Ligand Modified Poly Oxo-hydroxy Metal Ion Materials,
Their Uses and Processes For Their Preparation
Field of the Invention
The present invention relates to ligand-modified poly oxo-
hydroxy metal ion materials and their uses, in particular
for nutritional, medical, cosmetic or biologically related
applications for example for the treatment of a deficiency
related to a component of the material or for the removal
of an endogenous substance capable of binding to the
material The present invention further relates to
processes for preparing the materials and optimising their
physico-chemical properties and their medical uses
Background of the Invention
Iron deficiency is the most common micronutrient
deficiency in the world today, affecting more than 4
billion people globally It is estimated that 2 billion
people - over 30% of the world's population - are anaemic
(WHO, http //www who int/nut/ida htm, accessed 20 December
2005) Iron deficiency is not a problem solely confined
to the developing world Epidemiological surveys
performed m European countries show that iron deficiency
concerns 10-30% of menstruating women and iron deficiency
anaemia (IDA) 1 5 to 14% (Hercberg et al , 2001, Goddard
et al , 2005) Iron deficiency anaemia can result in
decreased intellectual performance, decreased physical
capacity, alterations in temperature regulation,
alterations in the development of gestation, and
compromised immune and metabolic functions, all of which
impact upon quality of life and health economics (Edgerton
et al, 1979, Hercberg et al, 2001, Scholz et al, 1997)
The standard first line treatment for simple mild IDA is,
commonly, supplementation with oral ferrous sulphate

More complex or severe iron deficiencies may be treated
with intravenous iron or blood transfusions, but
subsequent management is with oral iron preparations In
spite of the widespread use of oral iron preparations
their effectiveness is poor This is due to (i) variable
absorption characteristics and (11) side effects resulting
in poor compliance Strategies for the prevention of iron
deficiency include the use of iron-fortified foods
Commonly used fortificants include ferrous sulphate,
ferric chloride, ferric sodium EDTA and ferric
pyrophosphates However, despite fortification
strategies, iron deficiency remains a common global
problem and, thus, cheap and effective supplements are
required
WO 2005/000210 describes the synthesis of high molecular
weight iron saccharidic complexes formed when freshly
precipitated iron hydroxides are subsequently aggregated
with sugar molecules to form secondary complexes These
complexes are acknowledged to be agglomerated mixtures
WO 03/031635 relates to an enzymatic method to prepare
calcium gluconate where the crystals are high purity and
high solubility
US 2005/0209322 describes a process for making sodium
ferric gluconate complexes for I v iron administration
that requires the initial step of preparing ferric
hydroxide with a subsequent step of reacting with the
ligand, sodium gluconate US 2005/0209187 relates to a
similar process for making iron sucrose complexes rather
than iron gluconate complexes
US 2003/0049284 describes a method for increasing the

solubility of salts of alpha hydroxy carboxylic acids, by
reaction with an alpha amino acid, such that the material
would have improved nutritional supplementation
properties
US 3,679,377 relates to the provision of an agronomically
effective source of iron in a plant nutrient solution as a
soluble ferric sulfato-hydroxyl complex anion The
materials produced are conventional ligand-metal ion
complexes
DE 20 2005 014332 Ul discloses metal-organic nanopowders
for use m materials engineering such as the formation of
polymeric composites through infection spraying or coating
of the nanopowders into or onto an existing material
Jugdaohsmgh et al (2004) describes a critical
precipitation assay that utilises a solution phase
reaction m which, at peri-neutral pH, organic acids
compete with the formation of the oxo-bridges between
aluminium atoms in the polymerisation process, limiting
the growth and decreasing the branching of the polyhydroxy
aluminium species (Jugdaohsmgh et al (2004), Powell et
al (2004)) The assay is usable because the efficiency
of the ligand in interrupting this process is related to
its affinity for aluminium It was also noted m this
work that during solution-phase growth of polyhydroxy
aluminium species, the * competing ligand' becomes
incorporated within the polymer
Summary of the Invention
Broadly, the present invention relates to processes for
preparing solid ligand-modified poly oxo-hydroxy metal ion
materials and optimising their physico-chemical

properties The compositions generally comprise solid
ligand-modified poly oxo-hydroxy metal ion materials
represented by the formula (MxLy (OH) n) ,wherein M represents
one or more metal ions, L represents one or more ligands
and OH represents oxo or hydroxy groups, and may be used
in nutritional, medical, cosmetic or other biologically
relevant applications These include delivery of the
materials per se, or the use of the materials for the
delivery of a component of the material, such as the metal
ion, as a supplement or fortificant or food additive, or
the use of the material to remove or inhibit a component
and ameliorate any undesirable effects that it may cause
The solid ligand-modified poly oxo-hydroxy metal ion
materials disclosed herein constitute new forms of matter
that have not been described previously in the art for
such uses and which can be defined, inter alia, with
reference to structural, spectroscopic or compositional
parameters (i e using the analytical signatures of the
materials) or by the processes by which the materials have
been obtained Thus, while metal oxo-hyaroxide powders
are very well known in the field of inorganic chemistry,
m the present invention they are modified by biologically
compatible ligands {i e other than oxo or hydroxy groups)
to alter their physical and/or chemical properties to
produce new materials and for use m new applications As
part of the unique processes used to optmise and produce
the materials, it is notable that (1) the materials are
recovered as a solid following precipitation from solution
(e g aqueous solution) and (11) that the ligand
incorporation into the poly oxo-hydroxy metal ion solid
phase is, for at least one of the ligands involved,
through formal, identifiable bonding

Thus, by way of example, the present invention differs
from the critical precipitation assay disclosed in
Jugdaohsmgh et al (2004) because that assay was carried
out in solution and the precipitated material was not
subsequently isolated or further employed In contrast,
in the present invention, the formation of the polymers
continues to the point of precipitation and it is the
solid materials that are then characterised and used in a
variety of applications Furthermore, the present
inventors have found that the dried solid phase materials
exhibit physico-chemical properties that are sensitively
dependent upon the exact solution conditions used in the
production of the material, for example the choice of
ligand(s) and their concentration versus that of the metal
ion These materials are not, as might be expected,
simply metal oxides/hydroxides with subtly differing
degrees of crystallisation, and therefore subtly differing
material properties, but instead the ligand(s) incorporate
within the matrix of the poly oxo-hydroxy metal ion
precipitate through substitution of oxo or hydroxyl
groups This is generally non-stoichiometric but,
nonetheless, occurs through formal bonding, and leads to
distinct and novel alterations in the chemistry,
crystallinity and material properties of the solid Thus,
the compositions produced according to the present
invention are chemically novel entities and are not simply
the results of altering the degree of crystallinity of the
metal oxides/hydroxides Surprisingly, the conditions of
precipitation do not easily predict the properties of the
solid, such as the conditions of its re-dissolution and,
for example, using this system it is perfectly possible to
precipitate a material at pH 7 which can also be
completely re-aguated at pH 7 using only a slightly larger
volume of solution or by making a subtle change to the

solution chemistry Nonetheless, under the exact same
reaction conditions, material is formed with highly
reproducible properties Thus, the idea underlying the
present invention is that this process can be used to
produce M L OH solids with precisely tailored physico-
chemical characteristics for multiple biological
applications such as in medicine, nutrition or cosmetics,
where specific material characteristics are required
This approach has not been previously disclosed and it is
surprising that such subtle changes in the precipitation
process allow suitable changes in the solid phase that can
be used to produce such precisely tailored physico-
chemical (e g dissolution) characteristics or properties
Accordingly, m a first aspect, the present invention
provides a process for producing a solid ligand-modified
poly oxo-hydroxy metal ion material (MxLy(OH)n) , wherein M
represents one or more metal ions, L represents one or
more ligands and OH represents oxo or hydroxy groups, and
wherein the gross solid ligand-modified poly oxo-hydroxy
metal ion material has one or more reproducible physico-
chemical properties and displays M-L bonding for at least
one ligand that can be detected by physical analytical
techniques,
the process comprising
(a) mixing the metal ions M and the ligands L at a
first pH(A) at which the components are soluble,
(b) changing the pH(A) to a second pH(B) to cause a
solid precipitate of the solid ligand-modified poly oxo-
hydroxy metal ion material to be formed, and
(c) separating, and optionally drying, the solid
ligand-modified poly oxo-hydroxy metal ion material
produced in step (b)

By way of example, the materials produced by the processes
of the present invention may be employed in nutritional,
medical, cosmetic or other biologically relevant
applications A preferred example of such an application
is the use of the material to deliver the material, or a
part thereof such as a metal ion or a ligand, to a
subject, for example to correct a deficiency in the
component or so that the component provide a beneficial
effect to the subject An alternative example is the use
of a material to bind or sequester a component that may be
present in the system into which the material is
introduced, thereby to remove or inhibit that component
and ameliorate any undesirable effects that it may cause
In view of this, the process may comprise the further step
of formulating the solid ligand-modified poly oxo-hydroxy-
metal ion material in a composition for administration to
a subject
In any aspect of the present invention, the processes
disclosed herein may be employed to engineer or optimise
the physico-chemical properties of the material, for
example to control the dissolution profile or the
adsorption profile, or a similar property of the material,
and it is a considerable advantage of the processes
described herein that they are highly amenable to such
optimisation studies
Accordingly, m a further aspect, the present invention
provides a process for producing a solid ligand-modified
poly oxo-hydroxy metal ion material and optimising a
desired physico-chemical property of the material to adapt
it for a nutritional, medical, cosmetic or biologically
related application, wherein the solid ligand-modified
poly oxo-hydroxy metal ion material is represented by the

formula (MxLy(OH)n), wherein M represent one or more metal
xons, l> represents one or more ligands and OH represents
oxo or hydroxy groups, wherein the gross solid ligand-
modified poly oxo-hydroxy metal ion material has one or
more reproducible physico-chemical properties and displays
M-L bonding for at least one ligand that can be detected
by physical analytical techniques,
the process comprising
(a) mixing the metal ion(s) M and the ligand(s) L in
a reaction medium at a first pH(A) at which the components
are soluble,
(b) changing the pH(A) to a second pH(B) to cause a
solid precipitate of the ligand-modified poly oxo-hydroxy
metal ion material to be formed,
(c) separating, and optionally drying, the solid
ligand-modified poly oxo-hydroxy metal ion material
produced m step (b)
(d) testing the desired physico-chemical
characteristic(s) of the precipitated solid ligand-
modified poly oxo-hydroxy metal ion material, and
(e) repeating steps (a) to (d) as required by varying
one or more of
(i) the identity or concentration of the metal ion(s)
(M) and/or the ligand(s) (L) supplied m step (a), and/or
(11) the ratio of metal ion(s) (M) to ligand(s) (L)
supplied m (a), and/or
(ill) pH(A), and/or
(iv) pH(B), and/or
(v) the rate of change from pH(A) to pH(B) , and/or
(vi) the presence or concentration of a buffer
thereby to produce a solid ligand-modified poly oxo-
hydroxy metal ion material having the desired physico-
chemical property

