OVOTRANSFERRINS FOR USE IN THE TREATMENT OF IRON DEFICIENCY ANAEMIA
20240148837 ยท 2024-05-09
Assignee
Inventors
- Valerio Maria Ferrari (Milano (MI), IT)
- Maria Carla Baggio (Saronno (VA), IT)
- Paride GRISENTI (Milano, IT)
Cpc classification
A61K38/40
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
International classification
Abstract
Ovotransferrins with a degree of iron saturation from 0 to 15% are useful in the treatment of iron deficiency and iron deficiency anaemia, increasing the cell viability on gastric and intestinal cell models with respect to the holo-OvT 100%, when tested at the same concentrations, showing also a surprisingly better time dependent absorption of iron ions and a physiological trend allowing a single oral administration in 24 hours.
Claims
1. A method of treating iron deficiency and iron deficiency anaemia in a subject in need thereof, said method comprising administering to said subject a pharmaceutical effective amount of ovotransferrin with an iron content from 1-10 ppm to 201-246 ppm.
2. The method according to claim 1, wherein said ovotransferrin has an iron content from 1 to 10 ppm (apo-ovotransferrin).
3. The method according to claim 1, wherein said ovotransferrin has an iron content from 20 to 25 ppm to 201-246 ppm.
4. The method according to claim 3, wherein said ovotransferrin has an iron content of 221-227 ppm.
5. The method according to claim 1, wherein ovotransferrin is administered at a daily dose ranging from 100 to 400 mg in a single dose or twice a day.
6. A composition comprising as active ingredient an ovotransferrin with an iron content from 1-10 ppm to 201-246 ppm in combination with pharmaceutically or alimentary acceptable ingredients.
7. A composition according to claim 6 further comprising inorganic and/or organic iron compounds up to a maximum iron content equivalent to 74 mg/day.
8. A composition according to claim 7 wherein the iron compound is selected from ferrous sulphate, ferrous carbonate, ferric sodium diphosphate, ferric diphosphate, ferrous phosphate, elemental iron.
9. A composition according to claim 7 wherein the iron compound is selected from ferrous fumarate ferrous gluconate, ferrous citrate, ferric ammonium citrate, ferrous lactate, ferric saccharate, ferrous bisglycinate, ferrous L-pidolate, ferrous ammonium phosphate, ferric sodium EDTA.
10. A composition according to claim 6, in form of fortified food, food intended for infants and children, food for special medical purposes, total diet replacement for weight control, food supplements, food intended for sportsmen.
Description
DESCRIPTION OF THE FIGURES
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MATERIALS AND METHODS
[0046] Purified ovotransferrin from hen egg white have been supplied by BIOSEUTICA BV (NL) with different degree of iron saturation: apo form (not iron-bound) and holo form (iron-bound), the latter having 2 different degrees of iron saturation: 15% and 100% saturation. bLact employed in this study was purchased from the market (Biocon (Japan) LTD) and further analyzed by ICP showing an iron content of 157 ppm, which corresponds to a 11% iron saturation.
[0047] Caco-2 cell line, supplied by the American Type Culture Collection (ATCC), was cultured in DMEM-F12 (Sigma-Aldrich, St. Louis, US) containing 10% FBS (Sigma-Aldrich), 2 mM L-glutamine (Sigma-Aldrich), and 1% penicillin-streptomycin (Sigma-Aldrich) at 37? C. in an incubator at 5% CO.sub.2 (Christides, T et al. In-vitro assessment of iron availability from commercial Young Child Formulae supplemented with prebiotics. Eur. J. Nutr. 2016, 9, 1-10). Caco-2 cells were plated at 1?10.sup.4 cells in 96-well plates to study cell viability via the MTT test and to analyze the intracellular pathways through western blot analysis, cells were plated on 60-mm culture dishes until confluence. For absorption study, cells were plated 2?10.sup.4 cells on 6.5 mm Transwell? with 0.4 ?m pore polycarbonate membrane insert (Sigma-Aldrich) in a 24-well plate and cells were maintained in complete medium, changed every other day, first basolaterally and then apically for 21 days before the stimulations.
