Title of Invention

A METAL CHELATE COMPOUND

Abstract The invention discloses a metal chelate of the formula (CH3SCH2CH2C(OH)COO)2Fe.2H2O.
Full Text The present invention relates in general to a composition comprising water
and at least one complex of Methionine Hydroxy Analogue and a method for
preparing such composition and in particular to the use in human and animal
nutrition (monogastric and polygastric animals) of known chelates of
bivalent metals Mg, Ca, Mn, Co, Cu, Zn and Fe with methionine hydroxy
analogue. The present invention further relates to a method for preparing
new chelates with methionine hydroxy analogue, both in solid form with iron
(II), vanadium (IV) and (V) and molybdenum (V) and (VI), and in liquid form
in aqeuous solution with iron (II) and (III) and chrome (III). Eventually,
the present invention relates to the use of said new chelates, both in
solid form with iron (II), vanadium (IV) and (V) and molybdenum (V) and
(VI), and in liquid form in aqueous solution with iron (II) and (III) and
chrome aqueous solution with iron (II) and (III) and chrome (III), in human
and animal nutrition. This application is divided out of patent application
no. 511/KOLNP/2005 dated 28/03/2005. ^^^""
It is known that a metal chelate is a compound originating from an organic
molecule (such as an amino acid or a peptide chain or an alpha-keto acid or
an alpha-hydroxy acid) and a metal ion through strong coordination bonds.
Some known metal chelates are used in the field of human nutrition. Their
use is due to the biological action performed by the metal element
involved, as activator in several enzymatic reactions, and as regulator in
various metabolic functions in all living organisms. The presence of a
chelating molecule promotes the absorption, availability and use of the
metal element since the latter is carried by the organic component in all
areas of the organism.
This results also in a strong reduction of losses of unused metals in
dejections, and therefore in a significant economic saving and in an
environmental advantage.

The general features that should characterize a highly
bioavailable metal chelate are: (a) neutrality of
complex (the positive charge of metal is balanced by
the negative charge of ligands; (b) absence of
negative counter-ions (chlorides, sulfates); (c) low
molecular weight of complex ( defined metal/chelating agent (bidentate) ratio,
possibly Moreover, metal chelates should be obtained from
simple and clean processes with high yields starting
from raw materials that can be easily found. These
features can be implemented by means of suitable
ligands, which should be easy to deproton 'and have at
least two donor atoms in such a position to carry out
chelation. Examples of ligands are amino acids and
other organic acids suitably functionalized.
As far as iron is concerned, its administration in
chelated form is particularly efficient against
anemia. Anemia is a pathologic status of blood due to
the reduction of the number of red blood cells, to the
decrease of the amount of hemoglobin or to both
deficiencies. Iron is fundamental for hemoglobin since
it is the center in which oxygen is fixed into heme.
Classes of people who need a higher amount of iron are
menstruated or pregnant women, children under two
years of age, vegetarians, people suffering from
hemorrhoids, people suffering from ulcers and
eventually blood donors. Symptoms felt by an anemic
person are weariness, a higher sensibility to cold,
irritableness, loss of concentration and heart
palpitations. .._._
Furthermore, iron can protect from viral or bacterial
infections since it promotes the stimulation of the
immune system. In addition, iron promotes the

metabolism of vitamins belonging to B group.
It is known that the lack of these vitamins can result
in diseases such as dermatitis or even more serious
diseases such as pellagra (due to the deficiency of
vitamin B3). Iron intervenes in processes of- synthesis
of adrenaline and noradrenaline. Eventually, the lack
of iron leads to a slow cicatrisation of wounds.
A first aim of the present invention is to suggest an
integrator for administratxon in human nutrition. Said
integrator is also administered to patients suffering
from a deficiency of metal oligoelements such as: Mg,
Ca, Mn, Co, Cu, Zn and Fe.
A second aim of the present invention is to suggest an
integTatTor for agro-zootechnical nutrition to be
administered to monogastric or polygastric animals.
Said integrator is also administered to monogastric or
polygastric animals needing an administration of metal
oligoelements such as: Mg, Ca, Mn, Co, Cu, Zn and Fe
with a higher bioavailability.
A third aim of the present invention is to suggest a
method for preparing 'metal chelates with methionine
hydroxy analogue or one of its salts, both in solid
form with iron (II), vanadium (IV) and/or vanadium (V)
and molybdenum (V) and/or molybdenum (VI), and in
liquid form in aqueous solution with iron (II) and
(III) and chrome (III), in which said metals are
bonded to a bifunctional organic ligand with a strong
coordination bond so as to form a stable metal
chelate.
A further aim of the present invention is to suggest
the use of said new chelates, both in solid form with
iron (II), vanadium (IV) and/or vanadium (V) and
molybdenum (V) and/or molybdenum (VI), and in liquid
form in aqueous solution with iron (II) and (III) and