Examples of possible metal ions and ligands are provided
below In some embodiments, the materials of the present
invention may employ more than one species of metal ion or
ligand, for example two, three, four or five different
species of metal ion or ligand In addition, m some
embodiments, the ligand(s) L may also have some buffering
capacity as described m more detail below
As part of the process for optimising a desired physico-
chemical property of the material to provide for its
application, it may be desirable to vary physical or
chemical reaction conditions used m the process for
making the solid ligand-modif led poly oxo-hydroxy metal
ion material, for example the temperature of the reaction,
the ionic content and strength of the solution, buffering
capacity of the solution (e g using a buxfer such as MOPS
as in the examples) , or the conditions and apparatus used
to mix the reactants, to determine whether and how this
affects one or more properties of the material
In a further aspect, the present invention provides a
process for making solid ligand-modified poly oxo-hydroxy
metal ion materials for administration to a subject, the
process comprising having optimised a solid ligand-
modified poly oxo-hydroxy metal ion material according to
the process as disclosed herein, the further step of
manufacturing the solid ligand-modified poly oxo-hydroxy
metal ion material m bulk and/or formulating it in a
composition
In one embodiment, the processes of the present invention
have been employed by way of example to optimise and
produce ferric iron compositions, e g for use as iron
supplements, fortificants or therapeutics As is

generally used in the art, supplements are nutritional
compositions that are taken by subjects to correct,
prevent or insure against a deficiency m a mineral or
other dietary component A fortificant is somewhat
similar to a supplement but is generally applied to
compositions that are added routinely to foodstuffs to
improve their nutritional value, for example the addition
of iodide to table salt, B group vitamins to breakfast
cereals or iron to cereal products In addition,
compositions may be used therapeutically, usually m the
context of preventing or treating a pathology Or condition
caused by the deficiency in a mineral or other dietary
component In the case of iron, the ferric iron
compositions disclosed herein may be employed as
supplements, fortificants or as therapeutic compositions,
for example in the treatment of iron deficiency in
pregnant or pre-menopausal women, cancer or inflammatory
disease Such therapeutics are typically administered
orally or intravenously
Accordingly, in one aspect, the present invention further
provides a ferric iron composition for administration to a
subject which comprises a solid ligand-modified poly oxo-
hydroxy metal ion material represented by the formula
(MxLy(OH)n) , wherein M represents one or more metal ions
that comprise Fe3+ ions, L represents one or more ligands
and OH represents oxo or hydroxy groups m which the
ligands L are substantially randomly substituted for the
oxo or hydroxy groups, the solid ligand-modif led poly oxo-
hydroxy metal ion material having one or more reproducible
physico-chemical properties and demonstrable M-L bonding
using physical analysis
Generally a useful dietary iron supplement needs to share

some characteristics of simple ferrous salts, namely cost
relatively little and be reasonably well absorbed, but at
the same time, be less redox active and hence lead to a
low incidence of side-effects Some ferric salts do not
suffer from this disadvantage as they are already
oxidised, and are therefore less prone to redox activity
because the initiation of iron reduction in the
gastrointestinal lumen is less favourable than the
initiation of iron oxidation Moreover, the controlled
mucosal reduction of ferric iron, via the mucosal protein
DcytB, may provide a rate-limiting step for the entry of
iron to the circulation, which would lower the production
of circulating non-transferrin bound iron (NTBI) NTBI
may lead to oxidative damage in the circulation,
endothelium and the more vascular organs However, simple
ferric salts are not efficient supplements because their
rapid dissolution m the stomach is followed by
concentration-dependent oxo-hydroxy polymerisation in the
small bowel which inhibits their absorption Thus, while
ferric iron salts, typically ferric chloride, have been
tried as fortificants in certain foods, these are poorly
absorbed at supplemental or therapeutic doses due to
uncontrolled delivery of ferric ions into the small bowel
at bolus doses Chelation of ferric iron, for example
with maltol, may help overcome this small bowel solubility
issue for bolus doses, but has not proven commercially
viable due to production costs (WO 03/097627) In
addition there are concerns over the safety of chelators
such as maltol The compositions disclosed herein are
engineered to overcome such absorption, safety, side
effect and production cost problems Thus, these solid
ligand-modified poly oxo-hydroxy metal ion materials can
be tailored to have distinct dissolution profiles m the
stomach environment compared to the small bowel

environment In this way, rapid dissolution in the
stomach that then leads to undesirable bolus delivery of
iron in the small bowel, as occurs for simple ferrous and
ferric salts, can be avoided in the design of these
materials Both the pH of dissolution and the rate of
dissolution can be engineered to match requirements
Potentially, these solid phase ligand-modified poly oxo-
hydroxy ferric iron materials could be tailored to * sense'
iron requirements Absorption of iron from the gut lumen
and into the circulation occurs in individuals who require
iron In those who do not require iron there will be
little or no absorption and more iron will remain in the
lumen The dissolution or disaggregation of these solid
phase ligand-modified poly oxo-hydroxy ferric iron
materials could be vset' such that they dissolve or
disaggregate efficiently m an environment that is low in
aquated iron, but inefficiently in an environment that is
high in aquated iron This again would help to reduce
side effects without compromising absorption in those who
need iron Whether these materials are designed to
dissolve or disaggregate under gastrointestinal conditions
depends upon the optimal mode of iron absorption in the
gut as both soluble iron and very small aquated
particulate iron could both be absorbed but, either way,
the ligand-modified poly oxo-hydroxy ferric iron materials
could be so designed
In a further aspect, the present invention provides the
use of a composition of a solid ligand-modified poly oxo-
hydroxy metal ion material (MxLy(OH)n) as obtainable by the
processes disclosed herein for the preparation of a
medicament for therapeutic delivery of the metal ion to
the subject Alternatively, the present invention
provides a solid ligand-modified poly oxo-hydroxy metal

ion material (MxLy(OH)„) as obtainable by the processes
disclosed herein for the delivery of the metal ion to a
subject
Examples of the uses of the solid ligand-modified poly
oxo-hydroxy metal ion materials disclosed herein include,
but are not limited to, uses as dietary mineral
supplements and fortificants, therapeutic mineral
supplements {e g as administered by i v and oral
routes), drugs, nutrients or cosmetic carners/co-
complexes, phosphate binding agents, other binding or
sequestering applications, food additives, anti-
perspirants, sun-protection agents, vaccine adjuvants,
immuno-modulatory agents, direct cosmetic applications
including exfoliating agents, bone and dental
filler/cements, implant materials including brachytherapy,
and imaging and contrast agents
Embodiments of the present invention will now be described
by way of example and not limitation with reference to the
accompanying figures and examples
Brief Description of the Figures
Figure 1 Examples of the effects of weak (succinate,
closed square), intermediate (malate, open circle) and
strong {maltol, closed triangle) ligands on the formation
of solid ligand-modified poly oxo-hydroxy metal ion
material (A) and the disaggregation of the wet solid
materials in buffers at pH 6 (black bars) and pH 4 (grey
bars) (B), using the method described in "screening
assay" The ratios indicated are M L ratios that were
selected for formation of the materials The iron
concentration m the initial solution (prior to
precipitation) was 27mM

Figure 2 Effect of different ligands on the evolution of
precipitation of solid ligand-modified poly oxo-hydroxy
metal ion materials with increasing pH as described in
titration protocol no ligand {open circle), tartaric acid
(closed square) and malic acid (closed triangle) All
were prepared in 50mM MOPS and 0 9% w/v NaCl The iron
concentration in the initial solution (prior to
precipitation) was 27mM
Figure 3 Example of the effect of varying the pH of the
final solution during the preparation of the solid ligand-
modified poly oxo-hydroxy metal ion materials on the
disaggregation of these wet materials at different pHs m
the buffers indicated The materials, namely FeOHM-1 2-
MOPS50, were prepared following the preparation protocol
described in Methods with 0 9% w/v NaCl and final pH 6
(grey bars), pH 7 (striped bars) or pH 8 (black bars) The
percentage precipitation obtained was 10%, 30% and 48%
respectively The iron concentration in the initial
solution (prior to precipitation) was 27mM
Figure 4 Example of how the presence of an electrolyte in
the preparation of the solid ligand-modified poly oxo-
hydroxy metal ion materials can affect the disaggregation
of the material at four different pHs m the buffers
indicated Materials were prepared following the
preparation protocol described in Methods and oven dried
The materials, namely FeOHT-4 1-MOPS50, were prepared at a
final solution pH of 6 5 and formed in the absence of
electrolyte (grey bars, n=2) or in the presence of 0 9%
w/v NaCl (stripped bars, n=l), the percentage
precipitation obtained was 97% and 98% respectively (A)
The material, namely FeOHT-2 1-Niacm50, were prepared at

a final solution pH of 3 2 in the absence of electrolyte
(grey bars, n=2) or in the presence of 0 9% w/v KC1 (black
bars, n=2), the percentage precipitation obtained was 88%
and 91% respectively (B) The iron concentration in the
initial solution (prior to precipitation) was 27mM
Figure 5 Example of how drying the solid ligand-modified
poly oxo-hydroxy metal ion materials can affect its
disaggregation at four different pHs in the buffers
indicated The materials, namely FeOHT-4 1-MOPS50, was
prepared following the preparation protocol described in
method with a final solution pH of 6 5 in the absence of
electrolyte The percentage precipitation obtained was
97% The solid phase was divided into three aliquots and
either oven dried (grey bars, n=2), or freeze-dried (black
bars, n=2) or used wet (stripped bars, n=2) Note some
error bars are too small to be viewed Data shown m grey
bars have been shown previously in Figure 4A The iron
concentration in the initial solution (prior to
precipitation) was 27mM
Figure 6 Example of the effect of "ligand B" on the
evolution of precipitation of the solid ligand-modified
poly oxo-hydroxy metal ion material with increasing pH in
presence (i) or absence (n) of "ligand A", namely
tartaric acid, at M LA ratio 4 1 "Ligand B" showed were
either 50mM adipic acid (squares) or 50mM MOPS
(triangles) All titrations were performed following the
protocol described in the methods and m the absence of
electrolyte The iron concentration in the initial
solution (prior to precipitation) was 27mM
Figure 7 Example of the effect of ligand B on the

disaggregation of oven dried solid ligand-modified poly
oxo-hydroxy metal ion materials in four different buffers
Tartrate-modified poly oxo-hydroxy ferric materials at
M LR ratio 4 1, with tartrate being ligand A (LA) , were
prepared in the presence of different ligands B being 50mM
MOPS (grey bars, n=2), 20 mM benzoic acid (black bars,
n=3) or 50mM niacin (stripped bars, n=3) following the
preparation protocol described in Methods in the absence
of electrolyte The percentage precipitation obtained was
97%, 94% and 100% respectively Note some error bars are
too small to be viewed The data indicated in grey bars
have been shown previously in Figure 4A and 5
Figure 8 Energy dispersive X-ray microanalysis (EDX) of a
ligand-modified poly oxo-hydroxy metal ion material
(FeOHT-3 1-Ad20) showing the composition of the material
to be predominantly Fe and O with incorporation of C plus
very small additions of Na and CI from the electrolyte
used (the Cu signal is due to the support grid)
Figure 9 Typical infrared spectra of solid ferric oxo-
hydroxide in (A) , the tartrate-modified ferric oxo-
hydroxide in (B) (l e the ligand-modified poly oxo-
hydroxy metal ion material, FeOHT-4 1) and tartaric acid
in (C) The band corresponding to the C=0 stretch of
tartaric acid (1712 cm"1 m spectrum C) is replaced by two
bands (1356 and 1615 cm"1 in spectrum B) showing the
presence of bonding between the carboxylate group of
tartaric acid and iron m the FeOHT-4 1 material Note
also the presence of a broad band circa 3350 cm"1 due to -
OH stretch m spectra A and B
Figure 10 Percentage of iron disaggregation (without
ultrafiltration, A) and dissolution (with ultrafiltration,

B) after simulated passage through the stomach for the
time indicated Prior art is shown m closed symbols,
namely ferric oxo-hydroxide (closed squares), Maltofer
(closed circles), ferrous sulphate (closed triangles)
The ligand-modxfied poly oxo-hydroxy metal ion materials
are shown in open symbols, namely FeOHT-3 l~Ad20 {open
diamonds) and FeOHM-4 1-Bic25 (open triangles) Error
bars represent STDEV (note that certain error bars are too
small to be visible)
Figure 11 Aberration corrected high angle annular dark
field scanning transmission electron microscopy
(superSTEM) high resolution images showing that organised,
crystalline regions are less frequently discernible m
ligand-modified poly oxo-hydroxy metal ion materials {e g
FeOH-TRP15 (B) and especially in FeOHT-2 1-TRP15 (C)) than
m similar sized unmodified ferric iron oxo-hydroxide (A)
Figure 12 X-ray diffraction pattern of Maltofer (A) and
the ligand-modified poly oxo-hydroxy metal ion material
FeOHT-3 1-Ad20 (B) showing a clear presence of iron oxo-
hydroxide crystal structure in Maltofer and a clear lack
of detectable crystalline structure m FeOHT-3 1-Ad20,
apart from the co-precipitated electrolyte, sodium
chloride Reference lines for iron oxide and sodium
chloride are shown below each graph for clarity
Figure 13 Examples of the serum iron increase (A) and
percentage iron absorption (B) in human volunteers
following ingestion of ferrous sulphate, ferric oxo-
hydroxide or different solid ligand-modified poly oxo-
hydroxy ferric materials A ferrous sulphate {open
triangle, n=30), FeOHT-3 1-Ad20 (+ symbol, n=4), FeOHT-
2 1-TRP15 (- symbol, n=4), FeOHAdipatelOO (x symbol, n=2),

