Patent Description:
Oral iron is often poorly absorbed and tolerated in mammals, and according to the World Health Organisation (WHO) iron deficiency affects more than two billion people in developed and developing countries. This can result in adverse effects on cognitive function, oxygen transport, metabolism and immune function.

Iron is predominantly supplemented orally as the ferrous (Fe <NUM>+) iron, which is absorbed actively in response to body need through the divalent metal transporter <NUM> (DMT-<NUM>), yet has poor oral bioavailability and tolerability. Ferric (Fe <NUM>+) iron is usually better tolerated from a gastrointestinal point of view but tends to have poorer bioavailability than ferrous iron. Ferrous iron continues to be the international gold standard for oral iron absorption and the only salt mentioned on the WHO essential medicines list is the ferrous salt. Ferrous sulfate is the best absorbed oral iron. However, it remains poorly absorbed. The pharmaceutical approach to poor oral iron absorption is to increase the dose. This, however, results in significant gastro-intestinal distress. Delayed release and/or gastroprotective formulations (for example enteric coated) with and without iron have been marketed but are long acknowledged to persons known in the art to be less bioavailable and, accordingly, are not recommended. See, e.g., <NPL>. Current forms of oral iron used for supplementation have significant limitations, helping to explain the high incidence of iron deficiency, the only nutritional deficiency prevalent in developing and developed countries. We have surprisingly shown that gastroprotective, palatable, formulations, for example microencapsulated formulations, of iron entrapped in denatured protein, for example denatured whey protein, can be generated with improved bioavailability over ferrous sulfate. These are particularly suitable for preparation of products for oral ingestion, for example food products or supplements, where available iron causes palatability and stability problems. However, suitable formulations tend to require high protein/iron ratios, tend to be bulky, are relatively expensive to produce and do not easily lend themselves to formulation of supplements in the form of an oral dose such as capsules or tablets.

Therefore one issue to be addressed is providing a formulation for oral delivery, for example a tablet or capsule, that provides good iron absorption in a low cost, non-bulky, product. Such a formulation must lend itself to being made in a scalable manner. Such a formulation should provide effective delivery of iron so that it is effectively increases available iron in the body. Desirably such a formulation does so even with a lower total dose administered. Desirably such a formulation has good gastric tolerability. Desirably such a formulation achieves reduced iron passage into the lower GI tract. Suitably such a formulation demonstrates reduced adverse effects compared with other iron formulations.

<CIT> discloses compositions of iron with one or more buffering agents between a pH of <NUM> and <NUM>, preferably <NUM> and <NUM>. The buffering agents are constituted to maintain or reduce the pH of the duodenal fluid in the proximal, mid and distal duodenum. It further relates to methods of improving the absorption of an iron compound or an active pharmaceutical compound within the small intestine. The compositions and methods are said to improve absorption or bioavailability.

<CIT> discloses a carbonate-bicarbonate-iron-whey proteolysate complex. <NPL>, discloses an iron-milk-protein complex which is formed in the presence of citrate phosphate solution.

<NPL>, discloses the impact of protein pre-treatment conditions on iron encapsulation of whey-protein cold-set gel particles.

<NPL>, discloses the enhancement of Fe2+ bio-accessibility using ascorbate as a buffer.

The invention provides a composition comprising iron, buffering agent and a carrier comprising denatured protein, wherein the composition releases at least <NUM>% of the total load of iron as ferrous iron over the course of one hour in simulated gastric fluid at pH <NUM>, and wherein the composition when placed in simulated gastric fluid at pH <NUM> buffers the pH to at least <NUM> after <NUM> minutes, and wherein the composition when placed in a simulated intestinal fluid at pH <NUM> buffers the solution to at most pH <NUM> after <NUM> minutes, and wherein the composition, when administered orally to a human, has a relative trough to peak ratio of serum iron over <NUM> hours of at least <NUM>% that of an equimolar dose of an orally administered immediate release ferrous sulfate composition;.

Compositions of the invention are suitable for administration to mammals including humans.

It is desirable that in a composition of the invention that the release of the iron and release of the buffering agent occurs in a mole ratio. For example in a <NUM>:<NUM> ratio. So, for example where the mole ratio is <NUM>:<NUM> the amount by weight of the buffering agent released may substantially match that of the amount by weight of the iron released.

A composition of the invention is predominantly an immediate release formulation characteristics. Surprisingly, even though it has immediate release characteristics, it is well tolerated and does not induce adverse side-effects of the type one would expect with compositions which release high amounts of iron. Other formulations, which are slower release are taught to be well tolerated because the dose is released slowly over time and allowing for better tolerance. These have the disadvantage of being poorly absorbed. A composition of the invention has gastroprotective characteristics.

A composition of the invention may release at least <NUM>% for example at least <NUM>%, for example at least <NUM>%, such as at least <NUM> % of its buffering agent over the course of <NUM> minutes in simulated gastric fluid at pH <NUM>.

A composition may have an iron: protein ratio, by weight, of <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM>.

A composition of the invention has a total iron content of <NUM>% to <NUM>% by weight, for example <NUM>%-<NUM>%.

A composition may have a buffering agent/pH modifier (for example acetate/acetic acid): protein ratio, by weight, of <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM>.

A composition may have a buffering agent/pH modifier (wherein the buffering agent comprises acetate and acetic acid): iron ratio, by weight, of <NUM>:<NUM> to <NUM>:<NUM>, such as <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM>.

A composition may be (substantially) amorphous. This has been confirmed by XRD.

The denatured protein may be at least <NUM>%, <NUM>% or <NUM>% denatured.

The denatured protein may contain at least <NUM>%, <NUM>% or <NUM>% denatured beta lactoglobulin.

The denatured protein may have been subjected to a divalent metal iron removal process.

The water content of the composition may be less than <NUM>% such as less than <NUM>%, or less than about <NUM>% by weight.

The composition of the invention may have a core, which comprises a denatured aggregated protein matrix.

The carrier may comprise a core and a skin, wherein the skin comprises a denatured aggregated protein. Optionally, the skin may further comprise a gelling agent.

The core may comprise a denatured aggregated protein matrix. Optionally, the denatured protein contains, excluding iron, less than <NUM> divalent metal ions per <NUM> protein, such as less than <NUM> divalent metal ions per <NUM> protein, for example <NUM> divalent metal ions per <NUM> protein.

A composition may have, when administered orally to a human, a Trough To Peak ratio of serum iron over <NUM> hours of at least <NUM>%, at least <NUM>%, at least <NUM>% or <NUM>% that of an equimolar dose of an orally administered immediate release ferrous sulfate composition.

The compositions are administered to subjects and the serum iron is measured in subjects who have been fasting. Fasting means fasting for <NUM> hours.

The composition, when administered orally to a human, may have a trough to peak ratio of serum iron over <NUM> hours of at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>%, at least <NUM>% that of an equimolar dose of an orally administered immediate release ferrous sulfate composition.

The composition releases more than <NUM> wt% of the total iron content as ferrous iron over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated gastric fluid at pH <NUM>; the composition may release more than <NUM> wt% of the total iron content as ferrous iron over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated gastric fluid at pH <NUM>; the composition may release more than <NUM> wt% of the total iron content as ferrous iron over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated gastric fluid at pH <NUM>; the composition may release more than <NUM> wt% of the total iron content as ferrous iron over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated gastric fluid at pH <NUM>; the composition may release more than <NUM> wt% of the total iron content as ferrous iron over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated gastric fluid at pH <NUM>; the composition may release more than <NUM> wt%, <NUM> wt%, <NUM> wt%, <NUM>% wt, <NUM>% wt or <NUM>% wt% of the total iron content over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated intestinal fluid at pH <NUM>; and/or the composition may release more than <NUM> wt% of the total iron content over the course of <NUM> minutes, or <NUM> minutes, or <NUM> minutes, or <NUM> minutes in simulated intestinal fluid at pH <NUM>.

The composition may further comprises a stabilizer, such as ascorbic acid, and ascorbates, for example it may be selected from L-ascorbic acid, sodium L-ascorbate, calcium L-ascorbate, ascorbyl palmitate (palmitoyl L-ascorbic), erythorbic acid (D-isoascorbic acid), and sodium erythorbate (sodium D-isoascorbate) or combinations thereof.

