COMPOSITION FOR USE IN THE PREVENTION AND IN THE TREATMENT OF IRON DEFICIENCY

Abstract

The present invention relates to a composition for use in a method for the preventive or curative treatment of an iron deficiency and pathologies and disorders connected thereto in a subject, wherein said use comprises the administration of the composition to said subject.

Claims

1. A composition comprising: (a) a group D vitamin, or complexes, metabolic precursors and protection derivatives thereof, and (b) an iron (III) oxide, complex, or salt, formulated in association with a sucrose fatty acid ester or sucrester, and a lecithin (sucrosomial iron); and optionally, pharmaceutical or food grade excipients, additives or co-formulants, or combinations thereof.

2. The composition according to claim 1, wherein the iron (III) oxide, complex, or the salt formulated in association with said sucrose fatty acid ester or sucrester and said lecithin is further formulated with a gelatinized or pregelatinized vegetable starch.

3. The composition according to claim 1, wherein said group D vitamin is vitamin D3 or cholecalciferol.

4. The composition according to claim 1, wherein said iron (III) salt is iron pyrophosphate.

5. The composition according to claim 4, wherein the iron pyrophosphate is formulated in association with sucrose fatty acid esters E473 or sucresters E473, lecithin E322 and, optionally, pregelatinized starch.

6. A method for the preventive or curative treatment of a pathology or disorder connected to iron deficiency, the method comprising administering an effective amount of the composition of claim 1 to a subject in need thereof.

7. The method of claim 6, wherein said pathology or disorder connected to iron deficiency is an anaemia of inflammation.

8. The method of claim 6, wherein the pathology or disorder connected to iron deficiency is selected from among chronic kidney disease (CKD), nephropathy, neoplasms, chronic inflammatory intestinal diseases, ulcerous colitis, Crohn's Disease, coeliac disease, fibromyalgia and heart failure.

9. The method of claim 6, wherein said pathology or disorder connected to iron deficiency is nephropathy.

10. The method of claim 6, wherein the composition is formulated for oral administration.

11. A- non-therapeutic method for treating a pathology or disorder connected to iron deficiency, the method comprising administering an effective amount of the composition of claim 1 to a subject in need thereof.

12. The method of claim 11, wherein the pathology or disorder is chronic kidney disease (CKD), nephropathy, neoplasms, chronic inflammatory intestinal diseases, ulcerous colitis, Crohn's Disease, coeliac disease, fibromyalgia or heart failure.

13. The method of claim 11, wherein the pathology or disorder is nephropathy.

14. The method of claim 9, wherein the nephropathy is chronic kidney disease (CKD).

15. The method of claim 13, wherein the nephropathy is chronic kidney disease (CKD).

Description

EXPERIMENTAL PART

I) In Vitro Studies

[0128] In order to better understand the role of iron (III) (as a dietary source of iron) and the intake of vitamin D in the inflammatory process, an assessment was made of the expression of hepcidin and other inflammatory markers in treated human hepatocellular carcinoma and monocytic cell lines. The markers studied were interleukin-6 (IL-6), C-reactive protein (CRP) and hepcidin. IL-6 was assessed in the U937 cell line (monocytic cell line), whilst CRP and hepcidin were assessed in the HepG2 cell line (hepatocellular carcinoma cell line).

[0129] IL-6 and CRP are typically expressed and evaluated markers during acute inflammation (Rincon, M. Interleukin-6: from an inflammatory marker to a target for inflammatory diseases Trends in Immunology November 2012, Vol. 33, No. 11, pages 571-577, Pepys, M. P; Hirschfield, G. M. C-reactive protein: a critical update J. Clin. Invest. 2003, 111:1805-1812). Since lipopolysaccharide (LPS) is a well-known inducer of hepcidin expression in macrophages and hepatocytes (Zughaier, S. M. et al. The role of vitamin D in regulating the iron-hepcidin-ferroportin axis in monocytes J Clin Transl Endocrinol (2014) e9-e25), in order to stimulate inflammation the cells were treated with lipopolysaccharide (LPS) and then analysed for the production of markers in terms of protein expression in the supernatants of the cells. The aim of this study was to assess the ability of LPS to simulate inflammation in the selected cell lines and to investigate the effect of the compounds containing an iron (III) oxide, complex or salt and D vitamin according to the invention compared to other iron-containing compounds in the stimulated cell lines in terms of IL-6, CRP and hepcidin expression.