FeOHHistidxnelOO (closed square, n=2), FeOHM-4 1-Bic25
(open square, n=3) , FeOHGlucomc20 (closed triangle, n=3) ,
FeOHT-2 1-Niacm50 (open circle, n=3) , FeOH {closed
circle, n=2) B Percentage iron absorption (calculated
as the red blood cell incorporation of 58Fe divided by
0 80) from ferric oxo-hydroxide or the solid ligand-
modified poly oxo-hydroxy ferric materials (black bars)
compared with estimated absorption of iron from ferrous
sulphate for the same group of study participants (open
bars) Error bars represent the SEM Number of each
pairing vary from 2 to 4, except for ferrous sulphate in
the FeOHHistidinelOO group which was 1
Figure 14 Disaggregation of iron during simulated passage
through the stomach and duodenum from (A) prior art
compounds ferric pyrophosphate {Closed diamond), ferric
chloride (Closed square), ferric tri-maltol {Closed
triangle) , ferrous bisglycmate (Open square) , and (B) a
selection of compounds tested in our m vivo study in
Figure 13 ferrous sulphate (Open square), FeOHT-3 1-Ad20
(Open diamond) and FeOHM-4 1-Bic25 (Closed circle) For
details of the protocol see In vitro gastrointestinal
digestion assay in the Methods
Figure 15 Examples of the effect of different ligands, at
differing M L ratios, on the percentage of iron
disaggregation (A) and on the percentage of iron
dissolution (B) of solid ligand-modified poly pxo-hydroxy
metal ion materials, after 30 minutes incubation at
gastric pH 1 2 (black bars, n=3) or 60 minutes incubation
at intestinal pH 7 0 (open bars, n=3), error bars
represent standard deviations
Figure 16 Evolution of the formation of the ligand-

modified poly oxo-hydroxy ferric materials, namely FeOHT-
2 1-Ad20, with increasing pH, as described m the
titration protocol in Methods, and expressed as the
percentage of total iron in the starting solution
Percentage iron in the aggregated material is shown by the
closed triangles while the percentage of iron m both the
aggregated and aquated particulate materials is shown by
the closed square Note the remaining iron (1 e the
iron that is not in the aggregated or aquated particulate
form) is in the soluble phase
Figure 17 Example of the effect of ligand, M L ratio, and
final solution pH of formation on the disaggregation of
the tartrate-modified poly oxo-hydroxy ferric materials
through the modified in vitro gastrointestinal digestion
assay described in methods Bars represent the particle
size distribution of the disaggregated materials as a
percentage of total iron in solid phase Size ranges
determined were <5nm (stripped section), 5-20nm(grey
section), 20-300 nm (black section), and 1-10 um (white
section)
Detailed Description
The Metal Ion (M)
The solid ligand-modified poly oxo-hydroxy metal ion
materials may be represented by the formula (MxLy(OH)n) ,
where M represents one or more metal ions Normally, the
metal ion will originally be present in the form of a salt
that m the preparation of the materials may be dissolved
and then induced to form poly oxo-hydroxy co-complexes
with ligand (L) some of which is integrated into the solid
phase through formal M-L bonding, i e not all of the
ligand (L) is simply trapped or adsorbed in the bulk
material The bonding of the metal ion in the materials

can be determined using physical analytical techniques
such as infrared spectroscopy where the spectra will have
peaks characteristic of the bonds between the metal ion
and the ligand (L), as well as peaks characteristic of
other bonds present m the material such as M-0, o-H and
bonds in the ligand species (L) Preferred metal ions (M)
are biologically compatible under the conditions for which
the materials are used and are readily precipitatable from
aqueous solution by forming oxo-hydroxides Examples of
metal ions include iron, calcium, magnesium, zinc, copper,
manganese, chromium and aluminium ions A particularly
preferred metal ion is ferric iron (Fe3+)
By way of reference to the ferric iron compositions
disclosed herein, the presence of formal bonding is one
aspect that mainly distinguishes the materials from other
products such as "iron polymaltose" (Maltofer) in which
particulate crystalline iron oxo-hydroxide is surrounded
by a sugar shell formed from maltose and thus is simply a
mixture of iron oxo-hydroxide and sugar at the nano-level
(Hemrich (1975), Geisser and Muller (1987), Nielsen et al
(1994, US Patent No 3,076,798), 0S2OO6O2O5691) In
addition, the materials of the present invention are metal
poly oxo-hydroxy species modified by non-stoichiometric
ligand incorporation and should therefore not be confused
with the numerous metal-ligand complexes that are well
reported in the art (e g , see WO 03/092674, WO
06/037449) Although generally soluble, such complexes
can be precipitated from solution at the point of
supersaturation, for example ferric trimaltol, Harvey et
al (1998), WO 03/097627, ferric citrate, WO 04/074444 and
ferric tartrate, Bobtelsky and Jordan (1947) and, on
occasions, may even involve stoichiometric binding of
hydroxyl groups (for example, ferric hydroxide saccharide,

US Patent No 3,821,192) The use of hydroxyl groups to
balance the charge and geometry of metal-ligand complexes
is, of course, well reported in the art (e g iron-
hydroxy-malate, WO 04/050031) and unrelated to the solid
ligand-modified poly oxo-hydroxy metal ion materials
reported herein
Without modification, the primary particles of the
materials have metal oxide cores and metal hydroxide
surfaces and within different disciplines may be referred
to as metal oxides or metal hydroxides The use of the
term 'oxo-hydroxy' or *oxo-hydroxide' is intended to
recognise these facts without any reference to proportions
of oxo or hydroxy groups Hydroxy-oxide could equally be
used therefore As described above, the materials of the
present invention are altered at the level of the primary
particle of the metal oxo-hydroxide with at least some of
the ligand L being introduced into the structure of the
primary particle, 1 e leading to doping or contamination
of the primary particle by the ligand L This may be
contrasted with the formation of nano-mixtures of metal
oxo-hydroxides and an organic molecule, such as iron
saccharidic complexes, in which the structure of the
primary particles is not so altered
The primary particles of the ligand-modified poly oxo-
hydroxy metal ion materials described herein are produced
by a process referred to as precipitation The use of the
term precipitation often refers to the formation of
aggregates of materials that do separate from solution by
sedimentation or centrifugation Here, the term
"precipitation" is intended to describe the formation of
all solid phase material, including aggregates as
described above and solid materials that do not aggregate

but remain as non- soluble moieto.es in suspension, whether
or not they be particulate, colloidal or sub-colloidal
(nanoparticulates) These latter solid materials may also
be referred to as aquated particulate solids
In the present invention, reference may be made to the
modified metal oxo-hydroxides having polymeric structures
that generally form above the critical precipitation pH
As used herein, this should not be taken as indicating
that the structures of the materials are polymeric in the
strict sense of having a regular repeating monomer unit
because, as has been stated, ligand incorporation is,
except by co-incidence, non-stoichiometric The ligand
species is introduced into the solid phase structure by
substituting for oxo or hydroxy groups leading to a change
in solid phase order In some cases, for example the
production of the ferric iron materials exemplified
herein, the ligand species L may be introduced into the
solid phase structure by the substitution of oxo or
hydroxy groups by ligand molecules in a manner that
decreases overall order in the solid phase material
While this still produces solid ligand modified poly oxo-
hydroxy metal ion materials that in the gross form have
one or more reproducible physico-chemical properties, the
materials have a more amorphous nature compared, for
example, to the structure of the corresponding metal oxo-
hydroxide The presence of a more disordered or amorphous
structure can readily be determined by the skilled person
using techniques well known in the art One exemplary
technique is X-ray diffraction (XRD) which will produce an
X-ray diffraction pattern for the ferric iron materials
exemplified herein having poorly identifiable peaks for L
or M0/M0H, XRD relying on a regular arrangement of atoms
to diffract the X-rays and produce a pattern

Alternatively or additionally, a decrease in the
crystallmity of the structure of the material may be
determined by high resolution transmission electron
microscopy High resolution transmission electron
microscopy allows the crystalline pattern of the material
to be visually assessed It can indicate the primary
particle size and structure (such as d-spacing) and give
some information on the distribution between amorphous and
crystalline material Using this technique, it is
apparent that the chemistry described above increases the
amorphous phase of our described materials compared to
corresponding materials without the incorporated ligand
This may be especially apparent using high angle annular
dark field aberration-corrected scanning transmission
electron microscopy due to the high contrast achieved
while maintaining the resolution thus allowing the surface
as well as the bulk of the primary particles of the
material to be visualised
The reproducible physico-chemical property or
characteristic of the materials of the present invention
will be dependent on the application for which the
material is intended Examples of the properties that can
be usefully modulated using the present invention include
dissolution (rate, pH dependence and pM dependence),
disaggregation, adsorption and absorption characteristics,
reactivity-inertness, melting point, temperature
resistance, particle size, magnetism, electrical
properties, density, light absorbing/reflecting
properties, hardness-softness, colour and encapsulation
properties Examples of properties that are particularly
relevant to the field of supplements, fortificants and
mineral therapeutics are physico-chemical properties
selected from one or more of a dissolution profile, an

adsorption profile or a reproducible elemental ratio In
this context, a property or characteristic may be
reproducible if replicate experiments are reproducible
within a standard deviation of preferably ± 10%, and more
preferably ± 5%, and even more preferably within a limit
of ± 2%
The dissolution profile of the solid ligand-modified poly
oxo-hydroxy metal ion materials can be represented by
different stages of the process/ namely disaggregation and
dissolution The term dissolution is used to describe the
passage of a substance from solid to soluble phase More
specifically, disaggregation is intended to describe the
passage of the materials from a solid aggregated phase to
an aquated phase that is the sum of the soluble phase and
the aquated particulate phase (I e solution plus
suspension phases) Therefore, the term dissolution as
opposed to disaggregation more specifically represents the
passage from any solid phase (aggregated or aquated) to
the soluble phase
Preferred specific examples of the metal ions (M) include,
but are not restricted to, Groups 2, 3 and 5 metals of the
periodic table, the transition metals, heavy metals and
lanthanoids Examples include, but are not restricted to
Ag2+, Al3+, Au3\ Be2+, Ca2, Co2, Cr3+, Cu2+, Eu3+, Fe3\ Mg2+,
Mn2+, Ni2+, Sr2+, V5+, Zn2+, Zr2+ Moreover, many of these
metal cations take on different oxidation states so it
will also be appreciated that these examples are not
restricted to the oxidation states shown In many cases,
the solid ligand-modified poly oxo-hydroxy metal ion
materials comprise a single species of metal ion, for
example Fe3+

The Ligand (L)
In the solid phase ligand-modified poly oxo-hydroxy metal
ion- species represented by the formula (MxLy(OH)n), L
represents one or more ligands or anions, such as
initially in its protonated or alkali metal form, that can
be incorporated into the solid phase ligand-modified poly
oxo-hydroxy metal ion material Typically, this is done
to aid in the modification of a physico-chemical property
of the solid material, e g as compared to a poly oxo-
hydroxylated metal ion species in which the ligand(s) are
absent In some embodiments of the present invention, the
ligand(s) L may also have some buffering capacity
Examples of ligands that may be employed m the present
invention include, but are by no means limited to
carboxylic acids such as adipic acid, glutaric acid,
tartaric acid, malic acid, succinic acid, aspartic acid,
pimelic acid, citric acid, gluconic acid, lactic acid or
benzoic acid, food additives such as maltol, ethyl maltol
or vanillin, "classical anions' with ligand properties
such as bicarbonate, sulphate and phosphate, mineral
ligands such as silicate, borate, molybdate and selenate,
amino acids such as tryptophan, glutamme, proline,
valine, or histidine, and nutrient-based ligands such as
folate, ascorbate, pyridoxme or niacin Typically
ligands may be well recognised in the art as having high
affinity for a certain metal ion in solution or as having
only low affinity or not be typically recognised as a
ligand for a given metal ion at all However, we have
found that in poly oxo-hydroxy metal ion materials,
ligands may have a role m spite of an apparent lack of
activity in solution Typically, two ligands of differing
affinities for the metal ion are used in the production of
these materials although one, two, three, four or more
ligands may be useful m certain applications