The iron : protein ratio may be <NUM>:<NUM> to <NUM>:<NUM>, for example <NUM>:<NUM> to <NUM>:<NUM>, such as <NUM>:<NUM> to about <NUM> to <NUM> such as about <NUM>:<NUM> to about <NUM>:<NUM>.

The composition may consist of particles having an average particle size of <NUM> microns or less, <NUM> microns or less, <NUM> microns or less, <NUM> microns or less, or <NUM> microns or less, or <NUM> microns or less or <NUM> microns or less, or <NUM> microns or less, or <NUM> microns or less or <NUM> microns or less.

In one embodiment, the iron in the composition comprises <NUM>% to <NUM>% ferrous iron by weight.

The composition is desirably stable with respect to ferrous iron content and microbiological burden, for at least <NUM> months when stored in a sealed container at accelerated storage conditions (<NUM> and <NUM>% Relative Humidity).

In one embodiment, the composition is stable with respect to ferrous iron content when stored in a sealed container at ambient conditions for at least <NUM> months.

The denatured protein comprises denatured whey protein, denatured whey protein isolate, denatured beta lactoglobulin, or combinations thereof.

In one embodiment of the invention the composition comprises a salt. For example a salt of a short chain fatty acid, for example a C<NUM> to C<NUM> fatty acid. A salt which is included in the composition of the invention may have a buffering effect, for example in solution. A salt which is included in the composition of the invention may be a pH modifier, for example when a composition of the invention is in solution.

In a composition of the invention the buffering agent/pH modifier is present in an amount (by weight) of greater than about <NUM>%, such as greater than about <NUM>%. It may be greater than about <NUM>%, for example greater than about <NUM>%, such as greater than about <NUM>%.

A composition of the invention can be formed from denatured protein, a buffering agent such as a salt, and iron, wherein the iron is encapsulated by the denatured protein. In such composition the denatured protein is desirably present in an amount from <NUM>% to <NUM>%; such as <NUM>% to <NUM>%, for example <NUM> to <NUM>% by weight based on the total weight of the composition.

In such a composition the buffering agent is present in an amount from <NUM>% to <NUM>% by weight based on the total weight of the composition.

In such a composition the iron is desirably present in an amount from <NUM>% to <NUM>%, for example <NUM>% to <NUM>% by weight based on the total weight of the composition The denatured protein is a protein selected from whey protein, denatured whey protein isolate, denatured beta lactoglobulin, a milk protein composition containing beta lactoglobulin, or a protein containing beta lactoglobulin, or pea protein, or combinations thereof. One suitable protein is a denatured whey protein.

The iron is provided as ferrous sulphate.

Desirably the denatured protein contains less than <NUM> Calcium per <NUM> of total protein, <NUM> Calcium per <NUM> of total protein, for example, less than <NUM> Calcium per <NUM> of total protein, for example less than <NUM> Calcium per <NUM> of total protein.

One suitable form of composition of the invention is a dried form.

The dried form of a composition of the invention will include denatured protein, a buffering agent such as a salt, and iron.

Desirably the dried form of the composition is formed from a liquid composition without separation of any material from the liquid. It is desirable that the liquid and the materials carried by it are dried together. So there is no separation such as filtration, centrifugation etc. So for example there is no removal of buffering agent before drying. So there is a mother liquid in which the composition is carried there is no separation from that mother liquid before drying, for example no separation of the gel and/or buffering agent.

It has been surprisingly found that retaining the amount of the buffering agent in the liquid form in the dry material formed from the liquid form results in a dry material that is more effective and better tolerated than other forms such as ferrous sulphate. Because the amount of the buffering agent is not depleted (or not substantially depleted) it is thought that not only does it have a buffering effect in the liquid form, it may play a role in the stability and bioavailability of the iron that is present in the dry material. For example it may protect the ferrous form of iron with respect to oxidation. Typically then the dry material will have a higher buffering agent, for example salt, for example sodium acetate, concentration.

It is desirable that neither the dried material nor the gel is subjected to washing. Accordingly, it is useful to use a process, which does not require a washing step. It is thought that washing the dried material may remove buffering agent, for example salt such as sodium acetate. This in turn can affect the release profile of iron from the composition. So too can separation of the gel from a mother liquid for subsequent drying.

The invention also relates to a process for producing a dry material for delivery of iron according to claim <NUM>; the process comprising:.

The invention also relates to the above composition for use in the treatment of iron deficiency.

Disclosed herein is also a process for producing a dry material for delivery of iron; the process comprising:.

While it may not be necessary a composition of the invention can comprise a gelling agent to form, or to assist in forming a suitable gel.

By subjecting the liquid containing gel particles to drying, for example spray drying, both the liquid and the gel particles are dried together. This contrasts with processes where the liquid (for example mother liquid) is first removed and then the remaining material is processed, for example it is formed into particles.

Use of the process means that material which would otherwise be removed with removal of the liquid, may end up in the dried material. This results in an advantageous compositional change in the dried material.

For example with a higher amount of the buffering agent, for example salt, for example sodium acetate, present in the dried material, this may raise the pH of the stomach providing gastro-protection. This may help preserve iron in the <NUM>+ state (see above). It may also reduce the pH of the duodenum, which may assist with absorption of the iron.

Where a process of the invention forms dried particles the dried particles may have an average particle diameter of <NUM> to <NUM> microns, or <NUM> to <NUM> microns, or <NUM> to <NUM> microns, or <NUM> to <NUM> microns.

Desirably subjecting the liquid containing the gel particles to drying comprises spray drying to form dried particles. It is possible to use other drying techniques such as using tray drying, vacuum drying, drum drying, UV drying controlled to a maximum of <NUM>, or <NUM>, or <NUM> or <NUM> or <NUM>.

Desirably a buffering agent is present and maintains a pH of <NUM> to <NUM>, or <NUM> to <NUM>, or <NUM> to <NUM>. The pH is maintained when the composition is in the liquid form.

The buffering agent is a sodium acetate/acid buffer system.

The dried particles contain on average an amount of <NUM>% to <NUM>% by weight based on the total weight of the composition by weight of iron.

The dried particles contain denatured protein and the iron is encapsulated by the denatured protein.

The denatured protein is denatured whey protein, denatured whey protein isolate, denatured beta lactoglobulin, or combinations thereof.

The denatured protein may optionally be a protein subjected to a divalent metal ion removal process.

The iron may be present in the liquid as predominantly ferrous iron.

Desirably the shearing action is carried out for a relatively small period of time. For example the liquid formulation may be subjected to a shearing action for up to <NUM>, or <NUM> or <NUM>, or <NUM> minutes.

It is desirable to allow the gel particles that are formed by the shearing action to increase viscosity and become more dense before they are subjected to drying. For example the liquid composition containing the gel particles may be allowed to stand for a minimum period of time, for example <NUM>-<NUM> minutes, for example <NUM>-<NUM> minutes, such as about <NUM> minutes. After standing, the liquid composition containing the gel particles are then subjected to drying. Again, this results in a dried material which has a better bioavailability than ferrous sulfate.

It will be appreciated that the shearing is applied in the wet phase and before drying rather than after dried material is formed.

It is desirable to carry out the drying process over a shorter period of time, for example <<NUM> minutes, or <<NUM> minutes, or <<NUM> minutes, or <<NUM> minutes, or <<NUM> minutes, or <<NUM> minute. It is desirable to carry out the drying process so that the material being dried is exposed to an elevated temperature <NUM> to <NUM>, such as <NUM> to <NUM>, for example <NUM> to <NUM>.

Desirably the process of the invention forms particles with an average diameter in the range from <NUM>-<NUM> microns.

The lack of a washing step, which is a process feature but also increases the buffer capacity of the composition, also increases scalability, yield, reduces cost of goods and results in a composition which can be rapidly dried for example spray dried.

A composition of the invention achieves at least as good bioavailability as a formulation with > three times the dose level of iron.

