Present invention provides a process for preparation of iron sucrose complex.

Particularly the invention provides iron sucrose complex as an injectible compound.

Iron carbohydrate complexes, which have favorable properties for therapeutic use, are of great interest. Iron complexes with Dextran, Dextrose, maltose, Sucrose, Fructose are some of the important complexes Iron Sucrose, which belongs to the therapeutic class of haemitinic, is a complex of polynuclear iron (III) hydroxide in sucrose having molecular weight approximately between 34,000 and 60,000 Daltons, and structural formula [Na2Fe508(OH) -3(H20)]n TYHC^HteOn) where: n is the degree of polymerization and m is the number of sucrose molecules associated with the iron(lll) hydroxide. Iron sucrose containing 30% sucrose (w/v) is an injectable, which is administered intravenously for replenishing body iron stores in patients with iron deficiency on chronic hemodialysis and receiving erythropoietin especially in pregnant women. The injection iron sucrose acts like this in the patient .Iron sucrose is dissociated into iron and sucrose by the reticuloendothelial system and iron is transferred from the blood to a bone marrow. The Ferritin, the iron storage protein binds and sequesters iron into a nontoxic iron that is easily available. The iron binds to plasma transferring iron to extracellular fluid to supply to tissues. The transferrin receptors presented in membrane binds transferrin iron complex which is then internalized in vesicles. Iron is released within the cell and transferrin-receptor complex is returned to the cell membrane. Transferrin without iron is then released to the plasma. The intracellular iron becomes hemoglobin on circulating red blood cells. Transferrin synthesis increased ferritin production reduced in iron deficiency.

Reference is made to a US patent 3821192. This discloses a process for preparing a stable, soluble complex of iron with glucose, maltose or dextrin. This, however, does not include sucrose. The disclosed invention used ferric hydroxide derived from ferric chloride and polymerized in presence of carbohydrate at pH 11.0-14.0 .This is followed by concentration with organic solvent, filtration.

U.S. Pat. No. 4,746,730 (assigned to Medinianum Farmaceutci) discloses a method for preparation of complexes of iron and various carbohydrates like fructose and saccharose. The method comprises addition of an aqueous solution of ferric chloride to a fructose solution followed by addition of aqueous potassium hydroxide solution to get pH between 7.8 and 8.5. The complex is obtained by a process of lyophilisation which is very expensive on an industrial scale.

U.S. Pat. No. 4,994,283 (assigned to Procter and Gamble) discloses a method for preparing iron-sugar carbohydrate complex, which comprises initial preparation of a complex of calcium and a sugar followed by preparation of the iron-sugar complex by reaction with an iron source such as ferrous ammonium sulfate and treating the resultant iron-sugar complex with malic acid to give the desired iron-sugar complex. The method is quite lengthy as it does not involve the direct preparation of the iron-sugar complex and rather involves the intermediary of a calcium-sugar complex from which the product is obtained. Further, there is no surety that the iron-sugar complex obtained will be free from the calcium-sugar complex.

CA 1 253 821 discloses a method for preparation of water soluble iron dextran com lex involving formation of dextran utilizing enzyme and bacteria and subsequently reacting the same with freshly prepared iron (III) hydroxide. The preparation of iron dextran utilizing enzyme and bacteria is very selective and not convenient for industrial production

U.S.Pat. No. 7674780B2 discloses a method for preparation of iron and carbohydrate complexes. The method involves reacting ferric hydroxide and sucrose in aqueous reaction mixture comprising sodium ions at selected molar ratio of sucrose to ferric hydroxide, for selected time interval at pH 6.5 to 13. Isolating iron sucrose complex from aqueous reaction mixture and precipitating the mixture by freeze drying resulting in 60,000 Daltons molecular weight. The method disclosed in this patent application utilizes freeze drying for isolating iron sucrose, which is however not suitable for industrial purpose, since the isolation method is very expensive.