MATERIALS

1.1 Cells and Cell Cultures

[0130] The U937 cells (monocytic leukaemia cell lines) [Sundstrm,C.; Nilsson, K. Establishment and characterization of a human histiocytic lymphoma cell line (U-937) International Journal of Cancer 1976 May 15; 17(5):565-77] were grown in suspension in the medium RPMI 1640 (Sigma) supplemented with 2 mM glutamine, 10% FBS (EU-approved Euroclone), 100 U/ml penicillin and 100 g/ml streptomycin and were purchased from Sigma. The cells were maintained between 2-9100,000 cells/ml with 5% CO.sub.2 at 37 C.

[0131] The HepG2 cells (human hepatoblastoma cell lines) [U.S. Pat. No. 4,393,133] were grown in an adherent culture in ATCC medium, with a supplement of 10% FBS, 100 U/ml penicillin and 100 g/ml of streptomycin and were purchased from ATCC. The cells were maintained between 5-15100,000 cells/ml with 5% CO.sub.2 at 37 C.

[0132] PMA (Phorbol 12-myristate 13-acetate) was purchased from Sigma-Aldrich (catalogue number P1585-1MG), dissolved in 1 ml of DMSO (Sigma-Aldrich catalogue number D8418-50ML) and further diluted to a final concentration of 100 micrograms/ml in a sterile phosphate buffer saline solution (PBS) (Lonza catalogue number 17-516-F).

[0133] LPS (Lipopolysaccharides of Escherichia coli) was purchased from Sigma-Aldrich (Sigma catalogue number L4391), dissolved in sterile PBS and then diluted in sterile PBS and in the culture medium.

[0134] ELISA kit: Diaclone human IL-6 Elisa kit (catalogue number 950.030.096, sensitivity 2 pg/ml); DRG CRP HS ELISA (catalogue number EIA-3954, sensitivity range: 0.1-10 pg/ml); Invitrogen human C-reactive protein ELISA kit (catalogue number #KHA0031, sensitivity range: 10-1200 pg/ml); DRG Hepcidin-25 (bioactive) ELISA (catalogue number EIA-5258, sensitivity range: 0.35-80 ng/ml).

2. Test Setup

[0135] In order to establish the best experimental condition, preliminary tests were performed to investigate the cell response to the iron compounds and vitamin D, the optimal concentration of the compounds and the optimal incubation period. The selected experimental condition determined after this step was then applied to assess the response of the inflammatory markers.

2.1 Treatment of the Monocytic Cells and Induction of Macrophages

[0136] The U937 cells were treated with PMA as previously described (Shelley, C. S. et al. During Differentiation of the Monocytic Cell Line U937, Puran Mediates Induction of the CDllc 2 Integrin Gene Promoter J Immunol 2002, 168, 3887-3893): in the T25 tissue culture buffers, the U937 wells were treated with 20 ng/ml, 50 ng/ml, 100 ng/ml, 150 ng/ml and 200 ng/ml of PMA in an incomplete RPMI 1640 medium without FBS (without antibiotics or glutamine). After 24 h the medium was replaced with a fresh complete medium, without PMA, and the cells were left to adhere for 72 h (Garrelds, I. M. et al. Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids Mediators Inflamm 1999, 8, 229-235). With the aim of choosing the right concentration to be used for the test, after 72 hours the cells were checked for adhesion and morphological change.

2.2 Stimulation of U937 Cells with LPS

[0137] The U937 cells previously treated with 150 ng/ml of PMA were detached with the aid of a cell scraper (Garrelds, I. M. et al. Time dependent production of cytokines and eicosanoids by human monocytic leukaemia U937 cells; effects of glucocorticosteroids Mediators Inflamm 1999, 8, 229-235) and seeded in 6 wells with a cell density of 650,000 cells/ml. The cells were left to adhere for 24 h at 37 C., 5% CO.sub.2 and then the medium was replaced with an FBS-free incomplete one (without antibiotics or glutamine) containing 10 ng/ml, 20 ng/ml and 40 ng/ml of LPS from E. coli (Liu, H.-Z. et al. The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation Hepatobiliary Pancreat. Dis. Int. 2005, Vol. 4, No. 1, 84-89). The cells were checked after 6 hours and 20 hours in order to select the best concentration to use for the experiments.

