Casein succinylate of iron (iii) and method of its production

FIELD: metallurgy.

SUBSTANCE: invention relates to casein succinylate of iron (III) wherein iron content varies from 4.5 wt % to 7 wt %, water solubility exceeds 92% while phosphorus-to-nitrogen ratio exceeds 5 wt %.

EFFECT: additionally, invention relates to production of iron (III) and to pharmaceutical composition containing casein succinylate of iron (III).

17 cl, 4 tbl, 9 ex

 

The present invention relates to caseinsensitivity iron (III), characterized in that the iron content in it is from 4.5 wt.% up to 7 wt.%, and the fact that its solubility in water is about more than 92%.

The present invention additionally relates to a method of obtaining caseinsensitivity iron (III).

Prior art

Derived iron is widely used in medicine for the prevention and treatment of anemia and iron deficiency during pregnancy, in case of malabsorption syndrome, breastfeeding and growth process. Among them containing succinylate iron, often denoted by reducing IPS, positively differs in its characteristics, bioavailability and tolerability.

Chemical, physical and biological properties, as well as getting IPS, named Irontom (Ironlat), which was obtained based on milk proteins, described in Italian patent IT1150213. In addition, the solubility Irongate specified at pH>5, and it is incomplete for complex containing 6.7% of iron, and full of complex, containing 4.6% of iron. In the work Cremonesi et al. International Journal of Clinical Pharmacology Therapy and Toxicology, vol.31 (1993) pages 40-51, identified the chemical and pharmacological properties of caseinsensitivity iron (III), iron (III)complex derived from succinylamino casein. In experimento vivo this product, with the iron content, the equivalent of 5%, found a better gastrointestinal tolerability compared to other derived iron.

European patent EP939083B1 describes the method of obtaining caseinsensitivity iron (III), which provides for the use of specific procedures of splitting and drying to solve presence of insoluble components in the product. It also describes the use of edible casein as raw materials used in the process of obtaining caseinsensitivity iron (III).

To facilitate maintaining the product within certain limits under part microbiological contamination during the process add preservatives methylparaben and propylparaben; presence at the level of 3% and 1.05%, respectively, determine, by analyzing the final product. Such use of preservatives is also described in the patent EP1776382B1, which presents the use of moist granules in combination with a spray drying or lyophilization of the product to facilitate solubilization in the implementation of the method.

In the patent application EP319664 proposed a method based on the enzymatic degradation of caseinsensitivity iron (III) to solve the problems of solubility and viscosity observed in the complex, specifically, when a relative indicator of iron exceeds 10%.

Between Hrodna patent application WO2007/065812 describes obtaining caseinsensitivity iron (III), through reaction succinylcholine and subsequent complexation using operations with ion iron(III) in suspension, this does not address the aspects related to difficulties solubilization of caseinsensitivity iron (III). In fact, this patent application does not provide information relating to solubilize the resulting complex, and the viscosity may get solution is not measured.

Such problems solubilization particularly evident when the relative iron content exceeds the 5% limit.

The phenomenon of increasing the viscosity, which is observed with increasing concentration of the product in aqueous solution, hinders or even prevents the use of caseinsensitivity iron (III) beyond certain concentrations.

The combination of these effects limit the dose of product value, not exceeding 800 mg, which corresponds to approximately 40 mg of iron (III) per dose except for the use of the compounds in excess of 15 ml of solution.

In addition, the viscosity of an aqueous solution of caseinsensitivity iron (III) makes it difficult surgery to reduce the microbial content of the product by filtration or microfiltration, and requires the use of high amounts of preservatives.

Thus it is obvious that in this area does not offer simple solutions for overcoming the Oia problems solubilization of caseinsensitivity iron (III), exceptional medicines in the treatment of various forms of iron deficiency.

Description of the invention

It was discovered that can be obtained unexpected advantages in obtaining caseinsensitivity iron (III)if the quality of raw materials to use edible casein, which was subjected to purification process to remove impurities, inorganic impurities and/or to increase the ratio of phosphorus/nitrogen ratio (P/N). Actually, so perhaps getting caseinsensitivity iron (III), which is not only protein profile, containing fewer impurities, but also with an unexpected increase solubility in water. This increased solubility entails the possibility of obtaining aqueous solutions and/or suspensions, detecting a lower viscosity relative to the viscosity of the products obtained from commercial edible casein.

Relative to casein, the expression "protein impurities according to the invention is used to refer to proteins other than casein in its various variants, as measured by one-dimensional electrophoresis with staining using Kumasi blue and using densitometrical analysis. Preferably these protein impurities according to the invention are proteins other than casein alpha-S1, casinoriva-S2, the beta casein, Kappa casein and/or casein pseudo-Kappa.

Similarly, regarding caseinsensitivity iron (III), the phrase "protein impurities according to the invention is used to refer to proteins other than succinylamino casein in its various variants, as measured by one-dimensional electrophoresis with staining using Kumasi blue and using densitometrical analysis. Preferably these protein impurities according to the invention are succinylcholine proteins, other than products of succinylcholine casein alpha-S1 casein alpha S2, beta casein, Kappa casein and/or casein pseudo-Kappa.

The expression "inorganic impurities according to the invention is used to denote the sulfated ash.

The term "P/N" is used to denote the ratio between the mass amount of phosphorus present in the sample, which is measured by ICP method, and the mass quantity of nitrogen, which is measured by elementary analysis.

According to the present invention, the values expressed in weight percent (wt.%), will have in mind the weight relative to the total mass of caseinsensitivity iron (III) or casein.

Thus, the aim of the present invention is the proposal of caseinsensitivity iron (III)than those who, the iron content ranges from 4.5 wt.% up to 7 wt.%, and the fact that its solubility in water is more than about 92%. Preferably such solubility in water should be considered as a solubility of about 11.5 parts of water.

This caseysalinger iron (III) further comprises a protein impurities in amounts less than about 15%, preferably less than about 10%, and/or the ratio P/N is more than about 5%, preferably more than about 5.5%.