U.S. Pat. No.8030480B2 (assigned to Emcure pharmaceuticals Ltd, Pune) discloses a method of cost effective preparation of iron and sucrose complex. The process involves preparation of ferric hydroxide between pH 3.5 to 7 by addition of inorganic base sodium carbonate to get ferric oxyhydroxide. This is added to sucrose to obtain iron sucrose complex. Organic solvent is added to precipitation and concentration followed by filtration and dried. The obtained molecular weight is 34,000 to 60,000 Daltons.
US 7964568B2 assigned to Chromaceutical Advanced Technologies discloses a general method for the preparation of iron saccharidic complexes, including iron sucrose. The preparation of iron sucrose disclosed involves mixing of the aqueous solution of the ferric salt and sucrose followed by addition of sodium hydroxide solution to give ferric hydroxide sucrose complex. This method has the disadvantage of the inability to monitor whether the ferric salt initially added has been completely converted to ferric hydroxide for further reaction with sucrose solution. There are chances of iron sucrose complex being contaminated by residues of ferric hydroxide which may result in high molecular weight of the resultant product.

WO 2005/094202 A2 (assigned to Navinta LLC) discloses another method for the preparation of Iron sucrose comprising of addition of an inorganic base in a phased manner to an aqueous solution of ferric salts to obtain ferric hydroxide followed by addition to an aqueous solution of sucrose and heating at a temperature of 100-105.degree. C, followed by freeze drying of the resulting product. The iron sucrose thus obtained has to be purified to obtain the product conforming to desired specifications. The method disclosed in this patent application utilizes freeze drying for isolating iron sucrose, which is however not suitable for industrial purpose, since the isolation method is very expensive.

WO 2005/000210 A2 discloses a general method for the preparation of iron-saccharidic complexes, including iron sucrose. The preparation of iron sucrose disclosed in this patent application involves mixing of the aqueous solution of the ferric salt and sucrose followed by addition of sodium hydroxide solution to give ferric hydroxide sucrose complex. This method has the disadvantage of the inability to monitor whether the ferric salt initially added has been completely converted to ferric hydroxide for further reaction with sucrose solution. Therefore, there is every possibility of the iron sucrose thus formed being contaminated with the ferric salts employed initially and secondly, due to the possible incomplete formation of ferric hydroxide, the yield of iron sucrose will be lower, rendering the process unsuitable for industrial applications. Further, iron sucrose thus obtained has a molecular weight around 1,570,000
Daltons as described in Example 2 of the published application This high value of the molecular weight does not conform to the specification for molecular weight desired by the regulatory authorities (34,000 to 60,000 Daltons) for Iron sucrose.

Thus the prior art methods have several shortcomings such as i) utilization of freeze drying, which is expensive on industrial scale, ii) preparing iron sugar complex through the intermediary of the calcium complex of the carbohydrate, which is lengthy and more expensive, iii) glucose is required in conjunction with sucrose for preparation of the iron sucrose complex. Utilization of another carbohydrate like glucose makes the process more costly, iv) selection of an inorganic base for preparation of ferric oxyhydroxide is critical as inorganic bases like ammonium hydroxide or sodium hydroxide utilized for preparation of ferric oxyhydroxide fails to give the sodium ferric gluconate complex with sodium gluconate.

According to main object of the present invention there is provided a process for preparation of iron sucrose complex which obviates the drawbacks of the prior art.

According ti another object of the invention it is to provide injectable iron sucrose complex complying with the USP Injection norms and bacterial endotoxin limits not more than 50 CFU/mg.

The results substantiating the claim of effect of freeze drying are shown in the drawing accompanying the specifications.

Fig 1 shows the complete process in the making of iron sucrose complex.

Fig 2 Shows the variation of molecular weight with time of iron oxyhydroxide without the freeze storing.

