WO2019062071A1

WO2019062071A1 - Use of liposome in preparing pharmaceutical preparation for removing protein-bound toxin

Info

Publication number: WO2019062071A1
Application number: PCT/CN2018/082552
Authority: WO
Inventors: 丁峰; 史媛媛; 李玉林; 王宜峰
Original assignee: 上海交通大学医学院附属第九人民医院
Priority date: 2017-09-30
Filing date: 2018-04-10
Publication date: 2019-04-04
Also published as: CN109589306A

Abstract

A use of a liposome in preparing a pharmaceutical preparation for removing a protein-bound toxin. The liposome has a diameter of 50-500 nm. The present invention uses the liposome to remove the protein-bound toxin, such that the cost is lower than that of using albumin.

Description

Use of liposomes in the preparation of pharmaceutical preparations for scavenging protein-bound toxins

Technical field

The present invention relates to the use of liposomes in the preparation of pharmaceutical preparations for the clearance of protein-bound toxins, in particular during hemodialysis in patients with renal dysfunction.

Background technique

Uremic disease refers to a substance that continuously accumulates in the blood and tissues and is toxic with a decrease in renal function and a decrease in the clearance rate of solute by the kidney. The European uremic toxin working group is divided into three categories according to its biochemical characteristics and removal methods: 1) water-soluble, non-protein-bound small molecular substances, usually less than 500, such as urea, creatinine, etc. It is easy to be removed by hemodialysis; 2) medium molecular substances, the molecular weight is usually greater than 500, such as parathyroid hormone, the conventional hemodialysis removal effect of such substances is not ideal, can only be removed by blood purification method of large pore size dialysis membrane; 3) protein Binding toxins, such as sulphuric acid phenol, p-cresol sulphur, most blood purification methods have a poor effect on the removal of such substances. Current studies have shown that protein-bound toxins are closely related to cardiovascular events in the primary cause of death in patients with CKD (chronic kidney disease).

The main mode of removal of protein-bound toxins in prior art hemodialysis procedures is the addition of albumin to the dialysate, but this mode of removal is effective but costly.

A liposome is an artificial membrane. The hydrophilic head of the phospholipid molecule in water is inserted into the water, and the hydrophobic tail of the liposome extends to the air, and a spherical liposome which forms a double-layered lipid molecule after agitation, ranging from 25 to 1000 nm in diameter. Liposomes can be used for transgenic, or prepared drugs, using the characteristics of liposome fusion with cell membranes to deliver drugs into the interior of cells.

There have been no reports or literature on the use of liposomes for the removal of protein-bound toxins from uremic patients.

Summary of the invention

In view of the disadvantages of the prior art described above, it is an object of the present invention to provide a use of a liposome for the preparation of a pharmaceutical preparation for scavenging a protein-bound toxin for solving the cost of adding albumin in a dialysate in the prior art. Too high, poorly performing problems.

Based on experimental findings, Applicants have discovered that liposomes are capable of binding protein-bound toxins with good results.

To achieve the above and other related objects, the present invention provides a use of a liposome for preparing a pharmaceutical preparation for scavenging a protein-bound toxin, the liposome having a diameter of 50 to 500 nm.

The protein-binding toxin may be a protein-bound toxin produced by a plurality of protein-bound toxins, such as severe acute kidney injury with multiple organ failure.

Further, the protein-bound toxin refers to a protein-bound toxin in a uremic patient.

The liposomes can be prepared according to methods in the prior art. The liposomes may be monolayer or multi-layered, they may be of different sizes, and the charge is not limited, and vesicles of different characteristics may encapsulate the aqueous medium.

Preferably, the main component of the lipid layer membrane in the liposome is selected from the group consisting of a natural phospholipid or a synthetic phospholipid.

Preferably, the phospholipid is any one or both of soybean lecithin and hydrogenated soybean lecithin.

Further, the phospholipid is any one or more of distearoylphosphatidylglycerol (DSPG), dioleoyl lecithin (DOPC), and distearoylphosphatidylcholine (DSPC).

The pharmaceutical preparation can be directly added to the prior art hemodialysis solution for scavenging protein-bound toxins during hemodialysis.

Preferably, the liposome has a diameter of from 100 to 200 nm.

Preferably, the liposome has a diameter of from 200 to 300 nm.

Further, the pharmaceutical preparation refers to a preparation used in any of the following processes: interstitial hemodialysis, peritoneal dialysis, continuous renal replacement therapy.