[0048] GTL-16 cell line is a clonal line derived from a poorly differentiated gastric carcinoma cell line (Giordano S et al. p145, a protein with associated tyrosine kinase activity in a human gastric carcinoma cell line. Mol Cell Biol. 1988; 8:3510-7) and is widely used as a model of gastric epithelial cells. This cell line was donated by the Laboratory of Histology of the University of Eastern Piedmont (Novara, Italy). Cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 1% penicillin-streptomycin in incubator at 37? C., 5% CO.sub.2. The cells for the experiments were plated in different ways: to study cell viability (MTT test) and to analyze the intracellular pathways through Western blot analysis the cells were plated on 60 mm dishes until confluence. Before stimulation, the cells were maintained in DMEM without red phenol and FBS and supplemented with 1% penicillin/streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate in incubator at 37? C., 5% CO.sub.2, and 95% humidity for 18 h. Then the cells were treated with different agents in DMEM without red phenol and supplemented with 0.5% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine and 1 mM sodium pyruvate. GTL-16 cells were plated 2?10.sup.4 onto 6.5 mm translucent polyethylene terephthalate (PET) Transwell? insert 0.4 ?m in a 24 well-plate to study absorption and were maintained in complete medium, changed every other day, first basolaterally and then apically for 21 days before the stimulations.
[0049] Cell Proliferation Kit I (MTT). MTT dye (Sigma-Aldrich) was used to determine cell viability. After stimulation, cells were incubated with 1% MTT dye for 2 h at 37? C. in incubator as described in literature (Natoli M et al. Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. J Cell Physiol. 2011 June; 226(6):1531-43). The purple formazan crystals (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) were dissolved in DMSO. Cell viability was determined by measuring the absorbance through a UV spectrometer (VICTOR X3 Multilabel Plate Reader) at 570 nm with correction at 690 nm, and cell viability calculated by comparing results to control cells (100% viable).
[0050] Gastric model. A first model has been setup to mimic a gastric system. An in-vitro gastric barrier model has been created using gastric epithelial cells GTL-16 subclone derived from MKN-45 cells. This model employs human gastric epithelial cells, derived from squamous adenocarcinoma that maintains a wild type phenotype, thus preserving both the kinase activities and the phenotypic expression unchanged. The employed model has been validated in literature (Fernandes I et al. A new approach on the gastric absorption of anthocyanins. Food Funct. 2012 May; 3(5):508-16; Uberti F et al. Iron Absorption from Three Commercially Available Supplements in Gastrointestinal Cell Lines. Nutrients. 2017 Sep. 13; 9(9).). This model allows to measure the permeability and the integrity of the gastric barrier. At the end of 21 days, it has been possible to start the treatment to evaluate the passage to the underlying layer in terms of cell viability (in order to exclude any toxicity), free radical production (to exclude adverse effects), and concentration of iron using a special kit (Iron Assay). This data is relevant to determine the proportion of the OvT products (100% and 15% holo-OvT over the limit of efficacy comparable to 11% holo-bLact from the market; apo-OvT) that can reach the intestine or be readily available, compared to commercial product. The treatment has been evaluated for 4 hours (maximum absorption time of the molecules) and on-time points as follows: 1 h-2 h-3 h-4 h. The same conditions have been reproduced in presence of iron 3+ to define the mechanism under conditions of food supplementation. In addition, the iron transport mechanism has been analyzed by Western blot [2,3] for ferritin, ferroportin, DMT-1.
[0051] Intestinal model. A second model has been set up for the bioavailability study after the transition from the intestinal system. For this reason, an in-vitro intestinal barrier model has been created, according to the model below reported, using CaCo-2 cells. After 21 days of growth, cells were ready and it was possible to start treatments, evaluating the transition to the underlying layer in terms of cell viability or layer integrity (to exclude any toxicity), free radical production (to exclude adverse effects) and iron concentration physiologically present by means of a special kit (Iron assay). These tests have been maintained at the same experimental conditions and treatments of the first model (i.e. mimic a gastric system). The treatment has been evaluated over a period of 6 h (maximum time of absorption of the molecules), with on-time points of 1 h-2 h-3 h-4 h-5 h-6 h. The same conditions have been reproduced in presence of iron 3+ to identify the mechanism in conditions of food supplementation.
