STABLE IRON OLIGOSACCHARIDE COMPOUND

Abstract

The invention relates to n iron oligosaccharide compound with improved stability comprising a hydrogenated oligosacharride in stable association with ferric oxyhydroxide, the content of dimer saccharide in said hydrogenated oligosaccharide being 2.9% by weight or less, based on the total weight of the hydrogenated oligosaccharide. In further aspects is provided a process for preparing said compound as well as the use of said compound for preparation of a composition for treatment of iron deficiency anaemia.

Claims

1. An iron oligosaccharide compound comprising a hydrogenated oligosaccharide in stable association with ferric oxyhydroxide, the hydrogenated oligosaccharide having a weight average molecular weight (Mw) of less than 3,000 Daltons, characterized in that the content of dimer saccharide in said hydrogenated oligosaccharide is 2.9% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

2. A compound according to claim 1, characterized in that the content of monomer saccharide in said hydrogenated oligosaccharide is 0.5% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

3. A compound according to claim 1, wherein said hydrogenated oligosaccharide is hydrogenated dextran having a weight average molecular weight (Mw) between 500 and 3,000 Daltons, a number average molecular weight (Mn) above 500 Daltons, wherein 90% by weight of said dextran has molecular weights less than 3,500 Daltons, and the Mw of the 10% by weight fraction of the dextran having the highest molecular weights is below 4,500 Daltons.

4. A compound according to claim 1, wherein said hydrogenated oligosaccharide is hydrogenated dextrin having a number average molecular weight (Mn) higher than or equal to 500 Daltons, wherein the 10% fraction of said hydrogenated dextrin having the highest molecular weight has a weight average molecular weight (Mw) of less than 4,500 Daltons, and 90% of the dextrins have a molecular weight of less than 3,500 Daltons.

5. A compound according to claim 1, wherein (i) the weight average molecular weight (Mw) is 1,600 Daltons or less and the number average molecular weight (Mn) is 1,600 Daltons or less and/or (ii) the apparent molecular weight (M.sub.P) of said compound is 120.000 to 180.000 Daltons, measured on an autoclaved aqueous solution prepared by dissolving in 1,000 ml water 400 g powder of hydrogenated oligosaccharide in stable solution with ferric oxyhydroxide, the amount of iron (Fe) of the powder being 25% by weight.

6. (canceled)

7. A compound according to claim 1, wherein the amount of iron (Fe) in the iron oligosaccharide compound is 50% by weight or less.

8. A compound according to claim 1, wherein the content of dimer saccharide in the hydrogenated oligosaccharide is 2.5% by weight or less or is 2.3% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

9. (canceled)

10. Composition comprising a pharmacologically effective amount of a compound according to claim 1, and at least one pharmaceutically acceptable carrier.

11. A process for preparing an iron oligosaccharide compound, comprising the steps of: (a) hydrolysing a polysaccharide so as to reduce its molecular weight, (b) hydrogenating the resulting oligosaccharide to convert functional aldehyde groups into alcohol groups, (c) fractioning the hydrogenated oligosaccharide according to molecular weight, so that the purified fraction has a weight average molecular weight equal to or less than 3,000 Daltons, (d) combining the resultant fractionated hydrogenated oligosaccharide as an aqueous solution with at least one water-soluble ferric salt, (e) adding base to the resulting aqueous solution to form ferric hydroxide, and (f) heating the resultant basic solution to transform the ferric hydroxide into ferric oxyhydroxide in association with said oligosaccharide; characterized in that step (c) comprises a procedure of purification by one or more membrane processes having a cut-off value between 340 and 800 Daltons, which procedure is continued until the content of dimer saccharide in the purified fraction of oligosaccharide has been reduced to 2.9% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

12. (canceled)

13. The process according to claim 11, wherein said polysaccharide is dextran, starch or dextrin.

14. The process according to claim 13, wherein said polysaccharide is dextran and (i) said dextran in step (c) is purified by one or more membrane processes having a cut-off value suitable for holding back dextran of molecular weight above 2,700 Daltons and/or (ii) in step (e) the resulting solution is adjusted to a pH above 10 by addition of said base and/or (iii) in step (f) said heating is carried out at a temperature above 100° C. until the solution turns into a black or dark brown colloidal solution, which after neutralisation is filtered through a filter, whereupon one or more stabilizers are added.

