DRUG BASED ON MAGHEMITE FOR SIMULTANEOUS REDUCTION OF GASTROINTESTINAL SODIUM RESORPTION AND PHOSPHATE RESORPTION

Abstract

The invention includes a substance based on nanocrystalline maghemite having a crystal size of between 0.5 and 4 nm, which is defined by a proportion of divalent iron ions less than five percent by weight of the total iron, and the transport of sodium and simultaneously phosphate in the gastrointestinal wall from the gastrointestinal contents into the bloodstream is reduced and thus can improve the imbalance of electrolyte and water and mineral balances of patients having impaired renal function when used orally in combination with suitable pharmaceutical adjuvants.

Claims

1. A medicinal product for the simultaneous reduction of gastrointestinal resorption after oral application of sodium and phosphate based on maghemite with a magnetite proportion defined by the weight proportion of divalent iron ions on the total iron less than 5 parts by weight of a hundred and crystal sizes less than 4 nm, preferably 0.5 and 4 nm.

2. The substance according to claim 1, which is mixed with known pharmaceutical adjuvants suitable for oral administration.

3. A method for the preparation of a dispersion of maghemite crystals as claimed in claim 1, characterized in that alditols and one or more carbohydrates are mixed with an aqueous sodium hydroxide solution having a pH value of 10-15 and this mixture is then admixed with an aqueous solution of iron-II salts and iron-III salts having chloride ions, and wherein all mixtures and solutions have a temperature of 0-10° Celsius.

4. The substance according to 1, which is orally administered and is used for the treatment and improvement of disorders of water, electrolyte and mineral balance in humans with such imbalances from restricted renal function.

5. The substance of claim 2, wherein the adjuvants comprise at least one selected from the group consisting of alditols and monomeric and polymer carbohydrates from hexoses and pentoses.

Description

DESCRIPTION OF THE DRAWINGS

[0004] Description Drawing 1:

[0005] The result of the in vivo tests according to Example 2 in healthy rats is shown. Active ingredients were admixed to the feed and urine was collected in a metabolic cage for hours and blood was collected from the animals. Drawing 2A shows the sodium balance calculated from sodium urine excretion minus the feed sodium intake. Drawing 2B shows the serum phosphate level. Compared to known active ingredients for regulating the mineral content in patients having impaired renal function, only the active substance according to Example 1 simultaneously leads to a significant influence on the sodium balance and the serum phosphate level.

[0006] Description Drawing 2:

[0007] A highly-resolved transmission electron microscope image of a typical octahedral maghemite crystal according to the invention of the substance produced according to Example 1 is shown, wherein the longest extension of the maghemite crystal is only 3.5 nm. The selective electron diffraction (SAED) leads to a diffraction pattern which is typical for magnetite-maghemite crystals.

[0008] Description Drawing 3:

[0009] In comparison, the size distribution of the crystals is shown based on a size evaluation of the longest diameter of the crystals based on transmission electron microscope images. Drawing 3A shows the size distribution for the substance according to Example 1 and this shows a very uniform size distribution without a proportion of very large crystals. In comparison, Drawing 3B shows the size distribution of the substance according to Comparison Example 2 and here it can be seen that a considerable proportion of crystals is present over a size of 4 nm—

[0010] Description Drawing 4:

[0011] Drawing 4A shows a representative section of a transmission electron microscope image of the substance according to Example 1, which demonstrates that the substance according to Example 1 is a dispersion of very uniform maghemite crystals without large crystals or aggregates. Drawing 4B shows a representative section of a transmission electron microscope image of the substance according to Comparison Example 1. Here, very large individual crystals are found in a not inconsiderable proportion. Overall, the crystallinity is shown to be inferior to that of the substance according to Example 1.