Fig 3 shows the effect of storing in a freezer of variation of molecular weight with time of iron oxyhydroxide

Accordingly the present invention provides as the main embodiment, a process of preparing Iron Sucrose complex comprising the steps of:

a. reacting a Ferric Salt as the base in water at pH between 1.8 to 2.0 and maintaining for one hour, increasing the pH to between 4 and 5 maintaining the pH for one hour followed by increase in pH between pH 6 - 7 and maintaining for one hour resulting in ferric oxy hydroxide,

b. washing ferric oxy hydroxide using pharmaceutical grade water to remove excess chloride,

c. storing of purified Ferric Oxy hydroxide complex in deep freezer in a temperature range of -5 to 0° C up to 24 Hours

d. palletizing deep frozen Ferric Oxy hydroxide complex for effective reaction with sugar,

e. reaction of Ferric Oxy hydroxide from step (d) with pharmaceutical grade sugar between pH 10-
11.0 using base solution at a temperature in a range of 100 - 105° C for a time in a range of 50 minutes to 120 minutes,

f. dilution of Iron Sucrose complex obtained in step (e), using pharmaceutical grade water, passing the solution through micron filters,

g. concentrating the filtrate in step (g) under vacuum not less than 500 Hg/mm and at a temperature in a range of 60 - 80 ° C, or by reverse osmosis technique,

h. isolation of concentrated mass using organic solvents , drying, thereby yielding iron sucrose complex.

In another embodiment of the present invention wherein sterile micron filter may be selected from a group of 20, 10, 0.5 0.2 and 0.1 microns.

In a further embodiment of the present invention wherein, organic solvent may be selected from a group of alcohol and ketone groups.

In another embodiment of the invention, wherein addition of concentrated filtrate to organic solvents may be at room temperature, between 40 - 75° C by means of high speed stirring.

In still another embodiment of the invention, wherein stirring time is in a range of 15 -30 min.

In still another embodiment of the invention wherein drying iron sucrose complex may be obtained by vacuum tray drier, fluid bed drier, rotary cone vacuum drier at a temperature between 80-100 °C at a vacuum of NLT 500 Hg/mm. In a further embodiment of the present invention , wherein the weight average molecular weight of iron sucrose complex may be in a range of 40,000 to 55, 000 Daltons, In a further embodiment of the present invention, wherein Bacterial Endo Toxins limit may be less than 50 CFU/mg.

The invention is now described as hereunder. As shown in Figure 1 the whole process of preparation of iron sucrose complex comprises the steps of : preparation of iron hydroxide from an iron salt, preferably ferric chloride but without any limitation of use of any other iron salt. This strep essentially is a multi step process of reacting a Ferric Salt, preferably anhydrous ferric chloride as the base particularly in pharmaceutical grade water like that of USP grade water at pH between 1.8 to 2.0 and maintaining for one hour, increasing the pH to between 4 and 5 maintaining the pH for one hour followed by increase in pH between pH 6 - 7 and maintaining for one hour. This is followed by thorough washing in pharma grade water to remove residual chloride thereby yielding pure iron hydroxide. The chloride free iron hydroxide is next stored in freezer with temperature maintained in a preferred range of -5 to 0° C. The storing may be upto about 24 hours, but more preferably for a time interval in a range of 8 to 10hrs. The effect of storing iron hydroxide in a deep freezer is clearly seen in Figs 1 and 2. Fig 1 shows the variation with time of molecular weight for iron hydroxide not stored in a freezer. Its very clear that the molecular weight shows a sharp decline with time , whereas the change in molecular weight is almost minimal for a deep frozen iron hydroxide. The stored iron hydroxide becomes rock hard and needs to be broken into pieces to facilitate reaction with pharmaceutical grade sugar which is prepared in pure water, preferably a pharmaceutical grade water. The step involves reacting iron hydroxide palletized from deep frozen compound with pharmaceutical grade sugar between pH 10 - 11.0 using base solution at a temperature particularly in a range of 100 - 105° C for a time in a range of 50 minutes to 120 minutes, more particularly upto about 60minutes. This step yields iron sucrose complex in a bit impure form and needs further purification. This complex is next diluted in pharmaceutical grade water, and is filtered by passing the solution through micron filters, The filters are micron grade filters preferred of 20, 10, 0.5 0.2 and 0.1 microns. After this the sucrose complex filtrate is concentrated under vacuum not less than 500 Hg/mm and at a temperature in a preferred range of 60 -80 ° C, or by reverse osmosis technique. In the next process step the isolation of concentrated mass using organic solvents is effected , wherein the organic solvents are preferably selected from a group of alcohol and ketone groups. The organic solvents are added to the concentrated filtrate preferably at room temperature., or Concentrated mass is added to organic solvents at a temperature between 40 - 75° C by means of high speed stirring. The stirring may be preferably carried out for a period in a range of 15-30miniutes. This purified iron sucrose complex is then dried particularly by any of the means like vacuum tray drier, fluid bed drier, rotary cone vacuum drier, thereby yielding iron sucrose complex with a molecular weight in a range of 40,000 to 55, 000 Daltons.