[0052] The iron transport mechanism has also been analyzed for ferritin, ferroportin, DMT-1.
[0053] 3D gastro-intestinal model: To evaluate if the two models, gastric and intestinal, separately observed, are really able to promote the increase of plasma iron, a further three-dimensional (3D) model has been used, that allows to put the two cell populations (gastric and intestinal) in direct communication, mimicking what happens in a complex organism (gastro-intestinal model). The 3D model Vitvo? has been used for this experiment. Two fundamental parameters have been analyzed using this model: the concentration of OvT and iron
[0054] Caco-2 Permeability assay. After 21 days, two different concentrations of OvT and bLact were added to culture medium under different pH conditions, as reported in literature (Natoli M et al. A. Cell growing density affects the structural and functional properties of Caco-2 differentiated monolayer. J Cell Physiol. 2011 June; 226(6):1531-43; Uberti F et al. Iron Absorptionfrom Three Commercially Available Supplements in Gastrointestinal Cell Lines. Nutrients. 2017 Sep. 13; 9(9):1008). During treatments, the cells were maintained in incubator at 37? C., 5% CO.sub.2, and, at the end of stimulations, the iron quantity and OvT/bLact concentration were measured by specific kit.
[0055] GTL-16 Permeability assay. After 7 days, to study the effects on absorption of apical-to-basolateral pH gradients, the medium was changed on both the apical and basolateral sides adding HCl to the medium to obtain pH 3 at the apical side for 60 min, as reported in the literature [8,7]. At the defined time scheduled, the treatments were performed in the same conditions and manner as previously described, and then iron quantity and OvT/bLact concentration were detected with the kit.
[0056] bLact Determination. The concentration of bLact which crossed through gastric and intestinal barriers was measured according the method below described. At the end of stimulations, the basolateral volume was analyzed through a UV spectrometer (VICTOR X4, multilabel plate reader) at 320 nm, and the absorbance related to the standard curve was obtained. The results were expressed as means?SD (%) of absorption, normalized to the control.
[0057] OvT Determination. The concentration of OvT which crossed through gastric and intestinal barriers was measured according the method below summarized. Briefly, at the end of stimulations, the basolateral volume was analyzed through a UV spectrometer (VICTOR X4, multilabel plate reader) at 450 nm, and the absorbance related to the standard curve was obtained. The results were expressed as means?SD of absorption compared to the control.
[0058] Lactoferrin Ovotransferrin Quantification Assay. The Human Transferrin ELISA kit is designed to measure the amount of the target bound between a matched antibody pair following the manufacturer's instructions. A target-specific antibody has been pre-coated in the wells of the supplied microplate. Samples are added into these wells and bind to the immobilized (capture) antibody. The sandwich is formed by the addition of the second (detector) antibody, in particular a substrate solution is added that reacts with the enzyme-antibody-target complex to produce measurable signal. The intensity of this signal is directly proportional to the concentration of target present in the original specimen. Briefly, 100 ?L of diluted samples were incubated overnight at 4? C., washed 4 times with 1? Wash Buffer and then 100 ?L of biotin conjugate were added to each well and incubated for 1 hour at room temperature with gentle shaking. Then the wells were washed 4 times and 100 ?L of Streptavidin-HRP and 100 ?L of TMB Substrate were added. Finally, the plate was incubated for 30 minutes at room temperature under dark with gentle shaking and stopped with 50 ?L of Stop Solution. The absorbance was measured by spectrometer at 450 nm (VICTOR X4, multilabel plate reader).
[0059] Iron Quantification Assay. Iron Assay Kit (Sigma-Aldrich) which measures ferrous iron (Fe.sup.2+), ferric iron (Fe.sup.3+), and total iron (total iron-ferrous iron) in samples was used on astrocytes following the manufacturer's instructions. The absorbance at 593 nm was measured by a spectrometer (VICTOR X4, multilabel plate reader). Ferric iron concentrations are equal to the total iron (sample plus iron reducer)-Fe.sup.2+ (sample plus assay buffer). The results were expressed as means?SD (%) of absorption, normalized to the control.
