Glucose polymers for peritoneal dialysis

Abstract

The invention relates to a novel glucose polymer which is particularly useful for administration by the parenteral route, and to the method for the production thereof. The invention also relates to compositions comprising such a glucose polymer, and to the methods for the production thereof. The invention further relates to the use thereof as a medicament, for example as an osmotic agent for peritoneal dialysis.

Claims

1. A glucose polymer characterized in that it is obtained by branching and reduction of a starch having an amylose content of at least 10%, and in that said glucose polymer has an α-1,6-bond content of less than 20%, a weight-average molecular weight (Mw) less than 50,000 Da, this Mw being determined by liquid chromatography and detection by differential refractometry, and a polydispersity index (polyD) of less than 3.0 and greater than 0.5; and wherein reduction results in the conversion of carbonyl groups to hydroxyl groups, and in that it has a reducing sugar content of less than 0.5%, this percentage being expressed by dry weight of reducing sugars relative to the total dry weight of the glucose polymer.

2. The glucose polymer as claimed in claim 1, characterized in that said glucose polymer has an α-1,6-bond content of greater than 7%.

3. The glucose polymer as claimed in claim 1, characterized in that said starch has an amylose content of at least 20%.

4. The glucose polymer as claimed in claim 1, characterized in that it has a weight-average molecular weight (Mw) chosen in the range of from 20,000 to 50,000 daltons (Da); this Mw being determined by liquid chromatography and detection by differential refractometry.

5. The glucose polymer as claimed in claim 1, characterized in that it has a pH, after sterilization at 121° C. for 45 minutes, in a range of from 6 to 8, when it is formulated as an aqueous 5% solution.

6. The glucose polymer as claimed in claim 1, characterized in that it has an osmolality of between 200 and 300 mOsm/kg; when it is formulated as a 0.4% solution.

7. The glucose polymer as claimed in claim 1, characterized in that it has a reducing sugar content of less than 0.1%, this percentage being expressed by dry weight of reducing sugars relative to the total dry weight of the glucose polymer.

8. The glucose polymer as claimed in claim 1, characterized in that it is soluble to very soluble in water when it is at ambient temperature.

9. The glucose polymer as claimed in claim 1, characterized in that it is not substituted.

10. A composition, comprising the polymer as defined claim 1.

11. The composition as claimed in claim 10, characterized in that it is a solution.

12. The glucose polymer as claimed in claim 1 or the composition as claimed in claim 11, for use thereof as a medicament.

13. The glucose polymer as claimed in claim 1 or the composition as claimed in claim 11, for use thereof in peritoneal dialysis, and/or parenteral nutrition, and/or in plasma filling, and/or as an osmotic agent, and/or as a plasma expander, and/or in vaccinology, and/or as an adjuvant, and/or as a protein stabilizer, and/or as a protein carrier.

14. A method for producing a composition as defined in claim 10, which comprises mixing a polymer as defined in claim 1 with at least one other substance and/or a solvent.

15. A method for producing a glucose polymer as defined in claim 1, which comprises subjecting a starch having an amylose content of at least 10% to: (a) a branching step; and (b) a reduction step.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Influence of sterilization on the pH of solutions using various glucose polymers or glucose (positive control), the pH being measured after sterilization at 121° C. for 15 or 45 minutes.

(2) FIG. 2: Influence of sterilization on the production of GDPs, namely 5-hydroxymethylfuraldehyde (5-HMF), furaldehyde and 3,4-dideoxyglucosone-3-ene (3,4-DGE), of solutions using various glucose polymers or glucose (positive control).

(3) FIG. 3: Influence of sterilization on the reactivity with respect to proteins, of solutions using various glucose polymers or glucose (positive control) by measuring the absorbance at 284 nm.

EXAMPLES

(4) A. Substances Tested

(5) 1. Glucose Polymer According to the Invention [HSB-red])

(6) The starch used had an amylose content of 65% (EURLYON® 7, Roquette). A starch starch milk (suspension) containing 10% solids and at a pH of 7.5 was cooked at 160° C. The paste obtained was cooled to 75° C., and the pH was adjusted to 7.0.