The novelty of the invention lies in obtaining injectable iron sucrose complex complying with the USP Injection norms and bacterial endotoxin limits not more than 50 CFU/mg. The said novelties have been realized by the inventive steps of deep freeze storing of ferric hydroxide and reaction with pharmaceutical grade sugar in a time frame of less than 2 hours.

Following examples are given only to illustrate the invention and should not be construed to limit the scope of the invention

EXAMPLE 1

1. Preparation of Iron Sucrose from Ferric Chloride (Anhydrous)
Preparation of Ferric Hydroxide:

100 gm of Anhydrous FeCb (0.6172 moles) added into a beaker of 2 L volume, and 0.5 L of distilled water was added thereto, then the mixture was agitated to dissolve the solid at the temperature of 5°C. 30 % solution of sodium carbonate was added drop-wise with stirring till the pH adjusted to 1.8 - 2.0. After adjusting pH, the reaction mass is maintained for 1 hour. During the alkali addition, carbon dioxide was generated, and the color of the solution changed from light brown to dark brown. After 1 Hour maintenance the drop-wise addition was continued further to adjust pH to 4.0 to 5.0. Then again maintained for 1 more hour at this pH and temperature. After the 1 hour of maintenance, the reaction mass is adjusted to pH 6.5 - 7.0. Throughout the reaction temperature was controlled between 5. to 10 °C
Purification of Ferric Hydroxide:

The suspension obtained in the previous step was centrifuged and about 2 L of distilled water is used for washing to remove the chlorides. The washing procedure was repeated for 2 more times. Ferric hydroxide thus obtained has moisture content between 50 - 60 % and is stored in deep freezer between -5 to 0 °C for 8 -10 hours.

Preparation of Polynuclear Ferric Hydroxide-sucrose Complex:
The above-prepared deep freeze Ferric Hydroxide precipitate was made in to small pellets and put into a beaker. 1000g (2.91 moles) of sucrose was added, and 100 ml distilled water is added to the flask. This reaction mixture was kept under stirring for 30 min. Then using 30 ml_ of 20% (w/v) aqueous sodium hydroxide solution the pH of reaction mass was adjusted to 10 - H.O.This pH adjusted mass was transferred in to a 3 L RBF and was heated to 100 °C under stirring The heating is continued for 2 hours. After this heating was stopped and sample is taken for GPC analysis and allow the reaction mass to attain room temperature. A crude product of iron sucrose complex was obtained.