[0060] Western Blot for ferritin, ferroportin, DMT-1. Caco-2 and GTL-16 cells were washed and then lysed in ice with Complete? Tablet buffer (Roche, Basel, Switzerland) supplemented with 2 mM sodium orthovanadate (Na.sub.3VO.sub.4), and 1 mM phenylmethanesulfonylfluoride (PMSF; Sigma-Aldrich), 1:50 mix phosphatase inhibitor cocktail (Sigma-Aldrich), and 1:200 mix Phosphatase Inhibitor Cocktail. A quantity of 35 ?g of protein from each lysate was loaded on 15 or 5% SDS-PAGE gels and transferred to polyvinylidene fluoride membranes (PVDF, GE Healthcare Europe GmbH, Milan, Italy). These were incubated overnight at 4? C. with specific primary antibodies: anti-ferritin (1:1000, Santa-Cruz), anti-ferroportin-1 (1:2000, Santa-Cruz), anti-DMT-1 (1:400, Santa-Cruz). Protein expression was normalized and verified through ?-actin detection (1:5000; Sigma, Milan, Italy) and expressed as mean SD (% vs control).
[0061] Statistical analysis. For each protocol, at least five independent experiments were performed and the results are expressed as means SD of independent experiments performed on four technical replicates. One-way ANOVA followed by Bonferroni post hoc tests were used for statistical analysis, and pairwise differences compared by Mann-Whitney U tests. p-value<0.05 were considered statistically significant.
Experimental Section
[0062] Preparation of Ovotransferrin 15% Iron Saturated (Holo OvT 15%)
[0063] Load in a glass line reactor water (5 liters) and under stirring at room temperature Apo-Ovotransferrin (680 g; 8 mmol) and maintain the mixture under stirring at room temperature for 3 hours. Sodium bicarbonate (0.21 g) is the added to the reaction mixture, under vigorous stirring, to a final stable pH value of 8.54 then, after 30, FeCl.sub.3.6H.sub.2O (0.59 g; 2.2 mmol) was added and the final pH value maintained to 8,8?0,2 by addiction of a 10% sodium carbonate aqueous solution. After 15 under stirring at room temperature, Celite? (10.98 g) was added and the obtained mixture filtered under pressure on a 6 Filtrox AF6 panel model. The filtrate is then freeze dried to afford 580 g of Holo OvT 15%.
[0064] The ICP analyses of this product confirmed a final iron content of 210 ppm. The iron saturation of the obtained product was also confirmed to be 15% by a spectrophotometric titration at 465 nm against the apo OvT form saturated with and excess of FeCl.sub.3-6H.sub.2O in a 20 mM citrate/bicarbonate buffer at pH 9,0.
[0065] Preparation of Ovotransferrin 100% Iron Saturated (Holo OvT 100%)
[0066] The above-described procedure for the preparation of holo Ovt 15% was applied, with some modifications, also for the preparation of holo OvT 100% using 7 mol of FeCl.sub.3.6H.sub.2O for 1 mol of apo Ovt (relative ratio between OvT/iron 3+=1/7 instead of 1/0.28 utilized for the preparation of holo OvT 15%). In this process before the freeze drier treatment the mixture was ultrafiltered to remove the excess of iron salts on a 50 KDalton membrane and then filtered on a 3 ?m filter. The ICP analyses of this product confirmed a final iron content of 1400 ppm.
[0067] Dose-Response Study of Cell Viability on GTL-16 and Caco2 Cells
[0068] Different concentrations of OvT (apo and holo forms with different degree of iron saturation) and bLact have been tested for 24 h, and from this dose-response study, it was possible to identify the concentrations influencing the viability of GTL-16 and CaCo2 cells prior to start the experiments. Since the test products are different and could affect the cell viability, the MTT test was performed on gastric and intestinal cells. The selected concentrations for all the test products have been: 200 ?g/ml, 100 ?g/ml, 50 ?g/ml and 30 ?g/ml. All the OvT samples tested have shown to have a positive effect on cell viability, at least comparable to bLact (
[0069] These data confirmed that the two lower concentrations 50 ?g/ml and 30 ?g/ml of the three OvT tested have an effect comparable to bLact and no cytotoxicity on the gastric and intestinal cells, even though holo-OvT 15% saturated and apo-OvT has shown to be better in terms of mitochondrial metabolic well-being.