The next objective of the present invention is a method of obtaining caseinsensitivity iron (III), comprising the following stages:

a) reaction of casein and succinic anhydride in water to obtain succinylamino casein,

b) reaction succinylamino casein with iron chloride (III) obtaining caseinsensitivity iron (III)

characterized in that the casein according to stage a) is a purified casein having a low content of protein impurities and/or inorganic impurities and/or having a high ratio of P/N.

Preferably the content of said protein impurities is less than 15 wt.%, and the content of the above inorganic impurities is less than 1 wt.%, and the ratio between the mass of phosphorus and nitrogen is more than about 5 wt.%.

More predpochtitelney these protein impurities is less than 10 wt.%, and/or the content of these inorganic impurities is less than 0.4 wt.%, and the ratio between the mass of phosphorus and nitrogen is more than about 5.5 wt.%.

These protein impurities according to the invention are proteins other than casein alpha-S1 casein alpha S2, beta casein, Kappa casein and/or casein pseudo-Kappa.

Food caseine, generally identified by the method of deposition used when you receive them: as for acid casein, the deposition shall be implemented by acidification, while casein obtained with the use of rennet, rennet are, which are also often referred to as (rennet) rennet casein.

According to the present invention, edible casein is used as a raw material may be acid casein or rennet casein.

These commercial products, regardless of the type (acid or rennet) are usually accompanied by a few analytical documentation; the specified parameters, as a rule, represent the water content, protein content (as determined by applying a multiplier to the value of total nitrogen), the level of microbial contamination and grain size distribution. This level of characterization is fully suitable for use Caseins in PI the eve of industry, but we have found that the receipt of caseinsensitivity iron (III) requires an in-depth analytical evaluation of the raw material.

Commercial food casein and peeled casein used as raw materials according to the present invention for receiving caseinsensitivity iron (III), were analyzed by HPLC with reversed phase according to the method specified in the work Bonizzi et al. Journal of Chromatography A vol.1261(2009) pages 165-168, incorporated herein by reference.

Thus, it is possible to identify three types of casein alpha S1, alpha S2, beta, Kappa, and pseudo-Kappa.

It is also possible to estimate the presence of protein impurities through the analysis of electrophoresis as a one-dimensional and two-dimensional, using Kumasi blue as a reagent indicator, along with the separation of different types of casein and their different variants, the resulting post-transcriptional modifications (such as glycosylation and phosphorylation).

Specifically, for the quantitative determination of protein impurities used SDS-PAGE electrophoresis using a 14% polyacrylamide gel, stained with Kumasi blue, and densitometrically analysis on the basis of a calibration curve created using different amounts of BSA.

These caseine and peeled casein also analysed is ovale using the method using sulfated ash according to the European Pharmacopoeia (Eu method.Pharm. 2.4.14). Then determined the content of phosphorus (P) by ICP method and the content of nitrogen (N) by means of elementary analysis and, thus, the expected ratio P/N % by dividing the mass observed for phosphorus, mass number, observed for nitrogen, and by multiplying the result by 100. Table 1 below shows the results obtained for different types of Caseins, i.e. commercial food casein (acid and rennet) and purified Caseins according to the method of the present invention.

td align="center"> 0,4%
Table 1
CaseinRaw materials for theProtein impuritiesSulfated ashP/N%
Acid caseinComparative examples 1 and 215%1,6%
Rennet caseinComparative example 320%9,2%
Example 4Examples 8A and 9A5%5,5%
Example 5Example 8B9%0,9%5,1%
Example 6Example 8C8%0,2%5,6%
Example 7Examples 8D and 9B10%0,8%5,2%

Thus, observed that the analyzed commercial food casein detect the protein content of impurities, equivalent to 15-20% of the total protein in the sample, while the content of inorganic impurities, established by defining values corresponding to the amount of sulfated ash, which include about 1 wt.% - 2.5 wt.% in the commercial acid caseino and 7 wt.% - 10 wt.% rennet caseing that as you can see in the examples in table 1 above.

Therefore, the next aim of the present invention is also a method of cleaning these edible Caseins, leading to partial or total removal of protein impurities, inorganic impurities and/or to the increase in the ratio P/N.

Under this is obreteniyu specified food casein is treated with water, the polar organic solvent or its mixture, preferably with water.

Preferably the specified polar organic solvent is an alcohol C1-C4more preferably methanol, ethanol, isopropanol or a mixture.

According to the first embodiment of the present invention, the selected solvent used for the purification of casein, dissolving thus protein impurities and inorganic impurities for their separation from the insoluble casein by centrifugation or filtration. Characteristics of solubility of casein in the selected solvent as a function of temperature, pH and/or adding additives, such as, for example, sodium chloride, calcium chloride and/or ammonium acetate, is used for the implementation of this cleaning method.

Edible casein in the cleaning process can be directly brought into contact with the selected solvent under the conditions of dissolution of protein and inorganic impurities and after a suitable period of time of contact, with stirring, it is separated from the solution containing impurities by filtration or centrifugation.

According to the second embodiment of the present invention the cleaning carried out by first dissolving the casein in the selected solvent and subsequent deposition through a suitable VA is grovania pH, temperature and/or the addition of additives, such as, for example, sodium chloride, calcium chloride and/or ammonium acetate. Even in this case, the separation of casein protein and inorganic impurities will be carried out by filtration or centrifugation, and these impurities finish to dissolve in the solvent used for cleanup.

According to the aforementioned first embodiment of the present invention edible casein is brought into contact with the selected solvent, the pH of the mixture is not necessarily brought to a pH in the range of 3-5 or to pH in the range of 5-10. These pH adjustment can be effected, for example, using aqueous solutions of hydrochloric acid or sodium hydroxide. This mixture is brought to a temperature in the range of 0°C to 40°C, preferably in the range of 0°C-10°C. Optional can be added additives such as, for example, sodium chloride, calcium chloride and/or ammonium acetate. The resulting mixture is left to mix for a period of time 1-24 hours, preferably 2-10 hours.

The specified selected solvent thus used for dissolving protein impurities and inorganic impurities and separated from the insoluble casein, preferably casein is then separated from impurities by centrifugation or filtration. Filtering mo is et to be a standard type can be implemented by use of membranes suitable porosity according to the method of flow along a stream or using a type of dead-end filtration.

