Note: Descriptions are shown in the official language in which they were submitted.
WO91/07426 PCT/EP90/01878
~ ir.'~ ~ ~r 5 ~
BIOA~AIhABLE IRON-ALBUMIN COMPOUNDSL_~ PROCESS FOR TEE
PR~PARATION THER~OF AND PaARMACEUTICAL COMPOSITIONS
CONTAINING TEEM
~ ~ .
The present invention relates to albumins acylated
with dicarboxylic acid residues containing iron in a
~ioavailable form.
The use of complexes of iron with acylated
proteins for the oral martial therapy is known: see,
for examDle, Italian Patent N. 1,150,213, which
discloses an iron adducer comprising succinylated
proteins, which is obtained by reacting ferric salts
with carrier proteins of animal origin, such as
proteins from milk, organs or serum, or of vegetal
origin.
However, since said proteins have a variable
' composition, comoounds OL constant composition are
hardly obtained.
15Moreover, even though complexes of high iron
content (up to 20%) can be obtained, the high iron
amount in those derivatives also involves increases in
solution ~iscosity.
As a matter of fact, the Ironproteinsuccinylate
derivative (obtained according to -Italian Patent N~.
1,150,213) which is used for the appropriate
pharmaceutical formulation and is nowadays commercially
available contains 5% by weight iron The possibility
to obtain derivatives giving low viscosity solutions,
even when amounts well higher than 5% by weight iron
are supported by succinylated proteins, would represent
a marked improvement in the preparation of derivatives
for the medicinal use.
.
.
..,.,.
,.
WO 91/07426 PCr/EP90/01878
2~
Nevertheless the most important aspect to be
improved for this class of ~herapeutical agents
undoubtedly consists in reducing or completely removing
the gastroleslvity and diarrhoic effects related to the
S therapeutic principle.
Now it has been found, and it is the object of the
present invention, that using albumin as the protein to
obtain said acylated compounds, bioavallable iron
compounds are obtained which are highly soluble, even
when they contain high amounts of iron, said compounds,
moreover, being substantially devoid of toxicity and
showin~ reduced gastrolesivity and diarrhoic side-
ef~cts. Said disadvantages, which are characteristic
of these medicaments, can be relate~ to a poor
solubility of the iron derivatives or to precipitationat the intestine level or to the generation of hydroxy
radicals deriving from the release of the iron in the
ionic form.
Dif~erent albumins can be used in the preparation
of the compounds of the invention, particularly
pre~erred being highly pure bovine serum albumin and
lact albumin .
According to the invention, albumins are acylated
with dicarboxylic acid residues such as, for instance,
those deriving from succinic, glutaric, maleic, malic,
malonic, aspartic, glutamic acids and the like.
Particularly preferred is the residue from succinic
acid, which proved to be the most suitable for
impro~ing some protein characteristics, such as
solubility and degradability by proteases.
More particularly, the present invention relates
:: - ~ ,: ;,, ,
WO 91/07426 PCT/EP90/01878
to compounds comprising iron and succinylated bovine
serum albumin, supporting iron amounts from 3 to 10% by
weight, as well as compounds comprislng iron and
succinylated lactalbumin supporting iron amounts up to
20~ by weight, both these compounds being hlghly
soluble
Said compounds can be obtained in aqueous medium
by reacting the above mentioned succinylated or
acylated albumins~ with iron ions at a pH ranging from 2
to 12, preferably from 3 to 7.
The invention will be described in more detail in
the following non-limiting examples.
The compounds described in the Examples will be
named as follows :
Compounds of Examples 1-3 : Ironsuccinylalbumin.
Compounds of Examples 4-5 : Ironsuccinyl- -lactalbumin.
Com~ound of Example 6 : Ironaspartylalbumin.
Compound of Example 7 : Ironmaleylalbumin.
Compound of Example 8 : Ironglutarylalbumin.
EXANPLE 1
Preparation of Ironsuccinylalbumin wi~h 6% by weight Fe
content.
20 g of bovine serum albumin are dissolved in 400
ml of water. pR is adjusted to 8.0 by adding NaOH. 24 g
of su~cinic anhydride are added in small portions,
under stirring, keeping pH from 7.5 to 8 by addition of
2N NaOH and constant temperature below 25C, preferably
at 20~C. The mixture is left to react for one hour,
then pH is lowered to 3 + 0.5 by means of 2N HCl. A
white precipitate forms which is recovered by
centrifugation. The formed excess of succinic acid
is removed by dissolving the precipitate in 400
WO91/07426 PCT/EP90/0187B
ml of water at pH 8 . O, adjusting the solution pH to 3
0.5 with 2N HCl. A white precipitate forms which is
recovered by centrifugation. This procedure i5 repeated
until succinic acid is completely removed. Th~ obtained
final precipitate is dissolved ln 400 ml of water by
addition of 2N NaOH to pH 8Ø 7.20 g of FeC13.6H2O ,
equivalent to 1.48 g of Fe, are quickly added under
stirring. A red-brown precipltate instantly forms,
which is washed with 0.001N ~Cl, recovered by
filtration (or centrifugation) and dried under vacuum.
