CA2068655A1 - Bioavailable iron-albumin compounds, a process for the preparation thereof and pharmaceutical compositions containing them - Google Patents
Bioavailable iron-albumin compounds, a process for the preparation thereof and pharmaceutical compositions containing themInfo
- Publication number
- CA2068655A1 CA2068655A1 CA002068655A CA2068655A CA2068655A1 CA 2068655 A1 CA2068655 A1 CA 2068655A1 CA 002068655 A CA002068655 A CA 002068655A CA 2068655 A CA2068655 A CA 2068655A CA 2068655 A1 CA2068655 A1 CA 2068655A1
- Authority
- CA
- Canada
- Prior art keywords
- iron
- compounds
- ironsuccinylalbumin
- acylated
- albumins
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
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- 229940124344 antianaemic agent Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 239000000605 aspartame Substances 0.000 description 1
- IAOZJIPTCAWIRG-QWRGUYRKSA-N aspartame Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)OC)CC1=CC=CC=C1 IAOZJIPTCAWIRG-QWRGUYRKSA-N 0.000 description 1
- 229960003438 aspartame Drugs 0.000 description 1
- 235000010357 aspartame Nutrition 0.000 description 1
- 239000000305 astragalus gummifer gum Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 102000005936 beta-Galactosidase Human genes 0.000 description 1
- 108010005774 beta-Galactosidase Proteins 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- KDYFGRWQOYBRFD-NUQCWPJISA-N butanedioic acid Chemical compound O[14C](=O)CC[14C](O)=O KDYFGRWQOYBRFD-NUQCWPJISA-N 0.000 description 1
- 125000000484 butyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 229910000019 calcium carbonate Inorganic materials 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003638 chemical reducing agent Substances 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 238000004040 coloring Methods 0.000 description 1
- 235000018417 cysteine Nutrition 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008298 dragée Substances 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 239000003480 eluent Substances 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 125000001495 ethyl group Chemical group [H]C([H])([H])C([H])([H])* 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- VANNPISTIUFMLH-UHFFFAOYSA-N glutaric anhydride Chemical compound O=C1CCCC(=O)O1 VANNPISTIUFMLH-UHFFFAOYSA-N 0.000 description 1
- FETSQPAGYOVAQU-UHFFFAOYSA-N glyceryl palmitostearate Chemical compound OCC(O)CO.CCCCCCCCCCCCCCCC(O)=O.CCCCCCCCCCCCCCCCCC(O)=O FETSQPAGYOVAQU-UHFFFAOYSA-N 0.000 description 1
- 229940046813 glyceryl palmitostearate Drugs 0.000 description 1
- LEQAOMBKQFMDFZ-UHFFFAOYSA-N glyoxal Chemical compound O=CC=O LEQAOMBKQFMDFZ-UHFFFAOYSA-N 0.000 description 1
- 239000008187 granular material Substances 0.000 description 1
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 235000019447 hydroxyethyl cellulose Nutrition 0.000 description 1
- 229940071826 hydroxyethyl cellulose Drugs 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 239000003701 inert diluent Substances 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 150000002505 iron Chemical class 0.000 description 1
- 150000004698 iron complex Chemical class 0.000 description 1
- 235000014705 isoleucine Nutrition 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- 239000008101 lactose Substances 0.000 description 1
- 238000002356 laser light scattering Methods 0.000 description 1
- 235000010445 lecithin Nutrition 0.000 description 1
- 239000000787 lecithin Substances 0.000 description 1
- 229940067606 lecithin Drugs 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 235000005772 leucine Nutrition 0.000 description 1
- 229960003136 leucine Drugs 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229940072783 liquimat Drugs 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000314 lubricant Substances 0.000 description 1
- 125000003588 lysine group Chemical group [H]N([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])(N([H])[H])C(*)=O 0.000 description 1
- 235000019359 magnesium stearate Nutrition 0.000 description 1
- FPYJFEHAWHCUMM-UHFFFAOYSA-N maleic anhydride Chemical compound O=C1OC(=O)C=C1 FPYJFEHAWHCUMM-UHFFFAOYSA-N 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 229960002900 methylcellulose Drugs 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 231100000219 mutagenic Toxicity 0.000 description 1
- JMQITILRHSGUCB-BYPYZUCNSA-N n-[(3s)-2,5-dioxooxolan-3-yl]acetamide Chemical compound CC(=O)N[C@H]1CC(=O)OC1=O JMQITILRHSGUCB-BYPYZUCNSA-N 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 229940054441 o-phthalaldehyde Drugs 0.000 description 1
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 1
- OQCDKBAXFALNLD-UHFFFAOYSA-N octadecanoic acid Natural products CCCCCCCC(C)CCCCCCCCC(O)=O OQCDKBAXFALNLD-UHFFFAOYSA-N 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000020477 pH reduction Effects 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 1
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 1
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-M salicylate Chemical compound OC1=CC=CC=C1C([O-])=O YGSDEFSMJLZEOE-UHFFFAOYSA-M 0.000 description 1