15. (canceled)

16. (canceled)

17. A process according to claim 13, wherein the resulting solution is dried to obtain the resulting iron dextran compound or the resulting iron dextrin compound as a stable powder.

18. A process according to claim 17, wherein the amount of iron (Fe) in the iron dextran compound or iron dextrin compound is 50% by weight or less.

19. (canceled)

20. A process according to claim 13, wherein aid polysaccharide is dextrin, and (i) in step (c) the hydrogenated dextrin is purified to obtain a number average molecular weight (Mn) higher than or equal to 500 Daltons, wherein the 10% fraction of said hydrogenated dextrin having the highest molecular weight has a weight average molecular weight (Mw) of less than 4,500 Daltons, and 90% of the dextrins have a molecular weight of less than 3,500 Daltons, and/or (ii) in step (e) the resulting solution is adjusted to a pH above 8.5 by addition of said base and/or (iii) in step (f) said heating is carried out at a temperature above 85° C. until the solution turns into a black or dark brown colloidal solution, which after neutralisation is filtered through a filter, whereupon one or more stabilizers are added.

21. (canceled)

22. (canceled)

23. (canceled)

24. (canceled)

25. A process according to claim 11, wherein the hydrogenation in step (b) is performed using sodium borohydride in aqueous solution.

26. A process according to claim 11, wherein stabilization following step (f) is effected by addition of at least one salt of an organic hydroxy acid, wherein optionally said at least one salt of an organic hydroxy acid is selected from citrates.

27. (canceled)

28. A method for prophylaxis or treatment of iron-deficiency anaemia in an animal or human subjects, comprising parenteral or oral administration of a compound according to claim 1 to a said animal or human subject.

29. A hydrogenated oligosaccharide having a weight average molecular weight (Mw) of less than 3,000 Daltons, characterized in that said hydrogenated oligosaccharide has a dimer saccharide content of 2.9% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

30. (canceled)

31. A process for preparing a hydrogenated oligosaccharide according to claim 29, which process comprises the steps of: (a) hydrolysing a polysaccharide so as to reduce its molecular weight, (b) hydrogenating the resulting oligosaccharide to convert functional aldehyde groups into alcohol groups, and (c) fractioning the hydrogenated oligosaccharide according to molecular weight, so that the purified fraction has a weight average molecular weight equal to or less than 3,000 Daltons, characterized in that step (c) comprises a procedure of purification by one or more membrane processes having a cut-off value between 340 and 800 Daltons, which procedure is continued until the content of dimer saccharide in the purified fraction of oligosaccharide has been reduced to 2.9% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

32. (canceled)

33. A process for producing an injection liquid containing a compound according to claim 1, wherein (i) the compound as a dry powder is dissolved in an aqueous medium; pH is adjusted, if necessary; optionally, stabilizer is added; and the liquid is sterilized by filtration, before it is filled into ampoules or vials, or by autoclave treatment after filling into such ampoules or vials; or (ii) a liquid containing said compound is purified, adjusted as to iron content and pH, stabilized and sterilized by filtration before being filled into ampoules or vials or by autoclave treatment after being filled into said ampoules or vials.

34. (canceled)

Description

EXAMPLES

Hydrolysis and Hydrogenation of Dextran

[0080] An amount of pre-hydrolysed dextran collected as permeate from a membrane having a cut-off value <5,000 Daltons is further hydrolysed at pH 1.5 at a temperature of 95° C.

[0081] The hydrolysis is monitored using gel permeation chromatography (GPC) and is terminated by cooling, when the molecular weight of the material being hydrolysed is estimated to have achieved the desired value, i.e. a weight average molecular weight of 700-1,400 Daltons.

[0082] In the hydrolysis, low molecular weight dextran is produced but also glucose and iso-maltose are formed. Upon hydrolysis, the disaccharide content is at 7-8% by weight.

[0083] After cooling and neutralization, the amount of monomer and dimer is reduced by membrane processes having a cut-off value of 340-800 Daltons. The concentration of monosaccharide and disaccharide in the solution is monitored by gel permeation chromatography, and fractionation goes on until a dimer concentration of 2.9% by weight or less and a monomer concentration of 0.5% by weight or less is achieved.

[0084] Thereafter, the content of dextran is determined by optical rotation and the amount of reducing sugar is determined by use of Somogyi's reagent. To bring down the reducing capability below 3%, sodium borohydride is added to the solution at basic pH. The applied ratio of sodium borohydride:fractionated dextran is 1:34.7.