According to the aforementioned second embodiment of the present invention edible casein is dissolved in the selected solvent, subjecting it then settling with bringing to pH in the range 4-6, preferably in the range of 4.5-5. Any method known to the person skilled in the art, can be used to separate the casein from the solution containing the protein and inorganic impurities, preferably specified casein is separated by filtration or centrifugation.

Order to obtain the target level of cleaning these stages of purification may be repeated one or more times. Thus, the same purification method can be repeated or can be optimized for consistent implementation of the cleaning methods differ from each other.

Therefore, the next aim of the present invention is a purified edible casein is produced by the previously described methods of the present invention, and casein is the protein content of impurities, preferably comprising less than 15 wt.%, more preferably less than 10 wt.%; the content of inorganic impurities is preferably less than 1 wt.%, more preferably less than 0.4 wt.% and/or the ratio between the mass of phosphorus and nitrogen, which constitutes more than 5 wt.%, more preferably more than 5.5 wt.%.

the second purpose of the present invention is the use of this purified casein to obtain caseinsensitivity iron (III).

Getting caseinsensitivity iron (III) can be carried out according to methods known to the person skilled in the art, such as, for example, those described in patents IT1150213 and EP939083B1, incorporated herein by reference.

In one embodiment of the present invention is a method of obtaining caseinsensitivity iron (III), described above, involves the following stages:

a) reaction of casein and succinic anhydride in water to obtain succinylamino casein,

b) reaction succinylamino casein with iron chloride (III) obtaining caseinsensitivity iron (III)

where casein at the stage a) is a purified casein having a low content of protein impurities and/or inorganic impurities and/or having a high ratio of P/N, as described above, is as follows:

purified casein Succinimidyl in water with the use of succinic anhydride, while maintaining the pH during the reaction is preferably in the range of 7.5-8.5 by addition of aqueous sodium hydroxide solution.

The reaction temperature is about 20-25°C. after the reaction product is precipitated by adding with stirring an aqueous solution of hydrochloric acid to bring the pH to about 3.

Thus obtained succinylcholine casein is filtered and dissolved in water by adding an aqueous solution of hydroxy is and sodium. The solution is filtered to separate the possible insoluble solids; then an aqueous solution of ferric chloride (III) is added to a certain amount, which performs the function of the target iron content in the final product.

In the process of adding the lowering of pH and the precipitation of caseinsensitivity iron (III).

While adding a solution of ferric chloride (III) the pH may be adjusted using an aqueous solution of sodium hydroxide in order to avoid overly acidic conditions. In this case, after the addition of iron chloride (III) precipitation of the complex is performed by adding an aqueous solution of hydrochloric acid.

Formed solid is recovered and suspended in water. At this stage you can add preservatives (methyl - and propyl paraben). An aqueous solution of sodium hydroxide are added to obtain a pH of about 8.5, the insoluble matter is removed by centrifugation and caseysalinger iron (III) precipitates upon acidification using chlorothalidone acid in aqueous solution. The product is dried at low pressure.

Alternative drying at low pressure, caseysalinger iron (III) can be obtained from its aqueous solution by spray drying.

This method may also include the additional step microfiltration vodno the solution caseinsensitivity iron (III). The specified stage microfiltration according to the invention is designed to reduce microbial contamination.

Thus, another objective of the present invention is caseysalinger iron (III)obtained by the previously described method.

This caseysalinger iron (III) according to the invention has improved characteristics solubility in water compared to known in the art; preferably such caseysalinger iron (III) has a solubility in water of more than about 92%. It is meant, what is the solubility of preferably refers to the 11.5 parts of water.

Calculation of the solubility in water is carried out by solubilization of the sample at about pH to 8.0 using sodium hydroxide with the final ratio between water and caseinstallation iron (III), equivalent to 11.5 by weight, and using filtering of the drug on the filter with a porosity of 6 μm and with the definition of undissolved amount after drying by weighing the filter. The percentage solubility of the sample is calculated by subtracting from the mass of the sample mass, which refers to the component remaining on the filter, and by comparing the result with the original mass of the sample. The viscosity of aqueous preparations was measured at pH products about 8.0 to a concentration equivalent to 5 Mac is.%, 6 wt.% and 8 wt.%.

In addition, the filtration rate was measured on membranes with porosity, equivalent to 0.45 microns to create opportunities through microfiltration reduce microbial contamination for different samples. This calculation was carried out by measuring the time required for passing in the conditions of the vacuum system to 5 ml of the sample solution in water at about pH of 8.0 at a concentration of 5 wt.% through a membrane with a porosity of 0.45 μm to 3 cm2.

Table 2 below shows the results.

Example 9B
Table 2
SampleThe solubility of 11.5 parts of waterThe viscosity of the fluid (MPa·s)The filtering 5 ml of 5% in water at 0.45 µm (sec)
5% in water6% in water8% in water
EUR. Ave. 189%1754715∞ (blocked)
EUR. Ave. 284% 2167957∞ (blocked)
EUR. Ave. 385%1753863560
Example 8A97%1429207120
Example 8B94%1333351150
Example 8C98%112419590
Example 8D93%1544387180
Example 9A98%1228240180
92%1436411120

The next objective of the present invention is a composition containing caseysalinger iron (III) according to the present invention and at least one physiologically acceptable excipient.

This composition may be preferably included in the composition in solid or liquid form, more preferably in liquid form. Specified liquid form is preferably a solution or suspension, preferably an aqueous solution. The specified water solution can be administered orally or parenterally. This composition includes the number of caseinsensitivity iron (III) according to the invention, which preferably is in the range of 10-200 mg of iron, more preferably 20-100 mg of iron, even more preferably about 40 mg, 60 mg or 80 mg of iron.

According to a preferred embodiment of the invention, this composition is an aqueous solution comprising about 40 mg, 60 mg or 80 mg of iron dissolved in 15 ml of solution.