- YGSDEFSMJLZEOE-UHFFFAOYSA-N salicylic acid Chemical class OC(=O)C1=CC=CC=C1O YGSDEFSMJLZEOE-UHFFFAOYSA-N 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 229910000029 sodium carbonate Inorganic materials 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008117 stearic acid Substances 0.000 description 1
- 231100000456 subacute toxicity Toxicity 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 229940014800 succinic anhydride Drugs 0.000 description 1
- 238000010613 succinylation reaction Methods 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 239000004094 surface-active agent Substances 0.000 description 1
- 239000000375 suspending agent Substances 0.000 description 1
- 239000003765 sweetening agent Substances 0.000 description 1
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- 235000012222 talc Nutrition 0.000 description 1
- 238000004448 titration Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 1
- 230000036269 ulceration Effects 0.000 description 1
- 238000000108 ultra-filtration Methods 0.000 description 1
- 238000005353 urine analysis Methods 0.000 description 1
- 239000004474 valine Substances 0.000 description 1
- 239000000080 wetting agent Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K33/00—Medicinal preparations containing inorganic active ingredients
- A61K33/24—Heavy metals; Compounds thereof
- A61K33/26—Iron; Compounds thereof
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
- C07K1/113—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides without change of the primary structure
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
- A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/20—Pills, tablets, discs, rods
- A61K9/2004—Excipients; Inactive ingredients
- A61K9/2013—Organic compounds, e.g. phospholipids, fats
Abstract
Iron-albumin derivatives are described consisting of albumins acylated with dicarboxylic acid residues and containing high amounts of iron, insoluble at the stomach acid pH and highly soluble at the intestinal pH. The derivatives are characterized by a very low gastrolesivity and toxicity and by remarkable bioavailability and therapeutic effectiveness in the treatment of anaemic conditions.
Description
~ 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 :: - ~ ,: ;,, , 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.
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 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.
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.
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 :::
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.
: 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.
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-- : .
~ ~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 ~;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).
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 :: - ~ ,: ;,, , 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.
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 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.
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.
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 :::
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.
: 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.
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-- : .
~ ~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 ~;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.
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.
,~ 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.
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 : - , -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.
` 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 : - , -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.
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%.
. ~:
,: :: , : -.~
WO 91/07426 PCl/EP90tO1878 The following additional parameters were determined on Ironsuccinylalbumin, pr~pared as described in Exampl~ 3.
The obtained results prove that the end ~-amino groups and the -amino lysin groups are acylated by more than 95%.
. ~:
,: :: , : -.~
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 .
. ,, . .:
: ; :
~ ~ ~ 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 ~ :::.. : . . - . .
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.
~ , , '~
.
: : . : ~: : : . , 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
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 ~, . , . ; . - . .
~2~ r~ 5 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 , . . .
WO 91/07426 PCr/EP90/01878 2~ ;'~55 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).
': .
:
' , :
:. - , :
2f~
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) , : ~ :
WO 91/07426 PCr/EP9û/0187 Z~ 22 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.
~ ` ` ' WO 91/07426 PCr/EP90/01878 '':
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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.b . ~ . ., WO 91/074~6 Pcr/Ep9o/ol878 2~
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**
______ ______________________________________ _________ :
:
.~
~ ~ ~ r ~ 3 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 . .