Achieving good bioavailability with a lesser dose allows better tolerability even though the product is more immediate release.

For example as shown in <FIG>, a composition of the invention with an iron dose of <NUM>, outperformed a formulation sold under the name Tardyferon™ which has an <NUM> iron dose of ferrous sulphate.

In the present invention sodium acetate may be used as a buffering agent. It is postulated that sodium acetate forms a buffering agent on exposure to protons from acetic acid, another organic acid (e.g. butyric acid, ascorbic acid) or from protons present in gastric or intestinal fluid. This results from the equilibrium between sodium acetate and acetic acid. It is thought that it may form a buffer in the stomach at a pH range of <NUM> to <NUM>. It is thought that this buffering effect may contribute to the bioavailability of iron. Furthermore it is postulated that even though acetic acid may be present in the liquid composition from which the dried material is formed, much of it may evaporate during drying. When the material is exposed to protons in the low pH environment of the stomach, the pH rises providing gastroprotection. This local pH buffering action may extend into the intestine where the intestinal pH is maintained at a lower level than in the case of ferrous sulfate alone, preserving iron II for uptake at the DMT-<NUM>.

Furthermore, sodium acetate (and indeed acetic acid) is a food grade ingredient.

Compositions containing iron, buffering agent and denatured protein have been prepared that are capable of increasing serum iron in a subject. For example, spray dried microbeads have been prepared containing iron entrapped within a protein matrix and unbound iron in a buffered composition that provides a gastroprotective effect, preserves iron in the more available Fe<NUM>+ form and improves iron bioavailability in humans relative to previously known vehicles for delivering iron to a subject. This is achieved with small particle sizes, for example less than <NUM> microns.

As used herein, the term "calcium-depleted" or "decalcified" or "at least partially subjected to divalent metal ion removal" shall refer to protein raw material that has undergone a divalent metal ion removal process, including but not limited to the removal of calcium. Preferably, a decalcified protein comprises less than <NUM> calcium per <NUM> protein, less than <NUM> calcium per <NUM> protein, less than <NUM> calcium per <NUM> protein, less than <NUM> calcium per <NUM> protein, or only trace amounts of calcium. Alternatively, a decalcified protein may contain (excluding iron) less than <NUM>% divalent metal ions (w/w), less than <NUM>% divalent metal ions (w/w), less than <NUM>% divalent metal irons (w/w), or only trace amounts of divalent metal ions. There are standard methods of de-calcification of protein, apparent to those skilled in the art, including (a) acidification with dialysis and/or ultrafiltration and/or diafiltration, and or (b) using calcium chelating/sequestering agent(s) and/or (c) using cation exchange methods.

The term "protein-based carrier" as used in this specification should be taken to mean a substance at least partially derived from a protein-based source that is combined with a form of iron into a composition. The carrier may be used to render the composition suitable for its intended purpose. The purpose may be the effective delivery of iron to a mammalian subject. The protein carrier may provide advantages to the composition. Examples of such advantages include, but are not limited to, providing an advantageous modified iron-release profile to the composition, conferring additional anti-oxidative effects to the composition, reducing the level of gastrointestinal discomfort resulting from administration of the composition, and improving the level of iron uptake.

As used herein, the term "denatured protein" means a protein that is at least partially denatured, i.e., at least <NUM>% denatured.

As used herein "encapsulation" or "entrapped" means a process involving the complete envelopment (entrapment) of pre-selected material(s) within a matrix (usually referred to as a bead or sphere or microbead) or a core-shell capsule (usually referred to as a capsule), to give particles ranging from a few hundred nanometers up to a several centimeters in size.

"Bound iron", as used herein, refers to iron that is not easily washed off and "unbound iron" can be easily washed off. These terms are not intended to imply covalent or ionic bonding.

As used herein, the term "largely amorphous" means absence of evidence of short range order in the XRD associated with crystallinity. In other words, low crystallinity. - See, e.g. <FIG>.

As used herein, an "amorphous" substance includes a largely amorphous substance.

A capsule is made up of a defined and distinctive core (consisting of the encapsulated material) and shell part which are separated from each other. In preferred embodiments a microbead is a spherical structure which has (encapsulated) material distributed throughout the structure (i.e., a matrix). A microbead may have a surface layer ("skin") having the same composition as the interior but with different structure and chemical properties to the interior. The skin thickness and structure may influence microbead properties and behaviour - for example, swelling, pliability and payload diffusion.

Preferred embodiments of the invention reduce adverse effects in the stomach by modulating gastric pH unlike ferrous sulfate (<FIG>). This provides a way of maintaining iron intake without experiencing the adverse effects of medicinal products. It provides a way of more effectively maintaining iron intake using a supplement without other adverse effects, such as poor palatability associated with iron intake.

Thus, in one embodiment, the invention provides a preparation of microbeads comprising discrete microbeads, in which the microbeads comprise iron and denatured protein as described in the appended claims. In one embodiment, iron is entrapped within a denatured protein matrix core. Optionally the beads may also contain a gelling agent, such as a complex carbohydrate, such as alginate, or a protein, such as gelatin.

Optionally the beads may contain a glidant to help with processing. Suitable glidants include leucine, magnesium stearate, colloidal silicon dioxide, starch and talc and combinations thereof.

Additional iron loading can be achieved by applying a negative charge on the surface of a microdroplet prior to curing in an iron solution or by varying the curing temperature or level of protein denaturation or by incorporating substances such as vitamin C (ascorbic acid) known to chelate iron.

In one embodiment, the protein in the protein matrix has been subjected to a divalent metal ion removal process that results in calcium depletion.

Suitably, the protein in the protein matrix comprises whey protein, another milk protein composition containing beta lactoglobulin, or pea protein. Preferably the protein is denatured whey protein or calcium depleted denatured whey protein.

The microbeads comprise <NUM> to <NUM>% iron.

The percent iron can be estimated by instrumental or colorometric methods following digestion of the microbeads. Total residual inorganic content which reflects iron in calcium depleted microbeads can be estimated by high temperature thermogravimetric analysis. The microbeads have a ratio of iron:protein ranging from about <NUM>:<NUM> to about <NUM>:<NUM>.

The iron in the microbeads comprises ferrous (II) iron, which is derived from ferrous sulfate.

Preferably, the microbeads comprise acetate, citrate, phosphate, or ascorbate counterions. In preferred embodiments, these ions improve stability by reducing oxidation of the ferrous iron and/or improves release characteristics.

The invention also provides the above composition for use in a method for treating or preventing iron deficiency, the method comprising the steps of administering a composition according to the invention (preferably microbeads) to the mammal.

A composition according to the invention can be administered by any delivery vehicle known in the art. A preferred embodiment is an edible formulation, such as a powder (such as infant formula), prenatal vitamin formulation, multivitamin formulation, supplement, chewable supplement, gummy, food (such as chocolate or fat/oil), beverage, animal feed, tablet, capsule, or suspension. Lower-palatability embodiments are preferably in the form of capsules or coated tablets.

Compositions of the invention are preferably administered at a dosage sufficient to deliver an effective amount. One of ordinary skill in the art can determine the needs of a particular subject and take into account the bioavailability of the composition of the invention to determine an appropriate dosing regimen.

The composition is prepared by the following process:.

Drying may be done in an oven at <NUM>-<NUM>, preferably at about <NUM>. Alternatively, drying can be done at lower temperatures, such as room temperature, under vacuum. Preferably the drying is performed under an atmosphere of nitrogen or argon.

In another embodiment, drying occurs between <NUM> and <NUM>, between <NUM> and <NUM>, or at room temperature. In some embodiments, the step of drying may be performed under atmospheric pressure. In other aspects of some embodiments, the step of drying may be performed in at least a partial vacuum.

In aspects of some embodiments, the drying step results in the loss of between <NUM>% and <NUM>% of total weight of the composition, or between <NUM>-<NUM>% of total weight of the composition.

Drying can be performed in a rotating drum dryer under vacuum to reduce exposure to atmospheric oxygen while keeping particles in a constant motion to prevent sticking of the drying particles. Other techniques used for drying include using a vibrational fluidized bed dryer or rotary evaporator devices, which allow drying under controllable atmospheric conditions will keeping the particles in motion, or spray drying to achieve rapid drying of particles. Drying can also be performed by supplying a constant airflow or nitrogen flow over the microbeads.

