to make tablets, powders, granules or capsules, gummies, liquids, fortified foods, snacks, bread, yogurt, ice cream, rice, etc., for example, with conventional additives, such as lactose, mannitol, corn starch or potato starch; with binders, such as crystalline cellulose, cellulose derivatives, acacia, corn starch or gelatins; with disintegrators, such as corn starch, potato starch or sodium carboxymethylcellulose; with lubricants, such as talc or magnesium stearate; and if desired, with diluents, buffering agents, moistening agents, preservatives and flavoring agents. A dietary supplement can be administered in one, or more than one doses per day. A dietary supplement can be administered at various frequencies, e.g., four times daily, three times daily, twice daily, once daily, every other day, three times per week, twice per week, or once per week.

[0048] A “therapeutic composition” as used herein means a substance that is intended to have a therapeutic effect such as, for example, a pharmaceutical composition, a nutraceutical, a dietary supplement, and other substances. A therapeutic composition may be configured to contain a pharmaceutically acceptable carrier. The therapeutic composition may contain pharmaceutically acceptable excipients, such as vehicles, adjuvants, carriers or diluents, as well as pharmaceutically acceptable auxiliary substances, such as pH adjusting and bufferingAttorney Docket No. 61130-3PCTagents, tonicity adjusting agents, stabilizers, and wetting agents.

[0049] As used herein, the phrases “therapeutically effective amount” and “prophylactically effective amount” refer to an amount that provides a therapeutic benefit in the treatment, prevention, or management of a disease or an overt symptom of the disease. The therapeutically effective amount may treat a disease or condition, a symptom of disease, or a predisposition toward a disease, with the purpose to cure, heal, alleviate, relieve, alter, remedy, ameliorate, improve, or affect the disease, the symptoms of disease, or the predisposition toward disease. The specific amount that is therapeutically effective can be readily determined by ordinary medical practitioner, and may vary depending on factors known in the art, such as, e.g. the type of disease, the patient's history and age, the stage of disease, and the administration of other therapeutic agents.

[0050] The terms “treatment” or “treating” as used herein covers any treatment of a condition treatable by iron in a living organism, for example, a human, and includes:(i) preventing the condition from occurring in a subject which may be predisposed to the condition but has not yet been diagnosed as having it;(ii) inhibiting the condition, e.g., arresting or slowing its development; or (iii) relieving the condition, e.g., causing regression of the condition.

[0051] Ferritin is a form of dietary iron that contains a protein cage with iron oxide mineral inside the protein cage. In contrast with other iron supplements and dietary iron sources, ferritin’ s protein coat protects a user’ s intestine from free radical chemistry caused by iron and iron salts, which can irritate the intestine. In addition, ferritin iron is released slowly into the blood from the intestine, which also allows for protection of the intestine from free radical chemistry caused by iron.

[0052] Ferritin iron is absorbed by the intestine using a mechanism that is different from the mechanism of absorption of other iron sources such as non-heme iron supplements or heme iron from meat. Ferritin iron is absorbed in the intestine through a protein uptake pathway, rather than an ion pathway used by other iron supplements. Humans have been consuming ferritin iron for millennia in forms such as ferritin-rich legumes, which have been cultivated for 12,000 years. Contemporary cultures include legumes in their traditional and modern diets. In addition, phytoferritin can be eaten by vegans, which is important since many vegan diets are iron deficient and need to be supplemented with iron.

[0053] Phytoferritin contains an average of 1000 iron atoms per protein cage, and animal ferritin contains an average of 1500-2000 iron atoms per protein cage, which allows for more efficient absorption of ferritin iron by the body. In other words, for one transport event in theAttorney Docket No. 61130-3PCTintestine, the user’s body would obtain 1000 times as much iron as it would from non-heme iron salts and chelators.

[0054] Ferritin iron is well absorbed by animals. In a rat model, ferritin has been shown to cure iron deficiency. In humans, ferritin iron is absorbed on the order of 20-30%.

[0055] Plant material can be used as a starting material to isolate phytoferritin in a substantially pure form. A typical starting plant material is a legume such as a soybean. In addition to soybeans, yellow peas, green peas, lentils, chickpeas, peanuts, trefoil, pinto beans, Great Northern beans, navy beans, red beans, black beans, dark or light red kidney beans, fava beans, green baby lima beans, pink beans, mayocoba beans, small red beans, black-eyed peas, cranberry beans, white beans, rice beans, butter beans, or a combination thereof can be used as starting plant material.

[0056] The plant material used to isolate phytoferritin can include the whole plant, or any ferritin-rich portion of a plant, e.g., seed, stem, fruit, leaf, root (e.g., nodulating root), flower, stem, etc. In some cases, the source of the phytoferritin is one or more of a seed, a nodulating root, and a leaf. Where the source of the phytoferritin is a seed or a bean, the phytoferritin can be obtained from the whole seed or bean, or a part of a seed or bean, e.g., the hull.

[0057] The starting plant material can also be a processing stream or a waste stream resulting from the processing of soy or other beans. For example, the source of the isolated ferritin can be a waste stream from the production of tofu or soy milk from soybeans.Processing soy for soy milk produces an insoluble by-product of soy, called okara. Either wet or dried okara or other material from legume waste process streams can be used as starting plant material.

[0058] The plant material from the waste stream and / or the legumes themselves are treated to isolate the ferritin, followed by concentration of the ferritin. The concentrated ferritin can then be used to treat humans and other animals in need thereof, such as, for example, treatment of an iron deficiency.

[0059] Ferritin can be isolated from plant material using methods as those described in US Pat. S / N 8,476,061. To isolate ferritin from plant material, the plant material is separated into soluble and insoluble fractions. A neutral saline buffer is then added to the insoluble fraction to make an insoluble solution, which is clarified into soluble and insoluble solution fractions. The soluble solution is treated enzymatically with one or more glycosidase enzymes. The clarified soluble solution is fractionated to remove non-ferritin components and the isolated ferritin is concentrated.Attorney Docket No. 61130-3PCT

[0060] Alternatively, the ferritin in the clarified soluble solution can be further purified prior to concentration. One such purification method includes the steps of 1) centrifugation of the clarified soluble solution, followed by 2) tangential flow or crossflow filtration of the clarified material across a membrane, and optional 3) concentration of the clarified, filtered material such as by the spray dry method. The resulting purified ferritin, either the clarified material after step 2, or the powder after step 3, is more water soluble than ferritin from the clarified soluble solution.

[0061] Another method to isolate ferritin from plant material includes the steps of: (a) separation of the plant material into soluble and insoluble fractions; (b) enzymatic removal of non-ferritin components from the soluble fraction with one or more glycosidase enzymes, and (c) concentration of the isolated ferritin from step (b).

[0062] Another method to isolate ferritin from plant material is the addition of a neutral saline buffer to plant material followed by enzymatic treatment with one or more glycosidase enzymes. The enzymatically treated plant material substrate is then separated and clarified into soluble and insoluble fractions, which are then fractionated to remove non-ferritin components. The isolated ferritin from the fractionated soluble solution is then concentrated or further purified as described above.

[0063] In animals, ferritin is present in high amounts in the liver, kidney, spleen, and bone marrow. Ferritin may be derived from the tissues of animals. Iron-containing ferritin derived from animals can be also used by humans and other animals that need increased iron in their diet. Ferritin can be isolated from animal material as described in US Pat. S / N 8,476,061.

[0064] In nature, phyto ferritin contains a range of 1000-2700 iron atoms per ferritin protein cage. Ferritin proteins, including phytoferritin proteins, are composed of twenty- four identical or homologous subunits that assemble into a large spherical cage with a hollow cavity. The cage can accommodate up to about 4500 iron atoms.

[0065] The protein-caged iron mineral present in ferritin can be expanded in a ferritin protein containing less than the amount of iron possible in each of its protein cages or reconstituted in apoferritin. The starting ferritin protein (apoferritin or ferritin protein containing less than the maximum amount of iron) can be obtained by any means, including the ferritin protein isolation methods described above.

[0066] Using the calculation of the amount of ferritin iron contained in the starting solid material, a mineralization procedure can be done to incorporate additional iron atoms in each ferritin protein. The target of iron atom incorporation depends on the amount of iron atomsAttorney Docket No. 61130-3PCTcontained in the starting solid material. A target mineralization of between 2000-4500 iron atoms per ferritin cage is desirable.

[0067] The mineralization procedure is carried out as follows. First, iron atoms in the form of ferrous sulfate, ferrous fumarate, ferrous gluconate, ferrous bisglycinate, or any other iron chelate or iron salt, or naturally occurring spring water that is high in iron, is added to partially or fully purified or isolated ferritin protein, and the iron atoms are allowed to incorporate inside the cages of the ferritin protein.

[0068] After mineralization, the mineralized ferritin protein can be used in the various applications described above. For example, mineralized ferritin protein can be added into products for human or animal nutrition, such as food, beverages, nutraceuticals and supplements. Additionally, mineralized ferritin protein can be added to a heat-processed substance to increase the iron content of the heat-processed substance. A heat-processed substance can be food or a beverage. The isolated ferritin can be added to the heat-processed substance either before or after the substance is heat-processed. The mineralized ferritin can also be used in the methods of treatment or prevention of iron deficiency as described above.

[0069] Rough determination of the ferritin protein and iron concentration in plant extracts has been shown previously using quantitative Western blotting with a rabbit polyclonal anti- soybean ferritin antiserum, and / or pea ferritin antiserum, and / or horse ferritin antiserum. However, it is desirable to obtain an accurate quantitation of ferritin. The present invention provides methods to obtain an accurate quantitation of free iron, ferritin-bound iron and quantitation of ferritin protein in a sample.

[0070] Isolated plant or animal ferritin is particularly useful because it can be added into products for human or animal nutrition, such as food, beverages, nutraceuticals and supplements. A therapeutic or supplementary composition containing isolated plant or animal ferritin can be delivered to an organism in need of iron. The organism can be an animal such as a mammal, including a human. Alternatively, the organism can be a plant.