Also unexpectedly found that the liquid composition on the basis of caseinsensitivity iron (III) according to the present invention detects znachitelno.soderzhanie viscosity with respect to compositions, known in this field.

The viscosity of the liquid compositions containing caseysalinger iron (III) according to the invention is significantly reduced compared to the viscosity of compositions with equivalent content of iron, known in this area. This reduction of viscosity in preparations containing caseysalinger iron (III) according to the invention, seen in drugs derived from caseinsensitivity iron (III) with iron content equivalent to 5%, and even more obvious when comparing between products with a higher content of iron, in particular, between products with an iron content of 6% and beyond, as can be seen from table 2. Preferably the viscosity of the liquid composition containing solubilizing form about 8 wt.% caseinsensitivity iron (III) according to the invention is less than 400 MPa·s, more preferably less than 300 MPa·S.

These unexpected characteristics of the higher solubility of caseinsensitivity iron (III) according to the invention and a lower viscosity liquid compositions containing them, specifically used for receiving caseinsensitivity iron (III) when a high concentration of iron (such as, for example, equal to or higher than 40 mg 15 ml). The preferred solution according to the present invention contains a number of caseinsensitivity iron (III), and enclosed in the interval from 4 to 15 wt.%, preferably 4.5 to 12 wt.%, more preferably 5-8 wt.%, with respect to the total mass.

Such characteristics low viscosity also allow for filtration of liquid compositions containing caseinsensitive iron (III) according to the invention.

This operation is extremely useful for storing properties of the product, the subsequent composition and its conservation. In the operation of microfiltration and reducing microbial contamination is also possible to reduce or even avoid the use of preservatives, such as parabens, commonly used to prevent microbial proliferation.

In fact, it is possible to greatly reduce the microbial content of the compositions, preferably in liquid compositions containing caseysalinger iron (III) according to the invention.

Up to the present time high viscosity liquid compositions (e.g., aqueous solutions) on the basis of caseinsensitivity iron (III)derived from edible casein, not subjected to a suitable purification process, interfere with the conduct of such operations microfiltration.

Microfiltration according to the invention can be carried out on membranes with controlled porosity or tangential microfiltration on membranes with controlled porosity to reduce microbial contamination.

Specified Opera the Oia microfiltration, also associated with a lower viscosity liquid compositions (preferably aqueous) of caseinsensitivity iron (III), specifically useful for oral liquid compositions with a high shelf life, does not require the use of the considered high amounts of preservatives.

The following examples have the sole purpose of illustration of certain embodiments of the invention, and should not be construed as any limitation.

EXAMPLES

EXAMPLE 1

Comparative example to obtain caseinsensitivity iron (III) with iron content equivalent to about 5% of the commercial source of acid casein.

600 ml of demineralized water was added to 50 g of edible acid casein, and the mixture was stirred for 30 minutes and the pH was brought to a stable pH of 8.4 with 9.5% aqueous sodium hydroxide solution.

15 g of succinic anhydride was added in small portions with stirring. In the process of adding a maintained pH at about 8.4 by gradually adding a 9.5% solution of hydrochloride of sodium. An aqueous solution of 17% hydrochloric acid was added until a stable pH of 2.8. The selection was carried out by filtering the precipitated succinylamino casein, which resuspendable in 600 ml of demineralized water. the pH was brought to pH 8.5 by 9.5% vodno the solution of sodium hydroxide, it was supported by mixing a small insoluble fraction was filtered on filter paper and the solution made up 14.1 g of uranyl chloride iron (III) and 119 ml of water, was added under stirring. In the process of adding the lowering of pH and the precipitation of caseinsensitivity iron (III). After maintaining conditions stirring for 30 minutes, the pH was pH of 2.9, and the product was filtered. The product is suspended in 600 ml of water was gradually added 5% aqueous sodium hydroxide solution, and with stirring, brought the pH to a stable pH value of 8.5. Centrifugation was performed to remove insoluble component, equivalent to approximately 20 g wet matter; pH was adjusted by using hydrochloric acid in aqueous solution at 17% to a stable pH of 2.8. The precipitate was filtered and dried to obtain 54,1 g caseinsensitivity iron (III).

EXAMPLE 2

Comparative example to obtain caseinsensitivity iron (III) with iron content equivalent to about 6% of the commercial source of acid casein.

600 ml of demineralized water was added to 50 g of edible acid casein, and the mixture was stirred for 30 minutes and the pH was brought to a stable pH of 8.4 with 9.5% aqueous sodium hydroxide solution.

15 g of succinic anhydride was added a small portion of the under stirring. During the addition the pH was maintained at a level of about 8.4 by gradually adding a 9.5% solution of hydrochloride of sodium. An aqueous solution of 17% hydrochloric acid was added until a stable pH of 2.8. The selection was carried out by filtering the precipitated succinylamino casein, which resuspendable in 600 ml of demineralized water. the pH was brought to pH 8.5 with 9.5% aqueous sodium hydroxide solution, supported him by mixing a small nerastvorim fraction was filtered on filter paper, and the solution is composed of 17 g of uranyl chloride iron (III) in 150 ml of water, was added under stirring. While adding a solution of ferric chloride (III) the pH was maintained in the range of pH of 6.0 to 6.5 by adding sodium hydroxide in 5% aqueous solution. Upon completion of addition of solution of ferric chloride (III) the pH was adjusted to pH of 2.8 with the use of 17% aqueous solution of hydrochloric acid. The suspension is maintained in a state of agitation for 30 minutes, and the product was filtered. The product is suspended in 600 ml of water was gradually added 5% aqueous sodium hydroxide solution, and with stirring, brought the pH to a stable pH value of 8.5. Centrifugation was performed to remove insoluble component, equivalent to about 25 g wet matter; pH adjustment is ovale using hydrochloric acid in aqueous solution at 17% to a stable pH of 2.8. The precipitate was filtered and dried to obtain a 53.5 g of caseinsensitivity iron (III).

EXAMPLE 3

Comparative example to obtain caseinsensitivity iron (III) with iron content equivalent to about 5% of the commercial source of rennet casein.

