CN111773247B

CN111773247B - Application of iron sucrose in preparation of medicine for treating hyperphosphatemia-induced vascular calcification

Info

Publication number: CN111773247B
Application number: CN202010725568.5A
Authority: CN
Inventors: 汪平; 彭丹; 杨举红; 胡宁; 任慧敏; 郭成坤; 全正莉; 宋艳; 潘慧; 陈望善
Original assignee: Jingmen First Peoples Hospital
Current assignee: Jingmen First Peoples Hospital
Priority date: 2020-07-24
Filing date: 2020-07-24
Publication date: 2022-04-05
Anticipated expiration: 2040-07-24
Also published as: CN111773247A

Abstract

The invention belongs to the technical field of vascular calcification treatment, and particularly relates to an application of iron sucrose in preparation of a vascular calcification medicament for treating hyperphosphatemia induction. In the experiment, the invention finds that the iron sucrose can interfere the transmembrane transport of inorganic phosphorus in blood, inhibit the expression of phosphorus transmembrane transport protein and antagonize the oxidative stress injury induced by hyperphosphatemia, thereby playing a role in treating, relieving or inhibiting the vascular calcification induced by hyperphosphatemia.

Description

Application of iron sucrose in preparation of medicine for treating hyperphosphatemia-induced vascular calcification

Technical Field

The invention belongs to the technical field of vascular calcification treatment, and particularly relates to an application of iron sucrose in preparation of a vascular calcification medicament for treating hyperphosphatemia induction.

Background

Vascular calcification is mainly manifested by increased stiffness of the vessel wall, reduced compliance, and brittle and thin vessel wall, which makes the vessel easily burst. The incidence rate of vascular calcification in patients with chronic kidney diseases is high, the harm is large, but the current pathogenesis of vascular calcification is not completely clarified, and an effective prevention and treatment means is still lacking clinically.

Hyperphosphatemia is a common complication of patients with chronic kidney diseases and is an important cause of vascular calcification. Hyperphosphatemia causes oxidative stress injury in vascular smooth muscle cells, which in turn induces phenotypic transformation of vascular smooth muscle cells into osteoblasts and eventually into vascular calcification. At present, an effective prevention and treatment means is also lacked aiming at vascular calcification induced by hyperphosphatemia.

Disclosure of Invention

In view of the defects or the improvement needs of the prior art, the invention aims to provide the application of the ferrosucrose preparation in preparing the medicine for treating hyperphosphatemia-induced vascular calcification, the preparation mainly blocks the phenotypic transformation of vascular smooth muscle cells to osteoblasts by relieving or inhibiting oxidative stress injury caused by hyperphosphatemia so as to inhibit vascular calcification, and the invention aims to solve the problem that the prior art lacks an effective treatment means for hyperphosphatemia-induced vascular calcification.

In order to achieve the purpose, the invention provides an application of a ferrosucrose preparation in preparing a medicament for treating, relieving or inhibiting oxidative stress injury caused by hyperphosphatemia.

According to another aspect of the invention, there is provided the use of a sucron preparation in the manufacture of a medicament for the treatment, alleviation or inhibition of hyperphosphatemia-induced vascular calcification.

Preferably, the dosage form of the sucralfate preparation is injection.

Further preferably, the dosage form is an intravenous dosage form.

Preferably, the formulation is administered at a frequency ranging from once a month to once a day; each dose is in the range of 50mg to 200 mg; and when administered at a frequency of once daily, the number of consecutive administrations does not exceed 5.

Preferably, the concentration of sucroferric in the blood of the formulation does not exceed 5 μ g/mL.

Further preferably, the concentration of sucroferric in the formulation is from 2 μ g/mL to 5 μ g/mL in the blood.

Preferably, the hyperphosphatemia corresponds to a serum inorganic phosphorus concentration of greater than 2.0 mmol/L.