2.3 Stimulation of HepG2 Cells with LPS

[0138] The HepG2 cells were seeded in a 6-well plate with a cell density of 650,000 cells/ml and left to adhere for 24 hours at 37 C., 5% CO.sub.2. After 24 hours, the medium was replaced with an incomplete FBS-free one (without antibiotics) containing 10 ng/ml, 20 ng/ml and 40 ng/ml of LPS from E. coli (Sigma L4391) (Huang, Z. B. et al. HMGB1 release by human liver L02 and HepG2 cells induced by lipopolysaccharide Molecular Medicine Reports 8:103-112, 2013, Saliou, C. et al. Selective inhibition of NF-UB activation by the flavonoid hepatoprotector silymarin in HepG2, Evidence for different activating pathways FEBS Letters 1998, 440, 8:12; Chung, B. et al. Oncostatin M is a potent inducer of hepcidin, the iron regulatory hormone, The FASEB Journal 2010, Vol. 24, 2093-2103). The cells were checked after 6 hours and 20 hours in order to select the best concentration to use for the experiments.

2.4 Iron Compounds and Vitamin D

[0139] The compounds used for the experiments were: sucrosomial iron (prepared according to one of the methods described in WO 2014/009806 A1 (as previously noted), indicated as Sucro Fe in the figures), sucrosomial matrix (prepared according to one of the methods described in WO 2014/009806 A1 (as noted above) but without iron salts or oxides or complexes), iron pyrophosphate, iron sulphate, vitamin D and vitamin D with sucrosomial iron.

[0140] The iron content of the compounds (with the exception of the vitamin D and the sucrosomial matrix) was 30 mg/ml. A 1:100 dilution was performed for all of the compounds in sterile PBS in order to obtain an iron concentration of 300 micrograms/ml. For experimental purposes, the compounds that did not contain iron were diluted with the same dilution ratio as the iron-containing compounds (vitamin D was diluted at the same ratio as Vitamin D and sucrosomial iron and the sucrosomial matrix was diluted at the same dilution ratio as sucrosomial iron). The final iron concentration used in the experiments has been described elsewhere (Barisani et al. Iron overload and gene expression in HepG2 cells: analysis by differential display FEBS Letters 2000, 469, 208-212). The compounds (initial dilution of 300 pg/ml) were diluted in an incomplete FBS-free medium (RPMI 1640 for the U937 cells, EMEM for the HepG2 cells) with a dilution factor of 1:6 for a final iron concentration of 50 micrograms/ml. A test was conducted to assess the effect of the compounds on the cell cultures: induced FlepG2 and U937 were seeded a with a cell density of 650,000 cells/ml in 6 wells (final volume 1.5 ml/well). After 24 hours the medium was replaced with a new FBS-free (incomplete) one containing 50 g/ml of iron and after 24 hours the cells were macroscopically checked (visual inspection using a microscope). Given that after 24 hours the cells incubated with the iron treatment showed to be contaminated (black patch moving amidst the cells), the diluted compounds were irradiated under UV light for a total time of 150 minutes before further experiments were undertaken (Berrin zelik Fungi/Bactericidal and Static Effect of Ultraviolet Light in 254 and 354 nm wavelengths Research Journal of Microbiology 2007 2 (1): 42-49).

3. Marker Detection

3.1 Treatment of Cells and Analysis of the Markers

[0141] The U937 cells, induced with PMA as described above, were seeded in 6 wells at a concentration of 650,000 cells/ml (1.5 ml/well). The plates were incubated for 24 hours at 37 C. and 5% CO.sub.2. After the 24-hour medium was replaced with an FBS-free incomplete RPMI 1640 containing 20 ng/ml of LPS. A control well was prepared with an LPS-FBS-free incomplete medium. After 20 hours of incubation at 37 C. and 5% CO.sub.2, the medium was replaced with the iron treatment solutions (medium containing 50 g/ml of iron). In the control well, the medium was replaced with an incomplete FBS-free RPMI 1640 medium. Another control well without iron was prepared, replacing a container containing LPS with a fresh FBS-free incomplete medium. After 6, 18 and 24 hours, the supernatants were collected from the wells, centrifugated for 10 minutes at 1000 g, transferred into a new tube and stored a 20 C. for the analysis of IL-6 [Miki, K. et al. Extracellular activation of arginase-1 decreases enterocyte inducible nitric oxide synthase activity during systemic inflammation Am J Physiol Gastrointest Liver Physiol. 2009; 297(4): G840-G848]. The cells of each well collected were detached using a scraper and counted using Trypan blue with a Brker counting chamber (Strober W. Trypan blue exclusion test of cell viability Curr Protoc Immunol. 2001 May; Appendix 3: Appendix 3B).