[0071] The isolated ferritin agents and therapeutic compositions can be administered by continuous delivery, intermittent delivery, or through a combination of continuous and intermittent delivery. Many factors can influence the dosage and timing required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and / or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of a composition can include a single treatment or a series of treatments. In addition to their administration individually or as a plurality, the therapeutic compositions of the invention can beAttorney Docket No. 61130-3PCTadministered in combination with other known agents effective in treatment of diseases. In any event, the administering physician can adjust the amount and timing of administration of the therapeutic composition on the basis of results observed using standard measures of efficacy known in the art or described herein.

[0072] Ferritin-bound iron derived from plant sources can be administered orally to a subject in need thereof and produces sustained systemic iron availability characterized by prolonged elevation of serum iron levels relative to baseline. Measurable increases in serum iron concentration are maintained for at least about 12 hours, at least about 18 hours, or at least about 24 hours following administration. Such sustained systemic availability is consistent with controlled physiological uptake and regulated systemic delivery of iron.

[0073] Oral administration of plant-derived ferritin-bound iron produces a delayed peak serum iron concentration relative to conventional iron salts. Peak serum iron concentration occurs at least about 2 hours, at least about 3 hours, at least about 4 hours, or between about 3 hours and about 12 hours following administration. This delayed peak is consistent with uptake of ferritin-bound iron through protein-mediated cellular internalization mechanisms rather than rapid ionic absorption pathways.

[0074] Furthermore, administration of ferritin-bound iron produces sustained systemic iron exposure characterized by prolonged elevation of serum iron concentration and increased area under the concentration-time curve relative to conventional iron salts. Sustained systemic iron availability occurs without producing a rapid transient spike in serum iron concentration.

[0075] Administration of ferritin-bound iron results in a reduced hepcidin response relative to conventional iron supplements such as ferrous sulfate. Reduced hepcidin induction facilitates continued iron absorption and improved systemic iron utilization.EXAMPLE 1 - Determination of free iron versus ferritin-bound iron in a sample

[0076] Described herein is a method for quantitatively determining the concentration of free iron, which is iron that has not been encapsulated into a ferritin cage, versus ferritinbound iron. The methods of the present invention contemplate an accurate quantitation of iron derived from plant and animal ferritin, either natural or from recombinant apoferritin reconstituted with iron mineral. With this process, free iron passes through a crossflow filter and mass balance calculations are done to determine the amount of free iron. Using cross flow filter technology in this manner any small non-bound iron atoms pass through the filter and can be quantified.Attorney Docket No. 61130-3PCT

[0077] To determine the free iron present in isolated ferritin, two liters of ferritin isolated by any means were first diluted with eight liters of water, forming a slurry. The slurry was mixed, covered, for one hour in a sample tank.

[0078] Next, a crossflow system was briefly turned on (i.e. 2 minutes in this example) to mix the system’ s hold volume with the slurry sample, recirculating the slurry through the crossflow filter using a pump. A critical component to the crossflow system is the backpressure regulator controls. The system hold volume is critical, as detailed below. Once mixed, 500 mL was removed and frozen as starting material. This was used as Sample A and is the starting full iron amount in the mass balance Equation 1, shown below. This requires a valve to reduce the flow of the pump while increasing the back pressure across the filter membrane. There is also a pressure gauge on the backpressure controls to accurately measure this transmembrane pressure. In this system the operating back pressure was 15 psi.Equation 1A = B + C

[0079] Crossflow filtration effectively separates free iron from ferritin-bound iron using size-selective membranes. The setup ensures precise quantification and minimizes sample loss. The setup can be any style, but should have sufficient capacity and crossflow to be able to achieve at least 1,500 ml / hr. In this example, a 500 kilodalton (kDa) crossflow filter unit was used. The system used in this example was a Romicon HF UF Cartridge 1018-0.7-106-PM500 PN: 0720199 with a Simer 1 HP 9.8 AMP pump, model 2825ss-01. This is a centripetal pump with 10 gallons per minute (gpm) at zero feet of head pressure.

[0080] The system hold volume should be known, as this will be added diluent in the overall volume used to dilute the two liters of starting slurry. The system used in this example contained one liter hold volume. The permeate was collected in a permeate receiving vessel. A scale is used under the sample tank. This allows for monitoring of the permeate flow rate, as the weight in the sample tank decreasing over time is due to the permeate crossing the membrane filter. This monitoring is important in order to determine when the membrane begins to foul.

[0081] The system ran until 3 liters of permeate was collected in the receiving vessel. The system was stopped, the permeate (Sample B, free iron) and retentate (Sample C, ferritin-bound iron) samples and final volumes were recorded. Fresh water was used to flush the system and discard material in the system. This lost volume is considered in calculations.

[0082] 500 ml of permeate Sample B and 500 ml of retentate Sample C was packaged and frozen for future testing. Any remaining material was also packaged and saved as neededAttorney Docket No. 61130-3PCTfor future use.2L finished 8L IL hold 0.5L sampleA = product + water + volume - AB = 3L permeateC = 6.5L retentate1 L hold volume of systemremaining

[0083] The mass balance of iron content is done once the test results are obtained. Basic mass balance techniques are used to calculate the iron content of the permeate and retentate. The samples are analyzed for iron concentration using an appropriate analytical technique, such as atomic absorption spectroscopy (AAS), inductively coupled plasma (ICP), or other suitable methods as described further below. The present invention can include components required to determine the amount of ferritin iron in a sample, provided separately or in a kit. EXAMPLE 2 - Syringe Filtration Method

[0084] Alternatively, free iron may be separated from ferritin-containing material using a syringe-based filtration method. This method is particularly suitable for small-volume laboratory workflows and does not require pumps or crossflow filtration equipment.

[0085] In this embodiment, dried ferritin-containing material (e.g., Slolron) is reconstituted in water and placed on a shaker for approximately two hours to extract free iron. The mixture is then allowed to settle for about one hour, permitting ferritin aggregates to precipitate while unbound iron remains in the supernatant.

[0086] The supernatant is drawn into a Luer-lock syringe, and a 0.22 pm or 0.45 pm syringe filter is attached. Manual pressure is applied to pass the liquid through the filter, thereby removing ferritin particulates and generating a filtrate containing free iron. The filtrate may be collected directly into sterile sample tubes suitable for downstream analysis such as AAS, ICP, or ferrozine colorimetric assays.

[0087] This syringe-based filtration method minimizes equipment requirements, reduces processing time, and provides a rapid alternative to crossflow filtration for determining free-iron concentrations in ferritin-containing samples.EXAMPLE 3 - Production of custom rabbit ferritin polyclonal antibody

[0088] Custom rabbit anti- ferritin polyclonal antibodies were produced at GenScript (Piscataway, NJ) using a peptide-KLH conjugate antigen having the amino acid sequence spanning amino acids 109-136 of the soybean ferritin heavy chain (FTH1) protein (UniProtKB P19976). The polyclonal anti-ferritin antibody was affinity-purified. For long-Attorney Docket No. 61130-3PCTterm storage, the polyclonal antibody was aliquoted and stored at -20°C or below, avoiding repeated freezing and thawing cycles.

[0089] An ELISA was performed using the 109-136 amino acid soybean FTH1 antigen as the coating antigen at a concentration of 4 μg / ml, 100 μl / well. The coating buffer used was Phosphate Buffered Saline (PBS, pH 7.4) with 0.02% sodium azide. The purified anti-ferritin antibodies were used as the primary antibody. The secondary antibody used was an HRP conjugated goat anti-rabbit IgG (H+L). The results are shown in Table 1 below.Table 1. ELISA results for pre- immune serum and purified antibodyDilution Purified anti-ferritin antibody (A450 nm)NC 1:1,000 0.0511 1:1,000 3.4482 1:2,000 3.4313 1:4,000 3.0804 1:8,000 2.5975 1:16,000 2.4016 1:32,000 1.8897 1:64,000 1.3088 1:128,000 0.9099 1:256,000 0.50110 1:512,000 0.29811 Blank 0.05312 Blank 0.053Titer: 1:512,000Starting dilution: 1:1,000 (Equivalent to 1 μg / ml)The titer is the highest dilution with S / B (Sample / Blank) >=2.1NC is negative control (Pre-immune serum)

[0090] An indirect ELISA was also performed using the peptide of SEQ ID NO. 1 as the coating antigen at a concentration of 4 pg / ml, 100 pl / well. The coating buffer used was PBS (pH 7.4) with 0.02% sodium azide. The secondary antibody used was an HRP conjugated anti-rabbit IgG Fc monoclonal secondary antibody (GenScript, Piscataway, NJ, Cat. No.A01856). The results of two anti- ferritin antibody batches are shown in Table 2 below.Table 2. ELISA results of affinity-purified antibodyConcentration 1000 500 250 125 62.50 31.25 15.62 7.81 3.90 1.95 Blank (ng / ml)Sample\ 1:1000 1:2000 1:4000 1:8000 1:16,000 1:32,000 1:64,000 1:128,000 1:256,000 1:512,000 Blank Titer DilutionAntibody 2.749 2.763 2.690 2.565 2.470 2.110 1.708 1.150 0.698 0.455 0.060 1:512,000 Batch #3942Antibody 2.785 2.776 2.717 2.612 2.355 2.133 1.642 1.230 0.743 0.416 0.055 1:512,000 Batch #3944The titer is the highest dilution with S / B (Signal / Blank) >=2.1, the OD450in blank is the average of two technical replicates.The starting concentration of 1 mg / ml and the corresponding dilution ratio is calculated based on the actual concentration.Attorney Docket No. 61130-3PCT