The interstitial hemodialysis is a prior art, and specifically refers to one of renal replacement treatment methods for patients with acute and chronic renal failure. It diverts blood from the body to the outside through a dialyzer composed of a myriad of hollow fibers. The blood is exchanged with the electrolyte solution (dialysate) containing a similar concentration in the body inside and outside the hollow fiber. It removes metabolic wastes from the body, maintains electrolytes and acid-base balance; at the same time removes excess water from the body and returns the purified blood to the entire process.

Peritoneal dialysis is a dialysis method that uses the human's own peritoneum as a dialysis membrane. The dialysate which is poured into the abdominal cavity exchanges solute and moisture with the plasma components in the capillaries on the other side of the peritoneum, and removes the metabolites and excess water retained in the body, and at the same time replenishes the substances necessary for the body through the dialysate. By continuously updating the peritoneal fluid, the purpose of kidney replacement or supportive treatment is achieved.

Continuous renal replacement therapy, also known as continuous blood purification (CBP), is a new method of blood purification. In 1995, the first International Conference on Continuous Renal Replacement Therapy provided for continuous renal replacement therapy by replacing a damaged renal function with a continuous blood purification treatment for 24 hours or nearly 24 hours a day. Continuous renal replacement therapy includes continuous arteriovenous, intravenous venous hemofiltration (CAVH, CVVH), continuous arteriovenous, intravenous venous hemodialysis (CAVDH, CVVDH), continuous arteriovenous, intravenous venous hemodiafiltration (CAVHDF) , CVVHDF) and other modes.

Further, the only active ingredient or the main active ingredient of the scavenging protein-binding toxin in the pharmaceutical preparation is a liposome.

Further, the liposome is added at a concentration of 30 to 50 g/l at the time of use.

Specifically, it is to ensure that the concentration of the liposome in the use system is 30 to 50 g/l when the pharmaceutical preparation is added to the use system. The use system generally refers to dialysate.

It has been found through experiments that the above dose is the optimal dose, and the efficiency of further increasing the concentration of lipid globules to remove toxins will not increase further.

Preferably, the liposomes are added in an amount of 40 g per liter of dialysate.

Another aspect of the invention provides a dialysate comprising a liposome.

Preferably, the concentration of the liposome in the dialysate is from 30 to 50 g/l.

Preferably, the liposome has a diameter of from 100 to 200 nm.

Preferably, the dialysate is any one of interstitial hemodialysis dialysate, peritoneal dialysate or continuous renal replacement therapy dialysate.

Another aspect of the invention discloses the use of the above dialysate for scavenging protein binding toxins.

Another aspect of the present invention provides a pharmaceutical preparation for removing a protein-bound toxin, wherein the active ingredient in the pharmaceutical preparation is a liposome having a diameter of 50 to 500 nm.

Further, the liposome has a diameter of 100 to 200 nm.

Another aspect of the invention provides a method of removing protein-bound toxins in a patient, the method comprising providing liposomes to the blood of the patient, utilizing liposome clearance protein binding toxins.

Further, the main component of the lipid layer membrane in the liposome is selected from a natural phospholipid or a synthetic phospholipid.

Further, the method refers to adding liposome to the dialysate during interstitial hemodialysis, peritoneal dialysis, and continuous renal replacement therapy.

Preferably, the liposome is added in the dialysate in an amount of 30 to 50 g/l in use.

Preferably, the liposome has a diameter of from 50 to 500 nm, more preferably from 100 to 200 nm.

As described above, the use of the liposome of the present invention in the preparation of a pharmaceutical preparation for removing uremic toxin has the following

Beneficial effects:

Since the cost of liposomes is much lower than that of albumin, the cost of dialysis is greatly reduced, and the effect is good, which greatly reduces the cost of treatment for a wide range of patients. For example, in the current method of albumin dialysis, the cost of one dialysis is about 10,000 yuan. If liposome is used, the cost will be reduced to less than 1,000 yuan, which is very beneficial to the improvement of human health.

DRAWINGS

Figure 1 shows the adsorption rate of liposome to p-cresol PCS sulfate.

Figure 2 shows the adsorption rate of liposomes to indole sulfate IS.

Figure 3 shows the adsorption rate of liposomes to hippuric acid HA.

Figure 4 is a dose effect curve of liposome adsorption of p-cresyl sulfate.

Figure 5 is a dose effect curve of liposome adsorption of indoxyl sulfate.

Figure 6. Dose-effect curve of liposome adsorption of hippuric acid hippuric acid.

Figure 7 Percent removal of PCS in a one-time rapid equilibrium dialysis.

Figure 8 shows the percentage of IS clearance in a one-time rapid equilibrium dialysis.

Figure 9 shows the percentage of IS clearance in a one-time rapid equilibrium dialysis.

Figure 10 is a diagram showing the in vitro closed cycle pattern.