For many applications, ligands need to be biologically
compatible under the conditions used and generally have
one or more atoms with a lone pair of electrons at the
point of reaction The ligands include anions, weak
ligands and strong ligands Ligands may have some
intrinsic buffering capacity during the reaction Without
wishing to be bound by a particular explanation, the
inventors believe that the ligands have two modes of
interaction (a) substitution of hydroxy groups and,
therefore, incorporation with a largely covalent character
within the material and (b) non-specific adsorption (ion
pair formation) These two modes likely relate to
differing metal-ligand affinities (i e strong ligands for
the former and weak ligands/anions for the latter) There
is some evidence in our current work that the two types of
ligand are synergistic m modulating dissolution
characteristics of the materials and, perhaps, therefore,
in determining other characteristics of the material In
this case, two ligand types are used and at least one
(type (a)) is demonstrable as showing metal binding within
the material Ligand efficacy, probably especially for
type (b) ligands, may be affected by other components of
the system, particularly electrolyte
The ratio of the metal ion(s) to the ligand(s) (L) is also
a parameter of the solid phase ligand-modifiedpoly oxo-
hydroxy metal iron material that can be varied according
to the methods disclosed herein to vary the properties of
the materials Generally, the useful ratios of M L will
be between 10 1, 5 1, 4 1, 3 1, 2 1 and 1 1 and 12, 13,
14, 1 5 or 1 10
Hydroxy and oxo groups

The present invention may employ any way of forming
hydroxide ions at concentrations that can provide for
hydroxy surface groups and oxo bridging in the formation
of these poly oxo-hydroxy materials Examples include but
are not limited to/ alkali solutions such as sodium
hydroxide, potassium hydroxide and sodium bicarbonate,
that would be added to increase [OH] in an ML mixture, or
acid solutions such as mineral acids or organic acids,
that would be added to decrease [OH] m an ML mixture
Conditions used in the process
The exact conditions of mixing and precipitation of the
solid ligand-modified poly oxo-hydroxy metal ion material
will vary depending upon the desirable characteristics of
the solid material Typical variables are
(1) Starting pH (1 e the pH at which M and L are
mixed) This is always a different pH to that at which
oxo-hydroxy polymerisation commences Preferably, it is a
more acidic pH, more preferably below a pH of 2
(2) The pH at which oxo-hydroxy polymerisation
commences This is always a different pH to that of the
starting pH Preferably, it is a less acidic pH and most
preferably above a pH of 2
(3) Final pH This will always promote precipitation and
may promote agglomeration of the solid ligand-modified
poly oxo-hydroxy metal ion material and preferably will be
a higher pH than the pH at which oxo-hydroxy
polymerisation commences It will be appreciated by the
skilled person that where a pH difference exists between
commencement of oxo-hydroxy polymerisation and the final
pH value, addition of further M, L, OH", H+, excipients or
other substances may be undertaken before the final pH
value is achieved
(4) Rate of pH change from commencement of oxo-hydroxy

polymerisation to completion of reaction This will occur
within a 24 hour period, preferably within an hour period
and most preferably within 20 minutes
Concentrations of M and L While the concentration of OH
is established by the pH during oxo-hydroxy
polymerisation, the concentrations of total M and total L
m the system will be fixed by the starting amounts in the
ML mix and the final solution volume Typically, this
will exceed 10"6 molar for both M and L and more
preferably it will exceed 10"3 molar Concentrations of M
and L are independent and chosen for one or more desired
characteristics of the final material and especially so
that the concentration of M is not too high such that the
rate of oxo-hydroxy polymerisation occurs too rapidly and
prevents L incorporation Similarly the concentration of
L will not be too high to prevent metal oxo-hydroxy
polymerisation For example, the ligand-modified poly
oxo-hydroxy materials in which M is ferric iron are
produced preferably with iron concentrations of the
initial solution below 300 mM and most preferably below
200 mM, providing ranges of ferric iron concentrations
between between ImM and 300mM, more preferably between
20mM and 200mM, and most preferably of about 40mM
(5) Solution phase The preferred solution for this work
is aqueous and most preferably is water
(6) Buffer The solution may have a buffer added to help
stabilise the pH range of oxo-hydroxy polymerisation
Buffers may be inorganic or organic, and in some
embodiments will not be involved in formal bonding with
the metal ion(s) M of the solid phase material
Alternatively, one or more of the ligands L involved in
formal bonding with the metal ion(s) M of the solid phase
material may have some buffering capacity that is
additionally favourable in achieving the desired

composition of the final material Buffer concentrations
are less than 500 mM, preferably less than 200 mM and most
preferably less than 100 mM
(7) Temperature The preferred temperature is above 0
and below 100°C, typically between room temperature (20-
30°C) and 100°C, most typically at room temperature
(8) Ionic strength Electrolyte such as, but not limited
to, potassium chloride and sodium chloride, may be used in
the procedure The ionic strength of the solution may thus
range from that solely derived from the components and
conditions outlined m (l)-(8) above or from the further
addition of electrolyte which may be up to 10% (w/v),
preferably up to 2%, and most preferably <1%
(9) Extent of mixing of the components This issue
mainly relates to degree of stirring and preferably
stirring is achieved such that the starting solutions
(i e M, L and buffer) are rapidly mixed and maintained
homogenous throughout
It will be apparent to those skilled in the art that while
the above variables may all control the physico-chemical
nature of the precipitate, further variables such as the
collection system and/or excipients used for the recovery
of the precipitate, which may involve purposeful
inhibition of agglomeration, its drying and its grinding
may subsequently affect the material properties However,
these are general variables to any such system for solid
extraction from a solution phase After separation of the
precipitated material, it may optionally be dried before
use of further formulation The dried product may,
however, retain some water and be m the form of a
hydrated solid phase ligand-modified poly oxo-hydroxy
metal ion material It will be apparent to those skilled
m the art that at any of the stages described herein for

recovery of the solid phase, excipients may be added that
mix with the ligand-modified poly oxo-hydroxy metal ion
material but do not modify the primary particle and are
used with a view to optimising formulation for the
intended function of the material Examples of these
could be, but are not limited to, glycolipids,
phospholipids (e g phosphatidyl choline), sugars and
polysaccharides, sugar alcohols (e g glycerol), polymers
(e g polyethyleneglycol (PEG)) and taurocholic acid
Formulations and Uses
The solid phase materials of the present invention may be
formulated for use m a range of biologically relevant
applications, including formulation for use as
pharmaceutical, nutritional, cosmetic, or personal hygiene
compositions The compositions of the present invention
may comprise, m addition to one or more of the solid
phase materials of the invention, a pharmaceutically
acceptable excipient, carrier, buffer, stabiliser or other
materials well known to those skilled m the art Such
materials should be non-toxic and should not interfere
with the efficacy of the solid phase materials for the
application in question
The precise nature of the carrier or other component may
be related to the manner or route of administration of the
composition These compositions may be delivered by a
range of delivery routes including, but not limited to
gastrointestinal delivery, including orally and per
rectum, parenteral delivery, including injection, dermal
delivery including patches, creams etc, mucosal delivery,
including nasal, inhalation and via pessary, or by implant
at specific sites, including prosthetics that may be used
for this purpose or mainly for another purpose but have

this benefit
Pharmaceutical compositions for oral administration may be
in a tablet, capsule, powder, gel or liquid form A
tablet may include a solid carrier such as gelatin or an
adjuvant Capsules may have specialised properties such
as an enteric coating Liquid pharmaceutical compositions
generally include a liquid carrier such as water,
petroleum, animal or vegetable oils, mineral oil or
synthetic oil Physiological saline solution, dextrose or
other saccharide solution or glycols such as ethylene
glycol, propylene glycol or polyethylene glycol may be
included Where the solid ligand-modified poly oxo-
hydroxy metal ion material needs to be maintained in a
solid form, eg to control the delivery of a component of
the material, it may be necessary to select components of
the formulation accordingly, e g where a liquid
formulation of the material is made
For intravenous, cutaneous or subcutaneous injection, or
injection at the site of affliction, the active ingredient
will be m the form of a parenterally acceptable aqueous
solution or suspension which is pyrogen-free and has
suitable pH, isotonicity and stability Those of relevant
skill m the art are well able to prepare suitable
solutions using, for example, isotonic vehicles such as
Sodium Chloride Injection, Ringer's Injection, Lactated
Ringer's Injection Preservatives, stabilisers, buffers,
antioxidants and/or other additives may be included, as
required
The materials and compositions used in accordance with the
present invention that are to be given to an individual
are preferably administered in a "prophylactically

effective amount" or a "therapeutically effective amount"
(as the case may be, although prophylaxis may be
considered therapy), this being sufficient to show benefit
to the individual (e g bioavailability) The actual
amount administered, and rate and time-course of
administration, will depend on the nature and severity of
what is being treated Prescription of treatment, e g
decisions on dosage etc, is within the responsibility of
general practitioners and other medical doctors, and
typically takes account of the disorder to be treated, the
condition of the individual patient, the site of delivery,
the method of administration and other factors known to
practitioners Examples of the techniques and protocols
mentioned above can be found in Remington's Pharmaceutical
Sciences, 20th Edition, 2000, Lippincott, Williams &
Wilkms A composition may be administered alone or in
combination with other treatments, either simultaneously
or sequentially, dependent upon the condition to be
treated
Examples of the uses of the solid ligand-modified poly
oxo-hydroxy metal ion materials disclosed herein include,
but are not limited to, uses as dietary mineral
supplements and fortificants, therapeutic mineral
supplements (e g as administered by 1 v and oral
routes) , drugs, nutrients or cosmetic carners/co-
complexes, phosphate binding agents, other binding or
sequestering applications, food additives, anti-
perspirants, sun-protection agents, vaccine ad}uvants,
immuno-modulatory agents, direct cosmetic applications
including exfoliating agents, bone and dental
filler/cements, implant materials including brachytherapy,
and imaging and contrast agents

Ligand-modified poly oxo-hydroxide materials may be used
as supplements for nutritional or medical benefit In
this area, there are three main examples
(i) Therapeutic (prescription) supplements, which are
generally administered by the oral or 1 v routes for the
treatment of indications including iron deficiency
anaemia, iron deficiency and anaemia of chronic disease
The therapeutic administration of materials of the present
invention may be in conjunction with other therapies and
especially with the concomitant use of erythropoietin
(II) Nutritional (self prescribed/purchased supplements)
which are usually for oral delivery
(III) Fortificants These may be traditional forms- in
terms of being added to food prior to purchase - or more
recent fortificant forms such as 'Sprinkles' which are
added (like salt or pepper) to food at the time of
ingestion
In all formats, but most especially for fortificants,
subsequent formulation, such as addition of a protective
coating (e g lipid), may be necessary to make the
material compatible with its intended usage In addition,
any of these supplemental forms can be co-formulated,
either by incorporation within the material through use of
co-formulated material(s) as ligand(s) or through
trapping/encapsulation of said materials, or simply
through co-delivery of said materials
As described herein, one particular application of the
solid ligand-modified poly oxo-hydroxy metal ion materials
of the present invention is for the treatment of mineral

deficiencies, for example iron deficiency In an
alternative application the materials may be employed to
bind or sequester a component present in an individual
By way of example, the ferric iron compositions disclosed
herein may be used to deliver iron to an individual for
use in the prophylaxis or treatment of iron deficiency or
iron deficiency anaemia which may be suspected, or
diagnosed through standard haematological and clinical
chemistry techniques Iron deficiency and iron deficiency
anaemia may occur in isolation, for example due to
inadequate nutrition or due to excessive iron losses, or
they may be associated with stresses such as pregnancy or
lactation, or they may be associated with diseases such as
inflammatory disorders, cancers and renal insufficiency
In addition, there is evidence that the reduced
erythropoiesis associated with anaemia of chronic disease
may be improved or corrected by the effective delivery of
systemic iron and that co-delivery of iron with
erythropoietin or its analogues may be especially
effective in overcoming reduced erthropoietic activity
Thus, by way of further example, the ferric iron
compositions disclosed herein may be used to deliver iron
to an individual for use in the treatment of sub-optimal
erythropoietic activity such as in anaemia of chronic
disease Anaemia of chronic disease may be associated
with conditions such as renal insufficiency, cancer and
inflammatory disorders As noted above, iron deficiency
may also commonly occur in these disorders so it follows
that treatment through iron supplementation may address
iron deficiency alone and/or anaemia of chronic disease
It will be recognised by those skilled m the art that the
above examples of the medical uses of iron supplements are
by no means limiting