In one embodiment, the invention relates to a preparation of microbeads in which the microbeads comprise a polymerized matrix formed from denatured calcium depleted protein having iron microencapsulated and/or entrapped within the matrix.

Typically, the microbeads have a generally spheroid shape. In some embodiments the mean diameter is <NUM> microns or less, <NUM> microns or less, <NUM> microns or less, <NUM> microns or less, or <NUM> microns, or less than <NUM> microns. In some embodiments, the particle size distribution is narrow.

In some embodiments particles have an average diameter of between <NUM> and <NUM> microns. The particles may be in the form of beads with a particle size between <NUM> and <NUM> microns, between <NUM> and <NUM> microns, between <NUM> and <NUM> microns, or between <NUM> and <NUM> microns in diameter. In some embodiments particles have an average diameter of between <NUM> and <NUM> microns, between <NUM> and <NUM> microns, between <NUM> and <NUM> microns, or between <NUM> and <NUM> microns in diameter. In some embodiments beads over a certain size may be preferable because they may display better flow characteristics, reducing the likelihood of aggregation during handling and the need for the use of an anti-caking agent or the like. Alternatively, the particles may be nanoparticles with a size below <NUM> microns.

The composition could comprise particles per se, or the composition could comprise the end result of such particles that have undergone one or more additional processing steps. This can be advantageous because in use, the protein may form a protective coating around the outside of the bead. This may result in a staged-release profile.

The microbeads of the invention are preferably dried rapidly, at elevated temperature, under vacuum, or in a nitrogen atmosphere. The resulting microbeads (following drying) preferably have <<NUM> % moisture as indicated by thermogravimetric analysis (see for example <FIG>).

One embodiment of the invention is a composition comprising iron, buffer and a carrier comprising denatured protein. The iron in the composition preferably comprises ferrous iron. The denatured protein comprises whey protein, whey protein isolate, beta lactoglobulin, or combinations thereof. Preferably, the denatured protein is at least <NUM>% denatured. In one embodiment, the denatured protein contains at least <NUM>% denatured beta lactoglobulin. The iron : protein ratio, by weight, is about <NUM>:<NUM> to about <NUM>:<NUM>.

The moisture content of the composition is less than <NUM> % by weight, less than <NUM>% by weight, about <NUM>-<NUM>%, about <NUM>-<NUM>%, or about <NUM>-<NUM>%.

In one embodiment, the composition comprises a stabilizer, such as Ascorbic acid, or Ascorbate (Sodium ascorbate, Calcium ascorbate, Fatty acid esters of ascorbic acid), Tocopherols (Alpha-tocopherol, Gamma-tocopherol, Delta-tocopherol), Propyl gallate, Octyl gallate, Dodecyl gallate, Erythorbic acid, Sodium erythorbate, Tertiary-butyl hydroquinone, Butylated hydroxyanisole (BHA), Butylated hydroxytoluene (BHT), or combinations thereof.

In a preferred embodiment, the composition is more palatable than commercially available iron formulations, such as ferrous sulfate in acidified water.

In a preferred embodiment, the composition is stable in that its dissolution profile at pH <NUM> and pH <NUM> changes less than <NUM>%, less than <NUM>%, less than <NUM>%, less than <NUM>% or is substantially unchanged with respect to iron II release for at least <NUM> months, preferably at least <NUM> years, when stored in a sealed container at ambient conditions. In a preferred embodiment, the composition is stable with respect to microbiological burden for at least <NUM> months, preferably at least <NUM> years, when stored in a sealed container at ambient conditions. Stability with respect to microbiological burden means the composition is "free of objectionable microorganisms", as that phrase is interpreted by FDA of <NUM> CFR <NUM>. Preferably, this includes a Total Viable Count with a Maximum Tolerable amount of <NUM><NUM> cfu/<NUM>, Total Yeast and Moulds Maximum Tolerable <NUM><NUM> cfu/<NUM>, and an absence of E-Coli.

In preferred embodiments, the composition is in the form of microbeads for oral administration. Preferably, after oral administration the incidence of constipation, as assessed using the Bristol Stool Scale (described herein), is reduced by at least <NUM>% and/or the incidence of nausea, as assessed using the modified Gastrointestinal Symptom Rating Score (described herein), is reduced by at least <NUM>%.

The term "calcium depleted" as applied to a composition should be understood to mean that the composition comprises less than less than <NUM> divalent metal ions (such as calcium) per <NUM> protein, such as less than <NUM> divalent metal ions per <NUM> protein, for example less than <NUM> divalent metal ions per <NUM> protein. In some embodiments, the composition contains less than <NUM>% or only trace amounts of divalent ion/calcium measured by standard methods.

The microbeads of the invention comprise (as a dry weight %) <NUM>-<NUM>% denatured, optionally calcium depleted, whey protein or whey protein isolate; and <NUM>-<NUM>% iron.

Methods for denaturing whey protein will be known to those skilled in the art, and include heat denaturation and pressure-induced denaturation. In one embodiment of the invention, the whey protein is heat denatured at a temperature of <NUM> to <NUM>, preferably about <NUM>. The whey protein is heated at a temperature of greater than <NUM> for more than <NUM> minutes. Usually, the whey protein is agitated during denaturation. Several methods for monitoring the unfolding/denaturation and formation of soluble oligomers will be known. These include dynamic light scattering and size exclusion techniques. It is useful to monitor the extent of thiol exposure in whey protein solutions using <NUM>,<NUM>'-dithiobis-(<NUM>-nitrobenzoic acid) or DTNB which produces coloured adducts on reaction with exposed thiols. In a preferred embodiment, the extent of denaturation of the protein or beta lactoglobulin is greater than <NUM>% or greater than <NUM>%, which can be measured using DTNB.

In some embodiments, the protein employed in the process of the invention has at least <NUM>%, <NUM>% or <NUM>% protein content (on a moisture, carbohydrate and fat free basis).

Suitably, the concentration of the at least partially denatured protein solution/suspension is from <NUM> to <NUM>%, preferably from <NUM> to <NUM>%, and ideally from <NUM> to <NUM>% (w/v). Typically, the protein is whey protein, ideally, the suspension is passed through a series of filters having a gradually decreasing pore size.

In one embodiment, the ferrous iron solution has a pH of less than <NUM> or less than <NUM>.

The gelling solution is typically free of calcium ions. The gelling solution has a sodium concentration of <NUM>-<NUM> or typically <NUM>-<NUM>. Suitably, the solution has an organic acid concentration of <NUM> to <NUM>, typically <NUM> to <NUM>, and ideally about <NUM>. Typically, the solution has a pH of <NUM> to <NUM>, suitably less than <NUM>. Generally, the solution has a temperature of <NUM>-<NUM>, typically about <NUM>. Typically, the acidic gelling solution comprises a surfactant to prevent or inhibit agglomeration of the formed microbeads. Suitably, the surfactant is a polysorbate surfactant, ideally Tween <NUM>. The gelling solution may contain a glidant such as leucine or magnesium stearate. On mixing the denatured protein solution and the acidification/gelling solution, a hydrogel network forms immediately. This is broken up by high shear mixing forming hydrogel particulates which further cure in the acidic environment.

Suitably, the formed microbeads are subject to an extended curing period in the gelling solution, for a period of at least <NUM> minutes (after gelation), and preferably for a period of at least <NUM> minutes. In a preferred embodiment of the invention, the formed microbeads are cured for a period of time from <NUM> to <NUM>, <NUM> to <NUM>, or <NUM> to <NUM> minutes. Ideally, the curing solution is agitated during the curing process.

The microbeads of the invention are typically capable of surviving intact during passage through the mammalian stomach and capable of releasing the ferrous iron in the gastrointestinal tract distally of the stomach, for example in the small intestine. The term "surviving intact in the stomach" means that the microbeads are resistant to gastric and peptic break-down in the mammalian stomach during gastrointestinal transit.