[0091] Indirect ELISA results of pre-immune serum and affinity-purified antibody after the third immunization was also performed using the peptide of SEQ ID NO. 1 as the coating antigen at a concentration of 4 μg / ml, 100 μl / well. The coating buffer used was PBS (pH 7.4) with 0.02% sodium azide. The secondary antibody used was HRP conjugated anti-rabbit IgG secondary antibody. The results are shown in Table 3 below.Table 3. ELISA results of pre-immune serum and affinity-purified antibody after the 3 rd immunizationConcentration NC 1000 500 250 125 62.5 31.25 15.62 7.81 3.90 1.95 Blank / (ng / ml)Sample\ 1:1000 1:1000 1:2000 1:4000 1:8000 1:16,000 1:32,000 1:64,000 1:128,000 1:256,000 1:512,000 Blank Titer DilutionAntibody 0.085 2.664 2.641 2.639 2.483 2.344 2.106 1.749 1.276 0.843 0.517 0.061 >1:512,000 Batch #3942Antibody 0.067 2.809 2.731 2.684 2.618 2.511 2.340 2.054 1.654 1.186 0.762 0.068 >1:512,000Batch #3944The titer is the highest dilution with S / B>=2.1, the OD450in blank is the average of two technical replicates.The starting concentration of 1 mg / ml and the corresponding dilution ratio is calculated based on the actual concentration.NC is negative control (pre-immune serum)EXAMPLE 4 - Determination of ferritin concentration in a sample

[0092] The present invention contemplates a method for determining the amount of ferritin iron in a sample. As shown in FIG. 1, a capillary electrophoresis was done using the antiferritin antibody described in Example 3. The capillary electrophoresis was done using the automated WES system (Western Blot Service, Austin, TX). The WES system uses capillary electrophoresis with automated protein fractionation, immobilization and immunodetection to quantify protein in the sample.

[0093] Using the anti-ferritin antibody, differences between horse and legume ferritin sources were seen. The results of horse anti-ferritin antibody (13 exposure and 12 exposure lines) and the soy anti-ferritin antibody described in Example 3 (2 exposure and 3 exposure lines) using identical samples of soy ferritin are shown in FIG. 1.

[0094] The linearity of the concentration and fit were used to generate a standard curve from the data from the capillary electrophoresis, as can be seen in Table 4 and FIG. 2, which show samples spiked with 200 pg horse ferritin and isolated soy ferritin. The standard curve can be used to determine the amount of ferritin in a sample. The specificity of the specific antibody for ferritin and the accuracy of the capillary electrophoresis process with this antibody is a significant improvement over previous techniques used to quantify the amount of ferritin in a sample.Attorney Docket No. 61130 -3 PCTTable 4: Ferritin concentrationFerritin Ferritin + Total Adjusted 27 kDa 29 kDa Sample 200 ug Total(ug / mL) HorseFerritin(ug / ml)400 600 12,790,028 6,725,028 5,679,493 7,110,535 300 500 11,079,156 5,014,156 5,377,002 5,702,153 200 400 6,779,485 714,485 5,390,932 1,388,554 100 300 7,918,614 1,853,614 4,885,142 3,033,47250 250 5,506,088 -558,912 4,756,741 749,348 Equation from all Horse Ferritin y=30,325xx=200y=6,065,000Horse Ferritin offset 6,065,000EXAMPLE 5 - Ferritin concentration standard curve

[0095] Capillary electrophoresis using the WES system was performed in order to develop a standard curve for determining the ferritin concentration in a sample using the ferritin antibody described in Example 3. FIG. 3 depicts the image obtained from the WES system showing generation of the horse ferritin standard curve.

[0096] Table 5 shows the numerical values of the areas under the peaks and the peak molecular weights for the standard curve. These correspond with monomer, dimer and trimers respectively for denatured ferritin subunits. The linearity is very good with an R² value of 0.9885 as shown in FIG. 4.Table 5: Ferritin standard curveStandard Curve DataHorse 0.1 Buffer 95 for 5 min.Sample ug / ml Total 24 kDa 45 kDa 60 kDa 66 kDa 200 22,794,767 14,167,256 3,593,994 3,903,354 100 62,832,396 24,282,995 19,504,810 3,062,747 15,981,844 50 23,174,393 12,817,600 7,758,192 1,365,322 1,233,279 25 14,671,736 7,379,095 5,532,580 1,132,494 627,567 12.5 8,157,211 3,771,931 3,334,991 786,232 264,057 6,25 3,803,743 2,015,821 1,520,725 267,197EXAMPLE 6: Ferritin quantitation in the pea protein extractAttorney Docket No. 61130-3PCT

[0097] Ferritin was extracted from peas using the methods described above. Sample of starting pea concentrate was hydrolyzed for a minimum of 4 hours in a shaker table with water. Samples were spun in an ultra-centrifuge for 10 minutes at 35,000 G. For the second extraction the previously spun down sample was again re-hydrolyzed for a minimum of 2 hours and spun down again in similar manner. Two extractions were done. The WES system results from the first extraction are shown in FIG. 5, which shows the first water extraction of the starting pea protein concentrate at decreasing dilutions. The monomer, dimer, and trimers can be seen.

[0098] The results from the second extraction (compared to horse ferritin) are shown in FIG. 6. The data shows WES system results comparing 42.25 ug / ml of concentrated pea protein from the second extraction and 50 ug / ml horse ferritin standard.

[0099] The concentration of the ferritin from the starting pea protein can be determined from the WES system results. 240 grams total of pea protein extract was obtained. The initial sample was made at 16.7% solids, which is 40.08 g of pea protein extract as the starting solids mass. As an example, a dilution of 85 ug / ml would mean 85 ug / 1,000 ug buffer or 1,000 / 85 = 11.76 times dilution.

[0100] From the first extraction, the calculation of 595 ug / ml ferritin in the original sample (50.6 ug / ml x 11.76 (dilution)) was obtained as measured with the anti- ferritin antibody obtained in Example 2. After the second extraction, the calculation of 765.6 ug / ml of ferritin in the original sample (44.1 ug / ml x 11.76 (dilution)) was obtained.

[0101] When the sample from the first extraction was assayed again using a newly produced antibody as described in Example 3, 1,862 ug / ml ferritin was calculated to be in the original sample (158.3 ug / ml x 11.76 (dilution)). 131 ml of pea protein extract resulted in 243,871 ug (or 243.871 mg) of ferritin. This resulted in a yield of 0.610 % ferritin (243.871 mg Ferritin / 40.08 g pea protein x 100%). The extracted sample was concentrated in a convection air dryer by drying down the sample by 5.03% or 100% / 5.03% = 19.88 times more concentrated. This resulted in a calculation of 12.13% (0.610% x 19.88) of starting ferritin.

[0102] After the second extraction, the calculation of 518 ug / ml of ferritin in the original sample (65.1 ug / ml x 11.76 (dilution)) was obtained using the newly produced antibody. When the sample from the second extraction was assayed again using a newly produced antibody as described in Example 2, 765.6 ug / ml ferritin was calculated to be in the original sample (65 ug / ml x 11.76 (dilution)). 128 ml of pea protein extract resulted in 97,997 ug (or 97.997 mg) of ferritin. The extracted sample was concentrated as described above byAttorney Docket No. 61130 -3 PCTdrying down the sample resulting in 22% of ferritin in the combined first and second pea protein extracts.

[0103] FIG. 7 shows the WES system data from the first and the second water extraction. The two extractions have similar amounts of ferritin present.

[0104] Table 6 depicts the actual measured values of ferritin in starting pea protein concentrated extracts fit to the known standard curve of horse ferritin.Table 6: Ferritin concentration from pea extractsExtraction PeaConcentration (ug / ml) Ferritin ug / ml1 3401 3401 170 76.181 170 130.621 85 47.991 85 53.141 42.25 74.851 42.25 39.131 21.125 56.201 21.125 19.602 3402 3402 170 110.732 170 101.542 85 40.342 85 47.792 42.25 17.172 42.25 33.94Table 7: Calculation for the percent of solids in the first, second and third pea protein concentrate starting materialSample Tare Filled Solution Dry Product Precent Number Weight Weight Weight Weight Weight Solids Average 1stExtract 1 7.6 45.2 37.6 9.5 1.9 5.05% 5.03%2 7.6 43.6 36 9.4 1.8 5.00% 3 7.6 51.4 43.8 9.8 2.2 5.02% 4 7.7 54.9 47.2 8.3 0.6 1.27% 1.11% 2ndExtract 5 7.7 53.6 45.9 8.2 0.5 1.09%6 7.7 38.7 31 8 0.3 0.97% 7 8.5 48.1 39.6 8.5 0 0.00% -0.09% 3rdExtract 8 8.6 65.8 57.2 8.5 -0.1 -0.17%Attorney Docket No. 61130-3PCTEXAMPLE 7 - Toxicity study / safety data of ferritin in rats

[0105] A repeated dose 90-days oral toxicity and safety study of ferritin was performed in Sprague Dawley rats. This study was designed to provide information on the effects of repeated exposure of ferritin, to establish No Observed Adverse Effect Level (NOAEL), to provide information on reversibility, delayed toxicity, selection of concentration for longer term studies and target organ toxicity of the ferritin test item in Sprague Dawley rats.

[0106] 120 Rats (60 Male and 60 Female) were divided into 8 groups [G1 - Vehicle (0 mg / kg b.wt. per day), G2-Low Dose (650 mg / kg b.wt. per day), G3 -Mid Dose (1300 mg / kg b.wt. per day), G4- High Dose (1950 mg / kg b.wt. per day), G5- Vehicle Control Recovery (0 mg / kg b.wt. per day), G6- Low Dose Recovery (650 mg / kg b.wt. per day), G7- Mid Dose Recovery (1300 mg / kg b.wt. per day), G8- High Dose Recovery (1950 mg / kg b.wt. per day)]; 10 male + 10 female in main group and 5 male and 5 female in recovery group animals per group.