chrome (III), for preparing metal integrators for
human and animal nutrition.
In an embodiment of the present invention said first
and second aim are achieved by suggesting the use of
metal chelates [2:1] having the general formula (I):
(CH3SCH2CH2CH (OH)COO)2M«nH20 — (I)
in which the bifunctional chelating agent is 2-hydro-
xy-4-methylthiobutanoic acid, an alpha-hydroxy acid,
known as "methionine hydroxy analogue" (MHA); M is a
bivalent metal cation chosen from the group
comprising: Co, Ca, Mg, Zn, Fe, Cu and Mn, and n is
the number of water molecules; for preparing metal
integrators for treating patients suffering from a
deficiency of metal oligoelements or for
administration in the agro-zootechnical field to
monogastric or polygastric animals. In formula (I)
there are zero to twelve water molecules, preferably
zero to six. For instance, zero to four. 2-hydroxy-4-
methylthiobutanoic acid builds with iron ion a chelate
having a well defined stoichiometry, containing two
molecules of chelating agent pro iron atom, in the
same way as it builds chelates with bivalent metals
Mg, Ca, Mn, Co, Cu and Zn.
The method for preparing metal chelates having formula
(I) was described in international patent application
PCT/IT99/00225, and consists in the direct reaction of
MHA with the corresponding carbonates of bivalent
metals Mg, Ca, Mn Co, Cu and Zn.
In an embodiment according to the present invention
the Applicant has improved a method for preparing
metal chelates having formula (I), which envisages the
iirect reaction of MHA and metals (II) oxides such as:
1q, Ca, Mn, Co, Cu, Zn and Fe. ,
!n this new embodiment oxides of metals (II) are used

instead of carbonates as described in application
PCT/IT99/00225, and technical and operating conditions
are unchanged with respect to those referred to in
application PCT/IT99/00225, and therefore said
operating conditions are regarded as contained in the
present application.
Advantageously, the following are used: magnesium
oxide mixed with magnesium carbonate or alternatively
completely replacing magnesium carbonate, zinc oxide
mixed with zinc carbonate or alternatively completely
replacing zinc carbonate, and calcium oxide mixed with
calcium carbonate or alternatively completely
replacing calcium carbonate.
The Applicant has found it advantageous to use for
human nutrition the following metal chelates:
- (CH3SCH2CH2CH (OH)COO)2Zn»2H20
The two water molecules are not bonded to the metal.
- (CH3SCH2CH2CH (OH)COO)2Cu
It is an anhydrous complex without water molecules
bonded to the metal.
- (CH3SCH2CH2CH(OH)COO)2Co»2H20
The two water molecules are complexed.
- (CH3SCH2CH2CH(OH)COO)2Mn«2H20.
The two water molecules are complexed.
- (CH3SCH2CH2CH(OH)COO)2Ca»2H20
The two water molecules are not bonded to the metal.
- (CH3SCH2CH2CH(OH)COO)2Mg»2H20
The two water molecules are not bonded to -the metal.
In another embodiment of the present invention said
third aim is achieved by suggesting a method for
preparing metal chelates with methionine hydroxy
analogue or one of its salts, both in solid form with
iron (II), vanadium (IV) and/or vanadium (V) and
molybdenum (V) and/or molybdenum (VI), and in liquid