A red-brown powder (19 g) is obtained which is
insoluble in water and becomes soluble adjusting pH to
about neutrality by addition of 2N NaOH. The compound
shows at the analysi6 an iron content of 6Ol-6.2% by
weight. By electrophoresis on cellulose acetate (as
described in item 4 below) the compound moves as a
unitary spot, in which lron can be determined.
EXAMPLE 2
Preparation of Ironsuccinylalbumin with 3.8-4.1a~ by
2 0 weight Fe content.
The compound is prepared as described in Example
1, but adding 4.8 g of FeC13.6H2O (equivalent to 0.922
g of Iron).
The compound, moving under the electrophoretic
field as a unitary spot in which iron can be
determined, shows a 3.8-4.1% by weight Fe content.
EXAMPLE 3
Preparation cf Ironsuccinylalbumin with 9.5-10% by
weight Ye content.
20 1 of demineralized water and 1 kg of bovine
serum albumin are placed into a reactor, obtaining a
solution of pH 6.7-6.8. A 5N NaOH solution ls slowly
:::
WO91/07426 PCT/EP90/01878
2~ 555
dropped therein until pH 7.5, then 1.2 kg of succinic
anhydride are added in small portions, keeping pH from
7.2 to 7.5 by addition of 5N NaOH. At the end of the
addition, pH is ~ept to 7.5 for 30 min. then the
5 solution is acidified to pH 3 with 5N HCl, under
stirrin5. The obtained precipitate is centrifuged,
thoroughly washed with demineralized water and
redissolved in 20 1 of deminerallzed water with 5N NaOH
to pH 7.5 , to obtain a clear solution in 30 min. The
- 10 precipitation procedure at acld pH and the dissolution
procedure at basic pH are repeated twice. Then the
solution is slowly added with a solution of 578.5 g of
FeC13.6H2O in 10 1 o~ demineralized water keeping pH
from 6.5 to 6.8 by addition of 5N NaOH. Then the
lS mixture is stirred at pH 6.6-6.8 for 30 min., acidified
to pH 3 with 6N HC1 and left under stirring for 30
min.. The precipitate is centrifuged, thoroughly washed
with demineralized water adjusting p~ to 8.5 with 5N
NaOH. A~ter having kept at the pH of 8.5 for about one
20i hour, the obtained solution is filtered through Celite
(300 g). The precipi.tation procedure at acid pH and the
dissolution procedure at basic p~ are repeated for one
time. The solution is filtered on Celite (200 g), the
- ~ product is precipitated at p~ 3 with 6N HCl, stirring
25 for 30 min , then it is centrifuged and thoroughly
washed with demineralized water. The dark ~rown solid
is sieved and dried under vacuum at 3QC, to obtain 1
kg of Ironsuccinylalbumin.
The title compound is obtained.
~XA~PLE 4
Preparation of Ironsuccinyl-C~-lactalbumin with 11-
:. ~ . : -
~: --- , . .
WO ~1/07426 PCr/EP90/01878
2~
11.45% by weight Fe content.
1 kg of ~-lactalbumin dissolved in 20 1 of water
is placed into a reactor. A solution is obtained having
pH 6.7-6.8. The protein is succinylated and purified
from succlnic acid as described in the previous
Example. The solution of the succinylated protein is
added with 1.2 kg of FeC13.6H2O in 10 1 of water,
keeping pH from 6.5 to 6.9 by addition of 5N NaOH. The
reaction is carried out as described in the above
Example to obtai.n, at the end of the reaction, 1 kg o~
the title compound.
EX~?LE 5
Preparation of Ironsuccinyl-~-lactalbumin with a 18.3-
18.5% by weight Fe content.
1 kg of ~-lactalbumin dissolved in 20 1 of water
is placed into a reactor, to obtain a solution with pH
` 6.7-6.8. ~he protein is succinylated and purified as
: described in Example 3. The solution of succinylated
~-lactalbumin is added with ~.4 kg of FeC13.6H2O in 10
1 of water, keepi.ng pH from 6.5 to 6.9 ~y addition of
5N NaOH. The reaction is carried out as described in
Example 3, to obtain 1 kg of the title compou~d.
EXAMPLE 6
: Preparation of Ironaspartylalbumin with 7.5% by weight
Fe content.
10 g of bovine serum albumin are dissolved in 200
ml of water and pH is adjusted to 7.2 by addition of
NaOH. 10 g of acetylaspartic anhydride are added in
small portions, under stirring, keeping pH from 7.2 to
?. 3 bv addition of 2N NaOH. The solution is
ultrafiltered, keeping the volume constant by
continuous addition operating with an ultrafiltration
WO91/07426 PCT/EP9O/0187g
j;? ~< ~ ) ~r ~ 5
membrane with cut-off 10,000 Daltons, for 3 hours, so
as to remove acetylaspartic acid passing through the
membrane. The solution containing aspartylated albumin
is recovered from the ultrafilter; 7.0 g of FeC13.6H2O
are added : a red-brown precipitate forms which is
washed with water at pH 3, xedissolved adjus~ing pH to
7.1 and freeze-dried.
9.5 g of the title compound are obtained.
EXAMPLE 7
Preparation of Ironmaleylalbumin with 6~5~o by weight Fe
content.
The iron compound is prepared as described in
Example 6, but using maleic anhydride as the acylating
agent.
15The title compound is obtained.
E~CAMPI.E 8
Preparation of Ironglutarylalbumin with 7.0% by weight
Fe content.