Through the technical scheme, compared with the prior art, the invention can obtain the following beneficial effects:

the present invention surprisingly found that ferric saccharate can alleviate hyperphosphatemia-induced vascular calcification by the following mechanisms: the hyperphosphatemia causes oxidative stress injury in vascular smooth muscle cells, then induces the vascular smooth muscle cells to transform to osteoblast phenotype, and finally develops into vascular calcification, and the iron sucrose can block the pathway by interfering the transmembrane transport of inorganic phosphorus in blood, inhibiting the expression of phosphorus transmembrane transport protein in blood and antagonizing the oxidative stress injury induced by the hyperphosphatemia, thereby having the effect of treating, relieving or inhibiting the vascular calcification induced by the hyperphosphatemia.

Hyperphosphatemia is particularly common in patients with chronic kidney diseases, and the invention can be applied to the patients with hyperphosphatemia to reduce the incidence rate of cardiovascular events of the patients, prolong the life cycle of the patients, improve the life quality and reduce the huge economic and social burden of chronic kidney diseases on the world.

Drawings

FIG. 1 shows the effect of different concentrations of sucron on the stimulation of calcium and iron deposition in the vascular annulus by high phosphorus.

FIG. 2 is a graph showing the effect of different concentrations of sucron on oxidative stress injury in hyperphosphatically stimulated blood vessels.

FIG. 3 is a graph of the effect of different concentrations of sucron on hyperphosphatically stimulated vascular smooth muscle cell phenotypic transformation.

FIG. 4 is a graph showing the results of detection of protein expression of alpha-SMA, Runx2, Pit1 and FGF23 by Western blot.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Hyperphosphatemia (serum inorganic phosphorus > 2.0mmol/L) is a common complication of patients with chronic kidney diseases and is an important cause of vascular calcification. Hyperphosphatemia causes oxidative stress injury in vascular smooth muscle cells, which in turn induces phenotypic transformation of vascular smooth muscle cells into osteoblasts and eventually into vascular calcification.

Sucrose iron CAS number: 8047-67-4, EINECS No.: 232-464-7; molecular formula C₆H₈FeO₈And the molecular weight is 263.9679. Ferric saccharate is currently used clinically to treat iron deficiency anemia as an iron supplement. However, the experiments of the invention unexpectedly find that the iron saccharate can relieve and inhibit oxidative stress injury induced by hyperphosphatemia and obviously reduce the calcification index of blood vessels.

It is widely believed that ferric ions have a strong oxidizing property and may aggravate oxidative stress damage of tissues and organs. However, in some embodiments of the present invention, the vascular rings of SD rats are cultured in the normal blood phosphorus or hyperphosphatemia environment respectively, and the calcification index and the oxidative stress index of the vascular rings are detected after the intervention by adding different concentrations of ferric saccharate, so that the calcium deposition in the vascular rings is obviously reduced with the increase of the concentration of ferric saccharate; and the iron sucrose can obviously reduce the content of reactive oxygen species and inhibit the oxidative stress injury of vascular smooth muscle cells induced by hyperphosphatemia by analyzing the oxidative stress injury index of the blood vessels. Thus, ferric saccharate not only does not exacerbate oxidative stress injury, but also blocks this pathway by antagonizing hyperphosphatemia-induced oxidative stress injury, thereby acting to treat, alleviate or inhibit hyperphosphatemia-induced vascular calcification.

The prior art proposes the use of phosphate binders for treating hyperphosphatemia, such as PA21, which is based on ferric saccharate and is formulated as chewable tablet for use. PA21 is used to reduce blood phosphorus mainly by binding phosphorus in food in the intestinal tract to reduce phosphorus intake. Unlike PA21, the present invention adopts ferric saccharate preparation for intravenous administration to reach effective blood concentration in blood and inhibit the expression of P transmembrane transporter Pit 1. Therefore, the invention interferes the transmembrane transport of inorganic phosphorus in blood by intravenous injection of the iron saccharate, inhibits the oxidative stress injury of vascular smooth muscle cells induced by the inorganic phosphorus, blocks the phenotypic transformation of the vascular smooth muscle cells, further reduces the calcium deposition in the blood vessels, and achieves the purpose of treating, relieving or inhibiting the calcification of the blood vessels.