form in aqueous solution with iron (II) and (III) and
chrome (III).
In the case of vanadium and molybdenum the method for
preparing their chelates consists in the reaction of
their corresponding oxides with methionine hydroxy
analogue.
Alternatively, in the case of vanadium and molybdenum
the method for preparing their chelates consists in
the reaction of their corresponding metal salts with
methionine hydroxy analogue.
For instance, an amount of solid V2O5 cr M0O3 is added
with a solution of methionine hydroxy analogue at a
high temperature and under stirring. A transparent
solution and a solid precipitate of a vanadium or
molybdenum chelate are obtained from the reaction.
In a preferred embodiment the Applicant has arranged
in a receptacle vanadium oxide V205 and methionine
hydroxy analogue MHA. Vanadium oxide and methionine
hydroxy analogue are present in a molar ratio of 1:2
to 1:8, preferably 1:4 to 1:6 (Vanadium/MHA).
The receptacle is equipped with stirring means,
heating means and means for reaction reflux. The
reaction takes placed under mechanical stirring and
under reflux for a time between 20 and 60 minutes,
preferably 30 minutes. At the end of the reaction a
dark green solution is obtained, from which after
cooling a solid green precipitate is obtained. From
the chemical and physical analysis of the precipitate
the Applicant has found confirmation that it is a
vanadium (IV) complex having formula VOL2, where L =
deprotoned methionine hydroxy analogue.
During the reaction vanadium (V) oxide reduces to
vanadium (IV) .
En the case of iron (II) the method of preparation

takes place by reaction of sodium salt (or an alkali
metal or alkaline-earth metal salt) of MHA with
ferrous sulfate (or any other soluble iron (II) salt)
in water environment.
The molar ratio MHA/Iron (II) is of 2:1 for completing
the reaction. Ferrous chelate precipitates from the
reaction environment and is filtered and washed with
water so as to eliminate soluble sodium sulfate which
builds up.
After filtration and washing the chelated product is
then dried so as to reduce the amount of absorbed
water. The product is a pale yellow powder, little
water soluble, having formula:
(CH3SCH2CH2CH (OK) COO) 2Fe-2H20.
Water molecules are bonded directly to iron (TGA
data).
The infrared vibrational spectrum shows the occurred
chelation: as a matter of fact, the spectrum has a
series of characteristic bands according to the above
structure.
In particular, the band due to the asymmetric
stretching of carboxylic group can be observed at
1,596 cm"", significantly moved to low frequencies
with respect to free MHA (1,720 cm"1) as expected for
deprotonation and coordination. A pure and stable
product is thus obtained.
In an embodiment of the present invention stable solutions
of iron (III) and chrome (III) chelates with MHA can be
obtained by dissolution in water environment of soluble
salts of iron or chrome (III) and of MHA in a ration
MHA/M(III)> 2,preferably 3, and keeping pH at a suitable
value so as to prevent precipitation of the corresponding
hydroxides.
In a preferred embodiment the Applicant has improved

. the preparation of a stable solution of chrome (III)
in which a chrome salt, for instance chrome sulfate
Cr2(S04)3 is reacted with methionine hydroxy analogue
MHA under stirring, for instance by heating, for a
time between 20 and 120 minutes, preferably between 30
and 60 minutes. Chrome salt and methionine hydroxy
analogue are present in a molar ration of 1:2 to 1:30,
'preferably 1:2 to 1: 20. At the end of the reaction a
solution of chrome(III) complexes with methionine
hydroxy analogue is obtained.
In another preferred embodiment of the present
invention said fourth aim is achieved by suggesting
the use of said new chelates, both in solid form with
iron (II), vanadium (IV) and/or vanadium (V) and
molybdenum (V) and/or molybdenum (VI), and in liquid
form in aqueous solution with iron (II) and (III) and
chrome (III), for the preparation of metal integrators
for human and animal nutrition.
Metal chelates described in the present invention can
be used mixed one with the other in various
quantitative ratios for preparing metal integrators.
.Therefore, the integrators suggested both for human
and animal nutrition can contain one or more metal
chelates according to the present invention.
With reference to the metal chelates described above
and according to the present invention the Applicant
has made a series of experimental tests.
In vitro tests
For in vitro tests cells of human colon adenocarcinoma
(CACO-2) have been used, these being the most
frequently used in vitro system for studies on
intestinal functionality, in particular as far as
transepithelial transport is concerned, since said
cells (CACO-2) develop ultra-structural, functional