A preferred method of producing the microdroplets is a prilling by vibration technique, in which the denatured calcium depleted protein and iron salt are prepared separately and not mixed until just prior to or during extrusion through a nozzle and laminar break-up of the extruded laminar jet is induced by applying a sinusoidal frequency with defined amplitude to the nozzle with defined aperture size. Examples of vibrating nozzle machines are the ENCAPSULATOR (BUCHI Labortechnik AG, Flawil, Switzerland), a machine produced by Nisco Engineering AG, or equivalent scale-up version such as those produced by BRACE GmbH and the like.

Typically, the nozzle has an aperture of between <NUM> and <NUM> microns, preferably between <NUM> and <NUM> microns, suitably <NUM> and <NUM> microns, and ideally about <NUM> microns.

Suitably, the frequency of operation of the vibrating nozzle is from <NUM> to <NUM>,<NUM>. Optionally an electrostatic potential is added to the droplet, wherein the electrostatic potential between nozzle and curing solution is typically <NUM> to <NUM> V. Suitably, the amplitude is from <NUM>. 7kV to 7kV. Typically, the falling distance (from the nozzle to the acidification bath) is less than <NUM>, preferably less than <NUM>, suitably between <NUM> and <NUM>, preferably between <NUM> and <NUM>, and ideally about <NUM>. The flow rate of suspension (passing through the nozzle) is typically from <NUM> to <NUM>/min; an ideal flow rate is dependent upon the nozzle size utilized within the process.

In one embodiment, the process involves a step of monitoring the size of the initial microbeads generated for suitability.

Suitable compositions include comestible products such as food products and beverages, and food supplements in any form, for example unit dose products, powders, and the like. Typically food products include health drinks, yoghurts and yoghurt drinks, health bars, and the like. The composition may be a component of a formulation which is edible and orally active, e. g as an infant formula powder, prenatal vitamin, multivitamin, supplement, chewable supplement, gummy, food, beverage, animal feed, tablet, capsule, or suspension.

The preparation of microbeads of the invention may be provided in a dried form, for example a spray-dried, drum dried, dehydrated, or freeze dried form, or they may be provided as a suspension is a suitable solvent, for example water.

Denatured calcium depleted whey protein isolate (WPI) is preferable for producing microbeads of the invention. Whey protein concentrate (WPC) is also a possible encapsulation material.

One aspect of this technology involves the use of denatured calcium depleted whey protein isolate /concentrate. In some embodiments, reducing the divalent metal content of protein raw material reduces spontaneous gelation of the protein solution during processing, enhances its iron binding characteristics and reduces calcium release following administration to mammals, therefore enhancing iron uptake. Calcium inhibits iron uptake through DMT-<NUM>.

Dried calcium-depleted WPI is suitably dissolved in the optimum composition for iron microencapsulation. Calcium depleted whey protein isolate (WPI) can be initially denatured at appropriate environmental conditions (pH, salt, solid concentration) to enable the production of a soluble dispersion of protein aggregates suitable for extrusion and encapsulation in the presence of sodium acetate and ferrous sulfate. This process can be used to stabilize ferrous compounds in the matrix network of whey protein micro-spheres. This process occurs instantaneously when whey protein hydrogel solution comes into optimal conditions of electrolyte concentration, pH, agitation and temperature. Ferrous and sulfate ions in the curing solution can aid curing and allow iron uptake into the bead through diffusion and entrapment.

The preparation of calcium depleted whey protein (e.g., WPI) to form ferrous encapsulation material typically involves:.

The calcium depleted denatured protein suspension can be extruded through a concentric nozzle with a ferrous sulfate solution into a curing solution containing acetic acid/sodium acetate (<NUM>-<NUM>) buffering system with a pH <NUM>-<NUM>, with surfactant and continuous agitation to reduce coalescence/aggregation at high flow rates. It will be understood that bringing the pH of the denatured protein solution close to its isoelectric point ("PI") will promote aggregation by reducing repulsive coulombic forces.

A number of techniques can be used to obtain the microbeads of the invention. For simplicity the methods can be categorized as mechanical, chemical or physicochemical processes and include techniques such as: chemical; in-situ polymerization and interfacial polymerization; physiochemical; complex coacervation and mechanical; spray-drying and extrusion based methods.

Mechanical techniques are based on the principle of generating droplets from a polymer extruded through a nozzle (orifice) or from the breakup of a liquid jet. They work using mechanical means (i.e. cutting or vibration forces) to increase the normal dripping process at the orifice, or they break-up an extruded liquid stream produced by the polymer when it is passed through the nozzle. After production, the droplets are immediately solidified to spheres/capsules by either physical e.g. cooling or heating, or chemical means e.g. gelation. Several different mechanical based techniques can be used to encapsulate iron and other materials within whey protein matrices to produce particles with the final desired characteristics. Simple dripping is the oldest technology for the production of particles. The extrusion of a whey protein solution through an orifice (nozzle) at low velocities results in the extruded liquid sticking to the edge of the nozzle until gravitational force is high enough to overcome surface tension, resulting in the release of a drop. A small rise in the velocity increases the number of droplets formed, whilst further escalation amplifies droplet formation even further. After formation the droplets are immediately cured and the size of the resultant particles is mainly dependent on the orifice diameter. Produced beads usually have a size of more than <NUM>.

Spray-drying is a unit operation in which a liquid polymer is firstly atomized by a compressed air stream and subsequently dried by a separate hot gas current in a drying chamber, allowing the formation of the particles. A <NUM>-fluid nozzle is used in which air passes through an outside channel and atomizes the liquid stream passing through the inner channel. The liquid stream consists of a gel dispersion of microparticles of calcium depleted denatured whey protein, buffer and iron solution and is atomized into fine particles at the nozzle which are immediately dried by flash evaporation into whey protein beads entrapping the buffer and iron. The produced particles are collected using cyclone technology. This technique produces whey protein iron particles of between <NUM> - <NUM> microns. The dried particles can be further treated in additional curing solutions if required.

Two other techniques, which are known to persons versed in the art are three fluid nozzle techniques used in conjunction with a spray dryer and microfluidic devices.

One aspect comprises a composition comprising an amorphous preparation of iron salt associated with a protein-based carrier. The iron in the composition may comprise some ferric (Fe3+) iron. This may be advantageous because ferric iron, when delivered to the GI tract, may give rise to a reduced level of gastrointestinal discomfort compared to ferrous iron. Ferric iron is capable of undergoing reduction in the intestine to ferrous iron, the substrate for DMT1 activity. However, the amorphous preparation of iron salt associated with a protein-based carrier typically has at least <NUM>% ferrous (Fe2+) iron which facilitates adequate bioavailability via absorption mediated by intestinal enterocyte DMT-<NUM>. Furthermore, the ferrous iron release from the composition of the invention at low pH and in the presence of the components of gastric fluid such as pepsin, is limited in order to protect the stomach and limit nausea, vomiting and epigastric pain.

In one aspect, the composition may be formed by mixing an iron-containing composition with a protein-based composition, and by drying the resulting mixture. In another aspect, where the mixture comprises ferrous iron, the drying step may result in converting at least a portion of the ferrous iron in the mixture to an amorphous preparation of iron salt in the ferric form associated with a protein-based carrier. The drying process might make use of additional materials such as Silicon Dioxide to prevent "caking" of the composition during drying.

This dried composition may be advantageous over non-dry compositions (including gel preparations), which can be more variable and/or less stable on storage, especially with respect to oxidation, and inconsistent in their production, collectively presenting challenges for formulation, scale up and dose optimisation. Beads not subjected to heating/drying present additional formulation challenges due to their bulk. Furthermore, undried composition of iron present technical and cost challenges from a compatibility perspective if it is desired to incorporate iron into a multi-active supplement, for example a multivitamin and/or multi-mineral supplement.