EXAMPLES

[0012] Method for the determination of phosphate binding capacity in a simulated gastrointestinal passage with excess of inorganic phosphate A 100 mM solution of sodium dihydrogen phosphate (Sigma-Aldrich No. 04269) was prepared in 0.1 M hydrochloric acid. The solution was heated to 37° C. and maintained at this temperature. 40 ml of this solution was transferred with the iron-containing phosphate binder in a quantity to obtain a 40 mM solution based on iron and thus a molar ratio in the incubation solution of inorganic phosphate to iron of 1:0.4. The pH was adjusted to 1.2 by means of hydrochloric acid using a titrator. An aliquot of 0.5 ml was withdrawn after each incubation for one hour and then the pH value was determined in the steps pH 2.5, 4.5, 7.0, 7.5 by means of the ammonium molybdate method photometrically by means of titrator with the extinction of 880 nm in the filtrate after centrifugation through a 3 kD CentriPrep® filter (regenerated cellulose). The free complexable iron content of the filtrate was determined photometrically by means of the orthophenanthroline method with the extinction of 520 nm.

Comparison Example 1

[0013] A maghemite-based phosphate adsorbent was prepared according to Example 1 patent international publication number WO 2013/034267 A1. 7.55 g of iron (III) chloride hexahydrate (Sigma-Aldrich, No. 31232) was dissolved in 50 ml of bi-distilled water cooled to 4° C. (solution A). To solution A, 3.2 g of iron (II) chloride tetrahydrate (Sigma-Aldrich No. 44939) was added and dissolved (solution B). In addition, 25 g of D-mannose (Sigma-Aldrich, No. 63582) was dissolved in bi-distilled water cooled to 4° C. (solution C). Solution B and C were combined and stirred for 2 min (solution D). 100 ml of 1.5 M NaOH (cooled to 4° C.) was added to solution D, and the resulting mixture was stirred for 5 min at 4° C. until a homogeneous colloid was formed (about 5 min) and then heated to 60° C. and stirred again at 60° C. for 15 min. During 15 minutes, the solution was cooled to room temperature while stirring and reduced to 100 ml by ultrafiltration (10 kD, Spectrum, Hollow Fiber, PES). The solution was dialyzed by means of dialysis tubes (12-14 kD cutoff regenerated cellulose, Spectra Por) 5 times against 2 liters of bi-distilled water until no iron and chloride was detectable in the filtrate. The colloidal solution present after the dialysis in a total quantity of 200 ml was mixed with 0.1 g of mannose, 3 g of gum arabicum (Acaciabaum Reagent Grade, Sigma G9752) and 3 g of inulin (Sigmaaldrich 12255, Chicory), which together had been dissolved in 25 ml of bi-distilled water. This dispersion was stirred for 3 min and filled up to 1 1 with 100% ethanol. The nanoparticles were thus precipitated and additionally centrifuged at 800 rcf. The sediment was dried overnight at 60° C. The resulting dry substance was finely pulverized to a powder. The thus resulting powder has an iron content of 157 mg/g of dry substance at 2.04% content of bivalent iron relative to the total iron. The evaluation of 500 crystals leads to an average longest diameter of 3.4+1.9 nm with a proportion of 90% of the crystals smaller than 10 nm, however with a proportion of <20% of the crystals between 5 and 10 nm.

TABLE-US-00001 TABLE 3 Phosphate binding capacity at incubation 100 mM PO4 to 40 mM Fe for Comparison Example 1. Phosphate binding free iron capacity weight proportion % PH [mg PO4%/mgFe of initial weight 1.2 670 26 2.5 790 6 4.5 910 <1 7 950 <1 7.5 970 <1