Experimental Description
Introduction
Inorganic mineral-based materials have widespread
biological applications that include dietary supplements,
phosphate binding agents, antacids, immune adjuvants
(alum) and antiperspirants (alum) These are often co-
formulated in such a way that the mineral physico-chemical
properties, such as rates of dissolution and/or
disaggregation, are modestly altered in an attempt to
improve their efficacy We have however developed a
procedure whereby the actual structure, at the level of
the primary particle (the primary unit within the lattice
structure) , can be modified within oxide/.nydroxide
minerals This nano-structunng can lead to profound
changes in mineral characteristics and can be tuned to
provide mineral with precisely specified physico-chemical
characteristics Moreover the methodology is cheap and
can be applied on as large a scale as required The
modifying agents are all biologically compatible, food
grade ligands allowing rapid introduction of novel
materials to human streets An exemplar of these
materials is the production of a novel class of iron
supplements that may have therapeutic parenteral and oral
applications, as well as widespread roles as fortificants
and dietary supplements
With supplements, we believe that one desirable property
is that the rate of nutrient absorption mimics that seen
for the same nutrient when ingested in a food For
example, with iron, the rate of dietary iron absorption
can be controlled through the rate of iron dissolution
In the following examples, we have produced a number of
different solid ligand-modified poly oxo-hydroxy metal ion
materials using the process of the present invention, with

the aim of identifying compositions that release iron in a
controlled fashion The aim is that the rate of
dissolution will allow the ferric iron to be donated to
the mucosal reductase (DcytB) in a fashion that prevents
build up of iron m the lumen or bolus absorption into the
circulation- neither of which are desirable Thus the
ferric iron compositions of the present invention should
have lower gastrointestinal side effects as they will not
undergo facile redox cycling in the gut In addition,
there is scope to design the compositions to dissolve
differently at gastric pH versus intestinal pH There is
also the possibility of tailoring the compositions to
dissolve at different rates depending upon the
concentration of iron in the local solution (e g the gut
lumen), such that the compositions may *sense' iron
requirements of the environment and thus iron requirements
of the individual The remaining, unabsorbed luminal iron
would be largely unavailable for undesirable redox
reactivity within the lumen and would pass harmlessly into
the faeces
Nomenclature of materials
Throughout the examples the FeOHLA-i j-Lsk nomenclature
was adopted to describe the preparation for ligand-
modified poly oxo-hydroxy ferric iron materials, where Lfl
refers to the ligand with higher solution affinity and LB
to the ligand with lower solution affinity for iron The
ratio i 3 refers to the molar ratio between iron (Fe) and
ligand A (LA) and k refers to the concentration (mM)of
ligand B(LB)IZI solution prior to the precipitation of
ligand-modified poly oxo-hydroxy ferric materials Where
only a weaker ligand (ligand B) was present the
nomenclature used was FeOH Lsk For example, the material
defined as FeOHT-3 1-Ad20 was prepared using a molar ratio

of three Fe to one tartrate and a concentration of adipate
of 20 mM The iron concentration in solution was 40 mM
unless stated otherwise in the figure legends
Materials
All chemicals were purchased from Sigma-Aldrich, Dorset,
UK, unless otherwise specified All laboratory ware was
m polypropylene The materials used m the preparation
of the ligand-modified poly oxo-hydroxy ferric iron
materials for the an vivo study were prepared with food
grade chemicals or pharmaceutical grade chemicals also
from Sigma-Aldrich, with the exception of the 58Fe
elemental iron used in the preparation of the 5flFe ferric
chloride which was purchased from Chemgas, Boulogne,
France
Methods
Screening assay
A series of dietary ligands was tested in a screening
assay for their effects on the formation of solid ligand-
modified poly oxo-hydroxy metal ion materials Briefly,
in a centrifuge tube, a fixed volume of stock solution of
ferric iron (400mM FeCl3 with 50mM MOPS, pH 1 4) was mixed
with varying volumes of a stock solution of ligand (400mM
with the exception of maltol which was 200mM, plus MOPS at
50mM and 0 9% NaCl) to obtain the desired metal ligand
ratio The volumes were then equally adjusted to parity
with a solution of 50mM MOPS and 0 9% NaCl All the
solutions obtained at this stage were fully soluble at pH
< 2 0 A small aliquot was taken to confirm the starting
iron concentration and then the pH was raised to ~6 5 by
drop-wise addition of concentrated NaOH to avoid high
volume changes After centrifugation at 2500 rpm for 10
minutes, an aliquot of supernatant was taken to analyse

the iron remaining m solution The remaining supernatant
was discarded and a fixed volume of dissolution buffer at
pH 6 (MOPS lOmM) or pH 4 (Acetic acid lOmM) was then added
to the wet solid of each tube and incubated overnight at
room temperature The tubes were then centrifuged (2500
rpm for 10 minutes) and an aliquot of supernatant taken to
determine the iron that was disaggregated The iron
concentration in each aliquot was measured by ICPOES
analysxs
Titration experiments
An acidic concentrated stock solution of iron (as ferric
chloride) was added to a solution containing either the
ligand A, ligand B or both ligand A and B at appropriate
concentrations to obtain the desired M L ratios In some
cases 0 9% w/v of electrolyte (for example NaCl or KCl)
was also added The solution was mixed thoroughly and an
aliquot collected for analysis of the "starting iron"
concentration The pH of the solution was always <2 0 and
the iron fully solubilised Next the pH was slowly
increased by drop-wise addition of a concentrated solution
of NaOH with constant agitation until the mixture reached
a basic pH (generally >8 0) At different points during
the titration, a homogeneous aliquot (lmL) of the mixture
was collected and transferred to an Eppendorf tube Any
aggregate formed was separated from the solution by
centrifugation (10 minutes at 13000 rpm) The iron
concentration m the supernatant was assessed by ICPOES
In some cases the supernatant was analysed for the
presence of aquated particulate iron and the size
distribution was measured (see below) When aquated
particulate iron was present, the supernatant was
ultrafiltrated (Vivaspm 3,000 Da molecular weight cut-off
polyethersulfone membrane, Sartonus Stedium Biotech GmbH,

Goettingen, Germany) and the iron concentration in the
filtrate, 1 e "soluble iron", was analysed by ICPOES
Preparation of solid ligand-modified poly oxo-hydroxy
ferric iron materials
The materials were prepared following a protocol similar
to the titration experiment described above Briefly, an
acidic concentrated stock solution of iron was added to a
solution containing either the ligand A, ligand B or both
ligand A and B In some cases 0 9% w/v of electrolyte was
also added The "starting pH" of the solution was always
<2 0, and the iron fully solubilised The pH was then
slowly increased by drop-wise addition of a concentrated
solution of NaOH with constant agitation until reaching
the desired final pH
When preparing the solid material as a pellet, the entire
mixture was then transferred to a centrifuge bottle and
spun at 4500 rpm for 15 minutes The supernatant was
discarded and the aggregated solid phase collected in a
petri dish When necessary, the solid was then dried in
an oven at 45 °C for a minimum of 8 hours Alternatively,
the mixture (precipitate and supernatant) was freeze-dried
at -20 °C and 0 4 mbar
When preparing the solid material as aquated particulate
material, the total mixture was either freeze-dried as
above, or concentrated by ultrafiltration (Vivaspm 3000
Da molecular weight cut-off polyethersulfone membrane,
Sartorius Stedium Biotech GmbH, Goettingen, Germany) and
then air dried in an oven at 45 °C for a minimum of 8
hours In some cases the mixture was dialysed (1,000 Da
regenerated cellulose membrane Spectra/pro 7, Cole-Parmer,
London, UK) in water to remove excess iron, ligands and

electrolytes before undergoing one of the drying processes
described above
When using bicarbonate as ligand B a variation of this
protocol was used to avoid release of C02 from
transformation of bicarbonate at acidic pH The starting
solution containing ligand A (when applicable) and
bicarbonate was prepared at pH 8 5 The appropriate
volume of acidic concentrated stock solution of iron was
then added drop-wise in conjunction with NaOH pellets
(progressively added to the mixture as required) m order
to always maintain a pH >7 5 The final pH of the
preparation was 8 5
Disaggregation assay
Known amounts of solid ligand-modified poly oxo-hydroxy
ferric iron materials were added into tubes (about 3 mg
iron per tube) Then, 3 mL of buffer (see below) were
added and the tubes shaken vigorously and incubated at
room temperature overnight After centrifugation at 4500
rpm for 15 minutes to separate the aggregated solid phase
from the aquated phase, an aliquot of supernatant was
collected to measure the disaggregated iron concentration
The remaining supernatant was discarded The mass of
remaining material (i e the wet pellet) was recorded
Concentrated HN03 was added to this pellet and the new
mass recorded The tubes were left at room temperature
until all the pellet dissolved and an aliquot was
collected for ICPOES analysis to determine the iron
concentration in the wet pellet
The buffers were either 50mM MOPS with 0 9% NaCl at pH
7 0, 50mM Maleic acid with 0 9% NaCl at pH 5 8-6 0 and
1 8-2 2, 50mM sodium acetate/ 50mM acetic acid glacial

with 0 9% NaCl at pH 4 0-4 5
In vitro gastrointestinal digestion assay
An amount of the solid ligand-modified poly oxo-hydroxy
ferric iron materials or control iron materials namely
ferrous sulphate, ferric chloride, or unmodified ferric
oxo-hydroxide, equivalent to 60mg elemental iron, were
added to a synthetic gastric (stomach) solution (50 mL of
2 g/L NaCl, 0 15 M HC1 and 0 3mg/mL porcine pepsin) and
incubated at 37°C for 3 0 minutes with radial shaking
Then 5 mL of the resulting gastric mixture was added to 30
mL of synthetic duodenal solution (containing lOg/L
pancreatin and 2g/L NaCl in 50mM bicarbonate buffer pH
9 5) The final volume was 35 mL and the final pH was
7 0 The mixture was incubated at 37°C for 60 mm with
radial shaking Homogeneous Aliquots (lmL) were collected
at different time points during the process and
centrifuged at 13,000 rpm for 10 minutes to separate the
aggregate and aquated phases The supernatant was
analysed for iron content by ICPOES At the end of the
experiment, the remaining solution was centrifuged at
4,500 rpm for 15 min and the supernatant analysed for Fe
content by ICPOES The mass of remaining material (l e
the wet pellet) was recorded Concentrated HN03 was added
to this wet pellet and the new mass recorded The tubes
were left at room temperature until all the pellet
dissolved and an aliquot was collected for ICPOES analysis
to determine the quantity of iron that did not
disaggregate / dissolve The starting amount of iron was
calculated from the iron m the wet pellet plus the iron
in the supernatant
To differentiate between soluble iron and aquated
particulate iron in the supernatant, at each time point,