(7) The starch was then subjected to a branching step by means of a branching enzyme (BRANCHZYME®, Novozymes), used in a proportion of from 625 to 1000 U/g dry of starchy matter, for 22 hours at 65° C. and at pH 7.0. The reaction medium was then cooled to 48° C., and the pH was adjusted to 5.5.

(8) The branched starch thus obtained was subjected to a hydrolysis step by means of a β-amylase (OPTIMALT® BBA, Genencor International), used in a proportion of from 1 to 4 U/g dry of starchy matter, for 2 hours at 48° C. and at a pH of 5.5. The enzyme was then deactivated by heating for 1 hour at 85° C. The reaction mixture was cooled to 50° C. and the pH was adjusted to 3.5.

(9) The reaction product was centrifuged at 5000 rpm, and the supernatent was collected.

(10) A solution containing 30% of branched starch thus obtained was prepared. Dissolution was promoted by heating at 90° C., then the temperature was lowered to 40° C. The pH was adjusted to 10.5 with sodium hydroxide (3% NaOH).

(11) The starch in solution was subjected to a reduction step by means of 200 mol % of NaBH.sub.4 relative to the reducing functions. At the end of the reaction, the pH was adjusted to 6.5. 18% H.sub.2SO.sub.4 was added to the reaction product.

(12) The solution was dialyzed on a 1000 Da membrane overnight. The product thus obtained was dried in a rotary evaporator and milled with a knife mill.

(13) 2. Comparative Glucose Polymers ([ICO-Red], [ICO], [HBS])

(14) The [ICO] product corresponds to an unmodified icodextrin, that is to say a non-reduced icodextrin.

(15) In order to produce the [ICO-red] product, a reduced icodextrin of the prior art in accordance with U.S. Pat. No. 6,770,148 was prepared by reduction of an icodextrin. For the reduction, the process was carried out as described in point 1. above.

(16) The [HBS] product corresponds to a product obtained by branching of starch, but which has not undergone reduction. For the branching, and as described in point 1. above, the glucose polymer was branched by means of a branching enzyme and subjected to a hydrolysis step by means of a β-amylase.

(17) 3. Glucose (Reference)

(18) The glucose used was anhydrous dextrose (ROQUETTE).

(19) B. Characterization of the Glucose Polymers Used

(20) The characterization of the [HBS-red] glucose polymers according to the invention, and of the comparative [ICO-red], [ICO], [HBS] glucose polymers were determined according to the following methods.

(21) 1. Determination of the α-1,6-bond content. The α-1,6-bond content was determined by proton NMR. The α-1,6-glucosidic bond content, expressed as percentages, corresponds to the amount of signal of the proton carried by the C1 of an anhydroglucose unit which bonds another anhydroglucose unit via an α-1,6-bond, when a value of 100 has been given to all of the signals of the glucosidic protons carried by all the C1 of the residues of said glucose polymers.

(22) 2. Determination of the osmolality. The osmolality was determined on the basis of an aqueous solution prepared in deionized water comprising 0.4% of glucose polymer. The measurement of the osmolality of this solution was carried out using an osmometer (FISKE® ASSOCIATES MARK 3), according to the constructor's indications.

(23) 3. Determination of the weight-average (M.sub.W) and number-average (M.sub.N) molecular weights, and calculation of the polydispersity index (polyD). The average molecular weights M.sub.W and M.sub.N were determined according to two methods.

(24) Method 1: liquid chromatography (using pullulans of various M.sub.W for the calibration) and detection by differential refractometry.