Figure 11a shows the effect of three ways on the clearance of p-cresol p-cresol (A blood side).

Figure 11b shows the effect of three ways on the removal of p-cresol p-cresol (B dialysate side).

Figure 12a shows the scavenging effect of indoxyl sulfate on three ways (A blood side).

Figure 12b shows the effect of three ways on the clearance of indoxyl sulfate (B dialysate side).

Figure 13a shows the effect of three ways on the clearance of hippuric acid (A blood side).

Figure 13 b The effect of three ways on the clearance of hippuric acid (B dialysate side).

Figure 14a shows the clearance effect of indole-3-acetic acid from indoleacetic acid in three ways (A blood side).

Figure 14 b The scavenging effect of indole-3-acetic acid of indoleacetic acid in three ways (B side of dialysate).

Detailed ways

The embodiments of the present invention are described below by way of specific examples, and those skilled in the art can readily understand other advantages and effects of the present invention from the disclosure of the present disclosure. The present invention may be embodied or applied in various other specific embodiments, and various modifications and changes can be made without departing from the spirit and scope of the invention. It should be noted that the process equipment or apparatus not specifically noted in the following examples employ conventional equipment or apparatus in the art. In addition, it should be understood that one or more of the method steps recited in the present invention are not exclusive of other method steps that may be present before or after the combination step, or that other method steps can be inserted between the steps specifically mentioned, unless otherwise It should be understood that the combined connection relationship between one or more devices/devices referred to in the present invention does not exclude that other devices/devices may exist before or after the combined device/device or Other devices/devices can also be inserted between the two devices/devices unless otherwise stated. Moreover, unless otherwise indicated, the numbering of each method step is merely a convenient means of identifying the various method steps, and is not intended to limit the order of the various method steps or to limit the scope of the invention, the relative In the case where the technical content is not substantially changed, it is considered to be a scope in which the present invention can be implemented.

Example 1 Preparation of liposome

0.6 g of Tween-80, 0.6 g of cholesterol, 0.6 g of sodium deoxycholate and 2.4 g of soybean lecithin were weighed and completely dissolved by adding 100 mL of dichloromethane. The mixture was added to a clean anhydrous flask, and the film was spun under a reduced pressure of 30 degrees (the organic solvent was evaporated overnight).

Glucose having a total mass of 10% was weighed, dissolved in 100 mL of ultrapure water, and added to a film-forming flask, and rotated in a 37-degree water bath to hydrate the film.

After the mixture was completely hydrated and the mixture was taken out, the liquid was added to an elegant homogenizer (set pressure 400 bar, 30 min) for 30 min.

After taking out, freeze-drying was collected. The liposome was determined to have a diameter of 100 to 200 nm.

Example 2 Verification of liposome-adsorbing protein binding to uremic toxin concept

I. Preliminary selection of three representative proteins in combination with uremic toxins:

1. p-cresyl sulfate (PCS, molecular weight 188Da, albumin binding rate ~ 95%)

2. Indoxyl sulfate (IS, molecular weight 212Da, albumin binding rate ~ 90-95%);

3. Hippuric acid (HA, molecular weight 179Da, albumin binding rate ~ 50%)

II. Preliminary determination of liposome-adsorbing protein binding to uremic toxin:

Method

(1) Reagent

Bovine serum albumin BSA (sigma, purity ≥98%)

Phosphate Buffer 1×PBS (Beijing Solarbio Science & Technology Co.)

P-cresol sulfate PCS (APExBIO)

Potassium sulphate potassium salt IS (sigma)

Hippuric acid HA (sigma)

(2) Ultrafiltration tube

Ultra 0.5mL ultrafiltration tube (Millipore, molecular retention 3KD)

(3) Operation steps

a. Accurately weigh PCS 10mg, IS 11.4mg, HA18.9mg, dissolved in phosphate buffer (1xPBS, pH 7.2-7.4), dilute to 265ml, placed on a shaker, protected from light at room temperature, shake overnight Evenly, spare.

b. Take 16ml of the above solution, dispense 8 parts, 2ml each, add BSA and liposome respectively, and prepare the final concentration of BSA 40g/L, liposome 10g/L, 20g/L, 40g/L , 80g / L, 120g / L, 160g / L.

c. After 30 min, 60 min, 120 min and 240 min later, take the above BSA solution containing protein-binding uremic toxin or 0.4 ml of different concentrations of liposome solution, and add 0.5 ml ultrafiltration tube, 12000 rpm. Centrifuge for 30 minutes at 4 ° C and take the ultrafiltrate.

d. Calculate the BSA binding rate or liposome adsorption rate of the above toxins

BSA binding rate or liposome adsorption rate = 100x (total concentration - ultrafiltrate concentration) / total concentration

e. Repeat the above steps 3 times.