thxs fraction was also ultrafiltered (Vivaspin 3,000 Da
molecular weight cut-off polyethersulfone membrane,
Sartorius Stedium Biotech GmbH, Goettmgen, Germany) and
again analysed by ICPOES
The gastrointestinal digestion of commercial iron
preparations was also tested with this assay using the
dose of total iron recommended by the manufacturers
Ferric pyrophosphate 14mg (Lipofer, Boots), ferrous
bisglycinate 20mg (Gentle iron, Solgar), ferric-hydroxide
polymaltose complex 80mg (Maltofer, Ferrum Hausmann),
ferric tri-maltol 30mg {Trimaltol, Iron Unlimited)
Modified in vitro gastrointestinal digestion assay
The particle size of the ligand-modified poly oxo-hydroxy
ferric iron materials under simulated gastric and
intestinal conditions was determined using an adapted nin
vitro gastrointestinal digestion assay" in which no
protein was in solution The absence of proteins was
required to measure particle size as these interfere with
the measurement but the procedure was otherwise identical
to the nin vitro gastrointestinal digestion assay" with
extra aliquots being collected at various time points for
the determination of particle size
Inductively Coupled Plasma Optical Emission Spectroscopy
analysis (ICPOES)
Iron contents of solutions or solids (including wet
solids) were measured using a JY2000-2 ICPOES (Horiba
Jobm Yvon Ltd , Stanmore, U K ) at the iron specific
wavelength of 259 940 nm Solutions were diluted m 5%
nitric acid prior to analysis while solids were digested
with concentrated HN03 The percentage of iron in
solution or solid phase was determined by the difference

between the starting iron content and either the iron in
the soluble phase or the iron in the solid phase depending
on the assay
Determination of particle size
The size distribution of micron-sized particles was
determined using a Mastersizer 2000 with a Hydro-uP
dispersion unit (Malvern Instruments Ltd, Malvern, UK) and
nano-sized particles were determined with a zetasizer Nano
ZS (Malvern Instruments Ltd, Malvern, UK) Mastersizer
measurements required no sample pre-treatment whereas
centrifugation was needed to remove large particles prior
to Zetasizer measurements
Structural analysis
Transmission Electron Microscopy and Energy Dispersive X-
ray Analysis (EDX)
Powder samples were analysed by first dispersing the
powder in methanol and then drop-casting on standard holey
carbon TEM support films Commercial tablets were
similarly analysed but were first crushed to release the
powder Analysis were undertaken by the Institute for
Materials Research, University of Leeds, UK
Scanning Transmission Electron Microscopy
Powder samples were analysed by first dispersing the
powder in methanol and then drop-casting on standard holey
carbon TEM support films Commercial tablets were
similarly analysed but were first crushed to release the
powder Analysis were undertaken by aberration-corrected
scanning transmission electron microscopy (Daresbury,
superSTEM)
Infrared Analysis (IR)

IR spectra were collected using a DurasamplIR diamond ATR
accessory with a Nicolet Avatar 360 spectrometer with a
wavelength range of 4000-650cm-1 and resolution of 4cm"1
Analysis were undertaken by ITS Testing Services (UK) Ltd,
Sunbury on Thames, UK
X-Ray Diffraction Analysis
Samples were analysed as dry powders Commercial tablets
were crushed to release the powder Analysis was by X-ray
diffraction analysis at the University of Cambridge using
a Philips X'Pert PW3020 (theta/2theta, 2 motors) with up
to 14 hour scan time and 5-70° 2theta on CuKalpha
In vivo absorption study
Subjects
Healthy young women {aged 18-45 years) with mild iron
deficiency anaemia (defined as haemoglobin between 10-11 9
g/dL plus either serum ferritin below 20ug/L or
transferrin saturation below 10%), or clear iron
deficiency (defined as serum ferritin below 12ug/L) were
recruited to take part xn the study Exclusion criteria
were pregnancy or lactation and known coeliac disease,
moderate/severe anaemia (haemoglobin levels <10 g/dL),
cardiovascular disease, chronic respiratory disease,
chronic liver disease, renal disease, chronic infection,
or chronic inflammation Other exclusion criteria were
surgery in the past three months, cancer diagnosis m the
last ten years, known history of hereditary
haemochromatosis or haemoglobmopathies, current
medication that could alter iron metabolism, recent blood
donation/heavy blood loss (in the past 3 months)
Subjects who regularly consume vitamin and mineral
supplements were asked to discontinue supplementation 2
weeks before the screening for the study Written

informed consent was obtained from all subjects The study
protocol was approved by the Suffolk Local Research Ethics
Committee
Study design
The experimental treatment was either a single dose of
58Fe labeled ligand-modified poly oxo-h/droxy ferric iron
material (60 mg total iron) or ferrous sulphate (65 mg
total iron) Ferrous sulphate is used as a reference dose
to control for individuals who are poor absorbers (defined
as those who have no significant net area under the curve
(AUC) for plasma iron following ferrous sulphate
ingestion) A crossover study design was used with each
volunteer acting as her own control
Fe absorption was based on erythrocyte incorporation of
the 58Fe stable-isotope label 14 days after the intake of
labelled iron test compounds The test compounds and the
reference compound (ferrous sulphate) were taken (with or
without breakfast), under strictly standardised conditions
and close supervision, after an overnight fast with 14
days interval No intake of food or fluids (apart from
water) was allowed for 4 h after the iron compound intake
Ten blood samples (12 mL) were taken during each of the 2
visits to determine the absorption of Fe at the following
times before intake and 30, 60, 90, 120 180, 210 and 240
minutes after intake of the iron compound An additional
blood sample was taken at baseline (before intake) to
confirm iron status (full blood count, ferritin, soluble
transferrin receptor, transferrin saturation) and
determine erythrocyte 5BFe incorporation
Total serum iron concentration was analysed by a standard

clinical chemistry procedure based on the method by Smith
et al using the chromophore Ferene®
RBC incorporation of S8Fe was determined using an Elan DRC
Plus Inductively Coupled Plasma Mass Spectrometer (Perkm
Elmer Sciex, Beaconsfleld, UK) The sample introduction
system consisted of a V-groove nebuliser, a double-pass
spray chamber, a demountable quartz torch, and a quartz
injector (2 mm internal diameter) Platinum-tipped
sampler and skimmer cones (Perkm Elmer Sciex,
Beaconsfield, UK) were used for all analyses Baseline
whole blood samples were collected from participants in
the study immediately prior to administration of a 60 mg
oral Fe supplement labelled with 2mg 5SFe/ and a second
blood sample was collected 14 days after administration
Whole blood was diluted 100-fold with an aqueous solution
containing 0 5% Triton X-100, 1% butan-1-ol, 0 5% ammonia,
and 0 007 % nitric acid Instrument conditions were tuned
for optimum signal sensitivity {via the measurement of
24Mg, 115In and 238U isotopes) , minimum oxide formation (via
the measurement of the 140Ce and 155Gd isotopes to allow
monitoring of the degree of CeO formation at m/z = 155)
and minimum doubly charged ion formation (via the
measurement of the 138Ba and e9Ga isotope signals to allow
monitoring of the degree of 138Ba2+ formation at m/z = 69)
Further adjustment was then performed to reduce mass bias
between 5eFe and 57Fe (approximately 5%) Detector
voltages were dropped from the typical -2400 and 1550 V to
-1725 and 1050 V for analogue and pulse stages,
respectively
Preparation of S8Fe labelled ferric chloride solution
A solution of saFe labelled ferric chloride was prepared
by dissolving 100 mg 5BFe enriched elemental iron

(Chemgas, Boulogne, France) in 4 mL 37% HC1 in a pear-
shaped glass flask attached to a condenser and heated at
48°C in a water bath The temperature was raised
gradually over time to keep the solution boiling as the
concentration of chlorine dropped When the elemental
iron powder was dissolved, 0 5 mL of 30% hydrogen peroxide
were added to oxidize ferrous iron to ferric iron The
flask was then sealed, once the oxidation reaction
finished/ i e once the formation of 02 bubbles stopped
The concentration of iron in the final solution was
determined by ICPOES and the Ferrozine assay was used to
confirm the absence of ferrous iron
Preparation of the 5BFe labelled ligand modified poly
oxo-hydroxy ferric iron material
The chosen ligand-modified poly oxo-hydroxy ferric iron
materials enriched with 58Fe were prepared following the
protocol described above (see Preparation of solid ligand-
modified poly oxo-hydroxy ferric iron materials) using a
ferric chloride stock solution containing 3 5% w/w 58Fe <2
mg of 58Fe per 60 mg total iron in the ingested solid
material) from the 58Fe labelled ferric chloride solution
discussed above
Results and discussion
Effect of Ligand A
A series of ligands, namely maltol, succinic acid/ citric
acid, lactic acid, tartaric acid, malic acid, gluconic
acid, aspartic acid, glutamic acid, histidine and
glutamme, were studied for their effect on ferric poly
oxo-hydroxide precipitation from solution
Initially, the ligands were all tested using the screening
assay described above at ratios of 1 1 to 1 5 and

classified in three groups The first group, "strong
ligands", were ligands found to inhibit the formation of
80% of the solid material at ratio 1 1 and included
gluconic acid, citric acid and maltol The second group,
"weak ligands", were ligands found to have little effect
on the amount of solid material formed (<10% at all the
ratios tested) and included aspartic acid, succinic acid,
lactic acid, glutamic acid and histidine The third
group, "intermediate ligands", were ligands found to have
an influence, between strong and weak ligands, on the
amount of solid material formed at, at least, one of the
ratios tested and included malic acid, tartaric acid and
glutamine
In a second instance, six ligands from the three groups
described above were re-screened for their effects on both
the formation of ferric poly oxo-hydroxide precipitation
at varying M L ratios, and the dissolution of the solid
materials formed in pH 6 and pH 4 buffers (see screening
assay above) As expected, the ligands had variable
effects on the percentage of poly oxo-hydroxy iron that
was precipitated depending upon (a) the group the ligand
belonged to and (b) the M L ratio Yet, the solid
materials formed, showed variable re-aquation properties
that were not predictable from the precipitation
behaviours Examples of results using a strong affinity
ligand, namely maltol, a weak affinity ligand, namely
succinate, and an intermediate affinity ligand, namely
malate, are shown m Figures 1A and B Re-dissolution
clearly depends upon the ligand and its ratio to iron
which may be expected What is not expected is that the
strong ligand, maltol, did not promote any re-dissolution
of the iron at pH 6 0 in spite of the fact that soluble
iron-maltol complexes can be formed (for at least a

proportion of the iron) at this pH Moreover, the
intermediate ligand, malate, allowed greater dissolution
of iron from the solid phase at pH 6 0 than the strong
ligand maltol - even when ratios were matched (c f 1 1J
Examples of further results with other ligands or ratios
are shown in Table 1
Table 1 The effect of single ligands on poly oxo-hydroxy
iron precipitation and the re-dissolution of that iron

Two ligands, namely malate and tartrate, that showed most
effects in the screening assay, were chosen for study m
greater detail The re-dissolution profile was studied
using a more defined assay in four different buffers (see
Disaggregation Assay m Methods) The buffers contained
0 9% w/v electrolyte so that the results obtained would
reflect the behaviour of the material m a biological
ionic strength environment Also the pH environments were
chosen to reflect different parts of the gastrointestinal
tract from gastric (pH 1 8) to intestinal (pH 7 0)
Firstly, the results shown m Table 2 confirmed that the
two ligands affected not only the precipitation but also
the disaggregation profile depending on the ratio used m
the preparation of the ferric poly oxo-hydroxide materials
as seen in the screening assay above Generally,
increasing the ligand ratio decreased the formation of the

ligand-modifxed poly oxo-hydroxy feme iron solid
material and increased the disaggregation profile
However, the extent of the effect seen with one ligand did
not reflect the extent of the effect seen with another
ligand as illustrated here with malate and tartrate The
results observed were reproducible as indicated in table 2
with malate at ML ratio 1 2
Table 2 Effect of malate and tartrate ratios on the
percentage of iron precipitated as ligand-modified poly
oxo-hydroxy ferric iron materials and the disaggregation
of the materials