[0142] The HepG2 cells were seeded in 6 wells at a concentration of 650,000 cells/ml (1.5 ml/well). The plates were incubated for 24 hours at 37 C. and 5% CO.sub.2 first. Afterwards, the 24-hour medium was replaced with an incomplete EMEM without FEM, containing 40 ng/ml of LPS. A control well was prepared with an LPS-FBS-free incomplete medium. After 20 hours of incubation at 37 C. and 5% CO.sub.2, the medium was replaced with the iron treatment solutions (medium containing 50 g/ml of iron). In the control well, the medium was replaced by an incomplete FBS-free EMEM medium. Another control well without iron was prepared, replacing a container containing LPS with a fresh incomplete FBS-free medium. After 6, 18 and 24 ore, the hours, the supernatants were collected from the wells, centrifugated for 10 minutes at 1000 g, transferred into a new tube and stored a 20 C. for the analysis of hepcidin and CRP. The cells of each well collected were detached using trypsin and counted using Trypan blue with a Brker counting chamber.

[0143] An analysis of the marker was performed using commercially available ELISA kits. Before the ELISA analysis was started, the samples were thawed at room temperature for 1 hour and mixed. The ELISA analysis was performed following the instructions of the kit manufacturers. The plates were read using a Molecular Devices 340PC384 microplate reader and the data were analysed by means of SoftmaxPro software using a data reduction function with 4-parameter logistic curve fitting.

4. Results of the Setup

4.1 Induction of Macrophages

[0144] For the purpose of conducting an analysis on the inflammation markers, the U937 cells had to be induced to macrophages by means of a treatment with Phorbol-12-myristate-13-acetate (PMA). The treatment was directly performed in T25 tissue culture columns cultured with 5'10.sup.5/ml of cells. 24 hours after the treatment, the cells began to adhere and the medium was replaced by a complete one. 48 h after the treatment with PMA, the U937 cells began to change their morphology in terms of increase in cytoplasmic volume and cytoplasmic protrusions. Five concentrations of PMA were tested (20 ng/ml, 50 ng/ml, 100 ng/ml, 150 ng/ml and 200 ng/ml PMA) and the 150 ng/ml enabled a better induction compared to the other concentrations in terms of cell adhesion and change in morphology. This concentration was confirmed in 2 tests.

4.2 LPS Stimulation

[0145] It is well known that LPS is capable of stimulating HepG2 cells [Villaroel et al. Interleukin 6 and lipopolysaccharide modulate hepcidin mRNA expression by HepG2 cells Biol Trace Elem Res. 2012 December; 150(1-3):496-501] and U937 [Liu, H.-Z. et al. The U937 cell line induced to express CD14 protein by 1,25-dihydroxyvitamin D3 and be sensitive to endotoxin stimulation Hepatobiliary Pancreat. Dis. Int. 2005, Vol. 4, No. 1, 84-89]. In order to understand whether LPS is capable of stimulating the production of IL-6 in the U937 cells induced by PMA and CRP and hepcidin in the HepG2 cells, a treatment was performed on the 2 cell lines for 6 and 20 hours with 10 ng/ml, 20 ng/ml and 40 ng/ml of LPS. The U937 cells seemed to have suffered with the LPS concentration of 40 ng/ml at 6 and 20 hours (there was a small amount of cells in the wells at the end of the treatment) and were discarded.

[0146] The HepG2 cells were more resistant to the LPS treatment at all concentrations and at both times. After the visual check, the concentration chosen to be analysed via ELISA was 20 ng/ml for both cell lines.

[0147] The ELISA analysis showed an IL-6 concentration in the supernatants of the U937 cells of more than 200 g/ml for both time points in the treated wells and a concentration of 24-34 ng/ml in the untreated wells.

[0148] The ELISA analysis showed a CRP concentration of 0.416 pg/ml at 6 h and 0.295 pg/ml at 20 hours. This value was slightly higher than the lower sensitivity limit of the ELISA kit. The concentration of hepcidin was not detected by the ELISA kit at 20 ng/ml. The LPS concentration to be used with this cell line should be greater than 20 ng/ml (40 ng/ml).

4.3 Dilution and Test with Iron Compositions

[0149] Before the cells were treated with the selected compounds, they were tested to assess their dissolution properties. The compounds were diluted in sterile PBS and in the standard cell culture medium (EMEM). All of the compounds seemed to be viscous; the iron sulphate was separated in two phases and needed to be vigorously mixed again in order to be resuspended.

[0150] An initial ten-fold dilution of all compounds was performed (3 mg/ml of iron): the solutions were rather viscous and the iron sulphate and the pyrophosphate changed the colour (pH) of the medium from red to yellow. The matrix and the vitamin D showed no problems.

[0151] A further ten-fold dilution was performed (300 micrograms/ml of iron): the solutions seemed to be less viscous and there was no evident colour change in the medium with iron pyrophosphate; iron sulphate showed a moderate colour change in the medium.