2. Detection method

The concentration of protein-bound toxin was determined by high-performance liquid chromatography.

3. Statistical methods

The data were processed using SPSS 21.0 statistical software, and the data were expressed as mean ± standard deviation. The comparison between the two groups was performed by independent sample t test, and the comparison between groups was analyzed by one-way ANOVA. P < 0.05 was considered statistically significant.

4. Results

As shown in Fig. 1, when the concentration of BSA was 40g/L, the BSA protein binding rate of PCS was 93.80%±0.92% at 30 min. When BSA was combined with PCS, there was almost no dissociation with time. At 30 min, the adsorption rate of 10 g/L liposome to PCS was 79.1%±2.82%, and there was no significant change in the adsorption rate of PCS by liposome compared with 30 min at 60 min, 120 min and 240 min, respectively (p>0.05). . At 30 min, the adsorption rate of PCS increased with the increase of liposome concentration.

As shown in Fig. 2, when the concentration of BSA was 40 g/L, the binding rate of BSA protein of IS was 94.07%±2.09% at 30 min. When BSA was combined with IS, there was almost no dissociation with time. At 30 min, the adsorption rate of 10 g/L liposome to IS was 51.11%±1.40%, and the adsorption rate at 120 min was the highest, reaching 63.81%±3.73%. At 60 min, the adsorption rate at 30 min was not statistically comparable to that at 30 min. Difference (p>0.05). At 30 min, the adsorption rate of IS increased with the increase of liposome concentration.

As shown in Fig. 3, when the BSA concentration was 40 g/L, the BSA protein binding rate of HA at 30 min was 58.46% ± 4.18%. When BSA was combined with HA, there was almost no dissociation with time. At 30 min, the HA adsorption rate of 10 g/L liposome was 57.30%±1.40%, and the adsorption rate of liposome to HA was not significantly changed compared with 30 min at 60 min, 120 min and 240 min, respectively (p>0.05). . At 30 min, the adsorption rate of HA did not increase significantly with the increase of liposome concentration (p>0.05).

Example 3 Concentration dependence of liposome-adsorbed protein binding to uremic toxin

Method

(1) Reagent

Bovine serum albumin BSA (sigma, purity ≥98%)

Phosphate Buffer 1×PBS (Beijing Solarbio Science & Technology Co.)

P-cresol sulfate PCS (APExBIO)

Potassium sulphate potassium salt IS (sigma)

Hippuric acid HA (sigma)

(2) Ultrafiltration tube

Ultra 0.5mL ultrafiltration tube (Millipore, molecular retention 3KD)

(3) Operation steps

a. Take 10 ml of the above solution in the same manner, and dispense 10 parts, 1 ml each, and add liposomes respectively. The final concentration of the prepared liposomes is 5 g/L, 10 g/L, 20 g/L, 30 g/ L, 40 g/L, 60 g/L, 80 g/L, 100 g/L, 120 g/L, and 160 g/L.

b. After 120 min, 0.5 ml of the above-mentioned liposome solution containing different concentrations of protein-bound uremic toxin was taken, added to a 0.5 ml ultrafiltration tube, centrifuged at 12,000 rpm, and centrifuged at 4 ° C for 30 minutes to obtain an ultrafiltrate.

d. Calculating the liposome adsorption rate of the above toxins

Liposomal adsorption rate = 100x (total concentration - ultrafiltrate concentration) / total concentration

e. Repeat the above steps 3 times.

2. Detection method: Same as above.

3. Statistical methods

4. Results

As shown in Figure 4, when the concentration of liposome was 5g/L, the adsorption rate of PCS at 120min was 70.38%±3.73%, and the adsorption rate of PCS increased with the increase of liposome concentration. When the concentration of plastid was 60g/L, the adsorption rate to PCS was 90.70%±0.45%. When the concentration of liposome was increased to 160g/L, the adsorption rate to PCS was 98.58%±0.14%.

As shown in Fig. 5, when the concentration of liposome is 5g/L, the adsorption rate of IS at 120min is 39.60%±1.20%, and the adsorption rate of IS increases with the increase of liposome concentration. When the concentration of plastid was 60g/L, the adsorption rate to IS was 83.63%±1.59%. Then, the adsorption rate of IS gradually became gentle with the increase of liposome concentration, and the concentration of liposome increased to 160g/L. At the time, the adsorption rate to IS was 91.12% ± 1.76%.

As shown in Fig. 6, when the concentration of liposome is 5g/L, the adsorption rate of IS at 120min is 59.06%±2.74%. There is no obvious law on the adsorption rate of HA with the increase of liposome concentration. When the liposome concentration was increased to 160 g/L, the adsorption rate to HA was 54.78% ± 4.61%.