::
WO91/07426 pcT/Epso/ol878 '2`~
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 .,.:, . .. ....
- ~
- ~
WO 91/07426 PCr/EP90/01878 ~r;~ .5 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 One drinkable vial contains:
;' ~
WO 91/07426 PCl'/EP90~01878 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 ,' .
, '` ''' ~ ~
~ . ` ..... ..
WO 91/07426 PCl/EP90/01878 2 ~.i~ r r 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 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 :
:,
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 .
. ,, . .:
: ; :
~ ~ ~ 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 ~ :::.. : . . - . .
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.
~ , , '~
.
: : . : ~: : : . , 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
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 ~, . , . ; . - . .
~2~ r~ 5 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 , . . .
WO 91/07426 PCr/EP90/01878 2~ ;'~55 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).
': .
:
' , :
:. - , :
2f~
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) , : ~ :
WO 91/07426 PCr/EP9û/0187 Z~ 22 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.
~ ` ` ' WO 91/07426 PCr/EP90/01878 '':
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**
.. ,. ~......... . ~
, ~
, ~ :~:.
.b . ~ . ., WO 91/074~6 Pcr/Ep9o/ol878 2~
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**
______ ______________________________________ _________ :
:
.~
~ ~ ~ r ~ 3 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 . .
::
WO91/07426 pcT/Epso/ol878 '2`~
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 .,.:, . .. ....
- ~
- ~
WO 91/07426 PCr/EP90/01878 ~r;~ .5 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 One drinkable vial contains:
;' ~
WO 91/07426 PCl'/EP90~01878 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 ,' .
, '` ''' ~ ~
~ . ` ..... ..
WO 91/07426 PCl/EP90/01878 2 ~.i~ r r 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 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 :
:,
Claims
1. Albumins acylated with dicarboxylic acid residues, containing iron in a bioavailable form.
2. Compounds as claimed in claim 1, in which acylated albumins derive from bovine albumin or lactalbumin.
3. Compounds as claimed in claims 1 or 2, in which albumins are acylated with dicarboxylic acid residues selected from succinic, glutaric, maleic, malic, malonic, aspartic and glutamic acids.
4. Compounds as claimed in claim 3, in which albumins are acylated with succinyl residues.
5. Compounds as claimed in any one of claims 1 to 4, containing 3 to 20% by weight iron.
5. Bioavailable iron containing compounds, which can be obtained from albumins acylated with dicarboxylic acid residues by interaction with iron ions in aqueous medium at pH ranging from 2 to 12, preferably from 3 to 7.
7. Pharmaceutical compositions containing as the active ingredient one compound as claimed in claims 1 to 6 in admixture with a suitable carrier or excipient.
8. Pharmaceutical compositions as claimed in claim 7, containing the active ingredient in amounts from about 300 to about 1500 mg.
9. A method for the treatment of anaemic conditions in warm blooded animals, which comprises administering one therapeutically effective dose of one of the compounds as claimed in claim 1, which dose ranges from about 10 to about 50 mg/ kg body weight.
10. The use of the compounds as claimed in claim 1 in the preparation of a medicament for the treatment of anaemic conditions in the warm blooded animals.
2. Compounds as claimed in claim 1, in which acylated albumins derive from bovine albumin or lactalbumin.
3. Compounds as claimed in claims 1 or 2, in which albumins are acylated with dicarboxylic acid residues selected from succinic, glutaric, maleic, malic, malonic, aspartic and glutamic acids.
4. Compounds as claimed in claim 3, in which albumins are acylated with succinyl residues.
5. Compounds as claimed in any one of claims 1 to 4, containing 3 to 20% by weight iron.
5. Bioavailable iron containing compounds, which can be obtained from albumins acylated with dicarboxylic acid residues by interaction with iron ions in aqueous medium at pH ranging from 2 to 12, preferably from 3 to 7.
7. Pharmaceutical compositions containing as the active ingredient one compound as claimed in claims 1 to 6 in admixture with a suitable carrier or excipient.
8. Pharmaceutical compositions as claimed in claim 7, containing the active ingredient in amounts from about 300 to about 1500 mg.