The iron compound is prepared as described in
Example 6, but using glutaric anhydride as the
acylating agent.
The title compound is obtained.
The pxotein complexes prepared in the above
Examples were analyzed according to the analytic
methodologies described hereinbelow.
1) Determination of the iron content
~In all of the compounds prepared according to
'~ ~ Examples 1-8 the iron content was determined ~y
extracting iron from the sample with 2N HCl; the
quantitative determination was carried out according to
the method described in Standard Methods 14th ed.,
1975, page 208, APHA, A~WA, WPCF (reaction with o-
- : .
WO91/07426 PCT/EP90J01878
~ ~5~3
phenanthroline).
In said compound iron is present ln the trlvalent
state-, as evidenced since no Fe ions can be determined
-~hen the o-phenanthrol1ne reaction is carried out in
the absence of reducing agents.
2) Solubility as a pH function
All the compounds prepared according the above
~xamples are insoluble at acid pH and soluble at basic
pH.
10The solubility prorile of Ironsuccinylalbumin,
prepared according to the method described in Example
3, is reported by way of example.
The solubility profile is taken from
spectrophotometric measurements of the absorbance
15dif~erence at 500 nm and 280 nm of solutions obtained
treatins 20 mg of Ironsuccinylalbumin with 50 ml of
water, in a p~ range from 2.0 to 7Ø 5 ml of the
sample are diluted to 10 ml with water, centrifuged at
3,000 rpm during 10 min. and the sllpernatant is read at
spectrophotometer.
Ironsuccinylalbumin is completely soluble at pH
values equal or above 6.5, whereas it is insolu~le at
p~ below 6. The reversed precipitation curve, o~tained
by acid~.fying the Ironsuccinylalbumin solution (20 mg
of the compound in water at p~ 7), shows the
precipitation onset at pH 4.5.
3) Determination of the Protein content
The protein amount in the samples prepared
according to Examples 1-8 was determined by titration
of protein nitrogen (using the method described in
ITALIAN OFFICIAL PRARMACOPOEIA IX ed., vol. I, pages
WO91/07426 PCT/EP90/01878
~;3
191-192)
The found values range from a maximum of 85h ( in
the compound ~f Example 3 ) to a-minimum of 6 5% tin the
compound of Example 5).
4) Electrophoresis on cellulose acetate
~lectrophoresis was carried out for ~0 min. at 40
- V/cm, usin~ 0.05M Tris tricine buffer, pH 8.6, which
detects bands at the same electrophoretic distance for
the compounds of the lnvention and for the respective
acylated proteins. No bands which can be attributed to
the starting al~umins can be evidenced. The presence of
iron in the bands of the compounds of the invention can
be detected by means of o-phenanthrcline.
By way of example, in case of determinations
carried out on the compounds of ~xamples 1-3, the
~` distance of the spot from the origin is 37 mm.
5) UV-VIS Spectroscopic analysis (Shimadzu UV-160?
,~ W-VIS Soectrum in the range 200-600 nm, recorded
on aqueous solutions at pH 7 containing 1 mg/ml o~ th~
compounds of the invention evidences an absorbance
increase from 600 to 340 nm.
6) Fluorescence emission spectrum
(Apparatus: spectrofluorometer Kontron SFM25).
The fluorescence emission spectra at 400 and 200
nm (excitation at 236 nm) show a poor emission, equal
to about 1/10 that of the starting protein.
7) ESR SPectroscopY
` The spectra were recorded both at room temperature
and at -160C with a spectrometer Varian ElD, and they
are characterized by a single peak of 1200 G width
between the maximum and minimum slope with a value of g
; = 2.0Q. These signals can be attributed to polynuclear
: - ,
-
WO91/07426 PCT/EP90/01878
5~
complexes (in this instance Fe3+ complexes)
characterized by strong exchange interactions.
No other signals which could be attributed to the
presence of iron in the mononuclear form can be
evidenced.
8) Electrophoresis on Sodium Dodecyl Sulfate (SDS)
Electrophoresis was carried out on 7.5~
polyacrylamlde gel containing 1% by weight SDS.
The electrophoretic bands were evidenced with
Comassle srilliant slue~ The following products were
used as mo~ecular weight standards: myosin (200 KD),
- beta-galactosidase (116 KD), phosphorylase b (97 KD),
bovine albumin t68 KD), ovoalbumin (42 KD). In
representative determinations, Ironsuccinylalbumin
showed a single band corresponding to 100 XD molecular
weight. A similar electrophoretic behaviour was shown
by Ironsuccinyl-~-lactalbumin, but the molecular weight
thereof is 20 KD.
9) Determination of the acylation degree and
localization of the acylation
The acylation degree was determined both
spectrophotometrically, according to the reaction of
the ~rotein free amino groups with ninhydrin and by
fluorometric reaction with o-phthalaldehyde (G. Goodno
et al., Anal. Biochem., 115, 203, 1981). soth these
methods were used to e~aluate localization of the
acylation on the protein fraction co~stituting the
prepared compounds.
The obtained results prove that the end ~-amino
groups and the -amino lysin groups are acylated by
more than 95%.
. ~:
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WO 91/07426 PCl/EP90tO1878
The following additional parameters were
determined on Ironsuccinylalbumin, pr~pared as
described in Exampl~ 3.