Lxgand-modified poly oxo-hydroxy ferric iron materials
were prepared at pH 6 5 m 50mM MOPS and 0 9% NaCl
Starting iron concentrations were 26 7mM Precipitation
steps were either carried out in individual tubes (a) as
per the precipitation procedure described in Screening
Assay, or as a batch (b) as per the preparation of solid
ligand-modified poly oxo-hydroxy ferric iron materials
(see methods) Disaggregation of all materials was
performed according to the method outline in
Dissagregation assay {see methods)
Secondly, the effect of the ligand on the rate of

formation of the ligand-modified poly oxo-hydroxy ferric
iron materials was studied using the titration protocol
described m the methods section Figure 2 shows the rate
of formation of the solid material with increasing pH
The addition of malate was found to delay the formation of
the solid material compared to the absence of ligand
This scenario is to be expected when a ligand competes
with the polymerisation of the poly oxo-hydroxy ferric
iron entity that results in the formation of the solid
material However, unexpectedly, tartrate was found to
have a promoting effect on the formation of the solid
material at lower pH This does not correlate with the
competition scenario described above In this case the
ligand, tartrate, appears to be enhancing the
precipitation Another observation was that tartrate, at
basic pH (>7 5), did promote disaggregation of this
material Indeed, Figure 16 shows a typical profile of
the formation of two solid phases, namely aggregated and
aquated tartrate-modified ferric poly oxo-hydroxide with
increasing pHs following the titration protocol described
m Methods These results were also observed with other
ligands A and ligands B (results not shown)
The disaggregation profile of the ligand-modified poly
oxo-hydroxy ferric iron solid material formed at different
pHs was shown to vary as illustrated m Figure 3 for
malate As the pH of preparation of the material
increases, the disaggregation profile decreases This is
in accordance with an increase of polymerisation and
formation of oxo-bridges with increasing pH, probably
limiting the modification effect of the ligand on the
material
The presence of o 9% w/v electrolyte, as soaium (NaCl) or

potassium chloride (KCl), m the preparation of the
ligand-modified poly oxo-hydroxy ferric iron solid
materials was also studied Figure 4A shows that the
presence of 0 9% NaCl did not affect the disaggregation
profile of the tartrate-modified poly oxo-hydroxy ferric
iron material at M L ratio 4 1 compared to the same
material prepared without NaCl Similarly, Figure 4B
shows that the presence of 0 9% KCl did change the
disaggregation profile of the tartrate-modified poly oxo-
hydroxy ferric iron material at M L ratio 2 1 (solution
containing 50mM niacin)
Finally, the effect of drying ligand-modified poly oxo-
hydroxy ferric iron solid materials was studied with
respect to disaggregation Drying the material generally
lead to a modest reduction in its disaggregation as
exemplified by the tartrate-modified poly oxo-hydroxy
ferric iron material at M L ratio 4 1 which is illustrated
in Figure 5 Small, inconsistent differences were
observed between oven-drying and freeze-drymg methods
(Figure 5)
Effect of ligand B
Almost all of the studies described above were carried out
with ligand-modified poly oxo-hydroxy ferric iron solid
materials produced m MOPS buffer MOPS is often used in
metal speciation studies due to its very weak interaction
with most metal ions and hence it rarely interferes in the
formation of metal complexes However, MOPS has a pKa of
7 2 and so has a buffering capacity around neutral pH
Thus, although MOPS would not interact directly with iron
or prevent the formation of the solid material, it may
indirectly influence the formation of the solid by
controlling the rate of change in environmental pH In

addition, the buffer used in the preparation of the
ligand-modified poly oxo-hydroxy ferric iron solid
materials should be safe for human consumption which MOPS
is not Therefore, to study the influence of the buffer,
or ligand B, on the formation and re-dissolution
properties of the ligand-modified poly oxo-hydroxy ferric
iron solid materials, we selected a series of compounds
with buffering capacity at varying pH ranges, namely,
adipate, bicarbonate, acetate, glutarate, dimethyl
glutarate, pimelate, succinate, vanillin, tryptophan,
benzoate, propionate, borate, niacin and pyridoxme
hydrochloride Figure 6 illustrates the effect of
changing MOPS for adipate on the rate of formation of the
tartrate-modified poly oxo-hydroxy ferric iron solid
material at M L ratio 4 1 (Figure 6(i)), as well as its
effect on the otherwise un-modxfied poly oxo-hydroxy
ferric iron solid material (Figure 6(H)) In both cases,
adipate had a promoting effect on the rate of formation of
the solid material
Following these observations, the formation and
disaggregation profiles of the tartrate-modified oxo-
hydroxy ferric iron solid materials were studied using
varying M L ratios Adipate reduced the disaggregation
capacity of the materials formed (Table 3) compared to
MOPS (Table 2), except at gastric pH (pH 1 8) which showed
low disaggregation capacity with both buffers In
contrast, in the case of malate-modified poly oxo-hydroxy
ferric iron materials, bicarbonate had a negative
influence on the percentage of precipitation and the
disaggregation capacity of the material (Table 2 and 3)
These effects fell off with lower concentrations of
adipate but not bicarbonate (Table 3, data in bold)

The influence of ligand B on the disaggreagtion profile of
the tartrate-modified poly oxo-hydroxy ferric iron solid
material is further illustrated in Figure 7 with niacin
and benzoate

Tartrate-modified poly oxo-hydroxy ferric iron solid
materials were prepared following the protocol
"preparation of solid ligand-modified poly oxo-hydroxy
ferric iron materials" {see methods) at pH 4 0 in either
50mM adipate (Ad50) or 20mM adipate (Ad20) without the
presence of an electrolyte Malate-modified poly oxo-
hydroxy ferric iron solid materials were prepared
following the same procedure at pH 8 5 in either lOOmM
bicarbonate (BiclOO) or 25mM bicarbonate (Bic25) without
the presence of an electrolyte The disaggregation of the
materials was performed according to the method outlined
in Disaggregation assay (see method) using the non-dried
material for FeOHT-Ad50 and FeOHM-BiclOO and the oven-
dried material for FeOHT-Ad20 and FeOHM-Bic25

Structural analysis of the solid ligand-modified poly oxo-
hydroxy ferric iron materials
The solid ligand-modified poly oxo-hydroxy ferric iron
materials prepared above differ from currently available
iron formulations in that they are not a simple inorganic
ferrous ion salt (e g ferrous sulphate) , an iron complex
in which the metal is coordinated with organic ligand
(e g ferric trimaltol), nor an organic ligand coated iron
mineral particle (e g iron polymaltose or *Maltofer')
The elemental analysis of particles from our solid ligand-
modified poly oxo-hydroxy ferric iron materials measured
by Energy dispersive X-ray analysis (EDX) clearly shows
the presence of carbon atoms in the iron- and oxygen-
contammg particles {an example is shown in Figure 8)
Furthermore, the infrared spectrum of the material
demonstrates the presence of a covalent-like bond between
the ligand and the metal (Figure 9) in addition to the
abundant presence of hydroxy groups This illustrates
that the ligand is incorporated into the structure of the
metal oxo-hydroxide lattice through formal bonding and not
simply adsorption or 'entrapment' The changes to the
dissolution characteristics of the material can be readily
explained by the manner in which the ligand alters the
metal-oxo-hydroxide lattice In freshly precipitated iron
oxo-hydroxide a fernhydnte-like structure is observed
with some clear crystalline regions the addition of
ligand B, in this case tryptophan, reduces the extent of
crystallinity while the addition of ligand A and B, in
this case trypotphan and tartrate, almost negate the
crystallinity entirely (Figure 11) Maltofer, which is an
organic ligand coated iron mineral particle, appeared more
like freshly precipitated iron oxo-hydroxide, indicating

that the ligand had not signif xcantly modr ^^^^^B
structure This comparison is best observer ^^
diffraction where iron hydroxide peaks are nov
for a ligand-modified poly oxo-hydroxy ferric i
material, but they are seen m Maltofer (Figure ±
broad and noisy peaks due to the very small size o
primary particles (a few nanometres) _/
Gastrointestinal digestion of the solid ligand-modified
poly oxo-hydroxy ferric iron materials
We compared the disaggregation of some prior art and
commercial iron compounds to that of the ligand-modified
poly oxo-hydroxy feme iron materials under simulated
gastrointestinal conditions (see Methods) The gastric
disaggregation (pHl 2) and the gastric dissolution
profiles of two of the ligand-modified poly oxo-hydroxy
ferric iron materials, in comparison with ferrous
sulphate, ferric oxo-hydroxide and iron polymaltose
(Maltofer), are shown in Figure 10 Ferrous sulphate
disaggregates and dissolves very well at acidic pH as is
expected for a metal salt Conversely, Maltofer
disaggregates very rapidly m the gastric conditions
(after 5 minutes almost B0% of the iron is disaggregated)
but remains in an aquated particulate form (typically
around 20 nm diameter results not shown) {Figure 10)
Percentage iron dissolution from Maltofer was less than 5%
although it should be noted that there can be a loss of up
to 10% of iron through binding to the ultrafiltration
membrane In comparison, the two novel ligand-modified
poly oxo-hydroxy feme iron materials had an intermediate
disaggregation profile compared to ferric oxo-hydroxide
and ferrous sulphate In addition, the dissolution of
these materials closely paralleled the disaggregation
profile under gastric conditions although this need not be

the case for these novel materials These data show a
clear difference between un-modified ferric oxo-hydroxide,
Maltofer, ferrous sulfate, and our ligand-modified poly
oxo-hydroxy ferric iron materials
Disaggregation of some of our novel ligand-modified poly
oxo-hydroxy ferric iron materials under gastric and
intestinal conditions was also compared to dissagregation
of other commercially available iron compounds, namely
ferric pyrophosphate, ferric chloride, ferric trimaltol
and ferrous bisglycmate The commercial compounds either
failed to disaggregate properly (e g ferric
pyrophosphate), or they disaggregated very rapidly (Figure
14) This rapid disaggregation, if paralleled by
dissolution, is believed to be responsible for giving rise
to bolus delivery of iron ions m the gut lumen and
likely, therefore, the occurrence of side effects The
novel ligand-modified poly oxo-hydroxy ferric iron
materials showed a degree of controlled release although,
clear differences can be seen in the rates of
disagreggation for the novel materials, indicating that
their properties can be tailored as required (Figure 14)
It should be noted in Figure 14 that whether iron remains
in solution or not at pH 7 0 is merely a function of
whether chelators/ligands are present (as they will
naturally be m the gut) and so the data for ferrous
sulphate and ferric chloride (where no ligand is present
in the compound) should not be over-interpreted
Iron disaggregation and dissolution under both gastric and
intestinal conditions for some ligand-modified poly oxo-
hydroxy ferric iron materials that were tested further in
human volunteers (see below) are presented m Figure 15
Again we show a range of different disaggregation and

dissolution profiles for the novel materials, illustrating
the possibility of tailoring them as required
A study of the particle size distribution after passage
through the modified gastrointestinal digestive assay of
some tartrate-modified poly oxo-hydroxy ferric iron
material is shown m Figure 17 Changing the M L ratio
{first vs second bar), pH of preparation {second vs third
bar) and type of ligand B (fourth bar) clearly affects the
size of particles obtained and therefore
disaggregation/dissolution profiles There is especially
an increase in smaller particle sizes with increasing
tartrate concentrations indicating less aggregation of the
primary particles with increasing L content In addition,
the higher the pH of preparation, the smaller the
resulting particle size
Iron absorption in humans of the solid ligand-modified
poly oxo-hydroxy iron ferric materials
Seven ligand-modified poly oxo-hydroxy ferric iron
materials have been assessed further for their absorption
in human volunteers and the results compared with
unmodified ferric oxo-hydroxide A summary of the results
is shown in Table 4



Serum iron increase three hours after ingestion and
percentage iron absorption (calculated as the red blood
cell incorporation of 58Fe divided by 0 80) of solid
ligand-modified poly oxo-hydroxy ferric iron materials
Mean ± SEM (n ranges from 2-4), * for FeOH at 180mm there
was a decrease from the baseline serum iron value
The serum absorption profiles of the compounds (Figure 13)
show that the novel ligand-modified poly oxo-hydroxy
ferric iron materials have much lower rates of iron
absorption than ferrous sulphate which may be advantageous
as this will prevent systemic exposure and potential
damage from transiently high levels of iron There was
clear iron absorption from all formulations (Figure 13)
and for at least one preparation this is estimated to be
equivalent to ferrous sulphate It is especially
noteworthy that literature reports indicate that ferric
polymaltose yields no detectable rise m serum iron
following ingestion and, that absorption of iron is very
low (Kaltwasser et al, 1987) and would be consistent with
our data for ferric oxo-hydroxide
The compounds FeOHT-2 1-TRP15 and FeOHGluconic20 are
examples of how changing the composition of these novel
materials changes their serum iron profile but maintains
the same percentage of iron absorption (Figure 13) again
indicating that the materials can be tailored to achieve
desired outcomes