[0070] Time-Dependent Study on GTL-16 and Caco2 Cell Line
[0071] On the basis of these tests 30 ?g/ml and 50 ?g/ml concentrations have been selected for all subsequent experiments: GTL-16 and CaCo2 cells have been stimulated with the 3 different iron saturation OvT at the above selected concentration (i.e. 50 ?g/mL and 30 ?g/mL) for 1, 3, 6 and 24 hours. At the end of the stimulations, an MTT test was performed to evaluate cell viability. As shown in
[0072] In particular, for Holo-OvT 15% iron saturated the effects are maintained also at 24 hours indicating a slow-release mechanism with a better physiologic profile respect to holo-OvT 100%.
[0073] Regarding the Caco2 cell, a time-dependent effect has also been observed which has reached an apex at 3 h, that was preserved at 6 h, and tended to decrease towards 24 h.
[0074] These results obtained in both cell types, have confirmed that OvT has a time-dependent effect and have suggested that holo-OvT 15% saturated at concentration of 30 ?g/mL could be the best option compared to control (p<0,05) and also compared to the bLact tested. Furthermore, based on the results, one dosage in 24 h in humans can be assumed.
[0075] Time Dependent Permeability of Gastro-Intestinal Barrier after Stimulation with Ovotransferrin
[0076] In order to study the biological functions of OvT, some experiments were performed to evaluate the OvT absorption on GTL-16 cells, Caco-2 cells and in a 3D model mimic the gastro-intestinal barrier.
[0077] As shown in
[0078] The same quantification was performed at the basolateral level of intestinal barrier model (CaCo2 cell). In
[0079] Three-Dimensional (3D) In-Vitro Model Mimickin In Vivo Complexity of the Gastro-Intestinal Barrier
[0080] In order to collect more data on the compounds under investigation, respect the two experimental models (gastric and intestinal) till now used separately, we utilized a more integrated gastro-intestinal model able to replicate in vitro the complexity of the gastro intestinal barrier. For this purpose, a three-dimensional (3D) validated in-vitro model mimicking in vivo complexity of the gastro-intestinal barrier was used. An intestinal barrier was recreated in-vitro and stimulated with the basolateral part previously collected from the gastric barrier; subsequently, the basolateral part was collected and analyzed with special kits to evaluate the quantity of Ovt and bLact passed through the barrier. This model puts the two cell populations in direct communication, as in a living organism. In this context, 50 ?g/ml and 30 ?g/ml of holo-OvT 100% and 15% saturated have been tested at the same timeframe previously used (1, 3 and 6 hours). The data obtained by this study (
[0081] Analysis of Molecular Pathways Involved by Ovotransferrin: Evaluation on DMT-1, Ferritin and Ferroportin Expression
[0082] To better define the involved mechanisms, different markers such as transferrin, ferritin, and DMT-1 have been evaluated by Western blot. DMT-1 (divalent metal transporter 1) is a very important transporter of bivalent metals, with a fundamental role in transporting ferrous ions, its effects has been analyzed by stimulating the 3D gastro-intestinal barrier models with 50 ?g/ml and 30 ?g/ml of holo-OvT 100% saturated and 15% saturated. As shown in
[0083] With the aim to better understand the biochemistry of the molecules under investigation on the iron metabolism we evaluate the effects on the different iron transport chains particularly of the transmembrane protein ferroportin and of the intracellular protein ferritin responsible respectively for extruding iron ions from a cell and for sequestering the iron ions in a non-toxic form inside the cells. Particularly the ferritin expression has an important role in iron homeostasis acting a reservoir of iron in the organism. The obtained data (
[0084] Finally, we have analyzed the expression of ferroportin: after 24 h of treatment, as shown in
[0085] All these experiments confirmed that holo-OvT 15% iron saturated is able to exert its positive effects on the iron metabolism, and particularly on the main proteins involved in the transport and storage of iron ions in the cells, in a better way compared to the tested Holo-OvT 100% saturated and to bLact.