Example 4 in vitro static dialysis

Method

(1) Reagent

Bovine serum albumin BSA (sigma, purity ≥98%)

Phosphate Buffer 1×PBS (Beijing Solarbio Science & Technology Co.)

P-cresol sulfate PCS (APExBIO)

Potassium sulphate potassium salt IS (sigma)

Hippuric acid HA (sigma)

(2) Main equipment

Single-Use RED (rapid equilibrium dialysis) Plate (Thermo Scientific ^TM , molecular retention 8000 Da)

(3) Operation steps

a. Accurately weigh 0.8 g BSA, and make up to 20 ml with the above solution containing PCS, IS and HA protein combined with uremic toxin, and the final concentration of BSA is 40 g/L, and shaken at room temperature overnight.

b. As shown in Table 1, the following solutions were added to Sample Chambers and Buffer Chambers in turn, and each well was loaded twice:

c. After the addition, the RED plate was covered with a sealing strip, and the RED plate was placed on a shaker and incubated at 37 ° C, 250 rpm for 4 hours.

d. Remove the seal strip and transfer the sample from Sample Chambers to a 1.5 ml EP tube and calculate the concentration of each toxin in the sample after 4 h of incubation.

Percent removal (%) = (C pre-incubation-C post incubation) / C pre-incubation in Sample Chambers

e. Repeat the above steps 3 times.

2. Detection method: Same as above.

3. Statistical methods

4. Results

Taking sample (1) as the reference for dialysis equilibrium time, after incubation for 4 hours at 37 °C, the percentage of PCS concentration clearance in the Sample Chamber was 46.53%±2.24%, the percentage of IS concentration clearance was 53.30%±1.10%, and the percentage of HA concentration clearance was 49.09%. ±5.14%, indicating that the 4 hour incubation can reach equilibrium dialysis time.

As shown in Figure 7, the PCS clearance percentage was 2.34% ± 1.93 after incubation for 4 hours in the PBS group Sample Chamber, and the PCS clearance percentage was 6.42% ± 1.64% after 4 hours incubation in the 5 g/L liposome Sample Chamber. There was an increase in the PBS group (p<0.05). As the liposome concentration increased, the percentage of PCS clearance in Sample Chambers gradually increased, and the percentage of PCS cleared by 40g/L liposomes was 27.74%±1.57%, with 40g/L BSA. The effect of clearing the percentage of PCS was similar (27.74% ± 1.57% vs. 29.14% ± 5.01%).

As shown in Fig. 8, the percentage of IS clearance was 4.42% ± 1.39% after incubation for 4 hours in the PBS group Sample Chamber, and the percentage of PCS clearance was 7.41% ± 1.48% after 4 hours of incubation in the 5 g/L liposome Sample Chamber. There was an increase in the PBS group (p=0.03). As the concentration of liposomes increased, the percentage of IS clearance in Sample Chambers gradually increased, and the percentage of PCS cleared by 60g/L liposomes was 29.72%±4.66%, with 40g/L BSA. There was no statistically significant difference in the percentage of PCS clearance (29.72% ± 4.66% vs. 34.00% ± 2.55%).

As shown in Fig. 9, after incubation for 4 hours in the PBS group Sample Chamber, the percentage of HA clearance was 22.99% ± 2.97%, and the percentage of PCS clearance after incubation in the 5 g/L liposome group for 4 hours was 28.47% ± 4.31%. There was no statistical difference between the PBS group (p=0.08). As the liposome concentration increased, the percentage of HA clearance in Sample Chambers increased gradually, but the increase trend was not as good as PCS and IS. The percentage of PCS cleared by 60g/L liposome was 41.79%±4.69%, which was not statistically different from the percentage of BSA cleared by 40g/L BSA (41.79%±4.69% vs. 47.99%±7.84%).

Example 5 in vitro closure cycle

Method

(1) Reagent

Bovine serum albumin BSA (sigma, purity ≥98%)

Phosphate Buffer 1×PBS (Beijing Solarbio Science & Technology Co.)

P-cresol sulfate PCS (APExBIO)

P-cresol (sigma)

Potassium sulphate potassium salt IS (sigma)

Indole-3-acetic acid (3-IAA, molecular weight 175 Da, albumin binding rate ~ 75%)

Hippuric acid HA (sigma)

(2) Main instruments and equipment

Ultra 0.5mL ultrafiltration tube (Millipore, molecular retention 3KD);

Small peristaltic pump (VWR, USA)

Microfilter (Weigao Group Co., Ltd.)