10) Succinic ac_ a content
7-13%, determined by GLC (Perkin Elmer 3s, 3 m x 2 mm
column GP 10% SP 1200 H3PO4 on Chromo~orbR) after
hydrolysis of a known amount of a sample with 2.5N NaOH
at 100C for 5 hours and subsequent acidification to pH
2-3 with 6N HCl ~he released succinic acid is
quantitatively transformed into the corresponding
methyl succinate by reaction with BF3/CH30H.
Methyl succinate is determined under the following
conditions, using methyl glutarate as the internal
standard:
Mitrogen : 30 ml/min.
Air : 3 atm.
Hydrogen : 1.5 atm.
Temperature : injector 255C
column 140C
F~I.D. detector 275C
Injections : S ~1
11) Circular dichroism (~ASCO 500C apparatus)
The measurements recorded in the range from 300 to
200 nm on Ironsuccinylalbumin solutions at pH 7 in
comparisQn with the starting albumin evidence that the
succinylation reaction involves the disordered
structure of the protein. Figure 1 shows the dichroism
spectrum of bovine serum albumin, of the corresponding
acylated compound and of the compound of Example 3.
12a~ lH-NMR Spectrum (300 mEz; D20; Varian Gem_ i 200
apparatus)
The spectrum shows broad bands and a weak signal
at 2.4 ppm, which signal is also present in the
spectrum of the corresponding succinylated albumln, but
.
. ,, . .:
: ; :
WO91/07426 PCT/EP90/0187X
~ ~ ~ 5 -:
with a more neat and defined configuratlon. The
spectrum is reported in Figure 2.
12b) C-NMR spectrum (Varian XL300 a~aratus)
The spectrum shows signals at 15-70 ppm (aliphatic
CH), 110-140 ppm (aromatic) and 180 ppm (carboxylic).
The poor resolution and intensi~y of the slgnals at 29-
32 ppm (hemysuccinyl residue) due to the presence of
iron, suggests for a possible implication of the
succinyl groups in the bond Wlt~l lron The spectrum is
reported in Figure 3.
13) X-ray s~ectrum (Apparatus: Siemens 500D
diffractometer)
The spectrum in the angular interval 2 from 9 to
51 evidences no peaks which can be attributed to the
presence of crystallinity centres, thus indicating the
amorphous nature of the compound.
14) Determinations_of the molecular weight
By means of sel-filtration on Superose 6RR columns
a not gaussian molecular weight profile is obtained
with a main peak > 106 Daltons and a very broad
shoulder up to 104 D.
Using the Laser Light Scattering technique with a
He-Na source of 6328A, 25 mW output power, a molecular
weighit of 17.5 ~ 2.5 Mill was determined.
15) Amino acid analysis
The analysis was carried out upon decomplexation
of the iron which can interfere in the analysis, by
subjecting a sample to total acid hydrolysis with 6N
HCl at 105C for 24 hours and determining the amino
acid composition with an automatic analyzer (Liquimat +
III Kontron) using Na buffers as the eluents.
The amino acid profile is reported in Table 1.
: t ~ :::.. : . . - . .
W091/07426 PCT/EP90/01878
13
Table 1 - Amino acid composition (amino acid % by mole)
_______________________________________________________
Aspartic acid 9.1 Valine 6.8
Threonine 5.a Methionine . 0.6
Serine 4.3 Iso leuc ine 2.4
Glutamic acid 13.9 Leucine 10.5
Proline 6.0 Tyrosine 3.1
Glycine 3.2 Phenylala~ine 4.3
Alanine 7.9 Lysine 9.3
1/2 Cysteine 5.0 Histidine 2.8
Arginine 4.2
_______________________________________________________
16) Proteolysis
The enzyme hydrolysis was carried out by treating
lS 25 mg dissolved in S ml of O.OSM Tris-~Cl buffer pH 7.6
with chymotrypsin (5~ ~g) and trypsin (50 ~g). 1 ml is
withdrawn ~rom the reaction mixture at definite times;
2 ml of a 5% trichloroacetic acid (TCA) are added. the
mixture is centrifuged at 3000 rpm for 110 min. and the
supernatant is spectrophotometrically read at 280 nm
against a TCA blank.
The 2bsorbance change as a function of time
represents the hydrolysis rate, which turns out to be
comparable to that of the corresponding albumin used in
2S the preparation Oc the compound, to evidence that iron
present in the compound does not inhibit the action of
proteolytic enzymes.
17) Iron mobilization
Iron mobilization is tested by reaction with
desferrioxamine, which is controlled as a function of
time by measuring the absorbance increase, at 487 nm,
of a solution containing 7.0 ul of desferrioxamine, 500
ug of Ironsuccinylalbumin previously neutralized at pH
WO 91/07426 P~/EP90/01878
3~
1~
7 . 4 in 20 mM Tris-HCl buffer.
~The absorbance increase at 487 nm during time
confirms that iron can be mo~ zed from
Ironsuccinylalbumin. The release rate is 2.2 mA for 5
min.
18) Viscosity measurements
Ironsuccinylalbumin is dissolved in water at pH
7.2 at concentrations of 0.1, 1.2, 4.8. 10% w/v. The
obtained solutions are viscosimetrically analyzed with
a capillary viscosimeter or a Brook~ield viscoslmeter.