[0152] At the dilution used for the experiments (300 times the dilution, 50 g/ml of iron) the compounds were completely dissolved both in PBS and in the medium without emissions of viscosity or changes in colour (pH).

[0153] The compounds were tested on U937 and HepG2 cell cultures for 24 hours at a concentration of 50 micrograms/ml of iron. The first test showed a contamination in the cell culture media in all the treated wells, in terms of black patches moving in the culture media. After UV irradiation of the compounds, a second test was performed and revealed no signs of contamination.

[0154] At 50 micrograms/ml, the compounds demonstrated to have an effect on the cytoplasmic volume of the cells (loss of volume), in particular the iron sulphate in the U937 cells. Moreover, the compounds had an effect on cell adhesion in the U937 cells. This effect was less evident on the HepG2 cells. However, the quality and quantity of cells in the wells at the end of the treatment were acceptable compared to the untreated wells.

5 Results of Marker Identification

5.1 levels of IL-6 in the Supernatants of U937

[0155] The levels of IL-6 in the supernatants were measured using the ELISA technique. The measurement was performed at 6 hours. The IL-6 levels in the U937 cells at 6 h are summarised in FIG. 1, where one notes a decrease in the IL-6 level in cells treated with the combination of vitamin D+sucrosomial iron (according to the invention) compared to those treated with vitamin D alone or with sucrosomial iron alone.

5.2 Expression of Hepcidin in Supernatants of HepG2 Cells

[0156] The expression of hepcidin produced by HepG2 cells is shown in FIG. 2. After 18 hours of treatment, the hepcidin levels were higher in the case of treatment with LPD and vitamin D. The cells treated with a combination of vitamin D+sucrosomial iron (according to the invention) showed a lower expression of hepcidin compared to those treated with vitamin D alone or with sucrosomial iron alone.

5.3 Levels of CRP in Supernatants of HepG2

[0157] FIG. 3 shows the levels of CRP (C-reactive Protein) produced by HepG2 cells in supernatants of cells after 24 hours of treatment with iron compounds, vitamin D and the association thereof. The association of vitamin D and sucrosomial iron (according to the present invention) is capable of decreasing CRP expression in a more marked manner than treatments with sucrosomial iron alone and vitamin D alone.

5.4 Conclusions

[0158] The combination of vitamin D and an iron oxide, complex or salt according to the present invention produces a marked reduction in the inflammatory markers IL-10, hepcidin and CRP. These results indicate the presence of a synergistic anti-inflammatory effect between component (a), i.e. vitamin D, and component (b), i.e. iron compound, according to the present invention.

II) CLINICAL STUDY

Aim

[0159] The aim of this study was to assess the support of vitamin D in improving the absorption of iron in patients with sideropenic anaemia and a vitamin D deficiency.

[0160] Vitamin D has an anti-inflammatory effect which induces the response of Th2 and Tregs and the polarization of M2 macrophages and production of hepcidin.

PATIENTS AND METHODS

[0161] In group A, 20 patients (M/F: 1/2), mean age 55 years (R 50-70), with vitamin D <10 ng/ml and a median value of haemoglobin (Hb) of 8.5 g/dl (R 8-9.5) and a median ferritin value of 20 ng/ml (2-28), were supported with sucrosomial iron 30 mg tid (ter in die, three times a day) for 3 months and cholecalciferol (vitamin D3) 4000 IU/day (according to the invention). In group A the median C-reactive protein (CRP) was 35 ng/ml (R 20-47) and 6 patients had a documented bacterial infection.

[0162] In group B, 20 patients (M/F: 1/2), mean age 60 years (R 55-68), with vitamin D <10 ng/ml and a median value of Hb of 8.2 g/dl (R 8-10) and a median ferritin value of 18 ng/ml (10-30), were supported with sucrosomial iron 30 mg tid for 3 months. In group B the median CRP was 28 ng/ml (R 20-32) and 7 patients had a documented bacterial infection.

RESULTS

[0163] In group A after 3 months the patients reached a median haemoglobin level of 12.5 g/dl (R 10-13). The patients with a bacterial infection reached a median haemoglobin level of 10 g/dl (R 9-11).

[0164] In group B after 3 months the patients reached a median haemoglobin level of 11.5 g/dl (R 9.5-12). The patients with a bacterial infection reached a median Hb level of 9.5 g/dl (R 9-10.5).

CONCLUSION

[0165] The administration of vitamin D jointly with the administration of sucrosomial iron (according to the invention) improves iron absorption in patients with a vitamin D deficiency, except in patients with a bacterial infection.