References
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We Claim
1 A process for producing a solid ligand-modified poly
oxo-hydroxy metal ion material comprising metal ions (M) ,
ligands (L) and oxo or hydroxy groups (OH), wherein
M represents one or more metal ions selected from Ag2+,
Al3+, Au3+, Be2+, Ca2+, Co2, Cr3+, Cu2, Eu3, Fe3+, Mg2+, Mn2+,
Ni2+, Sr2+, V5+ and Zn2+,
L represents one or more ligands which comprise a
ligand selected from a carboxylic acid, maltol, ethyl
maltol, vanillin, bicarbonate, sulphate, phosphate,
silicate, borate, molybdate, selenate, tryptophan,
glut amine, proline, valine, histidine, folate, ascorbate,
pyridoxine, niacin, adipate, acetate, g_utarate, dimethyl
glutarate, pimelate, succinate, benzoate and propionate, and
wherein the material has a polymeric structure in which
the ligands are non-stoichiometrically substituted for the
oxo or hydroxy grojps and are distributed within the solid
phase structure of the metal oxo-hydroxy material and so
that the substitution of the oxo or hydroxy groups by the
ligands is substantially random,
wherein at least some of the ligand integrates into the
solid phase so that it displays formal M-L bonding that can

be detected by physical analytical techniques and the gross
solid ligand-modified poly oxo-hydroxy metal ion material
has one or more reproducible physico-chemical properties,
the process comprising
(a) mixing the metal ions M and the ligands L at a
first pH(A) at which the components are soluble,
(b) changing the pH(A) to a second pH(B) to cause a
solid precipitate of the solid ligand-modified poly oxo-
hydroxy metal ion material to be formed, and
(c) separating, and optionally drying, the solid
ligand-modified poly oxo-hydroxy metal ion material produced
in step (b)

2 The process as claimed in claim 1, wherein the step of
formulating the material comprises adding an excipient
3 The process as claimed in claim 1 or claim 2any one of
the preceding claims, wherein the pH(A) is above a pH at
which oxo-hydroxy polymerisation of the corresponding metal
oxo-hydroxide commences

4 The process as claimed in any one cf the preceding
claims, wherein the pH is changed from pH(A) to pH(B) by the
addition of alkali, and preferably wherein the alkali is
added as a solution of sodium hydroxide, potassium hydroxide
or sodium bicarbonate to increase the concentration of OH in
the mixture of step (b) and/or wherein pH(A) is less than or
equal to pH 2 and pH(B) is greater than or equal to pH 2
5 The process as claimed in any one of claims 1 to 3,
wherein the pH is changed from pH(A) to pH(B) by the
addition of acid, and preferably wherein the acid is added
as a mineral acid or an organic acid to decrease the
concentration of OH in the mixture of step (b), and/or
wherein pH(B) is less than or equal to pH 2 and pH(A) is
greater than or equal to pH 2
6 The process as claimed in any one of the preceding
claims, wherein the one or more reproducible physico-
chemical properties are selected from dissolution (rate, pH
dependence and pM dependence), adsorption and absorption
characteristics, reactivity-inertness, melting point,
temperature resistance, particle size, magnetism, electrical
properties, density, light absorbing/reflecting properties,
hardness-softness, colour and encapsulation properties

7 The process as claimed in claim 6, wherein the
reproducible physico-chemical property is reproducible
within a limit of preferably ± 10%, and more preferably +
5%, and even more preferably within a limit of ± 2%
8 The process as claimed in any one of the preceding
claims, wherein the metal ion (M) is Fe3+
9 The process as claimed in any one of the preceding
claims, wherein the carboxylic acid ligand is selected from
adipic acid, glutaric acid, tartaric acid, malic acid,
succinic acid, aspartic acid, pimelic acid, citric acid,
gluconic acid, lactic acid and benzoic acid
10 The process as claimed in any one of the preceding
claims, wherein the ligand has buffering properties or a
buffer is present n a medium for carrying out the process
11 The process as claimed in claim 10, wherein the buffer
is selected from an inorganic buffer, such as borate,
silicate or bicarbonate, or an organic buffer such as 3-

propanesulfonic acid (MOPS), 2-[4-(2-hydroxyethyl)piperazin-
l-yl]ethanesulfonic acid (HEPES) , piperazine-N,N'-bis
(PIPES) or 2-Amino-2-hydroxymethyl-propane-l,3-diol (TRIS),
or a buffer selected from adipic acic, pimelic acid,
tryptophan or hydroxymethylcellulose
12 The process as claimed in claims 1, wherein the first
pH(A) is a pH below the pH at which oxo-hydroxy
polymerisation of the corresponding metal oxo-hydroxide
commences, and optionally pH(A) is less than or equal to pH
2 and pH(B) is greater than or equal to pH 2
13 The process as claimed in claim 1, wherein the pH is
changed from pH(A) to pH(B) by the addition of acid, and
optionally wherein the acid is added as a mineral acid or an
organic acid to decrease the concentration of OH in the
mixture of step (b)
14 The process as claimed in claim 1, wherein pH(B) is
less than or equal to pH 2 and pH(A) is greater than or
equal to pH 2

15 The process as claimed in claim 1, wherein
(a) the pH change from pH(A) to pH(B) occurs in a 24
hour period or less, more preferably within an hour period
and most preferably within 20 minutes, and/cr
(b) the concentrations of total metal ions (M) and
total ligand (L) are greater than 10-6 molar and more
preferably are greater than 10"3 molar, and/or
(c) the reaction medium is an aqueous solution, and/or
(d) the buffer stabilises the pH range of oxo-hydroxy
polymerisation, and/cr
(e) the buffer is selected from an inorganic buffer,
such as borate, silicate or bicarbonate, or an organic
buffer such as MOPS, HEPES, PIPES or TRIS, or a buffer
selected from adipic acid, pimelic acid, tryptophan or
hydroxymethylcellulose and /or
(f) the buffer concentrations are less than 500 mM,
preferably less than 200 mM and most preferably less than
100 mM, and/or
(g) the temperature of the reaction is between 0 and
100°C, and more preferably between room temperature (20-
30°C) and 100oC, and/or

(h) the ionic strength of the reaction medium is varied
by addition of electrolyte, and/or
(1) the components are mixed in step (a) to for a
homogeneous solution
16 A composition comprising a solid ligand-modified poly
oxo-hydroxy metal ion material comprising metal ions (M),
ligands (L) and oxo or hydroxy groups (OH), wherein
M represents one or more metal ions selected from Ag2+,
Al3+, Au3\ Be2+, Ca2+, Co2, Cr3+, Cu2+, Eu3+, Fe3+, Mg2+, Mn2+,
Ni2, Sr2+, V5+ and Zn2+,
L represents one or more ligands which comprise a
ligand selected from a carboxylic acid, maltol, ethyl
maltol, vanillin, bicarbonate, sulphate, phosphate,
silicate, borate, molybdate, selenate, tryptophan,
glutamine, proline, valine, histidine, folate, ascorbate,
pyridoxine, niacin, adipate, acetate, glutarate, dimethyl
glutarate, pimelate, succinate, benzoate and propionate, and
wherein the material has a polymeric structure in which
the ligands are non-stoichiometrically substituted for the
oxo or hydroxy groups and are distributed within the solid
phase structure of the metal oxo-hydroxy material and so
that the substitution of the oxo or hydroxy groups by the
ligands is substantially random,
wherein at least some of the ligand integrates into the
solid phase so that it displays formal M-L bonding that can
be determined by physical analytical techniques and the
gross solid ligand-modified poly oxo-hydroxy metal ion
material has one or more reproducible physico-chemical
properties

17 The composition as claimed in claim 16, which comprises
a solid ligand-modified poly oxo-hydroxy metal ion material
comprising metal ions (M) , ligands (L) and oxo or hydroxy
groups (OH), wherein M represents one or more metal ions
that comprise Fe3+ ions, L represents one or more ligands
and wherein the material has a polymeric structure in which
the ligands L are non-stoichiometrically substituted for the
oxo or hydroxy groups and are distributed within the solid
phase structure of the metal oxo-hydroxy material and so
that the substitution of the oxo or hydroxy groups by the
ligands is substantially random, wherein at least some of
the ligand integrates into the solid phase so that it
displays formal M-L bonding that can be detected by physical
analytical techniques and the solid ligand-modified poly
oxo-hydroxy metal ion material having one or more
reproducible physico-chemical properties
18 The composition as claimed in claim _7, wherein M is
Fe3+ ions
19 The composition as claimed in claim 17 or claim 18,
wherein
(l) the substantially randomly solid phase structure of
the material produced by substitution of hydroxy or oxo
groups by the ligand L is determinable by an X-ray
diffraction pattern having no identifiable peaks for L or
MO/MOH, and/or

(110) the substantially randomly solid phase structure
of the material produced by substitution of hydroxy or oxo
groups by the ligand 1 is an increase in the amorphousness
of the structure of the material as determinable by high
resolution transmission electron microscopy, and/or
(111) the reproducible physico-chemical property is „
selected from one or more of a dissolution profile, an
adsorption profile or a reproducible elemental ratio, and/or
(IV) the reproducible elemental ratio is reproducible
within a limit of preferably ± 10%, and more preferably ±
5%, and even more preferably within a limit of ± 2%
20 The composition as claimed in any one of claims 17 to
19, wherein the infrared spectra further comprises one or
more peaks for the bonds between M-O, O-H, and L alone
21 The composition as claimed in any one of claims 17 to
20 wherein
(I) the ligand L comprises tartarate or adipate or
succinate, or
(ii) the ligand L comprises tartarate and adipate, or
(iii) the ligand L comprises tartrate and succinate
22 The composition as claimed in any one of claims 17 to
21, wherein the ratio M L is between about 1 5 and 5 1

23 The ferric iron composition as claimed in any one of
claims 17 to 22 which is FeOHAd100 and FeT-3 1-Ad20

ABSTRACT

Title A process for producing a solid ligand-modified poly oxo-hydroxy metal
ion material
A process for producing a solid ligand-modified poly oxo-
hydroxy metal ion material comprising metal ions (M),
ligands (L) and oxo or hydroxy groups (OH), wherein
M represents one or more metal ions selected from Ag2+, Al3+,
Au3+, Be2+, Ca2+, Co2+, Cr3+, Cu2+, Eu3+, Fe3+, Mg2+, Mn2+, Nl2+,
Sr2+, V5+ and Zn2+,
L represents one or more ligands which comprise a ligand
selected from a carboxylic acid, maltol, ethyl maltol,
vanillin, bicarbonate, sulphate, phosphate, silicate,
borate, molybdate, selenate, tryptophan, glatamine, proline,
valine, histidine, folate, ascorbate, pyridoxine, niacin,
adipate, acetate, glutarate, dimethyl glutarate, pimelate,
succinate, benzoate and propionate, and
wherein the material has a polymeric structure in which the
ligands are non-stoichiometrically substituted for the oxo
or hydroxy groups ana are distributed within the solid phase
structure of the metal oxo-hydroxy material and so that the
substitution of the oxo or hydroxy groups by the ligands is
substantially random,
wherein at least some of the ligand integrates into the
solid phase so that it displays formal M-L bonding that can
be detected by physical analytical techniques and the gross
solid ligand-modified poly oxo-hydroxy metal ion material
has one or more reproducible physico-chemical properties,
the process comprising
(a) mixing the metal ions M and the ligands L at a first
pH(A) at which the components are soluble,
(b) changing the pH(A) to a second pH(B) to cause a solid
precipitate of the solid ligand-modified poly oxo-hydroxy
metal ion material to be formed, and
(c) separating, and optionally drying, the solid ligand-
modified poly oxo-hydroxy metal ion material produced in
step (b).