In some embodiments, conversion of ferrous (<NUM>+) iron into ferric (<NUM>+) iron during production of the composition may be brought about during drying. In other embodiments, the conversion of ferrous (<NUM>+) iron into ferric (<NUM>+) iron can be limited during drying by anti-oxidative effects of the whey protein, by rapid drying (e.g. Spray Drying), by drying in an inert (e.g. nitrogen) atmosphere, and/or by incorporation of a stabilizer with anti-oxidative effects. This can include, but is not limited to, the following in whole or in part: beta-carotene and carotenoids; vitamin c; vitamin e; zinc; selenium; copper; manganese; astaxanthin; black pepper extract; co-enzyme Q10; lycopene; lysine based antioxidants, methylcobalamine; grape seed extract; lutein; ginseng; citrus bioflavonoids, orange peel extracts, green tea extract, ginko bilboa, spruline, wheat grass, barley grass, alfalfa, flax seed, banana leaf extract.

One embodiment of the invention is a method for making a composition comprising the steps of: preparing a buffered iron-containing composition; preparing a protein-based composition (preferably denatured, calcium depleted whey protein/beta-lactoglobulin); mixing said ferrous iron containing composition with said protein-based composition; and converting at least a portion of the iron content of the mixture into an amorphous preparation of iron salt associated with a protein-based carrier. The iron-containing composition may comprise ferrous iron. At least a portion of the iron content of the mixture may be converted into a largely amorphous preparation of ferric iron associated with a protein-based carrier.

In another embodiment the method may be further specified such that: the ferrous iron-containing composition is a solution; the protein-based composition is a suspension of protein-based material; and the mixing comprises extruding the suspension through a vibrating nozzle such that the suspension is extruded in the form of microdroplets, the microdroplets being extruded into a bath comprising the solution such that beads are produced, the composition comprising said beads.

In some embodiments the conversion is achieved during drying of the composition. If the drying process is carried out in air or in the presence of oxygen, it is believed that this drying process has the effect of oxidising at least a portion of the iron content such that it changes from a predominantly ferrous (<NUM>+) state to an amorphous preparation of iron salt associated with a protein-based carrier where a proportion of the iron is in a ferric (<NUM>+) state.

In another embodiment, a divalent metal ion is substituted for iron in a composition described herein. Such metal ions include zinc, manganese, copper, chromium, selenium, molybdenum, combinations thereof, or combinations thereof with iron. In certain embodiments the resulting beads have improved palatability (e.g. Iron sulfate, zinc sulfate).

WPI was treated with ion exchange resins to replace divalent (e.g. calcium) cations with monovalent cations.

The ferrous iron encapsulation system was prepared using the calcium-depleted WPI, which contains (per <NUM>) more than <NUM> elemental iron and up to <NUM> gram protein. A stock solution of whey protein solution (WPS) was prepared in a blade mixer or Ultra-Turrax in the presence of a surfactant in the range of <NUM>-<NUM>% (w/w) at pH range <NUM>-<NUM>. The solution is filtered through a <NUM> micron filter. Whey protein isolate (WPI) was subsequently heat-denatured at appropriate environmental conditions (pH <NUM>, ><NUM>; <NUM>-<NUM> % w/w protein content). Heat treatment was performed between <NUM>-<NUM> at pH in the range of <NUM>-<NUM>. Heat denaturation was performed under agitation (<NUM>-<NUM> rpm) to enable the production of a soluble suspension of protein aggregates. Heat denaturation was performed for a between <NUM> and <NUM> minutes to allow denaturation and exposure of hydrophobic sites.

After Protein Activation (i.e. heat denaturation), the solution of aggregates was rapidly cooled to room temperature. If necessary it can be stored overnight at <NUM> with constant agitation. <NUM> of the protein solution was used for production of microbeads of the composition.

The curing solution was prepared immediately before production as follows and involves preparation of two solutions (<NUM> <NUM> sodium acetate buffer pH <NUM> containing acetic acid and <NUM> <NUM> iron sulfate solution) prior to mixing and extrusion of the protein solution. Once both solutions are combined together to make <NUM> of curing solution, Tween-<NUM> and L-Ascorbic acid were added to the curing solution, this solution was then used to gel and precipitate the <NUM> of denatured whey protein prior to particle size reduction using a rotating stirrer at <NUM>,<NUM> RPM for <NUM> minute. The <NUM> sodium acetate buffer solution was prepared by dissolving <NUM> of anhydrous sodium acetate (MWt <NUM>/mol) in <NUM> Ultrapure water. After complete dissolution of the salt, add <NUM> glacial acetic acid slowly while stirring. Stir the buffer solution for at least <NUM> minutes and re-stir if left standing. <NUM> FeSO<NUM> solution was prepared by dissolving <NUM> iron (II) sulfate heptahydrate in <NUM> Ultrapure water. Both solutions were mixed together (<NUM> of each) to make up <NUM> of "curing solution". The solution was sealed to prevent evaporation in a LR-<NUM> and was heated to <NUM>. The solution pH was <NUM>. A total of <NUM> of Tween-<NUM> was added to the curing solution and mixed for at least <NUM> minutes. This was followed by the addition of <NUM> of L-ascorbic acid and again the solution was mixed for <NUM> minutes.

Gel formation was achieved by agitating the curing solution within the IKA LR-<NUM> and adding the denatured WPS (as prepared above) to the curing solution, making sure that the adequate agitation is performed to break up the gel as it is forming. Also the temperature of whey protein-curing solution is maintained at <NUM>. The WPS is added using a graduated cylinder slowly over a <NUM>-<NUM> period. If significant foaming of the whey protein-curing solution is occurring, reduce the agitation speed. Excessive foaming makes the spraying of the solution very difficult. A defoamer may also be added to the solution to reduce foaming. After all the WPS has been added continue agitating (blending) and allow the gel to sit in the curing solution for <NUM>. The D90 of the gel particles should be below <NUM> microns. This enables easier pumping of the particles through the <NUM>-Fluid nozzle and prevents clogging.

After <NUM> mins of curing of the gel suspension to a BUCHI B-<NUM> Mini Spray Dryer (lab-scale). The D90 of the gel particles should be below <NUM> microns. This enables easier pumping of the particles through the <NUM>-Fluid nozzle and prevents clogging. The Spray Dryer was used in standard open mode with a de-humidifier in place to treat the incoming drying air. The high performance cyclone was used to enable as much recovery as possible of the product. The following parameters were used as a starting point to spray the solution.

During the process, the inlet temperature was increased to <NUM>.

The resultant spheroid particles contained bound and unbound iron, provided a substantially immediate release profile (<FIG>) had a D90 <<NUM> (an SEM of a bead is shown in <FIG>), were amorphous (<FIG>) with an initial Loss on Drying of <NUM>% (<FIG>) reducing to <<NUM>% on further drying at <NUM> (<FIG>). These particles had <NUM>% w/w of iron. These particles had <NUM>% w/w of acetate. The FTIR traces shows the presence of characteristic sodium acetate peaks in <FIG>, in the <NUM> to <NUM>-<NUM>.

As above, the ferrous iron encapsulation system was prepared using the calcium-depleted WPI, which contains (per <NUM>) more than <NUM> elemental iron and up to <NUM> gram protein. A stock solution of whey protein solution (WPS) was prepared in a blade mixer or Ultra-Turrax in the presence of a surfactant in the range of <NUM>-<NUM>% (w/w) at pH range <NUM>-<NUM>. The solution is filtered through a <NUM> micron filter. Whey protein isolate (WPI) was subsequently heat-denatured at appropriate environmental conditions (pH <NUM>, ><NUM>; <NUM>-<NUM> % w/w protein content). Heat treatment was performed between <NUM>-<NUM> at pH in the range of <NUM>-<NUM>. Heat denaturation was performed under agitation (<NUM>-<NUM> rpm) to enable the production of a soluble suspension of protein aggregates. Heat denaturation was performed for a between <NUM> and <NUM> minutes to allow denaturation and exposure of hydrophobic sites.

The microbeads of the composition were then made by curing solution (<NUM> Ferrous Sulfate, <NUM> Sodium Acetate described above ) was placed into and IKA LR-<NUM> and heated to <NUM>. Approximately <NUM> of denatured whey protein solution (<NUM>%) was added over a <NUM> second period. Following addition of the whey protein solution and gel formation, the curing solution was agitated with a Turrax rotary-stirrer at <NUM>,<NUM> rpm for <NUM> minutes and the solution was cured for <NUM> minutes at low agitation speed (stirrer <NUM> RPM). The wet particles formed again were very small (Particle Size Results from Malvern shown) showing a bimodal pattern with D50 of <NUM> microns before tray drying and further size reduction at <NUM>.