and electrical properties that are similar to those of
small intestine.
The formation of intercellular links can be monitored
through measurements ' of transepithelial electric
resistance (TEER) of the single layer of cells. Since
intercellular links limit the (para-cellular) movement
of solutes, TEER alterations are commonly used as
permeability index of said links.
The equipment used for cell growth and differentiation
is shown in Figure 1.
Said cells, referred to with A in Figure 1, have been
grown and differentiated on a carrier (permeable
filter), referred to with B in Figure 1, until a
single layer of differentiated cells joined by
functional intercellular links is formed. Said filter
B separates the apical environment C, (AP),
(simulating intestine lumen) from the basolateral
environment D, (BL), placed in the lower chamber,
simulating capillary blood flow.
Said cells have been treated for 3 hours with two
different concentrations of Fe(III)/MHA (1:3) and
Fe(III)/NTA (1:2) (nitriltriacetic acid taken as
reference chelate), in a buffer solution at pH 5.5 and
37°C. Said solutions have been placed in the apical
compartment C (AP) , whereas the basolateral
compartment D (BL) contained an iron-free solution of
apotransferrin placed in a buffer solution at pH l.'A.
During said experiment, at pre-established time
intervals, TEER has been measured in Q»cm2, Figure 2
(every 30 minutes over 3 hours) and Figure 3 after 24
hours.
The results are shown in Figure 4, indicating the
content of intracellular iron after 3 hours of
treatment with Fe(III)/MHA and Fe(III)/NTA at

different concentrations. Data are expressed in nmoles
iron/filter.
As can be inferred from Figure 4, the passage of
iron/MHA chelate from the apical environment, C, to
the cell is higher than the one observed in the
control.
Moreover, from Figure 5 (showing iron transport from
apical environment C to basolateral environment D
after treatment with two different concentrations of
Fe(III)/MHA and Fe (III) /NTA) it can be inferred that
the concentration of transported iron is comparable.
Data are expressed in nmoles iron/filter.
Data shown in Figure 4 and 5 confirm that iron chelate
is strongly absorbed by cells of intestinal microvilli
and moves within blood flow.
From Figure 2 (showing TEER measurements) it can be
inferred that intercellular links are unchanged, thus
proving the non-toxicity of iron chelate towards
cells, contrarily to what happens in the case of
unchelated iron such as ferrous sulfate.
Figure 3 shows the measurement of TEER 24 hours after
the buffer solution at 5.5 containing iron/MHA or
iron/NTA has been removed keeping the cells in
culture. Said Figure 3 shows how iron chelate/MHA is
stable within cells causing no toxic effect.
Finally, the tests show that MHA/M chelates according
to the present invention are efficiently absorbed,
stable within intestinal cells and non-toxic.
The results shown above support the use of said new
chelates, both in solid form with iron (II), vanadium
(IV) and/or vanadium (V) and molybdenum (V) and/or
molybdenum (VI), and in liquid form in aqueous
solution with iron (II) and (III) and chrome (III),
for the preparation of metal integrators for human and

animal nutrition.
In vivo tests
Said tests have involved both monogastric animals
(such as pigs) and polygastric animals (such as
calves).
a) Monogastric animals (pigs)
Two test groups of pigs (Control and Test) of 35 days
of age and weaned at 19 days, were administered a food
differing only in the zinc source.
Said food consisted, pro kg of food as such, of 3,500
Kcal ED, 1.15 g of lysine, and a total amount of zinc
element of 81 mg (81 ppm) , of which 31 mg (31 ppm)
were given by raw materials, whereas 50 mg (50 ppm)
were respectively in form of zinc sulfate (Control)
and zinc chelate with MHA (Test) according to the
present invention.
The two groups of animals were balanced by nest,
living weight and sex and were fed for 27 days. Other
4 animals were immediately sacrificed and a sampling
was made of them according to the following procedure.
The two groups of animals were weighed before starting
the test and after 27 days. At the end of the test the
pigs were sacrificed and stomach, intestine, left
kidney and liver were removed from every pig. Stomach
and intestine were emptied and weighed again so as to
obtain the net weight of said organs. A sample of
kidney, liver and brain was taken and frozen. Stomach,
intestine, left kidney and liver were then put
together with the remaining carcass and homogenized
with a mixer. A sample was then taken from the mass
thus obtained and frozen. The samples were then
dehydrated with a dehydrating device and underwent
chemical analysis.
The level of zinc, copper and iron were determined on

