STABLE IRON OLIGOSACCHARIDE COMPOUND

Abstract

The invention relates to an iron oligosaccharide compound with improved stability comprising a hydrogenated oligosaccharide in stable association with ferric oxyhydroxide, the content of dimes 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 CD 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

1-34. (canceled)

35. A hydrogenated oligosaccharide, which hydrogenated oligosaccharide when combined with ferric oxyhydroxide is capable of forming a stable iron oligosaccharide compound, wherein said hydrogenated oligosaccharide comprises the following: (i) a weight average molecular weight (Mw) between 500 and 3,000 Daltons, (ii) a dimer saccharide content of 2.9% by weight or less, based on the total weight of the hydrogenated oligosaccharide, (iii) it is a hydrogenated dextran oligosaccharide having a number average molecular weight (Mn) above 500 Daltons; (iv) 90% by weight of said dextrans have a Mw less than 3,500 Daltons, and (v) the Mw of the 10% by weight fraction containing the dextrans having the highest molecular weights is below 4,500 Daltons.

36. The hydrogenated oligosaccharide of claim 35, wherein the reducing capability of the hydrogenated oligosaccharide is less than 3.0%.

37. The hydrogenated oligosaccharide of claim 36, wherein said reducing capacity is determined by the cupric oxidation method.

38. The hydrogenated oligosaccharide of claim 35, wherein 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.

39. The hydrogenated oligosaccharide of claim 35, wherein the Mw of said hydrogenated oligosaccharide is between 850 and 1150 Daltons.

40. The hydrogenated oligosaccharide of claim 35, wherein the Mw of the 10% by weight fraction containing the dextrans having the highest molecular weights is below 4,000 Da.

41. The hydrogenated oligosaccharide of claim 35, wherein the Mw of the 10% by weight fraction containing the dextrans having the highest molecular weights is below 3,200 Da.

42. The hydrogenated oligosaccharide of claim 35, wherein 90% by weight of said dextran has molecular weights less than 3,000 Daltons.

43. The hydrogenated oligosaccharide of claim 35, wherein 90% by weight of said dextrans have molecular weights less than 2,700 Daltons.

44. The hydrogenated oligosaccharide of claim 35, wherein the 10% fraction having the lowest molecular weight has a Mw of 800 Daltons or more.

45. The hydrogenated oligosaccharide of claim 35, wherein the content of dimer saccharide in the hydrogenated oligosaccharide is 2.5% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

46. The hydrogenated oligosaccharide of claim 35, wherein the content of dimer saccharide in the hydrogenated oligosaccharide is 2.3% by weight or less, based on the total weight of the hydrogenated oligosaccharide.

47. A composition comprising the hydrogenated oligosaccharide of claim 35 and at least one pharmaceutically acceptable carrier.

48. The hydrogenated oligosaccharide of claim 35, wherein said hydrogenated oligosaccharide is prepared by a process comprising: (a) hydrolysing a polysaccharide comprising glucose monomers so as to reduce its molecular weight, (b) hydrogenating the resulting oligosaccharides to convert functional aldehyde groups into alcohol groups, (c) fractioning the hydrogenated oligosaccharides according to molecular weight, so that the purified fraction has a weight average molecular weight (Mw) between 500 and 3,000 Daltons.

49. The hydrogenated oligosaccharide of claim 48, wherein 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, and wherein the hydrogenated oligosaccharide is hydrogenated dextran oligosaccharide having a number average molecular weight (Mn) above 500 Daltons, 90% by weight of said dextran has molecular weights less than 3,500 Daltons, the average molecular weight (Mw) of the 10% by weight fraction of the dextran having the highest molecular weights is below 4,500 Daltons.

50. The hydrogenated oligosaccharide of claim 48, wherein said process further comprises decreasing the reducing capacity of said hydrogenated oligosaccharide to less than 3.0%.

51. The hydrogenated oligosaccharide of claim 48, wherein said hydrolyzed polysaccharide comprises dextran.

52. A hydrogenated oligosaccharide having a weight average molecular weight (Mw) between 850 and 1,150 Daltons, wherein said hydrogenated oligosaccharide comprises the following: (i) a dimer saccharide content of 2.3% by weight or less, based on the total weight of the hydrogenated oligosaccharide, (ii) said hydrogenated oligosaccharide is a hydrogenated dextran oligosaccharide having a number average molecular weight (Mn) above 500 Daltons, (iii) 90% by weight of said dextrans have molecular weights less than 2,700 Daltons, (iv) the Mw of the 10% by weight fraction of the dextran having the highest molecular weights is below 4,500 Daltons, and (v) 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.

53. A composition comprising the hydrogenated oligosaccharide of claim 52 and at least one pharmaceutically acceptable carrier.

54. The hydrogenated oligosaccharide of claim 52, wherein the reducing capability of the hydrogenated oligosaccharide is less than 3.0%.

55. The hydrogenated oligosaccharide of claim 54, wherein said reducing capacity is determined by the cupric oxidation method.

56. The hydrogenated oligosaccharide of claim 52, wherein the Mw of the 10% by weight fraction of the dextran having the highest molecular weights is below 4,000 Da.

57. The hydrogenated oligosaccharide of claim 52, wherein the Mw of the 10% by weight fraction of the dextran having the highest molecular weights is below 3,200 Da.

Description

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0080] FIG. 1 shows a plot of the t.sub.1/2 vs. the dimer content of the iron oligosaccharide products.

EXAMPLES

Hydrolysis and Hydrogenation of Dextran

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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.

[0085] 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.

[0086] 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

[0087] 120 kg dextran as produced above is mixed as an 18% solution with 150 kg FeCl.sub.3, 6 H.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)).

[0088] 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 035% calculated on basis of a solution containing 5% (w/v) iron.

[0089] 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 m (or alternatively a 0.2 m) membrane filter.

[0090] The solution is spray dried and the iron-dextran powder is ready for marketing or for further processing.

[0091] 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.

[0092] 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

[0093] 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.

[0094] 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.

[0095] The hydrolysis of Fe.sup.3+ was measured by optical absorbance at 287.3 nm.

[0096] 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.

[0097] The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Assessment after keeping Iron Amount of at elevated oligosaccharide dimer in temperature compound no. oligosaccharide t.sub.1/2/hours M.sub.P/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

[0098] 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.

[0099] 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 126 hours.