TABLE-US-00002 TABLE 2 Size distribution of the iron oxide crystals based on the evaluation of 500 crystals in the transmission electron microscope image of the sample according to Comparison Example 1. Size distribution proportion in % Size longest of the total diameter [nm] count 0.0-<0.5 0 0.5-<1.0 0 1.1-<1.5 2 1.5-<2.0 8 2.0-<2.5 32 2.5-<3.0 22 3.0-<3.5 12 3.5-<4.0 4 4.0-<4.5 2 4.5-<5.0 1 5.5-<6.0 3 6.0-<6.5 2 6.5-<7.0 3 7.0-<7.5 3 7.5-<8.0 3 8.0-<8.5 1 8.5-<9.0 0 9.0-<9.5 1  9.5-<10.0 0 10.0-<10.5 1

Example 1

[0014] 50 ml of water are cooled in an ice-water bath to temperature equilibrium. This is followed by successively dissolving 3.2 g of iron (II) chloride tetrahydrate and 7.55 g of iron (III) chloride hexahydrate=Solution 1. In another vessel, 25 g of D-mannitol and 5 g of inulin are dissolved in 100 ml of equally cooled 1.5 M sodium hydroxide solution=Solution 2. Solution 1 is poured rapidly into solution 2 and stirred further in ice-water cooling for 15 min. 3 ml of 30% hydrogen peroxide solution are then added, stirred for 5 minutes and then heated to 60° C. with stirring and stirred for a further 15 minutes. The sample is spontaneously cooled to room temperature and purified by means of dialysis against water and the retentate is centrifuged for 10 minutes at 4500 rpm (rotor radius 15 cm). The supernatant is transferred with 3 g of gum arabic, and the resulting solution is concentrated in a rotary evaporator and then freeze-dried. The resulting reddish brown powder has an iron content of 190 mg/g and a proportion of divalent iron <1%. The crystal size detected by TEM is between 0.5 and 4 nm for over 90% of the crystals. The electron diffraction pattern reveals a diffraction pattern characteristic of maghemite according to HKL classification: 220 plane=0.297 nm; 311 plane=0.254 nm; 400 plane=0.214 nm; 511 plane=0.164 nm; 440 level=0.151 nm. The evaluation of 500 crystals gives an average longest diameter of 3.0±0.6 nm with a proportion of 90% of the crystals smaller than 4.5 nm.

TABLE-US-00003 TABLE 3 Phosphate binding capacity at incubation 100 mM PO4 to 40 mM Fe for Example 1. free iron Phosphate weight binding proportion % capacity of initial PH [mg PO4%/mgFe weight 1.2 1185 8 2.5 1502 <1 4.5 1734 <1 7 1577 <1 7.5 1605 <1

TABLE-US-00004 TABLE 4 Size distribution of the iron oxide crystals based on the evaluation of 500 crystals in the transmission electron microscope image of the sample according to comparative Example 1. Size distribution Size proportion in % Longest of the diameter [nm] total count 0.0-<0.5 0 0.5-<1.0 2 1.1-<1.5 17 1.5-<2.0 34   0-<2.5 27 2.5-<3.0 15 3.0-<3.5 3 3.5-<4.0 1 4.0-<4.5 1

Example 2

[0015] Male rats of the breed Sprague Dawley from Charles River were used at the beginning of the experiment with weight of 200 g (n=8) per group listed below. The animals received feed ad libitum Altromin 1324 (powder form) in the first week of experiment (week 1) without active ingredient (0.7% phosphorus and 0.2% sodium relative to weight). Then for another 4 weeks (week 2-5), the feed mentioned above ad libitum with active substance additive. On the last day of each experiment week, the animals were kept individually in a metabolic cage for 24 hours (day 6 to day 7). Feces and urine were collected. Blood was obtained only on the last collection day. Blood and urine samples were examined by Synlab GmbH. [0016] The groups received as follows from week 2-5 additives per 100 g feed Group A control—no additives [0017] Group B adjuvants—addition of 0.2 g of mannitol and each 0.9 g of inulin and gum arabic [0018] Group C Example 1—based on iron as an additive to the feed 250 mg of iron [0019] Group D Velphoro®—based on iron as an additive to the feed 250 mg of iron [0020] Group E Fosrenol®—based on lanthanum as an additive to the feed 250 mg lanthanum [0021] Group F Renvela®—based on Sevelamer carbonate 500 mg [0022] The results for the 24 hours collection period on the last day of the experiment (5 weeks total experiment and 4 weeks of active ingredient feeding) are as follows:

TABLE-US-00005 TABLE 4 Results of study according to Example 2 Data collection Day 7 Week 5 Group A Group B Group C Group D Group E Group F Body weight (g) 438 ± 29  391 ± 33*  375 ± 31** 404 ± 28  374 ± 42*** 466 ± 25  Feed intake 24 hours (g) 18.75 ± 4.0  23.5 ± 4.6  25.3 ± 2.6* 26.6 ± 4.5 21.6 ± 4.7  18.4 ± 2.9  Sodium intake 24 hours [mmol] 1.76 ± 0.38  2.20 ± 0.43*   2.37 ± 0.24*** 2.50 ± 0.42*** 2.03 ± 0.44 1.72 ± 0.27 Sodium excretion Urine 24 hours [mmol] 1.64 ± 0.19 1.53 ± 0.21  1.3 ± 0.36 1.43 ± 0.23 1.61 ± 0.26 1.73 ± 0.14 Sodium balance Intake feed minus excretion Urine 24 hours [mmol] −0.12 + 0.38    −0.58 + 0.34*    −1.07 + 0.40**   −1.15 + 0.46*** 0.42 + 0.44 0.01 + 0.37 Feces excretion 24 hours Weight [g] 14.1 ± 3.0  12.9 ± 1.6  14.6 ± 3.2  17.1 ± 6.2 13.1 ± 2.6  14.7 ± 3.1  Urine volumes 24 hours [ml] 24.9 ± 5.5  16.7 ± 5.6  17.4 ± 6.9  16.1 ± 3.6 21.3 ± 9.4   26 ± 8.3 Serum sodium [mmol]  141 ± 1.4   140 ± 1.5   140 ± 1.5  141 ± 1.5  140 ± 1.0   142 ± 2.2  Serum phosphate [mmol] 2.54 ± 0.40 2.42 ± 0.30 1.80 ± 0.37 2.41 ± 0.21 2.16 ± 0.39 2.39 ± 0.4 

[0023] A statistical comparison of the analysis values of the control group with the active substance groups was carried out using the Prism 5.0f® program with the test One-Way Anova and Dunnett's postprocessing (* p<0.05; ** p<0.01; *** p<0.005).

Example 3

Reduction of Renal Osteodystrophy on the Animal Model of the Uraemic Rat by Co-Feeding the Substance According to the Invention according to 1

[0024] An uremia in rats (Sprague-Dawley, male, n=6, Charles River) is generated by co-feeding adenine (0.3% additional weight fraction, feed Altromin C100, it 1.2% phosphorus and 1.2% calcium for a period of 10 weeks. After this 10-week period, the adenine was discontinued and 3 animals were still given the above-mentioned diet for 4 weeks without active ingredient (control group) and the other 3 animals then received the above-mentioned diet supplemented with 0.125% by weight of the iron according to the invention according to Example 1 (active ingredient group). [0025] After this second 4-week period, the animals were killed and the thigh bones removed and the length determined (by right and left sides a total of n=6 bones). There was a significant difference in the diaphyseal thickness of the corticalis by means of (micro-computer tomography determined) 0.42 +0.08 mm for the control group and 0.59 +0.12 mm for the active substance group.

CITATIONS INCLUDED IN THE DESCRIPTION

Citations Patent Literature

[0026] US 2012/0263670 [0027] DE 102011112898 [0028] WO 2012/0006475 A1 [0029] WO 97/22266

Citations Non-Patent Literature

[0030] Labonte et al. 2014 Journal of the American Society of

[0031] Nephrology 26, online publication doi: 10.1681/ASN.2014030317