Powder X-Ray analysis was performed using a Miniflex II Rigaku diffractometer with Ni-filtered Cu Kα radiation (λ=<NUM>. The tube voltage and tube current used were <NUM> kV and <NUM> mA respectively. Each sample was scanned over <NUM> theta range <NUM>-<NUM>° with a step size of <NUM> °/s. As can be seen from <FIG>, the powdered XRD trace shows that the powdered bead structure is essentially amorphous (Y axis is intensity and X axis is <NUM> theta scattering angle).

Thermogravimetric (TGA) of loss on drying of microbeads of the invention following spray drying alone (A) and following spray drying with further drying at <NUM>. Weighed, powdered samples (<NUM>-<NUM>) were analysed in open ceramic pans. For the TGA measurement a TA-Instruments Thermogravimetric Analyzer TGA-Q50 instrument was used with the following temperature program: sample heated to <NUM> (<NUM>/min) and <NUM> isothermic at <NUM> (<FIG>).

The infrared measurements were performed on a PerkinElmer Spectrum <NUM> FT-IR Spectrometer between <NUM>-<NUM>-<NUM> and using attenuated total reflection (ATR) sampling (<FIG>).

The scanning electron microscopy (SEM) images were recorded on a Zeiss Ultra Plus Field Emission SEM with a Gemini® column (Zeiss). The dry sample beads were placed on a conducting carbon tape without any further preparation or sample coating. Accelerating voltages between <NUM>-3kV was used to overcome the extensive discharge effect.

A solution of iron (II) sulfate in water (<NUM>) was serially diluted using pH <NUM> KCl buffer. Aliquots (<NUM>µl) of the diluted solutions were added to a <NUM>-well plate containing <NUM>µl of <NUM>,<NUM>-phenanthroline (<NUM>). The plate was read at <NUM> on a multiwall plate reader in order to construct a calibration curve. Dissolution samples were diluted ten-fold typically at pH <NUM> into phenanthroline (<NUM>) and the samples read rapidly under N<NUM> blanketing.

A <NUM> quantity of beads was transferred to a vial containing <NUM> HCl (<NUM>) and left overnight at room temperature. The resulting solution was shaken and then a <NUM>µl aliquot was transferred into <NUM>µl of <NUM> HCl. A 100µl aliquot of the diluted solution was added to a <NUM>-well plate containing <NUM> sodium thiocyanate (<NUM>µl). Absorbance was measured at <NUM> on a multiwell plate reader. The concentration of the iron III was estimated by reference to a series of iron III standard solutions.

An accurately weighed sample (approximately <NUM>) of microbeads was transferred into a three necked vessel into which had been placed <NUM> pH <NUM> buffers (containing <NUM> sodium bicarbonate, <NUM>/ml bile acid extract, <NUM>/ml pancreatin, adjust to pH <NUM> with <NUM> HCl) at <NUM>. Generally, at <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and occasionally at <NUM>, <NUM> time points, samples were taken for iron (II) and iron (III) measurement. For iron II measurement, <NUM>µl of the dissolution supernatant was diluted into <NUM>µl pH1. <NUM> buffer. For iron III measurement, a <NUM>µl aliquot of the dissolution supernatant was diluted to <NUM>µl in <NUM> HCl and left overnight at room temperature. After the final time point, all the buffer solutions were taken out and <NUM> <NUM> HCl was added to the flask and left overnight. The beads were fully dissolved overnight and <NUM>µl solution was added to <NUM>µl <NUM> HCl for total iron III level measurement.

An accurately weighed sample (approximately <NUM>) of microbeads was transferred into <NUM> of pH <NUM> buffer containing NaCl (<NUM>), sodium taurocholate (<NUM>), <NUM>/ml pepsin, and adjusted to pH <NUM> with <NUM> HCl at <NUM>. Samples were typically taken for Iron (II) and Iron (III) measurement at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>. For iron II measurement, <NUM>µl of the solution was removed and diluted into <NUM>µl pH <NUM> buffer. For iron III measurement, <NUM>µl of the solution was diluted to <NUM>µl in <NUM> HCl and left overnight at room temperature. After the <NUM> time point, all the buffer solutions were taken out and <NUM> <NUM> HCl was added to the flask and left overnight. The microbeads were fully dissolved after overnight. A <NUM>µl aliquot was added to <NUM>µl <NUM> HCl for total iron measurement and indirect estimation of residual iron after <NUM> dissolution.

The iron II and iron III dissolution methods were validated for accuracy and precision.

A sample of microbeads was crushed in a mortar and pestle or milled in a ball mill. A <NUM> sample was transferred to a glass vial equipped with a magnetic stirrer, to which was added <NUM> dilute aqueous HCl (<NUM>) which had been nitrogen sparged to remove oxygen. The suspension was heated to 50oC and then subjected to ultrasonication until the crushed beads dissolved. A <NUM> aliquot was removed under nitrogen and rapidly transferred for measurement of iron II using the phenanthroline method described above.

Samples (<NUM> approximately containing <NUM> Iron) were added to <NUM> of pH <NUM> buffer (containing NaCl (<NUM>), sodium taurocholate (<NUM>), <NUM>/ml pepsin). The pH value of the solution was twice within <NUM> mins.

Samples (<NUM> approximately containing <NUM> Iron) were added to10ml of pH <NUM> buffer (containing <NUM> sodium bicarbonate, <NUM>/ml bile acid extract, <NUM>/ml pancreatin). The pH value of the solution was twice within <NUM> mins.

Equimolar amounts of Ferrous Sulfate were also evaluated in the same solutions with the same procedure and the results are presented in <FIG>.

Subjects were healthy adult females and were in good health with serum ferritin > <NUM>µg/L. They gave written informed consent and were non pregnant. Each subject received to one of two oral treatment groups at a daily elemental iron dose of <NUM>: Ferrous Sulfate <NUM> elemental iron and ST1501 <NUM> elemental iron in a single dose, cross over evaluation. In a separate analysis, the product was compared as part of a case report to Tardyferon <NUM> elemental iron. The endpoint was Trough to Peak Ratio of fasting (><NUM> hours) serum iron <NUM> hours following administration of the investigational and test treatments.

A fasting blood sample (8mls) was collected at the screening visit, and a full blood count (FBC), Serum iron and ferritin was assessed. During the intervention days, blood was also collected at baseline (8mls), <NUM> hours (4mls) and <NUM> hours (4mls). Full Blood Count, serum iron, ferritin and iron binding capacity was measured at baseline, serum iron was assessed at <NUM>, and <NUM> hours and ferritin and iron binding capacity was also measured. All samples were shipped to an approved contract laboratory for analysis. A total of 24mls of blood was collected throughout the study.

In one embodiment of the invention, the maximum premix load of iron for mixing with <NUM>% whey protein isolate was <NUM>-<NUM> ferrous sulfate. In some embodiments, preprocessing of the protein-based material, solution pH and the form of iron used had an effect on the product. For example, in some embodiments, adequate hydration of the protein-based material was required and ferrous sulfate heptahydrate was found to be preferable to dried ferrous sulfate because of the better water solubility and purity.

In one embodiment of the invention, whey protein isolate (WPI) was dispersed in <NUM> sterile water <NUM>% w/v and left to hydrate for <NUM>-<NUM> hours at <NUM> under slight agitation (<NUM> rpm). The pH of the dispersion was adjusted to <NUM> using HCl. The pH adjusted dispersion was optionally filtered through successive filters and then optionally finally through Durapore®<NUM> HVLP. The protein dispersion was then heated to <NUM> (<NUM>-<NUM>)°C for <NUM> -<NUM> under agitation (<NUM> rpm). The dispersion was then cooled on ice and stored at <NUM> for <NUM>.