Extracorporeal circulation line (US VWR company)

(3) Operation steps

I. Comparison of the binding rate of p-cresyl sulfate and p-cresol to BSA

Method as before, use

The protein binding rate of p-cresyl sulfate to cresol was about 93%-95%, and the protein binding rate of p-cresol to p-cresol was 93%-95%. The protein binding of the two was measured by Ultra 0.5mL ultrafiltration tube. The rate is similar, so the in vitro closed cycle uses p-cresol to represent one of the protein-binding toxins.

II. In vitro closed cycle

a. Blood side (B side) solution preparation:

Accurately weighed 20 g of bovine serum albumin BSA, 10.8 mg of p-cresol, 17.0 mg of potassium sulfonate, 35.8 mg of hippuric acid, dissolved in phosphate buffered saline, accurately weighed 13.2 mg of indole acetic acid, dissolved in In 500 ml PBS, 50 ml was added to the above PBS solution containing BSA, p-cresol, decylphenol and hippuric acid, and finally adjusted to 500 ml, shaken at room temperature overnight on a shaker. The blood side volume per cycle was 50 ml.

b. dialysate (D side) components

The volume of dialysate per cycle was 100ml, divided into three groups according to their composition, each group was cycled three times, respectively, 1PBS as the control group, 240g/L BSA dissolved in PBS, as BSA group, 340g/L lipid The plastids were dissolved in PBS as a liposome group.

c. Dialysis parameter setting

As shown in Fig. 10, blood side (B side) blood flow rate Qb = 5.0 ml / min, dialysate side (D side) dialysate flow rate Qd = 5 ml / min, and each cycle was performed for 360 min. After the extracorporeal circulation device is connected, both sides are pre-flushed with physiological saline. The beakers on both sides are placed on the magnetic stirrer during the dialysis process to ensure that the solution is always evenly mixed.

d. Specimen collection and testing

150 μl samples were taken from both sides of the extracorporeal circulation at 0 min, 10 min, 30 min, 60 min, 90 min, 120 min, 150 min, 180 min, 210 min, 240 min, 270 min, 300 min, 330 min and 360 min, and placed at -80 ° C for testing. Sample processing method containing liposome: 100 ul of the solution was sampled, 300 ul of acetonitrile was added to precipitate the liposome, centrifuged at 12000 rpm for 30 min at 4 ° C, and the supernatant was taken for 100 ul.

2. Detection method: Same as above.

3. Statistical methods

4. Results

1) Purification effect of p-cresol in in vitro closed cycle

As shown in Figure 11, the initial concentration of p-cresol in the blood side BSA solution is about 200umol/L, and the protein binding rate of p-cresol to p-cresol can reach 93%-95%. At the beginning, the free level was very low at 12.80±1.44 umol/L. During the 6-hour dialysis cycle, the decrease rate of the blood side of the control group (PBS) and the increase rate of the dialysate side were always slow, and the blood side of the control group was 6 h. The p-cresol reduction rate was 8.01% ± 1.73% at the beginning, and the p-cresol concentration at the end of dialysis on the dialysate side was 7.01 ± 0.80 umol / L. The blood-side p-cresol of the BSA group and the liposome group had the fastest rate of decline during the 1 h before dialysis. The rate of decline at 1 h was 50.08%±5.04% and 49.44%±24.4%, respectively, and the rate of decline during the 2 h dialysis. The decrease rate of p-cresol on the blood side of BSA group and liposome group was 56.34%±2.47% and 62.18±16.08%, respectively, at 2h. After 2h, the decrease rate of p-cresol on the blood side of BSA group and liposome group was significantly slower, and the decrease rate at 4h was 64.89%±3.50% and 70.18%±10.7%, respectively. The concentration of p-cresol on the blood side of the BSA group and the liposome group became substantially stable after 4 hours of dialysis. Similarly, the concentration of p-cresol on the dialysate side of the BSA group and the liposome group increased the fastest during the first hour of the cycle, and the p-cresol concentrations on the dialysate side of the two groups were 40.26±.78umol/L and 43.16±10.69, respectively. Umol/L, the increase of p-cresol concentration on the dialysate side of the two groups was significantly slowed down during the 2h cycle. At 2h, the p-cresol concentrations on the dialysate side were 49.07±6.31umol/L and 49.83±13.20umol/L, respectively. After 2h, the concentration of p-cresol in the dialysate side of the two groups still increased slowly. At 4h, the p-cresol concentration in the dialysate side of the two groups was 60.06±7.66umol/L and 64.81±10.86umol/L, respectively. The concentration of -cresol tends to be flat.