In some embodiments, the ferric saccharate formulation of the present invention is in the form of an injection. The iron sucrose has great oral side effect, may cause damage to digestive tract, and through years of clinical observation, the iron sucrose is safe and effective for intravenous administration.

In the embodiment of the invention, the iron saccharate can treat, relieve or inhibit hyperphosphatemia-induced vascular calcification, and the effect is dose-dependent. The sucralfate preparation not only ensures an effective vascular calcification-inhibiting effect but also avoids unsafe iron deposition within a certain dosage range, however, exceeding a certain range may lead to unsafe iron deposition.

In some embodiments, the sucralfate formulation is administered in sub-doses ranging from 50mg to 200mg, at a frequency ranging from once a day to once a month, which varies depending on the patient's sucralfate concentration, serum phosphorus level, and iron deficiency level. If once daily, continuous administration is required for no more than 5 days, and once every half month or once a month is preferred instead after every 5 days of continuous administration.

In other embodiments, the formulation maintains a plasma concentration of sucroferric iron of no more than 5 μ g/mL, preferably within a dosage range of 2 μ g/mL to 5 μ g/mL, to avoid intravascular unsafe iron deposition.

The ferric saccharate injection (trade name "Sentieng", "Weilefu" and "Tietai") is clinically used for patients who have poor effect of oral iron preparation and need intravenous iron treatment at present. The invention unexpectedly finds that the iron saccharate can be used for treating, relieving or inhibiting vascular calcification induced by hyperphosphatemia, can obviously reduce the calcification index of the blood vessel and has good treatment effect. But also can directly adopt the preparation of the prior commercial iron sucrose injection and the dosage and the use method thereof, and has good safety. The specific usage amount can also be adaptively adjusted according to the blood concentration of sucrose and iron, serum phosphorus index and iron deficiency index of a patient, so as to ensure safety and effectiveness.

The following are specific examples:

example 1

Male SD (Sprague-Dawley) rats were euthanized 1 week after acclimation and the aortic vascular rings were placed in each experimental group for 14 days. The grouping situation is as follows: CNT, normal control (regular medium); HP, high phosphorus group (conventional Medium +2.5mM PO)₄ ^3-) (ii) a HPLFe, high phosphorus low iron group (conventional Medium +2.5mM PO)₄ ^3-+2. mu.g/ml sucron); HPMFe, high phosphorus medium iron group (conventional Medium +2.5mM PO)₄ ^3-+ 5. mu.g/ml sucron); HPHFe, high phosphorus and high iron group (conventional Medium +2.5 mMPO)₄ ^3-+ 10. mu.g/ml iron sucrose).

Wherein, the conventional culture medium comprises the following components: conventional DMEM medium (dulbecco's modified eaglemedium) + 10% FBS (total bone serum) + 1% double antibody (streptomycin qing mixture).

The vascular rings of each experimental group were subjected to calcium staining (Von Kossa staining), iron staining (Perls' Prussianblue staining), and the calcium and iron contents in the vascular rings of each experimental group were determined.

Von Kossa staining procedure:

1. paraffin section dewaxing to water: placing the slices in xylene I20 min-xylene II 20 min-absolute ethyl alcohol I10 min-absolute ethyl alcohol II 10 min-95% alcohol 5 min-90% alcohol 5 min-80% alcohol 5 min-70% alcohol 5 min-distilled water washing.