Solutions prepared with Ironproteinsuccinylate (a 5%
sample ob~ained as described in Italian Patent
1.150,213) are analyzed, by comparison and at the same
concentrations.
The ~iscosity of Ironsuccinylalbumin samples, at
all the tested concentrations, is markedly lower than
that of Ironproteinsuccinylate.
19) Hydroxy radical generation
The capability of the iron compounds of the
invention to generate OH radicalc was investigated
according to B. Halliwell, J. Gutteridge, o. Aruoma,
Anal. ~iochem. 165, 215-219 ~1987).
FeC13 and FeSO4, which are known to ~e capable of
generating OH radicals, are used as the controls.
Representative results are reported in Table 2.
~ , ,
'~
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WO91/n7426 PCT/EP90/01878
Table 2 - Deoxyribose degradation at pH 4.0
----_ _ _ _ _
Fe Ascorbate at 5-32 nm
[20 ~m] [100 ~M]
______________________
FeC13 - 0.156
FeSO4 - 1.044
Ironsuccinylalbumin - O.069
FeC13 + 0.625
~0 FeS04 + 1. 59
Ironsuccinylalbumin + 0~45
_______________________ _______________________________
I~ the absence of ascorbate, the formation of free
radicals is significant with FeSO4 and, at a lower
extent, with FeC13.
Generation of free radicals deriving from
~ Ironsuccinylalbumin, in the presence of ascorba~e, is
; significantly lower than that of FeSO4.
20) Generation of free radicals in biologic_fluids
The bleomycin test /Life Chemistry report, 1987,
vol. 4, pages 113-142 - J~ Gutteridge, B. Halliwell/
was used for determining free radicals, due to the
presence of fre~ iron, in biologic fluids.
Groups of rats are treated intraperitoneally with
Ironsuccinylalbumin and Ironproteinsuccinylate
~ prepared according to Italian Patent 1,150,213) (in
-: amounts corresponding to 100 mg iron/kg), and with
ferrous sulphate (in amounts corresponding to 25 mg/kg
iron).
Plasma samples are collected after 2, 8 and 15
` hours and the presence of iron is determined with the
bleomycin test (see Table 3).
.
WO 91/07426 PCl/EP90/01878
~ :
f~J ~ S
16
Table 3 - Determlnation of free iron ln plasm~
Product Fe ( ~M )
2 h 8h 15 h
S --____________________________
Fe++ sulphate 4.1 2.6 n . d .
502 0.7
6.2 n.d. "
4.6 0.7
Ironprotein-
succinylate 5~2 l.S n.d.
4.8 n.d. "
4.1 0.9 "
3.8 n.d. "
Ironsuccinylalbumin n.d.n.d. n.d.
,. .. -
,. .. ..
ll ll ll
_______________________________________________________
Controls n. d. n.d. n.d.
________________________________________________.______
Ironcuccin,vlalbumin, contrary to ferrous sulphate
and Ironproteinsuccinylate, induces no free iron in
plasma, thus proving to be 'ree from the injuring
: ef~ects induced by free radicals deriving from iron.
Toxicity stud~
: In the tests for gastrointestinal tolerance, the
animals were treated orally with Ironsuccinylalbumin (a
30 sample prepared according to Example 3, which sample
being significant due the high iron content thereof,
and therefore being potentially more toxic) or ferrous
.
, ~ - , . .
:; . . : .
WO 91/07426 ~ S PCr/EP90/01878
sulphate at dose levels of 200 mg/kg. The animals were
killed at different times, up to 24 hours. The
examination of stomach and intestine showed that the
intestinal and gastric injuries were significantly less
marked after administration of Ironsuccinylalbumin than
ferrous sulphate.
The acute toxicity of the sample was compared with
ferrous sulphate and the results are reported in Table
4.
10. Ironsuccinylalbumin proves to be much less toxic
than ferrous sulphate and does not induces toxicity
symptoms up to 2000 mg/kg.
Subacute toxicity with repeated doses by the oral
route was tested with doses of 100. 250, 600 mg/kg/die
of the derivative (equivalent to 1, 2.5, 6 times the
treatment with 10 mg/kg/die iron). No toxicity signs
are evidenced during the treatment.
Tests on the dog at doses of 100, 300 and lOOO
mg/kg/die (1, 3 and 10 times the therapeutic dosage)
also evidenced no toxicity signs. Increase in body
weight, food assumption, ophthalmic conditions, as well
as electrocardiographic, hematological, blochemical and
urine analysis data did not differ from the control
ones,
In the Ames test the derivative was tested using 5
bacterial strains (SaImonella Ty~himurium Th 1535, TA
1537, ~A 1538, TA 98 and TA 100) by treatment in the
absence and in the presence of metabolic activation (by
S9 liver fraction). No mutagenic activity was
evidenced.
Toxicoloqic studies prove that Ironsuccinylalbumin
'
WO 91/07426 Pcr/ Ep9o/o 1878
~a~ 55 18
is tolerated in the intestinal tract, has a low acute
toxicity and it is not mutagenic, in comparison with
ferrous sulphate which causes gastric ulcerations, is
more toxic after single administration and has
mutagenic properties.