[0085] The solution is neutralized to pH<7.0 and subsequently de-ionized. The average molecular weights and the molecular weight distribution are determined chromatographically. Chromatography reveals that the desired conditions, viz. that 90% by weight of the dextran has a molecular weight less than 2,700 Daltons and that the weight average molecular weight (Mw) of the 10% by weight fraction of the dextran having the highest molecular weight is below 3,200 Daltons, are fulfilled. The final yield of dextran after de-ionization is approximately 50% relative to the initial amount of pre-hydrolysed dextran.

Synthesis of Iron-Dextran

[0086] 120 kg dextran as produced above is mixed as an 18% solution with 150 kg FeCl.sub.3, 6H.sub.2O. To the agitated mixture, 93 kg Na.sub.2CO.sub.3 as a saturated aqueous solution is added, whereupon the pH is raised to 10.5 using 24 litres of concentrated aqueous NaOH (27% (w/v)).

[0087] The mixture thus obtained is heated above 100° C. until it turns into a black or dark brown colloidal solution. After cooling, the solution is neutralized using 12 litres concentrated hydrochloric acid to obtain a pH of 5.8. After filtration the solution is purified using membrane processes until the chloride content in the solution is less than 0.55% calculated on basis of a solution containing 5% (w/v) Iron.

[0088] If the chloride content of the solution is less than desired to obtain an isotonic solution, sodium chloride is added and pH is finally adjusted to 5.6, and the solution is filtered through a 0.45 μm (or alternatively a 0.2 μm) membrane filter.

[0089] The solution is spray dried and the iron-dextran powder is ready for marketing or for further processing.

[0090] As an alternative to spray drying, the solution may be used for direct production of injection liquids having an iron content of e.g. 10% (w/v), as described above.

[0091] When using the iron-dextran powder for producing injection or infusion liquids, the powder is re-dissolved in an aqueous medium, the pH is checked and, if necessary, adjusted, and the solution is filled into ampoules or vials after being sterilized by filtration. Alternatively, the sterilization can take place by autoclaving after filling into ampoules or vials.

Analysis of the Stability of Iron Oligosaccharide Compounds Relative to their Content of Dimer

[0092] A range of iron oligosaccharide compounds with differing contents of disaccharide were analysed with respect to their rate of hydrolysis and apparent molecular weight, both of which are indicative of the quality of the respective compounds.

[0093] The rate of hydrolysis of Fe.sup.3+ from the compound of FeOOH and oligosaccharide in acidic solution (0.24 M HCl; 0.9% NaCl) is assumed to be correlated to the rate of physiological release of iron. Therefore, the rate of hydrolysis as expressed by the half-life (t.sub.1/2) is an important parameter of the investigated compounds.

[0094] The hydrolysis of Fe.sup.3+ was measured by optical absorbance at 287.3 nm.

[0095] Besides, it was found that the thermostability of iron oligosaccharides is a function of their apparent molecular weight (M.sub.p). Thus, such compounds are unstable to an unsatisfactory degree, if the apparent molecular weight markedly exceeds a value of 160,000 Daltons upon storage at an elevated temperature for three months as test solutions.

[0096] The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Assessment after Iron Amount of keeping at elevated oligosaccharide dimer in t.sub.1/2/ M.sub.P/ temperature compound no. oligosaccharide hours Daltons for 3 months 1 0.5% 25.5 133,490 stable 2 0.75% 21.7 149,532 stable 3 0.75% 22.5 146,353 stable 4 1.6% 21.6 138,053 stable 6 1.6% 18.3 145,250 stable 7 2.0% 20.9 150,983 stable 8 2.0% 20.3 142,664 stable 9 3.0% 12.1 152,516 unstable 10 5.0% 12.2 185,433 unstable 11 8.0% 9.7 215,143 unstable

[0097] As appears from Table 1, the apparent molecular weight is in a desirable range, viz. about 130 kD to about 160 kD, when the amount of dimer in the oligosaccharide is less than 3%. The same holds true for the rate of hydrolysis as expressed by the half-life. The Half-life (t.sub.1/2) is defined as the time at which the absorbance is the half of the value compared the absorbance at t=0.

[0098] The results shown in Table 1 above may be arranged into two series. When the results are plotted into a diagram of t.sub.1/2 vs. the dimer content FIG. 1 appears. The two straight lines fitted into the two series show an interception at a dimer content of 3.5% by weight and t.sub.1/2 of 12.6 hours.