Purification of Iron Sucrose:
To the crude Iron sucrose complex 1500 ml of pharmaceutical grade water was added. This diluted complex was filtered through sparkler and then micron filter set up. Preferably 20 micron is used in sparkler filtration setup followed by 10, 0.5, 0,2 and 0.1 micron cartridge set up. This filtrate was concentrated under vacuum not less than 500 Hg/mm and at a temperature in a range of 60 -80 ° C up to thick mass, To this thick mass ethanol (5000 ml) was added and stirred for 30 min and allowed to settle the mass for 30 min. Superannuated ethanol solution was decanted . Ethanol treatment substantially removes the charring and excess of sugar in the reaction. Methanol (4000 ml) was added to this Iron sucrose complex at 30 ° C and stirred for 15 - 30 min using high RPM stirrers. The mixture was allowed to settle for 30 min. and iltered under nitrogen atmosphere. The product was washed with 200 ml of acetone. Finally it was dried using vacuum tray drier at 60°C for 3 hours to obtain 480 - 500 g of pure iron sucrose. The weight average molecular weight of the final product was 40,000 Daltons to 55,000 Daltons and Polydispersity index less than 1.7 as measured by gel chromatography GPC. Thus obtained iron sucrose complex meets specifications of Iron sucrose USP Injection norms along with Bacterial Endo Toxin limit less than 50 CFU/mg. The weight ratio of iron to sucrose was 1: 1 with the iron content being between 4.8 to 6.4% and the water content being less than 2%.

EXAMPLE 2
The procedure of example 1 was followed except that purification of Iron sucrose complex is as described below.

Purification of Iron Sucrose:
To the crude Iron sucrose complex 1500 ml of pharmaceutical grade water was added. This diluted complex was filtered through sparkler and then micron filter set up. Preferably 20 micron is used in sparkler filtration setup followed by 10, 0.5, 0.2 and 0.1 micron cartridge set up. This filtrate was concentrated using reverse osmosis technique to concentrate the diluted product. After the concentration Iron sucrose was isolated using ethanol and methanol as described in the example 1.

Table 1 : Molecular weight Trend against time (Without Deep Freezing the Isolated Iron Oxy Hydroxide)

Table 2 : Molecular weight Trend against time (Deep Freezing the Isolated Iron Oxy Hydroxide)

The comparison of change in molecular trends with and without deep freeze storing of iron hydroxide clearly shows the efficacy of the freeze drying step in the process of present invention

We claim

1. A process of preparing Iron Sucrose complex comprising the steps of:

a. reacting a Ferric Salt as the base in water at pH between 1.8 to 2.0 and maintaining for one hour, increasing the pH to between 4 and 5 maintaining the pH for one hour followed by increase in pH between pH 6 - 7 and maintaining for one hour resulting in ferric oxy hydroxide,

b. washing ferric oxy hydroxide using pharmaceutical grade water to free of chloride,

c. storing of purified Ferric Oxy hydroxide complex in deep freezer in a temperature range of -5 to 0° C up to 24 Hours

d. palletizing deep frozen Ferric Oxy hydroxide complex for effective reaction with sugar,

e. reaction of Ferric Oxy hydroxide from step (d) with pharmaceutical grade sugar between pH 10 - 11.0 using base solution at a temperature in a range of 100 - 105° C for a time in a range of 50
minutes to 120 minutes,

f. dilution of Iron Sucrose complex obtained in step (e), using pharmaceutical grade water, passing the solution through micron filters,

g. concentrating the filtrate instep (f) under vacuum not less than 500 Hg/mm and at a temperature in a range of 60 -80 ° C, or by reverse osmosis technique,

h. isolation of concentrated mass using organic solvents , drying, thereby yielding iron sucrose complex.

2. A process according to claim 1, wherein sterile micron filter is selected from a group of 20,10, 0.5 0.2 and 0.1 microns.

3. A process according to claim 1, wherein, organic solvent is selected from a group of alcohol and ketone groups.

4. A process as claimed in claims 1 and 3 wherein addition of concentrated filtrate to organic solvents is at room temperature, between 40 - 75° C by means of high speed stirring.

5. A process as claimed in claims 1, 3 and 4, wherein stirring time is in a range of 15-30min.

6. A process according to claim 1, wherein drying iron sucrose complex is obtained by vacuum tray drier, fluid bed drier, rotary cone vacuum drier at a temperature between 80-100 °C at a vacuum of NLT 500 Hg/mm.

7. A process as claimed in claims 1 to 6, wherein the weight average molecular weight of iron sucrose complex is in a range of 40,000 to 55, 000 Daltons,

8. A process as claimed in claims 1 to 7, wherein Bacterial Endo Toxins limit is less than 50 CFU/mg.