2) Removal effect of indoxyl sulfate in in vitro closed cycle

As shown in Figure 12, the changes in indoxyl sulfate on the blood side and the dialysate side during the dialysis were similar to those of p-cresol. In the prepared blood side BSA solution, the initial concentration of indoxyl sulfate is about 150umol/L, the protein binding rate is 95.48%±1.79%, and the free level on the blood side at the beginning of dialysis is 7.83±2.31umol/L. During the 6-hour dialysis cycle, the decrease rate of indoxyl sulfate on the blood side of the control group (PBS) and the increase rate on the dialysate side were always slow. At 6 hours, the blood-side p-cresol decline rate of the control group was 8.75%. ±1.80%, p-cresol concentration at the end of dialysis on the dialysate side was 8.55 ± 1.64 umol / L. The blood indoxyl sulfate in the BSA group and the liposome group had the fastest decline rate during the 1 h before dialysis. The decrease rate at 1 h was 48.69%±0.74% and 47.76%±17.19%, respectively, and the decline rate decreased during the 2 h dialysis. Slowly, the decrease rate of indoxyl sulfate on the blood side of BSA group and liposome group was 56.74%±2.26% and 55.82±9.44%, respectively. After 2h, the decrease rate of indoxyl sulfate in the blood side of BSA group and liposome group was significantly slower, and the decrease rate at 4h was 62.55%±1.70% and 59.32%±7.75% at the beginning. The concentration of indoxyl sulfate on the blood side of the BSA group and the liposome group also tended to be stable after 4 hours of dialysis. Similarly, the concentration of indoxyl sulfate on the dialysate side of the BSA group and the liposome group increased the fastest during the first hour of the cycle. At lh, the concentration of indoxyl sulfate on the dialysate side was 36.69±5.80umol/L and 31.81±12.58umol/ L, the increase of p-cresol concentration in the dialysate side of the two groups was significantly slowed down during the 2h cycle. At 2h, the concentrations of indoxyl sulfate in the dialysate were 41.14±8.14umol/L and 35.79±11.30umol/L, respectively. The concentration of indoxyl sulfate in the dialysate side still increased slowly. At 4h, the concentration of indoxyl sulfate in the dialysate group was 49.98±6.71umol/L and 43.08±4.75umol/L, respectively. After that, the concentration of indoxyl sulfate in the dialysate group basically tended to be higher. gentle.

3) Purification effect of hippuric acid in in vitro closed cycle

As shown in Figure 13, the changes in hippuric acid on the blood side and the dialysate side during the dialysis were different from those of p-cresol and indoxyl sulfate. In the prepared blood side BSA solution, the initial concentration of hippuric acid hippuric acid is about 400umol/L, the protein binding rate is 60.89%±3.82%, and the free level on the blood side at the beginning of dialysis is 156.42±15.29umol/L. During the 6-hour dialysis cycle, the decrease rate of the hippuric acid on the blood side of the control group (PBS) and the rate of increase on the dialysate side were significantly increased or decreased compared with p-cresol and indoxyl sulfate. The hepatic hippuric acid concentration in the PBS group, BSA group and liposome group was the fastest in the first hour of dialysis, and the hippuric acid concentration in the PBS group was 22.84%±6.70% at the beginning of the blood side. At 1h, the decrease rate of blood side hippuric acid concentration was 43.07%±12.27%, which was significantly increased compared with PBS group (43.07%±12.27% vs. 22.84%±6.70%, p<0.05), liposome group 1h. The blood-side hippuric acid concentration decreased from the initial 33.01%±11.69%, and the efficiency was between the other two groups. The decrease rate of blood on the 2h side of the three groups was slower than that of the first hour. The decrease rate of hippuric acid concentration was 32.08%±3.04% at the 2h blood side of the PBS group, and the decrease rate of the blood side hippuric acid concentration at 2h in the BSA group. For the initial 62.74%±10.58%, the decrease rate was also significantly increased compared with the PBS group (62.74%±10.58% vs. 32.08%±3.04%, p<0.05), and the blood side hippuric acid concentration decreased at 2 hours in the liposome group. For the initial 49.94%±9.45%, the efficiency was between the other two groups, and the decrease rate was also significantly increased compared with the PBS group (49.94%±9.45% vs. 32.08%±3.04%, p<0.05), and BSA. There was no statistical difference between the groups (49.94%±9.45% vs. 62.74%±10.58%, p>0.05). After 2h, the decrease of hippuric acid concentration in the three groups was significantly slowed down. At 4h, the blood group hiphipic acid concentration in the PBS group was decreased by 50.95%±5.51%, and the blood side hippuric acid concentration in the BSA group was decreased. 77.18%±4.43%, compared with the PBS group, there was still statistical difference (77.18%±4.43% vs. 50.95%±5.51%, p<0.05). The decrease rate of hippuric acid concentration in the liposome group was 56.23%±5.19%, which was better than that in the PBS group (56.23%±5.19% vs. 50.95%±5.51%, p<0.05), which was inferior to the BSA group (56.23%±). 5.19% vs. 77.18% ± 4.43%, p < 0.05). After 4 hours of dialysis, the concentration of hippuric acid in the three groups of the blood group basically stabilized. The concentration of hippuric acid in the three groups of dialysate was the fastest in the first hour of dialysis. The concentration of dialysate in PBS group was 69.16±20.97umol/L at 1h of dialysis, and 69.16± at 1h in BSA group. 20.97umol/L, the increase was statistically significant compared with PBS group (69.16±20.97vs.69.16±20.97umol/L, p=0.02), and the concentration of hippuric acid on the dialysate side was 61.88±21.39umol/L at 1h in liposome group. Between the PBS group and the BSA group. The increase rate of hippuric acid concentration in the dialysate side of the 3h dialysis group was slowed down. The hippuric acid concentration in the BSA group was 104.61±30.65umol/L at 2h, which was higher than that in the PBS group (104.61±30.65vs.64.50±7.59umol/L, p <0.01), the hippuric acid concentration was 87.68±25.45umol/L at 2h in the liposome group, which was better than the PBS group (p=0.014), and there was no statistical difference compared with the BSA group (p>0.05). After 2h, the concentration of hippuric acid in the three groups of dialysate increased more slowly. At 4h, the concentration of hippuric acid in the PBS group was 99.56±13.71umol/L, and in the BSA group was 139.24±18.95umol/L, compared with the PBS group. There was still a statistical difference (p < 0.05). The liposome group was 111.71 ± 21.16 umol / L, between the two. After 4 hours of dialysis, the concentration of hippuric acid in the three groups of dialysate was basically stable.