[0086] Dose-Response Study of Cell Viability on GTL-16 and Caco2 Cells after Pretreatment with Fe3+
[0087] For this study only the 2 best performing concentrations of OvT and bLact, 50 ?g/ml and 30 ?g/ml, have been tested. Using GTL-16 cells as an in-vitro gastric model, firstly the cells have been treated with Fe3+ (final concentration 75 ?M) for 24 h, then followed by the treatment with OvT and bLact for 24 h. The obtained results show (
[0088] Dose-Response Study of Cell Viability on 3D Gastro-Intestinal Barrier Model in Presence of Iron
[0089] In this study a 3D gastro-intestinal barrier model has been used as a model mimicking the human complexity in order to evaluate the effects in a complex system and exclude possible adverse reactions. First, a gastro-intestinal barrier model made of a Transwell? transporting set-up have been used, which was also treated with Fe3+ (final concentration 75 ?M) for 24 h, then followed by treatment with OvT and bLact for 24 h. The results of cell viability (
[0090] Ovotransferrin Absorption on 3D Gastro-Intestinal Barrier Model in Presence of Iron
[0091] Having assessed the cell viability with test Products in presence of iron, we have carried out further experiments to study the biological effect of OvT, evaluating its absorption in 3D gastro-intestinal barrier model in the presence of Fe.sup.3+. The quantitative analysis of the basolateral environment of the gastro-intestinal barrier model confirmed a sustained slow absorption and release over time (3 and 6 hours), confirming the slow-release effect (
[0092] Furthermore, apo-OvT (standard 50 and 30) has shown to be greatly absorbed over the time in presence of iron (p<0.05 vs control, p<0.05 vs tested bLact), suggesting that apo-OvT could be an optimal candidate for administration to human under physiological conditions in presence of food.
[0093] Iron Absorption on 3D Gastro-Intestinal Barrier Model in the Presence of Iron and Ovotransferrin
[0094] We have further used our gastro-intestinal 3D barrier model pre-treated with iron (Fe.sup.3+) to check the iron bioavailability and absorption in presence of OvT. The results of quantitative analysis of the basolateral environment (
[0095] More into details holo-OvT 15% iron saturated at the concentration of 30 ?g/ml gave better results in the absorption on the long term (after 6 hours) compared to control (p<0,05) and also respect to bLact (p<0,05). Moreover apo-OvT, particularly at the concentration of 50 ?g/ml, showed best results in enhancing the iron bioavailability over time, particularly if compared to the tested bLact (p<0,05).
[0096] Analysis of the involved molecular mechanisms on the 3D gastro intestinal barrier model We proceeded with our 3D gastro-intestinal barrier model to check the expression of DMT-1, ferritin, and ferroportin in order to have a complete image of what would happen in humans.
[0097] DMT-1
[0098] DMT-1 has been tested (densitometric analysis of DMT-1 expression) using the 3D gastro-intestinal barrier model pre-treated with Fe.sup.3+ (75 ?g/ml) followed by 24 h treatments with 50 ?g/ml and 30 ?g/ml of different OvT products and bLact. DMT-1 expression (
[0099] Ferritin
[0100] We investigated the ferritin expression in the 3D gastro-intestinal barrier model pre-treated with Fe.sup.3+ (75 ?g/ml) followed by 24 h treatments with 50 ?g/ml and 30 ?g/ml of different OvT products and bLact. The results obtained from both compartments (
[0101] Ferroportin
[0102] Ferroportin is the only known mammalian iron exporter for non-heme iron. After feeding, dietary iron is absorbed into the cells of the small intestine and ferroportin allows that iron to be transported out of those cells and into the bloodstream. After 24 h of iron treatment (Fe.sup.3+ 75 ?g/ml) of gastric-intestinal 3D barrier model followed by treatment with OvT and bLact (