[0107] Reverse Osmosis (R. O.) water was selected as a vehicle based on solubility testing. Required amount of the ferritin test item was received from the Test Item Control Office (TICO) for each concentration and dose groups separately and were formulated with R. O. water for low dose, mid dose, high dose, low dose recovery, mid dose recovery and high dose recovery groups. Samples of dose formulation were analyzed for homogeneity and dose concentration analysis once before commencement of dosing on dayl of dosing and twice thereafter at monthly intervals during the treatment period. Samples were collected from lower, middle and top layers of the formulation prepared in air tight containers for homogeneity for all dose group levels and dose concentration analysis. Triplicate samples were collected each from lower, middle and top layers of each dose concentration of dose formulations including vehicle control (total 12 samples) for homogeneity and active ingredient concentration analysis. Dose formulation analysis was performed using validated analytical method. Test item formulation was administered orally by using 16 gauge oral gavage cannula attached with a syringe in Sprague Dawley rats for 90 days, consecutively. Animals from G2 (low dose), G3 (mid dose), G4 (high dose), G6 (low dose recovery), G7 (mid dose recovery) and G8 (high dose recovery) received the test item at a dose of 650, 1300 and 1950 mg / kg b.wt. per day respectively. Animals from G1 (vehicle) and G5 (vehicle control recovery) received R. O. water alone. A dose volume of 10 mL / kg b.wt. was maintained for all control and dose treated group animals.Attorney Docket No. 61130-3PCT

[0108] All animals were observed for mortality / morbidity check twice in a day until terminal sacrifice with cage side observation of main and recovery group animals until day 91 and 118 respectively. The body weight of all animals from all 4 main groups and 4 recovery groups were measured once prior to treatment and weekly thereafter until terminal sacrifice. All animals from each group were observed for detailed clinical observations including discharge, locomotor activity, skin, mucous membrane, eyes, ears, oral cavity, fur, respiration, urine / feces, etc. once before treatment and weekly thereafter until terminal sacrifice. Feed consumption was measured after post treatment on weekly basis. All animals from the dose groups and vehicle group survived up to scheduled termination.

[0109] No mortality / morbidity was observed in any of the treated animals throughout the study period. No abnormal clinical signs were observed in any of the animals throughout the study period. No test item related significant changes were observed in body weight and feed consumption in the treated groups as compared to concurrent control group. No abnormal changes were observed in the detailed clinical examination prior to treatment and weekly during the treatment and ophthalmoscopy examination. All clinical pathology parameters in test item treated groups were comparable with the control group. No test item related significant changes were observed in clinical pathology parameters in treated group compared to control group in both sexes. No test item related significant changes were observed in absolute and relative organ weight in both sexes when compared to respective control group. No test item related findings were observed in any of the organ during gross (macroscopic) and microscopic observation.

[0110] Based on the results obtained, it could be concluded that the tested isolated ferritin of the invention did not produce any systemic toxicity or adverse effects up to the highest dose level (1950 mg / kg b.wt. per day) when administered orally for 90 consecutive days, under the conditions and procedures followed in the present study. Additionally, based on the results obtained from recovery group, it could be concluded that the test item did not produce any systemic toxicity or reversibility or delayed toxicity at highest dose level (1950 mg / kg b.wt. per day). Based on the findings, the No Observed Adverse Effect Level (NOAEL) of the tested ferritin was determined to be 1950 mg / kg b.wt. per day in Sprague Dawley rats. The tested ferritin is 5% iron, so the dose used in rats translates to 97.5 mg / kg of body weight of iron. (1950 mg / kg x 5% = 97.5 mg). The results can be extrapolated to humans. Since the average weight of a human male is 70 kg, 70 kg x 97.5 mg / kg of Fe = 6,825 mg / 70 kg human, or 6.825 grams of Fe per 70 kg human daily for 90 days with no adverse events.Attorney Docket No. 61130-3PCTEXAMPLE 8: Nutritional profile of isolated ferritin

[0111] A typical nutritional profile from 100 grams of the isolated ferritin of the invention is shown in Table 8.Table 8: Ferritin nutritional profileUnit ValueCalories Kcal / 100g 311.8Calories from fat Kcal / 100g 42.84Total Fat g / lOOg 4.76Saturated Fat g / 100g 1.25Protein g / 100g 66.92Cholesterol mg / 100g < 2Sodium mg / 100g 3,276.43Calcium mg / 100g 126.81Iron mg / 100g 5336.09Carbohydrates g / 100g 0.37Sugar g / 100g 0.36Total Dietary Fiber g / 100g 15.33Potassium mg / 100g 158.22Moisture g / 100g 5.37EXAMPLE 9: Restorative effect of iron from bean ferritin on low hemoglobin 2 level caused by menstruation in Japanese women: A randomized, double-blind placebo-controlled intergroup trial

[0112] Recently, bean ferritin has been attracting attention as a source of iron which is also available to vegetarians. Although high rates of iron absorption and bioavailability from this protein have been reported, the clinical data on its efficacy are still scarce. In this study, bean ferritin iron was administered to premenopausal Japanese women for nine weeksAttorney Docket No. 61130-3PCTstarting immediately after menstruation in order to evaluate their recovery from low hemoglobin level as one sign of anemia. Subjects in the test supplement group received an iron intake of 5 mg from one capsule containing bean extract (containing the isolated ferritin of the invention) for five weeks, which was increased to 10 mg (i.e., two capsules) from the 6th to 9th week. The study evaluated the change in hemoglobin levels as the primary endpoint, and hematocrit, red blood cell count, serum iron, mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), serum ferritin, TSAT (TIBC), serum zinc, serum copper, anemia symptoms questionnaire, OSA sleep inventory, and the anti-fatigue questionnaire as the secondary endpoints. The results showed a significant difference (P=0.03) in the change in hemoglobin levels between the groups after nine weeks of intake, confirming the restorative effect of bean ferritin on low hemoglobin level caused by menstruation. Moreover, a significant difference (P=0.01) was observed in the amount of change in MCHC between the two groups after five weeks of intake, and after nine weeks of intake, a significant difference in the change in both MCH (P=0.02) and MCHC (P<0.01) was observed between the groups. A significant difference (P=0.03) was observed in the change in the serum ferritin levels after nine weeks of intake. The study confirmed that iron supplementation from bean ferritin containing the isolated ferritin of the invention is an effective treatment for low hemoglobin level and low ferritin level caused by menstruation.

[0113] Iron is an important component of a healthy diet, especially in menstruating women. The recommended daily intake for iron in menstruating adult women in Japan is 10.5 mg. A survey conducted by the Japanese Ministry of Health, Labour and Welfare revealed that the daily intake of iron in Japanese adult women was 7.2 mg, which is approximately 70% of the recommended value, indicating a high prevalence of iron deficiency among Japanese adult women. It has been estimated that the prevalence of various types of iron deficiency from the results of a survey of 3,015 Japanese women were: iron-deficiency anemia: 8.5%; latent iron deficiency: 8.0%; storage iron deficiency: 33.4%; normal: 43.6%; and other: 6.5%. The study showed that half of the women who participated in the survey were iron deficient or potentially iron deficient. In this study, the criteria for diagnosing anemia were determined from the Hb levels derived from the average values of healthy adults (i.e., transferrin saturation >16%, and serum ferritin levels >12 ng / ml). One of the reasons for the high prevalence of iron deficiency among Japanese adult women may be that the recommended daily intake for iron in Japan is considerably lower than the reference iron intake in the U. S. (18 mg / day).Attorney Docket No. 61130-3PCT

[0114] As a result of this iron deficiency, several Japanese women are commonly prescribed iron preparations and iron supplements. Iron preparations contain ferrous sulfate and ferric citrate, while iron supplements contain ferric citrate, iron pyrophosphate, and heme iron. Ferritin, the most important iron-storing protein, was isolated from soybean seeds in 1987. In recent years, following a variety of basic studies, properties, such as iron absorption kinetics and bioavailability, as well as the practical evaluation of ferritin as a dietary iron source have also been explored. Because the iron absorption capacity and bioavailability from ferritin are like those from ferrous sulfate, a highly absorbable iron preparation used in pharmaceutical products, the protein was marketed as a new highly absorbable iron supplement. While most studies to date have been carried out on laboratory samples, human clinical trials using widely available iron sources have not yet been conducted.

[0115] Soybean- and pea-derived ferritin iron are on the market. Compared to the iron sulfate used in pharmaceuticals, ferritin iron has few adverse effects, including extremely low iron taste and gastrointestinal disorders. Moreover, because of its high absorbency despite being a non-heme iron, ferritin iron can be ingested by vegetarians and vegans.

[0116] According to the Dietary Reference Intakes for Japanese issued by the Ministry of Health, Labour and Welfare, blood iron loss during menstruation is estimated to be 3.06 mg / day for women aged 10-17 years, and 3.64 mg / day for those aged 18 years or older.Additionally, a decrease of 30% or more in ferritin levels during menstruation was reported. The severity of iron deficiency in adult menstruating women may vary depending on the timing of their menstrual cycle. For this reason, it is worth noting that an accurate evaluation in adult menstruating women who receive oral iron supplements is only possible if blood sampling is carried out within a set amount of time from the end of the menstrual cycle.Therefore, careful management of subjects is required.

[0117] In this study, the administration of trial supplements and hematologic evaluation in healthy premenopausal women who experienced anemia symptoms by low hemoglobin levels were performed at the same time during their menstrual cycle, while controlling for menstrual cycle phases, to assess the effects of bean iron from test supplements on low hemoglobin level caused by menstruation. Consumption of trial supplements was started within one week from the end of menstruation. Blood samples were collected after five and nine weeks from the late follicular to the luteal phase of the menstrual cycle to assess the restorative effect of bean ferritin (containing the isolated ferritin of the invention) on low hemoglobin level caused by menstruation. The amount of iron in the test supplement wasAttorney Docket No. 61130-3PCTincreased from 5 mg / day to 10 mg / day after five weeks of intake, and the dose dependency was also evaluated.