Table 4
_____________________________________ ____-____________
Product Administration route LD50
______ ______________________.__________________________
Ironsuccinylalbumin i.p.> 2000 mg/kg
Ferrous sulphate i.p.2~0 mg/kg
Ironsuccinylalbumin o.s.> 2000 mg/kg
Ferrous sulphate o.s.1622 mg/kg
~
In order to pharmacologically characterize
Ironsuccinylalbumin, the follo~ing parameters were
evaluated: iron release a~ the gastric level; iron
absorption and kinetic, and the an-tianaemic effect
after a prolonged treatment using ferrous sulphate as
the control (in animals with experimentally induced
anaemia). The Ironsuccinylalhumin used or these tests
is the one prepared in Example 3.
A. Iron release in the stomach
Wistar male rats (Charles ~iver-Calco) fasted for
24 hours are treated orally with 2 mg Fe equi~alents/kg
ferrous sulphate or Ironsuccinylal~umin.
10, 20 and 30 Minutes after treatment the animals
are killed, the stomach is withdrawn and the free iron
in gastric juice is dosed with the Fe kit (Wako).
The results reported ln Table 5 treferring to a
sample containing 9.5% iron, but which is
representative of all the other compounds) show that
~, . , . ; . - . .
WO91/07426 PCT/EP90/01878
~2~ r~ 5
19
Ironsuccinylalbumin causes an iron release more than l0
times lower than ferrous sulphate, which explalns the
lower gastrolesivity of the compound of the .invention
compared with FeSO4, which is an antianaemic agent
widely used in therapy.
Table S
Iron concentrations in gastric juice of rats treated
with ironsuccinylalbumin or ferrous sulphate at a dose
of 2 mg iron/kg (~g/ml+S.E. ) .
---- ________________________~_____
Treatment Treatment times
l0' 20' 30'
_______________________________________________________
Ironsuccinylalbumin 9.0+l.9 8.l l.8ll.3+3.0
Ferrous sulphate 118 . 4+7 . 6 116 . 7~4 . 7 122 . 3+10 . 2
Controls 5 . 0~2 . 2 2 . 9+1. 24. 9+2 . 6
______ ____________________________________ .__________
N = 7
ANOVA : Ironsuccinylalbumin vs. ferrous sulphate : p
0.0l
B . ~ ~ ~ ~
Sideremia was evaluated by the batophenanthroline
method (Test Combination Iron - Boehringer Mannheim) in
the anaemic rat (anaemia was induced by iron-free diet
25 in the Spra~ue-Dawley rat and bred with iron-free diet
animals ~orn from anaemic mothers) one hour after the
treatment with Ironsuccinylalbumin or ferrous sulphate
at different dosages. The results obtained in different
tests proved that ferrous sulphate causes a higher iron
charge than Ironsuccinylalbumin samples.
C. Determination of iron kinetics
The sideremia values were measured in the anaemic
rat at different times after a single oral treatment
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WO 91/07426 PCr/EP90/01878
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with ferrous sulphate and Ironsuccinylalbumin at a dose
of 0.3 mg of Fe equivalents. The results show for both
the compounds an iron absorption peak 1 hour after the
treatment (see Table 6).
': .
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W 0 91/07426 PCT/EP90/01878
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Table 6
Sideremia values in the anaemic rat at different times
after - an oral treatment with O ~ 3 mg/kg of . iron as
ferrous sulphate or ironsuccinylalbumin (~g/100 ml +
E.D)
_______________________________________________________
Treatment Treatment times
0,3 ~g Fe/~g 0 15' 30' 60'
_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _
1 0 Ironsucci-
nylalbumin / 62,5+9,2**117,3+17,6~* 208,6+20,3**
++ ~
FeS04 / 197,8+18,5++ 247,9+15,5++ 307,7fl9,2++
Anaemic 30,3+1,6
controls
Table 6 (continued)
_________ _ _ _ _ ___ _
Treatment Treatment times
20 0,3 n~ Fe/kg 2h 3h 6h 24h
_ _ _ _ _ ___ __ _
Ironsuccinyl- 118, 3+14,3*~ 104, 8+15,0** 55, 8+5,1 29,6*1,1
albumin ++ ++
FeS04 261,5+21,5++ 219,7_28,1++ 93,9f22,8~+ 23,8fO,8
- _ _ _ _ _
; . ANOV~ at 2 factors: ** p < 0,01 *p< 0,05: Ironsuccinylalbumin
.. vs. FeS04
++ p < 0,01 + p < 0,05 within the group vs~
time O (anaemic controls)
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D . ~ntianaemic ef f ect
A prolonged treatment was carrled out during 6
weeks, in the anaemic rat, by administering orally
Ironsuccinylalbumin and ferrous sulphate as the
control, at doses of 3, 10, 30 mg Fe/kg/die.
The animals in groups of 5 animals each were
killed on days 0, 7, 14, 21, 28, 35 and 42 from the
treatment .
The data of the hematlc hemoglobin and serum iron
levels (Test Combinatlon Iron - Boehringer Mannheim)
obtained with the compound prepared as describe~ in
Example 3, are reported in Table 7. These results show
that the compounds are equiactive in restoring tne
hemoglobin levels, even though ferrous sulphate is more
15 rapid and powerful in restoring serum iron values.