Following the method described in example 1, caseysalinger iron (III) is obtained from 50 g of edible rennet casein. After drying receive a 54.2 g of caseinsensitivity iron (III).

EXAMPLE 4

Cleaning commercial acid casein.

6 l of demineralized water was added to 180 g of edible acid casein, pH was adjusted to pH 7.0 using 1 M aqueous sodium hydroxide solution. The mixture was cooled to 4°C, the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid. The suspension is maintained in a state of agitation for 16 hours, and the solid was filtered through a Buechner funnel.

The wet product is suspended in 6 l of demineralized water, pH was adjusted to pH 7.0 using 1 M aqueous sodium hydroxide solution. The mixture was cooled to 4°C, the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid. The suspension is maintained in a state of agitation for 3 hours, and the solid was filtered through a Buechner funnel.

The wet product resuspendable in 3 l of demineralized water, pH was adjusted to pH 7.0 with what omashu 1 M aqueous sodium hydroxide solution. The mixture was cooled to 4°C, the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid. The suspension is maintained in a state of agitation for 3 hours, and 390 g of wet solid substance was filtered through a Buechner funnel, the product was dried on a drum dryer for 20 hours at 25°C and at 30 mm Hg with getting 146 g of purified casein.

EXAMPLE 5

Cleaning commercial acid casein.

6 l of demineralized water was added to 18 g of edible acid casein, pH was adjusted to pH 7.0 using 1 M aqueous sodium hydroxide solution. The mixture was cooled to 4°C, the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid. The suspension is maintained in a state of agitation for 16 hours, and the solid was filtered through a Buechner funnel. Received 408 grams wet substance was dried in a drum dryer for 20 hours at 25°C and at 30 mm Hg with getting 162 g of purified casein.

EXAMPLE 6

Cleaning commercial acid casein.

42 l of demineralized water was added 144 g of edible acid casein. the pH was adjusted until a stable pH of 7.0 using 8% sodium hydroxide solution in water. the pH was brought to pH 3.0 with aqueous solution of 0.5 M hydrochloric acid. The mixture was cooled to 2°C and the pH was brought to pH 4.0, using a 0.01 M aqueous solution of sodium hydroxide. The suspension is maintained in a state of agitation for one hour, and 318 g of wet solids were filtered through a Buechner funnel, the product was dried on a drum dryer for 20 hours at 25°C and at 30 mm Hg to obtain 126 g of purified casein.

EXAMPLE 7

Cleaning commercial rennet casein.

6 l of demineralized water was added to 180 g of edible rennet casein, and the mixture was stirred for 30 minutes, cooled to 4°C, the mixture was maintained in a state of agitation for 16 hours, and the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid solution. The suspension is maintained in a state of agitation for one hour, and the solid was filtered through a Buechner funnel.

The wet product is suspended in 6 l of demineralized water, pH was adjusted to pH 7.0 using 1 M aqueous sodium hydroxide solution. The mixture was cooled to 4°C, the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid. The suspension is maintained in a state of agitation for 3 hours, and the solid was filtered through a Buechner funnel.

The wet product resuspendable in 6 l of demineralized water, pH was adjusted to pH 7.0 using 1 M aqueous sodium hydroxide solution. The mixture was cooled to 4°C, the pH was brought to pH 4.5 with 1 M aqueous hydrochloric acid.The suspension is maintained in a state of agitation for 16 hours, and the solid is filtered through a Buechner funnel.

Received 290 grams wet substance was dried in a drum dryer for 20 hours at 25°C and at 30 mm Hg to obtain 126 g of purified casein.

EXAMPLE 8

Getting caseinsensitivity iron (III) with iron content equivalent to about 5% of the original treated Caseins defined in examples 4, 5, 6 and 7.

Following the method described in example 1, caseysalinger iron (III) was obtained from purified Caseins obtained as described in examples 4, 5, 6 and 7.

The table below shows the number relating to each of the drugs.

Table 3
Caseysalinger iron (III)Raw materialsThe amount of caseinThe resulting number of caseinsensitivity iron (III)
Example 8AAcid casein, purified in Example 450 g54.6 g
Example 8BAcid casein, purified in Example 550 g 55,2 g
Example 8CAcid casein, purified in Example 650 gto 54.3 g
Example 8DRennet casein, purified in Example 750 g52.7 g

EXAMPLE 9

Getting caseinsensitivity iron (III) with iron content equivalent to about 6% of the original treated Caseins defined in examples 4 and 7.

Following the method described in example 2, received caseysalinger iron (III) from purified Caseins obtained as described in examples 4 and 7.

The table below shows the number relating to each of the preparations of iron (III).

Table 4
Caseysalinger iron (III)Raw materialsThe amount of caseinThe resulting number of caseinsensitivity iron (III)
Example 9AAcid casein, purified in Example 450 g56,0 g
P the emer 9B Rennet casein, purified in Example 750 g52,5 g

1. Caseysalinger iron (III), characterized in that the iron content ranges from 4.5 to 7 wt.%, and the fact that its solubility is more than about 92%, and the fact that the ratio of phosphorus/nitrogen is more than about 5 wt.%, preferably more than about 5.5 wt.%.

2. Caseysalinger iron (III) under item 1, characterized in that it contains protein impurities in amounts less than about 15 wt.%, preferably less than about 10 wt.%.

3. The method of receiving caseinsensitivity iron (III), comprising the following stages:
a) reaction of casein and succinic anhydride in water to obtain succinylamino casein,
b) reaction succinylamino casein with iron chloride (III) obtaining caseinsensitivity iron (III),
C) characterized in that the casein according to stage a) is a purified casein containing protein impurities less than 15 wt.% and/or inorganic impurities are less than about 1 wt.%, and/or having the ratio of phosphorus/nitrogen more than about 5%.

4. The method according to p. 3, wherein the content of said protein impurities is less than about 10 wt.% and/or the content of these inorganic impurities pillar is t less than about 0.4 wt.%.

5. The method according to p. 3, characterized in that the said protein impurities are proteins other than casein alpha-S1 casein alpha S2, beta casein, Kappa casein, casein pseudo-Kappa.