[0118] Methods. Trial supplements. The participants of the study were divided into two groups. One group received a test supplement containing bean ferritin (the isolated ferritin of the invention) and the other group received a placebo control supplement. The bean ferritin in the test supplement used in the study contains approximately 5% iron. By contrast, the control supplement did not contain any iron. Both the test and control supplements were packed as porcine gelatin capsules with a content weight of 280 mg per capsule and colored white with titanium dioxide. Corn starch was used as a basic ingredient for both trial supplements. The test supplement contained 5 mg of iron per capsule (100 mg of bean extract), while in the control supplement, the bean extract was replaced with an equal amount of corn starch.

[0119] Subjects. The subjects were recruited among paid volunteers based on the inclusion and exclusion criteria outlined below, and 40 out of 80 women who gave their consent to participate in the study were selected based on the laboratory values of hemoglobin at screening and the inclusion and exclusion criteria described below. The target number of subjects was determined based on previous studies. The subjects were randomly assigned to two groups, through stratified randomization. The groups were kept secret until the end of the study to ensure the blinding of participants, intervention providers, and outcome assessors. The study was conducted in accordance with the CONSORT 2010 Statement (Consolidated Standards of Reporting Trials 2010 Statement) for reporting randomized controlled trials. In addition, an outline of the study was registered in the public database UMIN Clinical Trials Registry (ID: UMIN000045253).

[0120] After the completion of the study, the allocation order and numbering of the participants were disclosed, and the groups of subjects that received the bean extract and the control supplement were designated as S group and P group, respectively. Before starting the screening process, the subjects were fully informed on content and methods of the study and their written consent was obtained. The ethical, scientific, and clinical validity of the study was reviewed and approved by the Institutional Review Board of Tsukuji Futaba Clinic, Hikobae-kai Medical Corporation. The study was conducted in accordance with the ethical principles of the Declaration of Helsinki and the Ethical Guidelines for Medical and Health Research Involving Human Subjects.

[0121] Inclusion criteria were as follows: 1) women who experienced symptoms of low hemoglobin level (anemia) on a daily basis, 2) premenopausal women, 3) BMI of less than 30Attorney Docket No. 61130-3PCTkg / m2, 4) menstrual cycle timing falls within the following time frames: the timing of the menstrual cycle is for those who fall within the specified time period, 5) with a relatively stable menstrual cycle, 6) provided written consent to participate in the study, and 7) Hb levels of less than 13 g / dL on a previous blood test.

[0122] Exclusion criteria were as follows: 1) women who are allergic to beans (e.g., soybean and pea), 2) currently on medication for iron-deficiency anemia and receiving medication for any disease, 3) consume food (e.g., soy milk) and health food products containing the ingredients of the test supplement used in the study, 4) irregular menstrual cycles, 5) serious diseases affecting the glycometabolism, lipid metabolism, liver function, kidney function, heart, circulatory system, respiratory system, endocrine system, immune system, and nervous system, or mental disorders, and women with a history of such diseases, 6) medication for a disease or with a history of a serious disease requiring medication, 7) develop allergies related to the study, 8) participating in other clinical research at the time of initiation of the study, 9) pregnant or planning to get pregnant or breastfeed during the study period, 10) uterine myomas or endometriosis, and 11) judged by the principal investigator to be unsuitable to participate in the study.

[0123] Study design and methods. The study, which used two kinds of supplements, was designed as a randomized double-blind placebo-controlled intergroup trial and was carried out at Hasegawa Clinics, Seishukai Medical Corporation, on two groups of women selected based on the timing of their menstrual cycle. The study schedule and menstrual cycle of the subjects are shown in Figure 8.

[0124] Both trial supplements were distributed to the subjects at the time of arrival to maintain blindness and were administered as follows: one capsule per day (5 mg of iron / day), preferably taken at breakfast with water or lukewarm water on an empty stomach. The subjects received the supplements for nine weeks; however, from the end of the 5th week the dose was doubled to two capsules per day (10 mg of iron / day).

[0125] The reason for increasing the dosage was to verify the difference between the 5 mg / day dosage and the 10 mg / day dosage. The 5 mg / day dosage was intended to supplement the approximately 4.1 mg / day dosage that Japanese adult women lack, while the 10 mg / day dosage was intended to supplement the recommended intake for adult women in Japan.During the period of intake of trial supplements, the subjects were asked to record daily their menstrual cycle phases, living conditions, dietary composition, and whether they took medications or the trial supplements. Subsequently, the subjects were asked to visit the hospital before, and at five, and nine weeks after intake, or upon discontinuation, and as mayAttorney Docket No. 61130-3PCTbe necessary, and were then asked to complete a web-based questionnaire on the anemia symptoms before, and at five, and nine weeks after the intake of trial supplements. Next, blood samples were collected to evaluate the efficacy and gather observational items (e.g., adverse events, body weight, BMI, blood pressure, pulse, and biochemical and hematological tests) before, and at five, and nine weeks after intake. In addition, biochemical and hematological tests were also performed.

[0126] Efficacy endpoints. In this study, the amount of change in the Hb levels was used as a primary endpoint for screening and efficacy evaluation. In addition to hematocrit, the red blood cell count, serum iron, MCH, MCHC, serum ferritin, and TSAT (TIBC), which are common indicators of iron deficiency, serum zinc and copper were also measured as secondary endpoints to evaluate the zinc and copper levels in the blood as the absorption of these two elements may be antagonized by iron intake. Additionally, questionnaire surveys on anemia symptoms, OSA sleep, and anti-fatigue were conducted to evaluate changes in quality of life. In addition to statistical analysis, the mean, standard deviation, standard error, median, and minimum and maximum values were calculated for all endpoints.

[0127] Statistical analysis. Statistical analysis for efficacy evaluation was carried out by Kansai University of Welfare Sciences. Firstly, an intra-group comparison in the study population was performed before, and at five, and nine weeks after intake. Since subjects varied in age and severity of iron deficiency, differences in each endpoint were already observed at the pre-consumption stage. In order to evaluate the efficacy of the treatment, while also taking into account this variation, the amount of change before, and at five, and nine weeks after intake was also calculated. Next, an intergroup comparison was performed. Intragroup and intergroup comparisons were analyzed using a paired t-test, and Student's t-test, respectively. The tests were applied assuming the normality of each dataset (while multiplicity was not considered) with a significance level of 5% (two-tailed test). SAS9.4 (SAS Inc.) was used for statistical analysis.

[0128] Results. A flow diagram of the progress from screening to analysis is shown in FIG. 9. Two subjects who violated the exclusion criteria after inclusion were excluded from the analysis. The baseline characteristics of the subjects are shown in Table 9, and no significant differences between the two groups in terms of age, BMI, hemoglobin, ferritin were noted. In Table 10, the subjects were classified based on the six degrees of severity of iron deficiency. No bias was observed in any of the two groups, and the composition of data in the subjects’ low hemoglobin level was uniform.Attorney Docket No. 61130 -3 PCT

[0129] Since the study also considers the timing of the menstrual cycle phases, Table 11 shows the number of days from the end of menstruation. In this case also, no substantial bias was observed in either group. However, despite controlling for menstrual cycle phases (i.e, from the late follicular through the luteal phase), after nine weeks, the time that elapsed from the end of menstruation to blood sampling in the S group became shorter than in the P group, making it difficult to assess recovery from low hemoglobin level. No subjects with menorrhagia were observed.Table 9: Baseline characteristics of subjects and timing of blood sampling in the S and P groups.Variable Group P(u:;-i8j Group S (a-;20) p-valueAge (years) 34.9 * 9.3 32.4 a 9.4 0.3995 BMHkgsW) 20.6 * 2.7 19.4 & 2.5 0.1596l ib (g'dL) 1 1.4 * 1.7 11.2:1: 1.9 0.7623 RBC puL) 412.7 ~ 37.8 4178 » 27.4 0.63241111%) 37.3 st: 4.4 37.4 * 4.7 0.9917Ferritin (ngird) 1S.<> si 31.8 15.0 * 19.9 0.6705 TSAT (%) 13.4 * 7.8 15.2 -.i: 0.1 0.5482MeansrSDTable 10: Classification of iron deficiency»3rosp P (»" 48) Groap 8few. ssKsnifs 8 9Inters ceikueacy 2 3Ne-isiess fears deficiency 4 4TSAT 2 iNan ironOetki wy 0 8Others 2 0Attorney Docket No. 61130 -3 PCTTable 11: Number of days from end of menstruation to blood sampling8*Parts of 3 blood sampling (0, 5 and 9 week) could not be conducted in 2 subjects due to infection with COVID-19. These subjects were removed from the evaluation.

[0130] Evaluation of efficacy by blood test. Table 12 shows the values of primary and secondary endpoints before (0 week), and at five, and nine weeks after intake as well as the amount of change from before, to five and nine weeks after intake.

[0131] The amount of change in the primary endpoint of Hb levels showed no significant intergroup differences after five weeks of intake; however, significant intergroup differences were observed after nine weeks of intake. Among the secondary endpoints (e.g., hematocrit, red blood cell count, serum iron, MCH, MCHC, serum ferritin, TSAT, serum copper, and serum zinc), a significant difference (P=0.01) in the amount of change in the MCHC levels between groups was observed after five weeks of intake, and a significant difference in the amount of change in MCH (P=0.02) and MCHC (P<0.01) levels between groups was observed after nine weeks of intake. The other items showed significant intragroup differences (e.g., red blood cell count and MCV); however, no significant intergroup differences were observed.