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Table 7
Hematological parameters during a 6 week treatment in
anaemic rats with different compounds containing iron
5 (3, 10 and 30 mg Fe/kg/die)
(Mean ~ S.E) ANOVA: P < 0.01 P < 0.05 vs. anaemic
control s
_______________________________________________________
Hematologic~ TreatmentDays of treatment
parameters mg Fe/kg/die 0 7 14
__ ______ ____ __ _ ___
Eemoglobin Normal controls 13,3+1,0 13,3+0,5 13,7+0,6
~mg/dl) Anaemic controls 3,3+0,23,4~0,2 4,0~0,2
IronsuccinylaIbumin 3 / 7,9_0,6** 8,2~1,2**
Ironsuccinylalbumm 10 / 10,0+0,7 11,1+0,4
IronsuccinylaIbumin 30 / 11,2+0,3 12,8+0,2
I FegO4 3 /8,0~0,3** 7,9+0,9**
; FeSO4 10 / 11,2+0,1 11,2+0,8
FeSO4 30 / 11,5+0,3 12,1+0,2
----O---_---__ ________________
Seric Iron Norm~l controls / 128,7+19,5** 111,0+11,4**
(~g/100 ml) Anaemic controls / 19,9+6,5 38,9+3,7
Ir~nsuccinylalbumin 3 / 27,1+1,6 54,2+6,8*
Ironsuccinylalbumln 10 / 142,7+31,7** 123,5+14,4**
Ironsucclnylalbumln 30 / 184,6~9,0** 102,6+4,9**
FeS04 .3 /67,8+7,8** 81,8+14,8**
FeSO4 10 /n.d. 183,7+42,2**
4 30 /189,6+38,0** 147,3+26,2**
.. ,. ~......... . ~
, ~
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WO 91/074~6 Pcr/Ep9o/ol878
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Table 7 (continued)
___________ ___________________________________________
~ematolsgical Treatment Days of treatment
parameters mg Fe/kg/die 21 28 35 42
~e~oglobin Normal contr~ls 13,8+0,4 13,6+2,2 15,1+0,5 14,8+0,8
(mg/dl) AnaemQc cGntrols 4,8+0,8 3,6+0,5 7,1+0,8 9,1=1,4
Ironsuccinylalbumin 3 9,7_0,6*~ 10,2+1,0**
Ironsuccinylalbumin 10 12.1+0,3 13,0+0,1 13,g+0,1 14,9+o,l
Ironsuccinylalbumin 30 11,9+0,4 12,9+0,3 14,1=0,9 12,7_0,5
FeSO4 3 12,5+1,0*~ 11,5+0,8**
~eSO4 10 12,6+0,4 12,5+0,4 13,6_~ 15~+~,7
FeS04 30 11,7+0,2 12,7+0,1 13,1+0,4
_______________________________________________________
Seric l~an Normal cGntrols 121,1_7,3** 114,5+19,0** 139,3+9, 8*~140,5+16,1*~
(pg/100 ml) Anaemic ccntrols 34,2+3,0 30,7+2,8 qO,6+4,9 74,7+16,9
Ironsucelnyl- 77, 2+11,9* 143,5+38,0**
: aIbumin 3
Ir~nsueclnyl- 153,5+8,5** 87,6+4,5** 113,5+~,~** 202,8+16,9**
albu~m 10
Ir~nsueeinyl- 133,6+9,1** 112,4+8,0** 133,5+4,0** 140,1~25,3~*
albumin 30
F~504 3 103,2_23,2** 133,8 10,8**
FeS04 10 133,8+3,2** 113,8~9,2** 129, 3_23, 8** 219,6_3,3**
FeS04 30 105,2+7,4** 125,7+13,4**157,5*37,0**
______ ______________________________________ _________
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WO91/07426 PCT/EP90/01878
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The above data prove that Ironsuccinylalbumin is a
protein iron complex (the iron contents can vary from 3
to -20~ by weight), the protein component of which is
albumin or lactal~umin extensively acylated, mainly at
the lysine chain, as evidenced by compara~ive physico-
chemical analysis, particl-larly electrophoresis, amino
acid composition, succinic acid content, (N~R), with
disorganized structure (CD-circular dichroism)
The protein can complex with high amounts of iron
lo in form of a polynuclear hydrated complex (ESR) with
amorphous structure, in which aquo-complexes are
maintained in solution by the succinylated protein,
thus forming an aggregate structure with molecular
weights higher than 106 Daltons.
The obtained compounds are highly insoluble in
acid medium and highly soluble at neutral or basic pH
` values.
The compounds are characterized by a very low
toxicity in the animal, due to the release of iron
traces, whic~ cannot give raise to free radicals. The
above mentioned effects are completely unforeseeable
and they are due to the presence of albumin in the
compound.