2. Silver leaching: the prepared silver nitrate solution is dripped on the tissue and incubated for 45min in the dark.

3. Color development: the sections were placed in the sun or under UV light for 15 min.

4. And (3) film washing: washing with double distilled water for 5 min.

5. Counterdyeing: re-dyeing the core for 5min, and flushing with running water for 5-10S.

6. Dewatering and sealing: placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, air drying, and sealing with neutral gum.

Perls' Prussian blue staining procedure:

2. Dyeing iron element: a 2% aqueous solution of potassium ferrocyanide: and 2% hydrochloric acid solution is mixed according to the ratio of 1:1 and then is subjected to drop dyeing for 1 h. And (5) washing with water.

3. Nuclear fixed red dye liquor: and putting the slices into a nuclear fast red dyeing solution for dip dyeing for 5min, and washing with water.

4. Dewatering and sealing: placing the slices in 95% alcohol I5 min-95% alcohol II 5 min-absolute ethanol I5 min-absolute ethanol II 5 min-xylene I5 min-xylene II 5min to dehydrate and transparent in sequence, taking out the slices from xylene, air drying, and sealing with neutral gum.

As shown in FIG. 1, the content A in FIG. 1 is a staining pattern of calcium staining (Von Kossa staining, red staining representing positive calcium staining) and iron staining (Perls' Prussian blue staining, blue staining representing positive iron staining) of the blood vessels of each experimental group; in FIG. 1, the content B is the result of the calcium deposition and iron deposition in the blood vessel ring (the ordinate unit is mg/gprot, where "mg" represents the content of Ca or Fe in a certain sample, and "gprot" represents the content of protein in the same sample, and mg divided by gprot can more accurately reflect the content of Ca and Fe in a unit tissue, so as to eliminate the difference caused by different sizes of the materials).

In FIG. 1 Ca represents calcium; fe represents iron; CNT represents the normal control group (regular medium); HP stands for high phosphorus group (conventional Medium +2.5mM PO)₄ ^3-) (ii) a HPLFe represents the high phosphorus low iron group (conventional Medium +2.5mM PO)₄ ^3-+2. mu.g/ml sucron); HPMFe represents the high phosphorus medium iron group (conventional Medium +2.5mM PO)₄ ^3-+ 5. mu.g/ml sucron); HPHFe stands for high phosphorus and high iron group (conventional medium +2.5 mMPO)₄ ^3-+ 10. mu.g/ml iron sucrose). Denotes P<0.05vs. CNT; denotes P<0.01vs. CNT; delta represents P<0.05vs.HP；ΔΔ,P<0.01vs.HP；#；P<0.05vs.HPLFe；##，P<Hplfe at 0.01vs. Where P-values refer to statistical differences between the two sets of data. P<0.05 represents the sameThe difference is statistically significant.

In fig. 1, the content A and the content B respectively represent the calcium content and the iron content of the vascular rings of different experimental groups. As can be seen from fig. 1, significant calcium deposition occurred in the high phosphorus group, and gradually decreased as the concentration of iron sucrose increased; when the iron sucrose concentration was 5. mu.g/ml, significant iron deposition occurred in the vascular ring, and the iron deposition was further aggravated as the iron sucrose concentration was increased.

The results in FIG. 1 show that: the stimulation of high phosphorus induces the calcification of blood vessels, and the sucrose iron can relieve the calcification of blood vessels in a dose-dependent manner; 2-5 mug/ml is probably the more appropriate blood concentration of the iron sucrose with both curative effect and safety.

Example 2

Reactive Oxygen Species (ROS) fluorescent probe DHE staining, MDA and SOD detection were performed on the vascular rings of each experimental group of example 1.

DHE staining procedure:

1. the solution was dissolved in DMSO (dimethyl sulfoxide) to give a 5mM solution, and the solution was kept away from light for further use.

2. The solution is diluted for later use according to the ratio of 1:1000 before use.

3. The animals were sacrificed and the vascular rings were removed, washed gently with PBS and frozen.

4. The frozen vessel rings were sectioned with a cryomicrotome.

5. After the preparation of the slide, the diluted probe solution was added dropwise to the tissue. Incubate at 37 ℃ for 30 min.