[0132] Figure 10 shows changes in the serum ferritin levels for each subject before and after intake of trial supplements, and Figure 11 shows the amount of change after five and nine weeks of intake. A large number of subjects in the S group showed an increase in ferritin levels, indicating a significant difference between the two groups after nine weeks of intake.Attorney Docket No. 61130 -3 PCTS v=s F i!ri mary sutcnma' Mb Ow 552 s 5.8 11.4 a 1.6 ft.^s:<,,'61,: aa J 1 3 ♦ 1 8 11.a a 16 <>.>x 8w 11 8 > S:5,i a 1.6!s.?>,5ve0w 01 a 8.6 0.55 0.1 a 0.5 836 0.75 _ Ow-Ow _ Osa a 8.8 <.6.01 _ 0,5. a 0,7 _ 871 _ Q. QQ Secondary csstconm RBC Ob' -420.3 a 29.5 414.4 a 37.5 0.50 { / id.) 5w 422.3 6 25.3 426.8 a 32.3 0.88 9w 433.1 a 30.3 4:&f> a 31 8 0.16 Sw-Ow 2.0 a 22.6 0.66 8,2 a 20,6 0.11 0.38 9w-0w 28.3 a 23.2 <.3.05 5,9 a 28.6 0.36 0.25 ill Ow 37.4 a 4.5 37,4 a 4,3 6.83 {%; 5w 38.8 a 4.3 37,7 a 4.2 632 9w 38.2 a 4.8 37,5 a 43 8«8 5w-0w -0.8 a 1.9 0.53 6,3 a 2,2 054 838 8w-8w 0.9 a 22 0.00 0.1 a 2.3 882 88- MCV Ow.99.0 a 9.7 902 a 5.9 864 (10; 5w S7.0 a 9.9 89.4 a 6.2 835 9w 88.3 a 9.5 59.4 a 6.8 887 Swxlw -5.9 a 5.8 <.0.01 -1.0 a 2.4 0,10 0.19 9b-0»- -5.5 a 5.9 0.02 41.9 a 2.2 0,09 0.82. MCH Ob- 26.6 a 4.0 27.4 a 2.7 0,00 0.61 (pg) 5b- 26.7 a 4.0 22.3 i 2,9 0,00 0.61 9w 27.2 a 4.1 27.3 a 2.8 0.00 0.98 Sw-Ow 0.1 a 0.3 0.55 -6.2 a 0.5 0.07 0.08 9w-0w 0.5 a 1.0 0.05 -6.2 a 6.3 0.21 0.02 MCHC Ow 29.8 a 1.7 36,3 a 1,4 0.33 {%! 3w.30.5 a 1.8 363 a 1,3 9.92 9w 30.7 a 1.9 50.4 a 1,1 0.80 5w-0w 0.7 a 0.8 <0.05 0,1 a 0,0 0.56 001 9«?-0w 0.9 a 0.7 <i!.8> 6,1 a 67 826 <.0.01 Ow 13.3 a 19.8 19,0 a 3i,6 O. S7 (ng / rjd) atv 559 a?.> 4 21.9 a 47.6 888 9w 58.8 a 179 18.5 a 33.8 868 5vv-0w 1.5 a 1.3.5 0.58 3.3 a 18.3 0,95 872 9vb0w 3.8 a 53.9 0.23 41.1 a 6.2 0.154 8,28 TSAT Ow 55.1 a 50.0 13.5 a 7.6 0.57 (%• 5vv 56.6 a 55.5 16.5 a 9.9 0.71 9w 52.5 a 50.2 15.4 a 19.6 0.62 5*<0w 5.7 a 7.0 0.29 2.1 2 5.9 0,32 0.83 9*.«Ow 1.8 a 8.0 033 2.0 a 83 0.14 0.96 Sv'mm Ow S3.4 A SF.’E SI S < 2£4 0.73 irwi 5w &).<> i: <51.4 £ $<5,5 0<>7 <>w ’S4.9:* AF. F A 4:?v>; 0.0$ Sw-Ow 7.5:* 25.5 FJS -SS s 22;? 0.00 0.55 <>w4Hv n.5 * 32.f> -2S * 3<? J 0.16 0.54 Semsn Ow 102.0 a 9.9 104,7 a 23,8 9.6<- Copper 8w KJS.1 a 17.7 311,8 a 26,8 0.33 (pg / dU 8kv5875 a 152 314.2 a 23,4 838 SveOw a 8 a 54 a 0.34 6.4 a 1(1.7 862 842 9wX»w 5.3 a 548 0.13 8.6 a 9.4 <88- 842 Serum 8W9a9 a 545 95,1 a 11.9 872 Zinc,5w 907 a 538 S5.4 a 13.1 0.97 (pgsdi.) 9w 95.0 a 1.3.5 93.5 a 10.4 O. SS “nv-Ow -3:?. a 527 0.28 -19.1 a 10.6 <881 0.659»-0w> 2.0 a 532 0.31 -3.0 a 19.6 0,44 0.94 Table 12: Primary and secondary outcomes of subjects in the S and P groups.Attorney Docket No. 61130-3PCTVarious questionnaires. Significant intragroup differences in the amount of change in several items of the anemia symptoms questionnaire (e.g., subjective feeling of iron deficiency symptoms, sense of fatigue, sense of vertigo or dizziness, sleepiness, facial complexion, discomfort during exercise, unpleasant feelings of restlessness in the lower limbs, and taste perception) were observed. Notably, significant intragroup differences in such items, such as subjective feeling of iron deficiency symptoms, sense of fatigue, sense of vertigo or dizziness, and facial complexion were observed in both group S and P. Significant intergroup differences in taste perception were observed only after three and seven weeks of intake. No significant intergroup differences were observed in the OSA sleep inventory and anti-fatigue questionnaire (data not shown).

[0133] Safety and side effects. No adverse events were reported during the course of the study. In addition, no common side effects associated with iron supplementation (e.g., heartburn, nausea, abdominal pain, constipation, etc.) were reported. No significant differences were observed in such observational items as body composition, and biochemical and hematological tests, and changes for all the events were within acceptable values.

[0134] Discussion. The study showed significant intergroup differences in the primary endpoint of Hb levels, as well as in MCH and MCHC, which are used as a measure of red blood cell quality, and serum ferritin, an indicator of the body iron stores. Table 13 shows the change in iron anemia symptoms by low hemoglobin level before and after the consumption of trial supplements in subjects who were classified based on the six degrees of severity of iron deficiency. The results show that approximately half of the subjects in the S group had recovered from low hemoglobin level. Moreover, while some subjects in the P group recovered, although in a smaller number than in the S group, the number of subjects who deteriorated was significantly higher in the P than in the S group. These results confirm that the intake of iron from bean ferritin can effectively lead to recovery from low hemoglobin level or low ferritin level caused by menstruation.Attorney Docket No. 61130 -3 PCT

[0135] Table 13: Change of iron deficiency from 0 week to 9 week in S and P groups S group P group0 week 5 week 9 week 0 week 5 week 9 week Iron deficiency anemia 9 8 6 8 8 8 Latent iron deficiency 3 2 2 2 2 2 Pre-latent iron deficiency 4 5 4 4 4 3 Low TSAT 1 1 1 2 1 0 Non iron deficiency 3 4 7 0 1 3 Others 0 0 0 2 2 2 Recovery (vs 0 week) 7 6 2 4 Deterioration (vs 0 week) 1 0 - 3

[0136] The serum ferritin levels were slightly higher in the S group, which continued to receive bean iron at a dose of 5 mg / day for five weeks, but were not significantly different from those in the P group. After nine weeks of intake, on the other hand, there was a significant difference between the amount of change observed in the S group, which continued to receive bean iron at a dose of 10 mg / day for an additional four weeks (for a total of nine weeks), and that observed in the P group. At the same time, the serum ferritin levels of subjects in the S group increased approximately 1.6 -fold on average compared to the beginning of the study, also suggesting an increase in the body iron stores.

[0137] Some studies reported that the serum ferritin levels, during menstruation, decrease by 30% or more, and, in particular, the follicular phase during and immediately after the end of menstruation is thought to be characterized by a decrease in ferritin levels. It has been reported that the main cause of iron deficiency in Japanese women is not insufficient intake, but iron loss through menstruation. Although iron stores are expected to be restored after menstruation, through dietary intake or other sources, it is worth noting that the majority of absorbed iron is transferred to the blood and used for hemoglobin formation.Consequently, body iron stores are believed to remain unchanged or even slightly reduced as shown by the changes observed in the P group in our study.

[0138] Moreover, in patients with iron-deficiency anemia treated with oral iron supplements, reticulocyte crisis (i.e., the rapid increase of the number of reticulocytes) occurs within 7-10 days of the start of treatment after an increase in serum iron levels, and is followed by an increase in Hb levels. Generally, it takes from three to four months (12 to 16 weeks) to fully replete iron stores (i.e., serum ferritin)). To restore the serum ferritin levels after menstruation and recover from iron-deficiency anemia or latent iron deficiency, iron should be supplied regularly from sources other than food. In case of iron supplementsAttorney Docket No. 61130-3PCTderived from beans, such as the one used in this study, a daily intake of 10 mg or more is recommended to ensure a more effective recovery. Moreover, the slight increase in the amount of change observed at a daily intake of approximately 5 mg, albeit not significant, suggests that continuous iron intake over a prolonged period (e.g., 12 weeks or longer) may lead to gradual recovery from low hemoglobin level.

[0139] In patients with low hemoglobin level, such as women during menstruation, elevated expression of iron homeostasis proteins, such as divalent metal transporter 1 (DMT1), Dcytb, ferroportin, and hephaestin is observed, and iron absorption from the digestive tract tends to increase. According to the National Health and Nutrition Survey conducted by the Ministry of Health, Labour and Welfare, approximately 70% of the iron consumed by Japanese from food sources is non-heme iron. It also should be noted that higher consumption of iron resulting from the concomitant intake of supplements as well as of food sources is known to inhibit the intestinal uptake of zinc and copper due to the competitive antagonism between these elements on the DMT1. In order to evaluate the inhibitory effect of iron from bean ferritin on copper and zinc uptake during iron absorption, in addition to the serum iron, serum zinc and copper was evaluated. The results showed no clear inhibitory trends, with no significant intergroup differences, although significant intragroup differences were observed in the P group.