the lyophilized samples by atomic absorption
spectroscopy. The body levels of zinc were referred to
the product.
On the basis of the body's "net" weight (i.e. without
the content of the digestive tube) and of its content
of zinc at test beginning and end it was possible to
determine also daily zinc retention. The results of
the chemical analyses carried out on the samples are
shown in Table 1. The pigs were sacrificed at an
average weight of 16.2 kg. The average daily weight
increase was of 324 g.
As can be inferred from the data shown in Table 2
concerning daily zinc retention of the two different
sources, the integration of zinc chelate was retained
by the organism 26% more (P=0.07) than the integration
with zinc sulfate. Table 3 shows the data concerning
the effect of the zinc source on the content of zinc,
copper and iron in liver, kidney and brain and,
therefore, on the interaction with said elements
present in the diet under inorganic form. As a matter
of fact, it is known about the interaction exerted by
said free ions by reducing one the absorption of the
other.
The content of said three minerals in liver was not
affected by the diet and therefore by the zinc source.
Average values were 296 mg/kg for zinc, 63 mg/kg for
copper and 220 for iron. Conversely, kidney showed a
higher content of zinc (+18%, P=0.07), of iron (+36%,
P reach only for the latter value the threshold of
statistic significance, though it showed a tendency
towards an increase in the retention of said metal
element.
In brain there was a tendency towards a higher content

of zinc (+13%), of copper (+20%), of iron (+25%).
The obtained results point out a higher
bioavailability of the metal element in chelated form
with respect to inorganic sources such as sulfates,
and further a lower interaction with other ions, which
results in a higher retention of the latter.
As is known, pigs are one of man-closest animal models
and as such they are often used as model for
evaluations and studies in the human field.
b) Polygastric animals (meat cattle)
Two groups of female Charoiaise calves (30 months old)
comprising 6 animals each, with an average starting
weight of 567 kg (Control and of 565 kg (Test), were
fed for 90 days with the same diet. The only
difference was that the Control group was administered
zinc carbonate and the Test group zinc chelate
according to the present invention. Daily ingestion
was of 22 kg/animal and the total daily supply of zinc
element was of 700 mg.
Living weight at test beginning and end, dead weight
and slaughtering yield were determined for each
animal. Data are shown in Table 4.
Animals fed with zinc chelate with respect to those
fed with zinc carbonate have a significantly higher
final weight (652 kg vs. 642 kg, p significantly higher daily weight increase (1,039 g
vs. 934 g, p weight (377 kg vs. 366 kg, P significantly higher yield (57.83% vs. 57.03%,
p the presence of zinc chelate in the ratio administered
to animals is shown in Table 5.
Said results show an evident improvement of the
aforesaid zootechnical performances of zinc chelate

with respect to inorganic sources of said element.
Stability constants for the various Fe/MHA complexes
have been calculated with potentiometric titrations.
The stability of iron (III) complexes is very high and
chelated species form also at acid pH. Uncomplexed
Fe3+ ions are present only at very low pH values
( complexed as chelated species metal/ligand = 1:2.







WE CLAIM;
1. A metal chelate of the formula (CH3SCH2CH2C(OH)COO)2Fe.2H2O.


The invention discloses a metal chelate of the formula (CH3SCH2CH2C(OH)COO)2Fe.2H2O.