6. The method according to p. 3, characterized in that the purified casein is the ratio of phosphorus/nitrogen more than about 5 wt.%.

7. The method according to p. 3, characterized in that the purified casein is obtained from acid casein or rennet casein.

8. The method according to p. 3, comprising the additional step microfiltration of aqueous solution of caseinsensitivity iron (III).

9. Caseysalinger iron produced by the method according to any of paragraphs.3-8.

10. Pharmaceutical composition containing caseysalinger iron (III) PP.1-2 and/or 9 and at least one physiologically acceptable excipient.

11. The composition according to p. 10, characterized in that it is presented in liquid or solid form, preferably in liquid form.

12. The composition according to p. 11, characterized in that the liquid form is a solution or suspension, preferably an aqueous solution.

13. The composition according to p. 10, characterized in that the said composition is a solution containing about 8 wt.% caseinsensitivity iron (III)having a viscosity of less than about 400 MPa·s, preferably less than about 300 MPa·S.

1. Composition according to any one of paragraphs.10-13, where caseysalinger iron (III) is contained in a quantity ranging from 5 to 15 wt.%, preferably from 6 to 12 wt.%, more preferably from 7 to 9 wt.% in relation to the total weight of the composition.

15. Composition according to any one of paragraphs.10-13 for oral or parenteral administration, preferably for oral administration.

16. Composition according to any one of paragraphs.10-13 for use in the treatment of iron deficiency.

17. The composition according to p. 16 for use in the treatment of anemia, iron deficiency during pregnancy, malabsorption syndrome, iron deficiency in breast-feeding and iron deficiency in the growth process.



 

Same patents:

FIELD: medicine, pharmaceutics.

SUBSTANCE: presented group of inventions refers to biotechnology, and concerns a DLK1-Fc fused protein and using it for the metastases inhibition, a polynucleotide coding such a protein, an expression vector containing the polynucleotide, a host cell producing the above fused protein, a method for producing the fused protein by culturing the above host cell, a composition containing the above fused protein, and a method for the metastases inhibition. The characterised fused protein contains a DLK1 extracellular soluble domain consisting of the amino acid sequence SEQ ID NO:4 and Fc domain of a human antibody.

EFFECT: group of inventions can be used for preparing a therapeutic agent for reduction of cancer cell migration and the metastases inhibition.

11 cl, 36 dwg, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to biochemistry. Application of a fused protein to obtain a composition for the body weight reduction is described. The fused protein contains a domain of transduction, a signal of mitochondrial localisation and a domain of a mitochondrial factor of transcription A, binding polynucleotide (TFAM), containing a group with high mobility. Methods of treating obesity by means of the said protein are described.

EFFECT: invention extends an arsenal of means for treating obesity.

9 cl, 5 dwg, 2 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of production of casein calcium chloride of technical casein by precipitation, and can be used in microbiological studies for production of components of storing media of cultures of microorganisms, and also production of calcium co-precipitates for food industry.

EFFECT: improvement of the method.

2 cl, 1 tbl, 5 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology, namely to leukolectins, and can be used in medicine. What is prepared is the polypeptide leukolectin characterised by SEQ ID NO:1-8. The recombinant preparation is ensured by using a nucleic acid coding it and integrated into an expression vector which is used to transform a host cell. Testing absence-presence or determining an amount of the polypeptide leukolectin are ensured by using an antibody or an antigen-binding fragment of a variable region of the above antibody which is specifically bound to the polypeptide leukolectin. The polypeptide leukolectin or the nucleic acid coding it are used as ingredients of a pharmaceutical composition in therapy of pathological disorders of skin and mucous membranes.

EFFECT: invention enables treating or preventing autoimmune disorders of skin, inflammatory diseases of skin or mucous membrane, or injured skin in an animal effectively.

16 cl, 19 dwg, 3 tbl, 12 ex

Antibody to epha2 // 2525133

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of immunology, medicine and biotechnology. Claimed are versions of anti-EPHA2 antibodies. Claimed antibodies are bound with polypeptide, consisting of amino acids 426-534 in SEQ ID NO:8. Also described are hybridomes, which produce such antibodies, and pharmaceutical compositions and methods of application of said antibodies and compositions.

EFFECT: invention can be used in medicine.

74 cl, 14 dwg, 14 ex, 1 tbl

FIELD: chemistry.

SUBSTANCE: invention relates to field of biotechnology, in particular to immunogens based on antigenic tau-peptide, and can be used in medicine. Obtained is immunogen, which contains antigenic tau-peptide, consisting of amino acid sequence, selected from SEQ ID NO:6, 8-19, 21-26, 105 and 108-112, covalently bound with immunogenic carrier by means of linker, represented by formula (G)nC, where n equals 0, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10. Linker can be located either on C-terminal (peptide -(G)nC), or on N-terminal (C(G)n-peptide) of peptide. Obtained immunogens are used as base for creation of pharmaceutical compositions for treatment of tau-associated neurological disorders.

EFFECT: invention makes it possible to induce immune response against tau autoantigen in efficient way.

12 cl, 10 dwg, 5 tbl, 16 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel non-branched carbamate derivatives of some peptides Wnt-5a, in particular to N-butyloxycarbonyl derivative, their pharmaceutical compositions and their application for treatment of gastric melanoma and cancer.

EFFECT: obtaining novel non-branched carbamate derivatives of some peptides Wnt-5a.