[0140] Since both groups consumed iron from food sources on a daily basis, and the iron intake from test supplement in the S group was lower (5-10 mg) than that from commercially available supplements (prescription drugs supply an iron intake of 100-200 mg / day), the differences were not considered significant. In order to evaluate the inhibitory effect of iron from bean ferritin, an intake of 40 mg or more (i.e., the acceptable daily intake reported for Japanese women) would be necessary. In the anemia symptoms questionnaire, several items showed significant intragroup differences in both groups. Since these changes include the restorative effect of iron supplementation from dietary sources, the intragroup differences observed in both groups were considered to be significant. Moreover, while all P- values in the S group were smaller, indicating slightly stronger recovery trends, the difference between the groups was not large enough to be considered significant. Because responses to questionnaires are highly subjective and may easily reflect some placebo effect, statistically significant differences were difficult to derive due to the large number of subjects with no iron- deficiency anemia included in the analysis. According to the Nutritional and Dietary Guidelines for athletes issued by the Sports Medicine and Science Committee of the JapanAttorney Docket No. 61130-3PCTSports Association, the iron intake in athletes should be 1.5 times the recommended amount for general adult women (15 mg / day), considering iron loss due to sweating and intestinal bleeding caused by exercise.

[0141] As also mentioned above, the recommended iron intake for adult women in the U. S. is 18 mg / day. It might be preferable for women who are menstruating or immediately after menstruation, and iron-deficient women who had or experienced symptoms of iron deficiency, to consume iron at a dose of 15-18 mg / day, which is the recommended intake for athletes and adult women in the U. S. Since the average consumption of iron in adult Japanese women is 7.2 mg / day, supplementation with 8-10 mg / day of iron from sources other than food (e.g., supplements) would be advisable. These recommended values for iron-deficient women are also supported by the results of this study. In this study, subjects were recruited using hemoglobin as a primary endpoint, but after the end of the study, we found that some subjects who exhibited higher serum ferritin levels than the reference upper limit (80 ng / ml) and did not have primary anemia were erroneously included in the analysis. In future studies, reference values for serum ferritin levels should be specified in the selection / exclusion criteria to allow a more accurate evaluation of the restorative effects of iron intake in iron-deficient patients.

[0142] Conclusion. The significant difference in the amount of change in Hb levels observed in the study after nine weeks of intake of iron from bean ferritin confirmed that recovery from low hemoglobin level or low ferritin level caused by menstruation in Japanese women was possible. Also, an intake of iron of 5 mg / day from bean ferritin for five weeks has been shown to improve the red blood cell quality (MCHC). Significant differences were also shown in the change in ferritin levels after nine weeks of intake, suggesting that iron consumed at a dose of 10 mg / day is more likely to improve the low hemoglobin level and increase the body iron stores than at a dose of 5 mg / day. Therefore, pea-derived bean ferritin intake is effective against anemia.

[0143] EXAMPLE 10 - Oral Administration of Plant-Derived Ferritin-Bound Iron and Evaluation of Systemic Iron Availability

[0144] Iron deficiency is the most common micronutrient deficiency worldwide and remains a significant public health concern. Conventional oral iron supplements, such as ferrous salts, are effective but have important limitations, including gastrointestinal side effects, limited absorption efficiency at higher doses, and physiological regulatory mechanisms — particularly hepcidin-mediated suppression — that can reduce iron uptake.Attorney Docket No. 61130-3PCTThese challenges have led to increased interest in alternative iron delivery systems with improved tolerability and physiological compatibility.

[0145] Ferritin is a naturally occurring iron- storage protein found in plant and animal tissues, capable of safely storing thousands of iron atoms within a stable protein shell. In plant-based foods such as legumes, ferritin represents a major form of dietary iron.Substantial evidence from human, animal, and cellular studies demonstrates that ferritinbound iron is efficiently absorbed and provides bioavailability comparable to conventional iron supplements, even in the presence of dietary inhibitors. Mechanistic studies indicate that ferritin-bound iron is absorbed through receptor-mediated endocytosis rather than traditional ionic transport pathways, allowing ferritin to deliver iron in a controlled and regulated manner.

[0146] Although ferritin-bound iron is recognized as a bioavailable dietary iron source, its pharmacokinetic behavior following oral administration in humans has not been fully characterized. In particular, the timing of systemic iron appearance, peak serum iron levels, and associated physiological regulatory responses remain incompletely understood.Characterizing the pharmacokinetic profile of ferritin-bound iron is important for optimizing dosing strategies, improving efficacy, and minimizing regulatory feedback mechanisms that limit iron absorption.

[0147] Accordingly, the present study was conducted to characterize the pharmacokinetic profile and systemic availability of orally administered plant-derived ferritin-bound iron in human subjects, including both healthy individuals and iron-deficient individuals.

[0148] This study was a prospective, randomized, double -blind pharmacokinetic and pharmacodynamic clinical trial conducted in accordance with Good Clinical Practice and ethical standards. The analysis focused on female participants aged 18-55 years who received Slolron®, a plant-derived ferritin-bound iron supplement, and evaluated systemic iron responses in both healthy individuals and those with iron deficiency anemia (IDA).Participants received an initial oral dose of 60 mg elemental iron under fasting conditions, followed by daily supplementation for approximately 28 days, with lower daily dosing for healthy subjects and higher dosing for IDA subjects.

[0149] Healthy subjects were required to have normal hemoglobin concentrations at screening (>12 g / dL), while the IDA cohort included participants with hemoglobin values <12 g / dL. Key exclusion criteria included chronic disease, recent iron supplementation, pregnancy, or abnormal clinical laboratory findings unrelated to iron status.Attorney Docket No. 61130-3PCT

[0150] Both groups receiving Slolron® received an acute, in-unit oral bolus of 60 mg elemental iron administered as three 400 mg capsules (each providing 20 mg iron as ferritinbound iron) under fasting conditions on Day 1 (PK day). Following completion of pharmacokinetic assessments, participants were discharged and instructed to continue daily oral supplementation for approximately 28 days (short-term pharmacodynamics). Healthy subjects received 20 mg elemental iron once daily, while subjects with iron deficiency anemia received 60 mg elemental iron once daily, administered one hour before meals in accordance with the study protocol.

[0151] Venous blood samples were collected pre-dose and at 0.5, 1, 2, 3, 6, 12, and 24 hours post -dose on the PK day. Serum iron and biomarker concentrations were used for noncompartmental pharmacokinetic analysis. The following PK parameters were derived using Phoenix® WinNonlin® (version 8.4 or higher): maximum observed concentration (Cmax), time to maximum concentration (Tmax), area under the concentration-time curve from 0-12 hours (AUC0-12), and area under the concentration-time curve from 0-24 hours (AUC0-24). Terminal elimination half-life (t½) was not included in the present analysis.

[0152] Biomarkers of iron metabolism — including hemoglobin, serum iron, ferritin, hepcidin, transferrin, total iron-binding capacity (TIBC), and transferrin saturation — were measured at baseline (pre-dose), over the first 24 hours and at the end of the approximately 30-day supplementation period. All assays were performed by certified clinical laboratories using validated analytical methods as specified in the protocol.

[0153] Analyses were conducted on a within- group basis for the Slolron® cohorts only. Continuous variables are summarized as mean ± standard deviation unless otherwise noted. Within-group changes from baseline to end of study were assessed using paired t-tests or nonparametric equivalents where appropriate. PK parameters are presented descriptively. A two-sided p- value <0.05 was considered statistically significant. No between-group comparisons or multiplicity adjustments were performed for this focused analysis.

[0154] A total of 30 participants received Slolron®, 15 healthy subjects and 15 subjects with IDA. All participants completed the in-unit PK assessment, and with at-home dosing. No protocol deviations affecting PK or biomarker interpretation were identified.

[0155] Following administration of a single 60-mg oral dose of ferritin-bound iron (Slolron®), measurable 24-hour pharmacokinetic responses were observed in serum iron and across additional biomarkers of iron metabolism in both healthy participants and those with iron deficiency anemia (IDA) (Table 14).Attorney Docket No. 61130-3PCT

[0156] Table 14: Acute Pharmacokinetics of Slolron®PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter - IRONHealthy Slolron 95.11 630.568 1098.201 3.00 (0.00-24.00) IDA Slolron 116 806.182 1350.262 6.00 (2.00-24.00) PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter - UIBCHealthy Slolron 389.8 3977.19 8191.561 1.00 (0.00- 24.00) IDA Slolron 412.2 3834.362 7901.322 1.00 (0.00- 24.00) PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter - FERRITINHealthy Slolron 26.18 213.654 425.614 1.00 (0.00- 24.00)24.00 (0.00 - 28.03 261.518 536.918IDA Slolron 24.00) PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter TIBCHealthy Slolron 423.5 4604.874 9286.879 1.00 (0.00-24.00) IDA Slolron 448 4640.343 9251.383 1.00 (0.00-24.00) PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter - TRANSFERRIN SATURATION %Healthy Slolron 27.98 181.52 312.914 6.00 (0.00 - 24.00) IDA Slolron 32.48 229.13 378.482 6.00 (2.00 - 24.00) PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter - TRANSFERRINHealthy Slolron 290.2 2703.871 4802.642 2.00 (0.00 - 24.00) IDA Slolron 317.2 3214.357 6511.437 6.00 (0.00 - 24.00) PK Parameter Cmax AUC 0-12 hr AUC 0 - 24 hr Tmax Test Parameter - HEPCIDINHealthy Slolron 11.45 113.874 229.063 6.00 (1.00- 24.00)IDA Slolron 8.607 58.281 116.881 1.00 (0.00- 24.00)

[0157] In healthy participants, mean serum iron reached a Cmax of 95.1 μg / dL with corresponding AUC 0–12 and AUC 0-24 values of 630.6 and 1098.2 μg·h / dL, respectively.Median Tmax occurred at 3 hours (range 0-24). In the IDA cohort, higher systemic exposure was observed, with a mean Cmax of 116.0 μg / dL, AUC 0–12 of 806.2 μg·h / dL and AUC 0-24 of 1350.3 μg·h / dL, and a later median Tmax of 6 hours (range 2-24).