PBS wash off excess probe solution and block with anti-fluorescence quencher.

7. Observed with a fluorescence microscope and photographed.

The detection results are shown in fig. 2, and content a in fig. 2 is ROS (reactive oxygen species) staining (fluorescent probe DHE staining) of the blood vessel rings of each experimental group, wherein: red fluorescence represents that ROS stains positively, blue fluorescence represents that DAPI (4',6-diamidino-2-phenylindole) stains cell nucleus, and MERGE is a combined graph of ROS and DAPI; the content B is the detection result of the MDA in the vascular ring (malondialdehyde, the higher the MDA is, the more serious the oxidative damage is) and the SOD activity (superoxide dismutase, the lower the SOD is, the more serious the oxidative damage is). Wherein, ROS, active oxygenClustering; MDA, malondialdehyde; SOD, superoxide dismutase; CNT, normal control (regular medium); HP, high phosphorus group (conventional Medium +2.5mM PO)₄ ^3-) (ii) a HPLFe, high phosphorus low iron group (conventional Medium +2.5mM PO)₄ ^3-+2. mu.g/ml sucron); HPMFe, high phosphorus medium iron group (conventional Medium +2.5mM PO)₄ ^3-+ 5. mu.g/ml sucron); HPHFe, high phosphorus and high iron group (conventional Medium +2.5mM PO)₄ ^3-+ 10. mu.g/ml iron sucrose). A, P<0.05vs.CNT；**，P<0.01vs.CNT；Δ，P<0.05vs.HP；ΔΔ,P<0.01vs.HP；#，P<0.05vs.HPLFe；##，P<0.01vs.HPLFe。

Content A in FIG. 2 shows the results of ROS staining (fluorescent probe DHE staining) in the vascular rings of different experimental groups, and it can be seen that the ROS content in vascular smooth muscle cells is obviously increased by high phosphorus stimulation, and gradually decreases with the increase of iron sucrose concentration.

The left graph of content B in FIG. 2 shows the MDA concentration in the vascular rings of different experimental groups, and it can be seen that the MDA content in vascular smooth muscle cells is obviously increased by high phosphorus stimulation, and the increased MDA is gradually reduced along with the increase of the iron sucrose concentration. The right graph shows the SOD activity of different experimental groups, and the SOD activity in vascular smooth muscle cells is obviously reduced by high-phosphorus stimulation, and the inhibited SOD activity is gradually recovered along with the increase of the iron concentration of sucrose.

Figure 2 results show that: hyperphosphatemia stimulates oxidative stress damage in vascular smooth muscle cells, and sucron dose-dependently reduces this damage.

Example 3

Detecting the gene expression of alpha-SMA, Runx2, Pit1 and FGF23 by using an RT-qPCR method on the vascular rings of each experimental group in example 1; protein expression of alpha-SMA, Runx2, Pit1 and FGF23 was detected by Western blot method to examine the effect of different concentrations of iron sucrose on the phenotypic transformation of vascular smooth muscle cells stimulated by high phosphorus, and the results are shown in FIG. 3.