The present invention also relates to the use of
the novel compounds as agents effective in the
! ` ' treatment of anaemias, as well as to all of the
industrial aspects related thereto, including the use
thereof in pharmaceutical compositions, which are a
further specific object of the invention. The compounds
of the invention can be incorporated in pharmaceutical
compositions, particularly in formulations suitable to
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WO91/07426 pcT/Epso/ol878
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the oxal administration. For the oral administration,
the compounds are formulated in form of tablets,
dispersible powders, capsules, dragees, granulates,
suspensions, syrups, elixirs or solutions. The
preparations for the- oral use can contain one or more
of the usual excipients, such as sweetening,
flavouring, colouring, covering and preservative
agents, in order to obtain a pleasant and palatable
preparation. Tablets can contain the active ingredient
in admixture with the usual pharmaceutically acceptable
excipients, for example inert diluents such as calcium
carbonate, sodium carbonate, lactose and talc,
granulating and disintegrating agents, such as alginic
acid and sodium carboxymethyl cellulose, binding agents
such as starch, gelatin, gum arabic and
polyvinylpyrrolidone, preservative agents such as
methyl, ethyl, propyl, butyl, or al~ali metals
benzoates or hydroxybenzoates, and lubricants such as
talc, magnesium stearate, stearic acid, glyceryl
palmitostearate and the like.
Syrups, elixirs, suspensions and solutions are
prepared according to known methods. Together with the
active ingredient, they ~an contain suspending agents,
such as propylene glycol, methylcellulose,
hydroxyethylcellulose, tragacanth gum and sodium
alginate, wetting agents, such as lecithin,
polyoxyethylene stearate and polyoxymethylenesorbitan
monooleaté and the usual preser~atives, surfactants,
sweetening and buffering agents. The addition of an
alkali metal hydroxide can sometimes be necessary. The
most preferred forms of said pharmaceutical
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WO 91/07426 PCr/EP90/01878
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formulations consist or drinkable ampoules and sachets,
the content of which is extemporarily dissolved or
suspended in water. The active ingredient dosages-can
range within wide limits, depending on the nature of
the used compound. Effective results are generally
obtained by administering the compounds of the
invention at daily dosages ranging from about 5 to
about 50 mg/kg body weight. The dosage pharmaceutical
forms generally contain from about 300 to about 1500 mg
1~ fo the active ingredient together with one or more of
the conventional solld or liquid pharmaceutical
carriers ad they can be administered one or more times
a day.
Some examples of pharmaceutical compositions are
reported hereinbelow.
~XAMPLE 9
One tablet contains:
Ironsuccinylalbumin (Example 3) mg 400 mg 1200
Glicerylpalmitostearate mg SS mg 165
Sodium carboxymethylcellulose mg 27 mg 80
Methyl p-hydroxybenzoate mg 1 mg 3
Propyl p-hydroxybenzoate mg 1 mg 3
EX~MPLE 10
One tablet contains:
~5 Ironsuccinylalbumin (Example 1) mg 1200
Glicerylpalmitostearate mg 165
Sodium carboxymethylcellulose mg 80
Methyl p-hydroxybenzoate mg 3
Propyl p~hydroxybenzoate mg 3
EXANPLE 11
One drinkable vial contains:
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WO 91/07426 PCl'/EP90~01878
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Ironsuccinylalbumin
(Example 3) mg 400 mg 600 mg 1200
Propyleneglycol mg 1000 mg 1500 . mg 3000
Sodium benzoate mg 35 mg 52 mg 105
Methyl p-hydroxybenzoate mg 30 mg 45 mg 30
Propyl p-hydroxybenzoate mg 15 mg 22 mg 45
Sodium saccharine mg 10 mg 15 mg 30
: Caramel (E 150 ) mg190 mg285 mg570
1 N sodium hydroxide mg1000 mg1500 mg3000
Depurated water q.s. toml 10ml 15ml 30
. ~XAMPLE 12
.
One drinkable vial contains:
Ironsuccinyl~ ~-lactalbumin (Example 5) mg 400
Propyleneglycol mg 1000
Methyl p-hydroxybenzoate mg 50
Propyl p-hydroxybenzoate mg 40
Sodium saccharine mg 30
Caramel mg 400
1 N sodium hydroxide mg 1000
, 20 Depurated water q.s. to ml 20
EXA~PLE 13
.
one drinkable vial contains:
Ir~naspartylalbumin mg 1200
Methylcellulose mg 800
Sodium benzoate mg 500
Propyl p-hydroxybenzoate mg 20
Sodium saccharine mg 30
1 N sodium hydroxide mg 1000
Depurated water q.s. to ml 15
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WO 91/07426 PCl/EP90/01878
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ExAMæLE 14
One sachet contains:
Ironsuccinylalbumin ( Examp le 3 ) mg 4 0 0 . mg 12 0 0
Sodium carboxymethylcellulose mg 6~, 6 mg 200
Sodium laurylsulfatemg 6,6 mg 20
; - Methyl p hydroxybenzoatemg 1,O mg 3
Propyl p-hydro~ybenzoatemg 1, 0 mg 3
Sodium saccharinemg 6, 6 mg 20
Caramel mg 33,2 mg 100
Sorbitol q.s. tog 2, 0 g 6, 0
EXAMæLE 15
One sachet contains:
Ironsuccinyl-0~-lactalbumin (Esempio 5) mg 600
Sodium carboxymethylcellulose mg ~0
Sodium laurylsulfate mg 10
Propyl p-hydroxybenzoate mg 3
Sodium saccharine mg 50
. .
Sorbitol q.s. to mg 3000
EXAMPLE 16
One sachet contains:
; Ironmaleylalbumin mg 1200
Sodium carboxymethylcellulose mg 180
Methyl p-hydroxybenzoate mg 3
` Propyl p-hydroxybenzoate mg S
:~ 25 Aspartame mg 30
: Sorbitol q.s. to mg 2500
:
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