(25) A set of columns (Shodex OH pak SB—800 QH) composed of the following columns was used: a column with a particle size of 8 μm, a pore size of 100 Å, an internal diameter of 8.0 mm and a length of 300 mm (OH pak SB—802 HQ—Waters ref. JWE 034256) a column with a particle size of 6 μm, a pore size of 800 Å, an internal diameter of 8.0 mm and a length of 300 mm (OH pak SB—803 HQ—Waters ref. JWE 034257) a column with a particle size of 13 μm, a pore size of 7000 Å, an internal diameter of 8.0 mm and a length of 300 mm (OH pak SB—805 HQ—Waters ref. JWE 034259).

(26) The pullulan standards used (Waters kit—Ref. JWE034207) had the following M.sub.W (Da): 78 000, 40 400; 211 000; 112 000; 47 300; 22 800; 11 800; 5900.

(27) The elution solvent was an aqueous 0.2 M sodium nitrate solution containing 0.02% of sodium azide, filtered on a 0.02 μm filter. The flow rate of the mobile phase for the chromatography was 0.5 ml/min.

(28) Method 2: liquid chromatography with detection by light scattering (RI detector and light scattering detector, DAWN-HELEOS II).

(29) A set of columns composed of the following columns was used: a column with a particle size of 10 μm, a pore size of 100 Å, an internal diameter of 8.0 mm and a length of 300 mm (PSS SUPREMA 100—Ref. SUA0830101E2); a column with a particle size of 10 μm, a pore size of 1000 Å, an internal diameter of 8.0 mm and a length of 300 mm (PSS SUPREMA 1000—Ref. SUA0830101E3).

(30) The elution solvent was an aqueous 0.1 M sodium nitrate solution containing 0.02% of sodium azide filtered on a 0.02 μm filter, and the dilution solvent was dimethyl sulfoxide (DMSO) containing 0.1 M of sodium nitrate. The flow rate of the mobile phase for the chromatography was 0.5 ml/min. The calibration was carried out with a pullulan (Pullulan P50, Shodex).

(31) 4. Determination of the reducing sugar content. The reducing sugar content was determined by the Bertrand method. More specifically, the following were introduced into a 250 ml conical flask: 20 ml of solution to be titrated containing the equivalent of 0.5 to 5.0 mg of glucose per ml; 20 ml of cupric solution (4% of copper sulfate pentahydrate); 20 ml of sodium tartrate solution (20% of sodium potassium double tartrate and 15% of sodium hydroxide); some glass beads. The whole mixture was heated to moderate boiling for 3 minutes and then left to separate out for 2 minutes. The supernatent was removed, and the Cu.sub.2O precipitate was dissolved in 20 ml of ferric liquor (5% of ferric sulfate and 20% of sulfuric acid). The solution obtained was titrated with a solution of potassium permanganate at 0.1 N, and using the Bertrand table.

(32) The results are presented in Table 1.

(33) TABLE-US-00001 TABLE 1 [HBS- [ICO- red] red] [ICO] [HBS] Reducing     <0.05       0.299      1.9      1.93 sugars (% dry) α-1,6-bond    14    7    7    14 content (%) M.sub.W (Da, 34 920 13 830 13 550 34 930 method 1) M.sub.N (Da, 19 850   7670   7280 18 690 method 1) polyD      1.8      1.8      1.9      1.9 M.sub.w (Da, 125 000  22 000 21 000 130 000  method 2) Osmolality   240   241   240   240 (mOsm/kg)

(34) C. Glucose Polymer Tests

(35) 1. Effects of the Sterilization on pH

(36) In this example, the inventors studied the influence of sterilization on the pH of solutions using various glucose polymers or glucose (positive control).

(37) 20 ml solutions containing 5% by dry weight of each of the substances to be tested were prepared in deionized water. The pH was measured after sterilization at 121° C. for 15 or 45 minutes, on the stirred solutions.

(38) The results are presented in FIG. 1.

(39) The glucose polymer in accordance with the invention [HBS-red] is that which presents the pH closest to that of physiological pH after sterilization. This is not the case with the comparative polymers [ICO-red], [ICO] and [HBS], which present acidic pHs after sterilization, below 6.5, or even 6.0; that is to say differences of more than 1 pH unit compared to physiological pH.