7 cl, 9 dwg, 7 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of biotechnology, namely to muteins of human tear lipocalin, and can be used in medicine. Mutein of human tear lipocalin (hTLc) has identifiable affinity of binding with human receptor Met (c-Met) receptor tyrosine kinase, or its domain, or fragment of human c-Met. Mutein contains from 6 to 18 amino acid substitutions relative to amino acid sequence of mature lipocalin of human tear liquid (SWISSPROT DATABANK ENTRY P31025; SEQ ID NO:36), selected from group, consisting of Arg 26→Thr, Val, Pro, Ser, Gly; Glu 27→Gln, Gly, Val, Ser; Phe 28→Met, Asp; Pro 29→Leu, Ile, Ala, Trp; Glu 30→Leu, Gly, Arg, Phe; Met 31→Ser; Asn 32→Leu, Arg, Val, Gln; Leu 33→Tyr, Val, Ile, Thr, Phe; Glu 34→Val, Arg, Ala; Leu 56→Asn; Ile 57→Gln; Ser 58→Ile, Val; Asp 80→Tyr; Lys 83→Ala; Glu 104→Asp; Leu 105→Thr; His 106→Trp and Lys 108→Gly. Mutein can also additionally contain the following substitutions: Cys 61→Ser; Cys 101→Ser; Cys 153→Ser; Arg 111→Pro; Lys 114→Trp; Thr 37→Ser; Met 39→Ile, Leu; Asn 48→Ser; Lys 52→Thr, Met; Met 55→Leu; Lys 65→Arg, Leu; Ala 79→Leu, Ser; Ala 86→Thr; Ile 89→Ser, Gln, Thr, His; Thr 40→Cys; Glu 73→Cys; Arg 90→Cys; Asp 95→Cys; Lys 121→Cys; Asn 123→Cys and Glu 131→Cys.

EFFECT: invention makes it possible to efficiently treat pathological disorders, which involve pathway HGF/c-Met, as well as to perform identification of human c-Met in sample.

40 cl, 16 dwg, 9 tbl, 25 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to biotechnology. What is disclosed is a vaccine representing four RNAs coding a prostate-specific antigen (PSA), a prostate-specific membrane antigen (PSMA), a prostate stem cell antigen (PSCA) and a six-transmembrane epithelial antigen of the prostate (STEAP). The vaccine is applicable for treating prostate carcinoma, preferentially neo-adjuvant and/or hormone resistant prostate carcinoma, as well as related diseases or disorders. Using the vaccine and a kit are also disclosed. The invention can be used in medicine.

EFFECT: preparing the vaccine for treating prostate carcinoma.

16 cl, 23 dwg, 8 ex

FIELD: biotechnologies.

SUBSTANCE: physiologically active protein or polypeptide are fused with version of alpha-1-antitrypsin, which has at least one mutated aminoacid residue. Mutations are performed in the following positions: asparagine residue instead of proline residue in position 357; or asparagine residue instead of proline residue in position 357 and threonine residue instead of serine in position 359; or asparagine residue instead of proline residue in position 357 and serine residue instead of cysteine in position 232; or asparagine residue instead of proline residue in position 357, threonine residue instead of serine in position 359 and serine residue instead of cysteine in position 232.

EFFECT: invention allows increasing half lifetime of physiologically active protein or polypeptide in vivo by maintaining its stable circulation in blood.

7 cl, 13 dwg, 7 ex

FIELD: chemistry.

SUBSTANCE: invention represents a method of obtaining collagen from biological material, which includes milling of a raw material, liquid processing of the biological material with obtaining a collagen-containing substance, separated into a sediment and liquid fraction, characterised by the fact that as the biological material applied is a medusa, preferably Rhopilema, preferably its cupola, which is crushed preferably to 1-2 mm, and for obtaining the collagen-containing substance the material prepared in such a way is mixed with drinking water with a ratio by the raw material weight to water as 1:2 and extracted at a temperature preferably of 15-18°C for 6-12 hours with periodical mixing, after that, the obtained extract is separated into the liquid fraction and collagen-containing sediment, which is after that dehydrated to moisture weight in it not more than 10%, after which it is pre-packed and packed.

EFFECT: extension of an arsenal of methods for obtaining neutral collagen.

FIELD: chemistry.

SUBSTANCE: invention relates to a method for chemical conversion of a peptide chain into a peptide thioether. A -C(=X)-R1 group is incorporated into a thiol group of a cysteine residue and the obtained peptide then reacts in an organic solvent with a compound having a substituted group of formula: NH-C(=Y)NHR3, and a -NH-C(=Y)NHR3 group binds in an addition reaction with the carboxyl group of the peptide bond at the N-terminal side of the cysteine residue, through which the peptide bond is broken and the peptide moiety at the C-terminal end is cut off. When the obtained peptide chain, having a -NH-C(=Y)NHR3 group, reacts with thiol in a buffer solution, a thiol exchange reaction occurs, specifically the thiol group of the thiol compound binds with the carbon of the carbonyl to which the -NH-C(=Y)NHR3 was bound, thereby removing the -NH-C(=Y)NHR3 group.

EFFECT: achieving conversion to peptide thioether.

14 cl, 4 ex

FIELD: biotechnology.

SUBSTANCE: invention relates to a method of production of casein calcium chloride of technical casein by precipitation, and can be used in microbiological studies for production of components of storing media of cultures of microorganisms, and also production of calcium co-precipitates for food industry.

EFFECT: improvement of the method.

2 cl, 1 tbl, 5 ex

FIELD: chemistry.

SUBSTANCE: disclosed is a method of purifying a compound of formula 1 which includes the following steps: (1) adding a raw compound 1 to a macroporous adsorption resin, (2) washing the macroporous adsorption resin with an aqueous solution, an organic solvent or a mixed solution of an organic solvent and water, (3) elution using the aqueous solution, organic solvent or mixed solution of an organic solvent and water.

EFFECT: improved method.

9 cl, 7 dwg, 12 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of purifying daptomycin, which includes steps a) loading partially purified daptomycin into an anion-exchange chromatographic column and subsequent purification steps b) and c) in reversed-phase chromatographic columns, where the elution buffer at step a) is a monovalent salt solution and the elution buffer at step b) and c) is an aqueous alcohol.

EFFECT: improved method.

16 cl, 2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science and concerns a method for producing the purified antigen of Dirofilaria immitis. The presented method involves mechanical homogenisation, centrifugation of a homogenate, collection of a supernatant to be used as an antigen; the homogenisation involves a 2-cm head end of a mature female of Dirofilaria immitis placed in an aqueous solution of saccharose 0.25 M in a ratio of 1:3, frozen at a temperature of -18°C, that is followed by mechanical homogenisation and protein extraction in the aqueous solution of saccharose 0.25 M at 4°C for 12 hours, wherein 3 cycles of five 30-second ultrasonic homogenisations of the supernatant is performed at 70 kHz every 30 seconds at 0°C; the supernatant prepared after ultrasonic homogenisation is dissolved in cooled acetone at a temperature of 0°C in a ratio of 1:20 with exposition for 1 hour at a temperature of 4°C.