9. A method for the treatment of anaemic conditions in warm blooded animals, which comprises administering one therapeutically effective dose of one of the compounds as claimed in claim 1, which dose ranges from about 10 to about 50 mg/ kg body weight.
10. The use of the compounds as claimed in claim 1 in the preparation of a medicament for the treatment of anaemic conditions in the warm blooded animals.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
NL9301964A NL9301964A (en) | 1990-11-09 | 1993-11-12 | Method for producing potato chips having a low fat content suitable for preparation in a microwave oven |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
IT02239989A IT1237783B (en) | 1989-11-16 | 1989-11-16 | BIOAVAILABLE IRON-ALBUMINE DERIVATIVES, THEIR PREPARATION PROCEDURE AND RELATED PHARMACEUTICAL COMPOSITIONS. |
IT22399A/89 | 1989-11-16 |
Publications (1)
Publication Number | Publication Date |
---|---|
CA2068655A1 true CA2068655A1 (en) | 1991-05-17 |
Family
ID=11195773
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CA002068655A Abandoned CA2068655A1 (en) | 1989-11-16 | 1990-11-09 | Bioavailable iron-albumin compounds, a process for the preparation thereof and pharmaceutical compositions containing them |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP0593443A1 (en) |
AU (1) | AU7039691A (en) |
CA (1) | CA2068655A1 (en) |
IE (1) | IE904124A1 (en) |
IT (1) | IT1237783B (en) |
PT (1) | PT95904B (en) |
WO (1) | WO1991007426A1 (en) |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT1251702B (en) * | 1991-10-16 | 1995-05-19 | Mediolanum Farmaceutici Srl | IRON COMPLEXES WITH CONALBUMIN AND ITS DERIVATIVES |
IT1251725B (en) * | 1991-11-04 | 1995-05-23 | Italfarmaco Spa | BIOAVAILABLE IRON COMPOUNDS WITH ACILATED OVOTRANSFERRIN OR ITS ACILATED HYDROLYSIS DERIVATIVES |
IT1291289B1 (en) * | 1997-04-30 | 1999-01-07 | Derivati Organici Lab | COMPLEXES CONSISTING OF FE (III) A POLYHYDROXYLATE AND OVALBUMIN COMPOUND |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
IT1150213B (en) * | 1982-03-02 | 1986-12-10 | Italfarmaco Spa | BIOAVAILABLE IRON DERIVATIVES FREE FROM GASTROLESIVITY, PREPARATION PROCEDURE AND RELATED PHARMACEUTICAL COMPOSITIONS |
IT1222912B (en) * | 1987-10-14 | 1990-09-12 | Italfarmaco Spa | HIGHLY SOLUBLE AND HIGHLY SOLUBLE POLYPEPTIDIC DERIVATIVES SUITABLE FOR MARTIAL THERAPY PREPARATION PROCEDURES AND THEIR PHARMACEUTICAL FORMS |
-
1989
- 1989-11-16 IT IT02239989A patent/IT1237783B/en active IP Right Grant
-
1990
- 1990-11-09 CA CA002068655A patent/CA2068655A1/en not_active Abandoned
- 1990-11-09 WO PCT/EP1990/001878 patent/WO1991007426A1/en not_active Application Discontinuation
- 1990-11-09 EP EP90916292A patent/EP0593443A1/en not_active Withdrawn
- 1990-11-09 AU AU70396/91A patent/AU7039691A/en not_active Abandoned
- 1990-11-15 PT PT95904A patent/PT95904B/en not_active IP Right Cessation
- 1990-11-15 IE IE412490A patent/IE904124A1/en unknown
Also Published As
Publication number | Publication date |
---|---|
EP0593443A1 (en) | 1994-04-27 |
PT95904A (en) | 1991-09-13 |
AU7039691A (en) | 1991-06-13 |
IT1237783B (en) | 1993-06-17 |
IT8922399A0 (en) | 1989-11-16 |
PT95904B (en) | 1998-01-30 |
WO1991007426A1 (en) | 1991-05-30 |
IE904124A1 (en) | 1991-05-22 |
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