[0158] Transferrin saturation (TSAT) showed a parallel pattern. In healthy participants, mean Cmax was 28.0%, with AUC 0-12 and AUC 0-24 values of 181.5 and 312.9 %-h, respectively, and a median Tmax of 6 hours. IDA participants demonstrated higher overallAttorney Docket No. 61130-3PCTexposure, with a TSAT Cmax of 32.5% and corresponding AUC 0 12 and AUC 0-24 values of 229.1 and 378.5 %-h, respectively. Median Tmax was 6 hours.

[0159] Unsaturated iron-binding capacity (UIBC) declined transiently following dosing, consistent with increased iron availability. In healthy participants, mean UIBC Cmax (lowest value) was 389.8 μg / dL with AUC 0–12 and AUC 0-24 values of 3977.2 and 8191.6 μg·h / dL, respectively; IDA participants demonstrated similar exposure magnitudes (Cmax 412.2 μg / dL; AUC 0-24 7901.3 μg·h / dL), with median Tmax at 1 hour in both groups.

[0160] Total iron-binding capacity (TIBC) showed modest variability over the 24-hour interval. In healthy subjects, mean Cmax was 423.5 μg / dL and AUC 0-24 was 9286.9 μg·h / dL; in IDA participants, mean Cmax was 448.0 μg / dL with AUC 0-24 of 9251.4 μg·h / dL. Median Tmax occurred at 1 hour for both cohorts.

[0161] Serum ferritin concentrations demonstrated small- magnitude fluctuations over 24 hours. In healthy participants, mean Cmax was 26.2 ng / mL with AUC 0-24 of 425.6 ng·h / mL and a median Tmax of 1 hour. IDA subjects exhibited slightly higher exposure (Cmax 28.0 ng / mL; AUC 0-24536.9 ng·h / mL), with a later median Tmax of 24 hours.

[0162] Serum transferrin concentrations remained within expected physiological ranges. In healthy participants, mean Cmax was 290.2 mg / dL with AUC 0-24 of 4802.6 mg·h / dL and a median Tmax of 2 hours. In IDA subjects, mean Cmax was 317.2 mg / dL with higher cumulative exposure (AUC 0-24 6511.4 mg·h / dL) and a later median Tmax of 6 hours.

[0163] Hepcidin exhibited modest and variable changes across the 24-hour window. In healthy participants, mean Cmax was 11.45 ng / mL with AUC 0-24 of 229.1 ng·h / mL and median Tmax of 6 hours. In the IDA cohort, both peak concentration (8.61 ng / mL) and overall exposure (AUC 0-24 116.9 ng·h / mL) were lower, with a median Tmax of 1 hour.

[0164] In healthy subjects receiving Slolron®, mean serum iron increased from 48.7 ± 26.0 μg / dL at baseline to 55.1 ± 28.5 μg / dL at the end of the study, representing a modest, non-statistically significant within-group change. Ferritin concentrations and hemoglobin values remained within the normal range, with no evidence of iron overload or dysregulated iron homeostasis. See Table 15.

[0165] Table 15: Changes in Iron Metabolism BiomarkersChange FromGroup Baseline End of Study Baseline P -ValueTest Parameter - IRONHealthy Slolron 48.7400 55.1308 9.9538 0.3759IDA Slolron 42.6600 75.6357 33.9786 0.0016Test Parameter - UIBCAttorney Docket No. 61130-3PCTHealthy Slolron 351.2000 350.6000 -7.9538 0.7413IDA Slolron 352.3000 339.8000 -17.8643 0.2731Test Parameter - FERRITINHealthy Slolron 20.9733 37.0308 15.2385 0.0179IDA Slolron 21.9333 45.3429 25.4357 0.0036Test Parameter - TIBCHealthy Slolron 400.0000 405.7000 2.0000 0.9245IDA Slolron 394.9000 415.5000 16.1143 0.2463Test Parameter - TRANSFERRIN SATURATION %Healthy Slolron 13.1860 14.0846 1.8200 0.5146IDA Slolron 11.5620 18.7429 7.5693 0.0018Test Parameter - TRANSFERRINHealthy Slolron 220.2000 289.9000 78.6462 0.0076IDA Slolron 263.7000 309.9000 52.1500 0.0230Test Parameter - HEPCIDINHealthy Slolron 8.4820 4.1323 -4.3777 0.1391IDA Slolron 5.5793 5.6629 -0.0693 0.9775

[0166] In contrast, subjects with IDA demonstrated larger directional improvements across multiple biomarkers of iron metabolism. Mean serum iron increased from 42.7 ± 19.5 μg / dL at baseline to 75.6 ± 30.4 μg / dL at end of study. Ferritin concentrations increased over the 30-day period, and hemoglobin values showed early upward trends consistent with initial iron repletion. While the study was not designed to detect full correction of iron deficiency within 30 days, these changes provide an early signal of biological response.

[0167] Across both cohorts, hepcidin concentrations did not demonstrate disproportionate acute or chronic elevations, suggesting that ferritin-bound iron delivery did not elicit excessive regulatory suppression of iron absorption over the dosing period.

[0168] Slolron® was well tolerated in both healthy and IDA participants. No serious adverse events were reported, and no participants discontinued due to gastrointestinal intolerance or laboratory abnormalities. Clinical safety parameters remained within acceptable limits throughout the study.

[0169] This study characterized the acute (0-24 hours) and short-term (30-day) biological and pharmacokinetic responses to plant-derived ferritin-bound iron in healthy women and women with IDA. Following a single 60 mg oral dose, both groups exhibitedAttorney Docket No. 61130-3PCTmeasurable, time-dependent increases in serum iron, with moderate peak concentrations and sustained systemic exposure over 24 hours.

[0170] Participants with IDA showed greater systemic iron exposure than healthy participants, reflecting normal physiological regulation that enhances iron absorption during deficiency. Importantly, ferritin-bound iron did not provoke excessive hepcidin responses, suggesting improved compatibility with the body’ s regulatory mechanisms and potentially allowing more efficient iron utilization compared to ferrous salts.

[0171] Over the 30-day supplementation period, healthy subjects maintained stable iron biomarkers without evidence of iron overload, while participants with IDA demonstrated meaningful increases in serum iron, ferritin, and hemoglobin, indicating early restoration of iron status. These findings confirm that ferritin-bound iron is well tolerated, effectively absorbed, and capable of improving iron biomarkers while maintaining physiological regulatory control. Overall, the results demonstrate that plant-derived ferritin-bound iron provides sustained systemic iron availability and represents a physiologically compatible alternative to conventional iron supplements.

[0172] Although the present invention has been described in considerable detail with reference to certain preferred embodiments, other embodiments are possible. The steps disclosed for the present methods, for example, are not intended to be limiting nor are they intended to indicate that each step is necessarily essential to the method, but instead are exemplary steps only. Therefore, the scope of the appended claims should not be limited to the description of preferred embodiments contained in this disclosure. All references cited herein are incorporated by reference in their entirety.

[0173] Insofar as the description above discloses any additional subject matter that is not within the scope of the claims below, the inventions are not dedicated to the public and the right to file one or more applications to claim such additional inventions is reserved.

Claims

Attorney Docket No. 61130-3PCTWhat is claimed is:

1. A method of increasing systemic iron availability in a human subject, comprising orally administering to the subject a composition comprising plant-derived ferritin-bound iron in an amount effective to produce sustained systemic iron exposure characterized by measurable serum iron levels at least 24 hours after administration.

2. The method of claim 1, wherein serum iron levels remain elevated relative to baseline for at least about 24 hours following administration.

3. The method of claim 1, wherein peak serum iron concentration occurs between about 3 hours and about 12 hours after administration.

4. The method of claim 1, wherein administration produces sustained systemic iron exposure characterized by an increased area under the concentration-time curve (AUC) relative to ferrous sulfate.

5. The method of claim 1, wherein the ferritin-bound iron produces controlled systemic iron release without a rapid transient spike in serum iron concentration.

6. The method of claim 1, wherein iron absorption is regulated based on physiological iron status of the subject.

7. The method of claim 6, wherein iron-deficient subjects absorb more ferritin-bound iron than iron-replete subjects.

8. The method of claim 1, wherein the ferritin-bound iron is absorbed through receptor-mediated endocytosis.

9. The method of claim 1, wherein the ferritin-bound iron comprises a protein shell encapsulating iron biomineral.

10. The method of claim 1, wherein the ferritin-bound iron is derived from a legume.Attorney Docket No. 61130-3PCT11. The method of claim 1, wherein the ferritin-bound iron is administered once daily in an amount equivalent to about 5 mg to about 100 mg elemental iron.

12. A method of treating iron deficiency or iron deficiency anemia in a human subject, comprising orally administering plant-derived ferritin-bound iron in an amount effective to increase systemic iron availability, wherein administration produces a delayed peak serum iron concentration occurring at least about 3 hours after administration.

13. The method of claim 12, wherein the human subject has iron deficiency.

14. The method of claim 12, wherein the human subject has iron deficiency anemia.

15. The method of claim 12, wherein administration increases serum ferritin concentration in the subject.

16. The method of claim 12, wherein administration increases hemoglobin concentration in the subject.

17. A method of delivering iron to a human subject, comprising orally administering plant-derived ferritin-bound iron in an amount effective to increase systemic iron availability while producing a reduced hepcidin response relative to an equivalent dose of ferrous sulfate.

18. The method of claim 17, wherein the ferritin-bound iron produces reduced hepcidin induction relative to ferrous sulfate.

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