FIG. 3, content A shows the gene expression (RT-qPCR) of a-SMA, Runx2, Pit1 and FGF23 in each experimental group; content B and FIG. 4 show the protein expression (Western blot) of vascular ring alpha-SMA, Runx2, Pit1 and FGF 23. alpha-SMA is alpha-smooth muscle actin, the lower the alpha-SMA is, the lower the alpha-smooth muscle actin is expressedThe more pronounced the phenotypic transformation; runx2 is Runt-related transcription factor 2, and higher Runx2 indicates more obvious phenotypic transformation; pit1 is type III sodium phosphorus cotransporter 1, the higher the Pit1 is, the more obvious the phenotypic transformation is; FGF23 is fibroblast growth factor 23, with higher indicating more pronounced phenotypic transformation; CNT, normal control (regular medium); HP, high phosphorus group (conventional Medium +2.5mM PO)₄ ^3-) (ii) a HPLFe, high phosphorus low iron group (conventional Medium +2.5mM PO)₄ ^3-+2. mu.g/ml sucron); HPMFe, high phosphorus medium iron group (conventional Medium +2.5mM PO)₄ ^3-+ 5. mu.g/ml sucron); HPHFe, high phosphorus and high iron group (conventional Medium +2.5mM PO)₄ ^3-+ 10. mu.g/ml iron sucrose). A, P<0.05vs.CNT；**,P<0.01vs.CNT；Δ,P<0.05vs.HP；ΔΔ,P<0.01vs.HP；#,P<0.05vs.HPLFe；##,P<0.01vs.HPLFe。

From fig. 3, content a, it can be seen that high phosphorus stimulation down-regulates vascular smooth muscle cell alpha-SMA gene expression, and that Runx2, Pit1 and FGF23 gene expression are all up-regulated, and these changes gradually improve as the concentration of sucrose iron increases. From fig. 3, content B and fig. 4, it can be seen that high phosphorus stimulation down-regulates vascular smooth muscle cell α -SMA protein expression, and that Runx2, Pit1 and FGF23 protein expression are all up-regulated, and these changes gradually improve as the iron sucrose concentration increases.

This example demonstrates that the higher the concentration of ferric saccharate, the lower the expression of the phosphorus transmembrane transporter Pit1, indicating that ferric saccharate inhibits the transmembrane transport of phosphorus, thus indicating that ferric saccharate in blood can interfere with the transport of inorganic phosphorus into vascular smooth muscle cells under hyperphosphatemia, thereby alleviating oxidative damage to vascular smooth muscle cells.

The results of fig. 3 and 4 show that: the high phosphorus stimulation causes vascular smooth muscle cells to transform from a myocyte phenotype to an osteoblast phenotype, the degree of phenotype transformation is inhibited by iron sucrose, and the inhibition effect is dose-dependent.

The above examples show that the high phosphorus group undergoes significant calcium deposition, oxidative stress injury and phenotypic transformation of vascular smooth muscle cells; as the concentration of iron sucrose increases, calcium deposition, oxidative stress injury, and phenotypic transformation of vascular smooth muscle cells all gradually decrease. The method shows that hyperphosphatemia causes oxidative stress injury in vascular smooth muscle cells, then the vascular smooth muscle cells are induced to be transformed to osteoblast phenotype, and finally the vascular calcification is developed, and the ferric saccharate can block the pathway by antagonizing the oxidative stress injury induced by hyperphosphatemia, so that the effect of treating, relieving or inhibiting the vascular calcification induced by hyperphosphatemia is achieved.

Furthermore, it is noteworthy that when the iron concentration of sucrose is too high, significant iron deposition occurs in the vascular ring. It indicates that the blood concentration of the sucralfate needs to be noticed in clinical application, otherwise, the blood vessel is damaged due to iron overload.

It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.

Claims

1. The application of the iron sucrose preparation in preparing the medicine for treating, relieving or inhibiting vascular calcification induced by hyperphosphatemia is disclosed, wherein the iron sucrose preparation is an intravenous injection.

2. The use of claim 1, wherein the formulation is administered at a frequency ranging from once a month to once a day; each dose is in the range of 50mg to 200 mg; and when administered at a frequency of once daily, the number of consecutive administrations does not exceed 5.

3. The use of claim 1, wherein the concentration of ferric saccharate in the blood is not more than 5 μ g/mL.

4. The use of claim 1, wherein the concentration of ferric saccharate in the blood is from 2 μ g/mL to 5 μ g/mL.

5. The use of claim 1, wherein the hyperphosphatemia corresponds to a serum inorganic phosphorus concentration of greater than 2.0 mmol/L.