(40) Furthermore, the inventors noted that an unwanted brown coloring appeared when the [ICO-red] glucose polymer was sterilized.

(41) 2. Effects of the Sterilization on GDP Production

(42) In this example, the inventors studied the influence of sterilization on the production of GDPs, namely 5-hydroxymethyl furaldehyde (5-HMF), furaldehyde and 3,4-dideoxyglucosone-3-ene (3,4-DGE), of solutions using various glucose polymers or glucose.

(43) 20 ml solutions containing 4% of each of the substances to be tested were prepared in deionized water. The GDP content was measured after sterilization at 121° C. for 15 minutes.

(44) The 5-HMF and furaldehyde content was determined by liquid chromatography using 5-HMF standards (Merck—Ref. 8.206.78.001) or furaldehyde standards for the calibration respectively), and detection by UV spectrophotometry at 280 nm. For the chromatography, a column with a particle size of 9 μm, 8% crosslinking, an internal diameter of 7.8 mm and a length of 300 mm (HPX 87H column—Biorad—Ref. 125.0140) was used. The conditions were the following: eluent 5 mN H.sub.2SO.sub.4 (1N sulfuric acid), flow rate of 0.8 ml/min.

(45) The 3,4-DGE content was determined by liquid chromatography (using pyrazinecarboxamide (Sigma—Ref. P7136) for the calibration) and detection by UV spectrophotometry at 230 nm. For the chromatography, a column with a particle size of 5 μm, a pore size of 120 Å size, an internal diameter of 4.6 mm and a length of 15 cm (Supelcosil LC-18—Supelco—Ref. 58230) was used. The conditions were the following: elution solvent H.sub.2O/MeOH, flow rate of 1.0 ml/min.

(46) The results are presented in FIG. 2.

(47) As expected, the glucose produces a GDP content that is much higher than that of the glucose polymers. The [ICO] and [HBS] comparative glucose polymers exhibit contents which are lower but nevertheless high.

(48) The [ICO-red] glucose polymer of U.S. Pat. No. 6,770,148, exhibits detectable contents of 5-HMF.

(49) The glucose polymer in accordance with the invention [HBS-red] is the only one for which, after sterilization, the GDPs are not detectable.

(50) These observations are all the more surprising since the pH for sterilization of the glucose polymer in accordance with the invention [HBS-red] is particularly high. This goes against the prior art teachings, which recommend sterilizing the solutions at acidic pH, precisely to avoid the formation of GDPs.

(51) 3. Effect of the Sterilization on the Reactivity with Respect to Proteins

(52) In this example, the inventors studied the influence of sterilization on the reactivity with respect to proteins, of solutions using various glucose polymers or glucose.

(53) Solutions containing 0.5% of each of the substances to be tested were prepared in a phosphate buffer (pH 7, 200 mM), in the absence or presence of 0.5% of L-lysine.

(54) The absorbance of the solutions at 284 nm was measured before and after sterilization at 121° C. for 45 minutes.

(55) The phosphate buffer used to prepare the solutions was used as a negative control.

(56) The results obtained with the best two substances, that is to say [HBS-red] according to the invention and comparative [ICO-red] are presented in FIG. 3.

(57) The difference in absorbance observed between the substance to be tested alone, before and after sterilization (dashed-line histograms) can be attributed to the production of degradation products. The results confirm the fact that the [HBS-red] glucose polymer of the invention is the substance which exhibits the greatest resistance to degradation during sterilization.

(58) The difference in the absorbance observed between the substance to be tested in the presence and absence of lysine after sterilization (gray histograms) can be attributed to the reactions which occur between the substance to be tested and the lysine, thus reflecting the reactivity of these substances with respect to proteins (Maillard reaction). The higher the difference in absorbance, the more the substance can be judged to be reactive with respect to proteins. The results indicate that the [HBS-red] glucose polymer according to the invention is the substance that is the least reactive with respect to proteins. This very weaker reactivity points to an excellent tolerance of the glucose polymer of the invention.