EFFECT: presented invention enables producing the high-sensitivity and specificity antigen and can be used in diagnosis of dirofilariasis in humans and animals.

2 tbl, 1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: present invention refers to biotechnology, more specifically to allergen modifications to reduce an allergenic capacity thereof, and may be used in medicine. A modified allergen is prepared by sequential modification of all primary amino groups or a portion thereof of lysine and arginine residues of an allergen molecule with using potassium cyanate and phenylglyoxal and used as an ingredient of a pharmaceutical composition for treating allergy.

EFFECT: invention provides preparing the modified allergen possessing the reduced allergenic capacity as compared to a respective native allergenic material and allergoids prepared by modification by either cyanate, or phenylglyoxal.

9 cl, 12 dwg, 4 ex

FIELD: chemistry.

SUBSTANCE: invention relates to biotechnology and represents a method of obtaining a preparation of an antibody or its antigen-binding site with reduced content of host cell proteins (HCP) from a sample mixture. The claimed invention can be used to obtain the antibody preparation. The method includes bringing pH to values 3-4 to form an initial separated sample. After that, pH of the initial separated sample is brought to values 6-8. The initial separated sample is brought in contact with resin for protein A-based affinity chromatography. The resin for affinity chromatography is washed out with a buffer solution. Sampling is carried out after affinity chromatography. The sample after affinity chromatography is brought in contact with an ion-exchange resin, after ion-exchange sampling is carried out. After the ion exchange sample is brought in contact with the resin for hydrophobic interaction chromatography (HIC) with sampling after HIC.

EFFECT: invention makes it possible to obtain the preparation of the antibody or its antigen-binding site with reduced HCP content.

25 cl, 12 dwg, 8 tbl, 4 ex

FIELD: chemistry.

SUBSTANCE: group of inventions relates to the field of biotechnology. Claimed is a method of purification of a factor, contributing to wound healing, which represents a hepatocyte growth factor (HGF). All stages of purification are carried out in the presence of antithrombin III (AT-III). In accordance with the claimed method carried out are: defrosting of the frozen HGF-containing source and removal of sediment from the defrosted source. After that, the obtained solution, which contains a supernatant and AT-III, is brought in contact with a carrier for affinity chromatography on an immobilised heparin. Then, the solution is separated from the carrier for affinity chromatography. The carrier is brought in contact with a desorption buffer with ionic strength sufficient for HGF desorption. The desorption buffer, containing HGF, AT-III and histidine-rich glycoprotein (HRGP) is collected. Also claimed are wound-healing compositions, which contain HGF, AT-III and/or HRGP, purified by the claimed method.

EFFECT: inventions make it possible to increase step-by-step output of the hepatocyte growth factor, with the hepatocyte growth factor being concentrated in eluate in the presence of AT-III.

26 cl, 2 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to a method of producing glycoprotein which is uniform in terms of functions derived from a sugar chain (e.g., blood half-life) and physiological activity units, i.e., a glycoprotein, having uniform amino acid sequence, sugar chain structure and higher-order structure, physiological activity, a method for screening for glycoprotein and a method of producing a mixture of glycoproteins.

EFFECT: high efficiency of the method.

6 cl, 37 dwg, 4 tbl, 6 ex

FIELD: chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to benzoimidazole derivatives of formula (I), as well as to their enantiomers, diastereoisomers, racemates and pharmaceutically acceptable salts, where n equals from 2 to 4, each of R1 substituents is independently selected from H, halogen, -C1-4alkyl, -C1-4pergaloalkyl, trifluoro-C1-4alkoxy, -NO2, -CN, CO2H, -OC1-4alkyl, -SC1-4alkyl, -S(C1-4alkyl)-Rc, -S(O)2(C1-4alkyl)-Rc, -S(O)-C1-4alkyl, -SO2-C1-4alkyl, -S-Rc, -S(O)-Rc, -SO2-Rc, -SO2-NH-Rc, -O-Rc, -CH2-O-Rc, -C(O)NH-Rc, -NRaRb, benzyloxy, phenyl, optionally substituted with one-two Rd, cyanobiphenyl-4-ylmethylsulpfanyl, cyanobiphenyl-4-ylmethanesulphonyl, or -S-(CH2)2-morpholine and two adjacent groups R1 can bind with formation of an aromatic 5-6-membered ring, optionally substituted with one methyl group or two atoms of halogen, optionally containing one or two S or N; Ra and Rb each independently represents C1-4alkyl, -C(O)C1-4alkyl, -C(O)-Rc, -C(O)CH2-Re, C1-4alkyl-Re, -SO2-Rc, -SO2-C1-4alkyl, phenyl, benzyl; or Ra and Rb together with a nitrogen atom, which they are bound with, form a monocyclic 5-6- membered heterocycloalkyl ring, optionally containing one heteroatom, selected from O; Rc represents -C3-8cycloalkyl, phenyl, optionally substituted with one-two Rd, benzyl, optionally substituted with one-three Rd; morpholine; Rd independently represents halogen, -OH, -C1-4alkyl or -C1-4perhalogenalkyl, trifluorine C1-4alcoxy, -OC1-4alkyl, or -O-benzyl optionally substituted with halogen, Re represents -C6heterocycloalkyl, optionally containing one or two of O or N atoms, optionally substituted with a methyl group; R2 and R3 both represent H, -CF3 or C1-3alkyl; each of Z represents a C or N atom, on condition that simultaneously not more than two Z represent N. The invention also relates to particular compounds, a pharmaceutical composition, based on formula (I) compound or a particular said compound, a method of treating diseases, mediated by propyl hydroxylase activity.

EFFECT: novel derivatives of benzimidazole, possessing an inhibiting activity with respect to PHD are obtained.

11 cl, 1 tbl, 186 ex

Up!