Documents:

01949-kolnp-2007-abstract.pdf

01949-kolnp-2007-assignment.pdf

01949-kolnp-2007-claims.pdf

01949-kolnp-2007-correspondence others 1.1.pdf

01949-kolnp-2007-correspondence others.pdf

01949-kolnp-2007-description complete.pdf

01949-kolnp-2007-drawings.pdf

01949-kolnp-2007-form 1.pdf

01949-kolnp-2007-form 2.pdf

01949-kolnp-2007-form 3 1.1.pdf

01949-kolnp-2007-form 3.pdf

01949-kolnp-2007-form 5.pdf

01949-kolnp-2007-gpa.pdf

1949-KOLNP-2007-(03-01-2012)-FORM-27.pdf

1949-KOLNP-2007-(24-02-2012)-ASSIGNMENT.pdf

1949-KOLNP-2007-(24-02-2012)-CERTIFIED COPIES(OTHER COUNTRIES).pdf

1949-KOLNP-2007-(24-02-2012)-CORRESPONDENCE.pdf

1949-KOLNP-2007-(24-02-2012)-FORM-16.pdf

1949-KOLNP-2007-(24-02-2012)-PA-CERTIFIED COPIES.pdf

1949-KOLNP-2007-ABSTRACT 1.1.pdf

1949-KOLNP-2007-ABSTRACT.pdf

1949-KOLNP-2007-AMANDED PAGES OF SPECIFICATION.pdf

1949-KOLNP-2007-AMENDED CLAIMS.pdf

1949-kolnp-2007-assignment 1.1.pdf

1949-KOLNP-2007-CANCELLED PAGES.pdf

1949-KOLNP-2007-CLAIMS 1.1.pdf

1949-KOLNP-2007-CORRESPONDENCE 1.1.pdf

1949-KOLNP-2007-CORRESPONDENCE OTHERS 1.2.pdf

1949-KOLNP-2007-DESCRIPTION (COMPLETE) 1.1.pdf

1949-KOLNP-2007-DESCRIPTION (COMPLETE).pdf

1949-KOLNP-2007-DRAWINGS 1.1.pdf

1949-KOLNP-2007-DRAWINGS.pdf

1949-kolnp-2007-examination report 1.1.pdf

1949-KOLNP-2007-EXAMINATION REPORT REPLY RECIEVED 1.1..pdf

1949-kolnp-2007-form 1 1.1.pdf

1949-KOLNP-2007-FORM 1.1.pdf

1949-KOLNP-2007-FORM 1.pdf

1949-kolnp-2007-form 13 1.1.pdf

1949-KOLNP-2007-FORM 13.pdf

1949-kolnp-2007-form 18 1.1.pdf

1949-KOLNP-2007-FORM 18.pdf

1949-KOLNP-2007-FORM 2.1.pdf

1949-KOLNP-2007-FORM 2.pdf

1949-kolnp-2007-form 3 1.1.pdf

1949-KOLNP-2007-FORM 3 1.2.pdf

1949-KOLNP-2007-FORM 3.1.pdf

1949-kolnp-2007-form 5 1.1.pdf

1949-KOLNP-2007-FORM-27.pdf

1949-kolnp-2007-gpa 1.1.pdf

1949-kolnp-2007-granted-abstract.pdf

1949-kolnp-2007-granted-claims.pdf

1949-kolnp-2007-granted-description (complete).pdf

1949-kolnp-2007-granted-drawings.pdf

1949-kolnp-2007-granted-form 1.pdf

1949-kolnp-2007-granted-form 2.pdf

1949-kolnp-2007-granted-specification.pdf

1949-KOLNP-2007-OTHERS DOCUMENTS 1.1.pdf

1949-KOLNP-2007-OTHERS.pdf

1949-KOLNP-2007-REPLY TO EXAMINATION REPORT.pdf


Patent Number 247120
Indian Patent Application Number 1949/KOLNP/2007
PG Journal Number 13/2011
Publication Date 01-Apr-2011
Grant Date 28-Mar-2011
Date of Filing 30-May-2007
Name of Patentee AGRISTUDIO S.R.L.
Applicant Address VIA GRAMSCI 56, I-42100 REGGIO EMILIA
Inventors:
# Inventor's Name Inventor's Address
1 CINTI ENRICO VIA BARBIERI 9/A, I-29100 PIACENZA
2 CIRIBOLLA ANTONIO VIA MARTIRI DELLA BETTOLA, 96, I-42100 REGGIO EMILIA
PCT International Classification Number A61K
PCT International Application Number PCT/IT2003/000400
PCT International Filing date 2003-06-27
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 MI2003A000863 2003-04-29 Italy
2 RE2002A000067 2002-09-06 Italy