In some embodiments of the invention, the pH of the iron salt-containing curing solution (containing monovalent metal ions with buffer in the range <NUM> to <NUM>) was adjusted to between pH <NUM> and <NUM>. Ideally a pH of between <NUM> and <NUM> is used for the curing solution. Ferrous sulfate (<NUM> to <NUM>) was added to the curing solution and pH further adjusted. The solution was then heated to <NUM>. Optionally a low concentration surfactant was added. The solution was then maintained at <NUM>.

The gel beads are dried at <NUM> for <NUM> hours or at up to <NUM> for <NUM>-<NUM> hours to form dry, amorphous preparations of iron associated with protein beads. Thermogravimetric analysis is used to determine the water content of the amorphous iron. The beads are sampled (known weight) and iron content of beads confirmed per w/w dry bead for the batch using sodium thiocyanate method following dissolution in <NUM> HCl. The dry, amorphous iron-protein beads are sealed in an airtight container.

A standard sodium thiocyanate method was used to determine the total iron content of the protein beads and expressed as %w/w beads. Total iron was determined by treating approximately <NUM> beads with <NUM> of <NUM> HCl at <NUM> for two hours to fully dissolve the beads. Then solution was diluted to <NUM> times in <NUM> HCl. <NUM>µl of diluted solution was reacted with <NUM>µl <NUM> sodium thiocyanate. The concentration of the iron III ions was determined by measuring absorbance of the complex at <NUM> and comparing to the calibration curve. In addition to light microscopy, further image analysis was performed using a Leica TCS SP5 confocal scanning laser microscope (CSLM) for the purpose of microcapsule morphology assessment. The mean size distribution and D (v, <NUM>) (size at which the cumulative volume reaches <NUM>% of the total volume) was evaluated using fifty beads per batch, which were analysed using a bright-field light microscope at a magnification maximum ×<NUM>.

The dissolution profile of the beads was studied by incubating the beads in pH <NUM>, pH <NUM>, and pH <NUM> buffers at <NUM> degrees. The iron II and iron III levels were measured at <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> minute time points. Iron II level was measured by taking <NUM>µl of the solution at each time points into <NUM>µl water, Iron II ion was determined by the standard complexometric titration with <NUM> <NUM>,<NUM> phenanthroline by measuring absorbance of the complex at <NUM> and comparing to the standard curve. Iron II measurement was carried out with appropriate suppression of artifactual oxidation to iron III by performing analysis under a nitrogen atmosphere. For iron III measurement, <NUM>µl of the solution was diluted to <NUM>µl <NUM> HCl and left overnight at room temperature to oxide fully. Iron III content was determined using the standard sodium thiocyanate method described above.

An upper limit of approximately <NUM>% beta lactoglobulin - BLG - (<NUM>% denatured WPI equivalent to <NUM>% BLG) was used to avoid spontaneous gellification of the BLG/WPI. Bead production was performed using a curing solution comprising up to <NUM>-<NUM> sodium acetate along with up to <NUM>-<NUM> ferrous sulfate, with curing for <NUM> minutes. The gel beads produced contained between <NUM> and <NUM>% w/w iron and when dried using conditions ranging from <NUM> for <NUM> hours to <NUM> for <NUM> hours, the compositions had between <NUM> % w/w iron and <NUM>% w/w iron respectively.

The dry, amorphous iron-protein microbeads produced are durable and stable. It has been shown that dry, amorphous iron-protein microbeads left in ambient and accelerated stability storage conditions for several months show solid-state characterisation similar to the original beads and also perform as well as freshly made samples in terms of iron II release at pH <NUM>.

When the dry, amorphous iron-protein beads are dissolved in water, they absorb water within <NUM> minutes and a gel diffusion layer is formed surrounding the dry bead, which is responsible for the modified iron release profile.

Ground, freeze dried beads are much less effective in-vivo. Also, in-vitro dissolution of ground, poorly formed dry gel beads rapidly results in a more immediate release profile.

Known quantities of the beads containing approximately <NUM>-<NUM> of elemental iron were dissolved into <NUM> of buffered solution to ensure sink conditions with respect to the ferrous sulfate iron at pH <NUM> and maintained at <NUM> in a temperature controlled bath. The solutions were covered to prevent evaporation. At baseline and <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM> minute time points, <NUM> × <NUM>µL aliquots of the solution were removed for analysis of iron. One of the aliquots was immediately diluted into <NUM>µl water to measure the iron II content in the solution by the standard complexometric titration with <NUM>,<NUM> phenanthroline. The other aliquot was preserved for iron III measurement, where <NUM>µl of the solution was diluted to <NUM>µl <NUM> HCl and left overnight at room temperature to oxide fully. Iron III content was determined using the standard laboratory isothiocyanate method. Experiments were conducted in triplicate.

Powder X-Ray analysis was performed using a Miniflex II Rigaku diffractometer with Ni-filtered Cu Kα radiation (λ=<NUM>. The tube voltage and tube current used were <NUM> kV and <NUM> mA respectively. Each sample was scanned over <NUM> theta range <NUM>-<NUM>° with a step size of <NUM> °/s. As can be seen from <FIG> the XRD traces for the physical combination of whey protein and FeSO<NUM>. 7H2O in proportions similar to the composition of ST1501 (dry Fe2+ releasing beads) show the presence of peaks at scattering angles <NUM> theta (degrees) = <NUM>, <NUM>, <NUM>, <NUM>, <NUM> and <NUM> which are absent from ST1501, confirming that the ferrous sulfate composition is largely in an amorphous physical state.

It is important to note that intermediate gel beads are not stable with respect to oxidation and this is reflected in reduced release of ferrous iron (II) in dissolution media. In accordance with this, gel beads prepared for more than <NUM> hours have variable and poor performance clinically and are not scalable or commercially acceptable. Furthermore, these gel intermediates are prone to microbiological growth. ST1501 microbeads of the invention were found to be stable in that the dissolution profile at pH <NUM> and pH <NUM> was substantially unchanged with respect to iron II release for at least <NUM> months when stored in a sealed container at ambient conditions. For example, in one embodiment of the invention, when blister packed in a [<NUM>] hydroxyl propyl methyl cellulose (HPMC) capsule under ambient conditions and in a [<NUM>] HPMC capsule under further sealed in aluminium, under Nitrogen in a sealed chamber at room temperature, the composition released <NUM>% ± <NUM>% and <NUM>% ± <NUM>% of the iron II content released at baseline (set at <NUM>%) over <NUM> hour during dissolution experiments at pH <NUM> following long term storage. Furthermore, both compositions were free of objectionable microorganisms, including a Total Viable Count with a Maximum Tolerable amount of <NUM><NUM> cfu/<NUM>, Total Yeast and Moulds Maximum Tolerable <NUM><NUM> cfu/<NUM>, and E-Coli Absent.

Claim 1:
A composition comprising iron, buffering agent and a carrier comprising denatured protein, wherein the composition releases at least <NUM>% of the total load of iron as ferrous iron over the course of one hour in simulated gastric fluid at pH <NUM>, and wherein the composition when placed in simulated gastric fluid at pH <NUM> buffers the pH to at least <NUM> after <NUM> minutes, and wherein the composition when placed in a simulated intestinal fluid at pH <NUM> buffers the solution to at most pH <NUM> after <NUM> minutes, and wherein the composition, when administered orally to a human, has a relative trough to peak ratio of serum iron over <NUM> hours of at least <NUM>% that of an equimolar dose of an orally administered immediate release ferrous sulfate composition;
wherein the composition has a total iron content of <NUM>% to <NUM>% by weight;
wherein the iron is provided as ferrous sulphate;
wherein the water content of the composition is less than <NUM>, less than <NUM>%, or less than about <NUM>% by weight;
wherein the denatured protein comprises denatured whey protein, denatured whey protein isolate, denatured beta lactoglobulin, or combinations thereof;
wherein the buffering agent is present in an amount of from <NUM>% to <NUM>% based on the total weight of the composition;
wherein the buffering agent comprises sodium acetate;
wherein the composition consists of particles having an average particle size of <NUM> microns or less or <NUM> microns or less or <NUM> microns or less, as measured by laser diffraction particle size analysis;
wherein the iron is encapsulated by the denatured protein.