4) Clearing effect of indole-3-acetic acid in in vitro closed cycle

As shown in Figure 14, the changes in 3-IAA in the blood side and the dialysate side of the three groups during dialysis were also different from those of p-cresol and indoxyl sulfate. In the prepared blood side BSA solution, the initial concentration of 3-IAA was about 15 umol/L, the protein binding rate was 75%±2.38%, and the free level on the blood side at the beginning of dialysis was 3.75±1.29 umol/L. The 3-IAA concentration in the three groups was the fastest in the first hour of dialysis. The 3-IAA concentration in the PBS group was 30.19%±13.94% at the 30 min and the blood side at 30 min in the BSA group. The decrease rate of 3-IAA concentration was 53.27%±27.71%, and the decrease rate of 3-IAA concentration on blood side was 30.6%±24.46% at 30 minutes in the liposome group. There was no significant difference between the three groups (p >0.05). At 1h, the decrease rate of 3-IAA concentration in the three groups was 34.91%±12.07%, 55.45%±21.09%, and 47.28%±28.07%, respectively. There was no significant difference between the three groups (p> 0.05). The decrease rate on the blood side of the 3h group was slower than that in the 1st hour. At 2h, the decrease rate of the 3-IAA concentration in the blood side of the PBS group was 42.96%±9.20%, respectively. The BSA group was superior to the PBS group. The difference was statistically significant (60.75%±9.10% vs. 42.96%±9.20%, p=0.038). The decrease rate of 3-IAA concentration in the blood side of liposome group was 64.24±17.59%, which was higher than that of PBS group (64.24±17.59). %vs.42.96%±9.20%, p=0.03). After 2 h of circulation, the concentration of 3-IAA in the blood of the three groups decreased more slowly. At 4 h, the decrease rate of 3-IAA concentration in the blood side of the PBS group was 51.15%±7.95%, respectively, and the BSA group was higher than the PBS group (68.82±3.31). %vs.51.15%±7.95%, p=0.012) The liposome group was also higher than the PBS group (75.19±10.24%, p=0.01), but there was no significant difference between the BSA group and the liposome group (p>0.05). After the 4th cycle, the concentration of 3-IAA on the blood side of the three groups tended to be stable. Similarly, the 3-IAA concentration on the three groups of dialysate increased the fastest during the first hour of the cycle, and the 3-IAA concentration on the dialysate side of the three groups was 2.83±1.01umol/L, 2.71±1.23umol/ at 1h. L, 3.06±1.81umol/L, the difference between the three groups was not statistically significant (p>0.05). The concentration of 3-IAA in the dialysate side of the three groups was slower in the 2h cycle. At 2h, the concentration of 3-IAA in the dialosome group was 4.33±1.06umol/L, which was higher than that in the PBS group (4.33±1.06vs.3.36±0.66). , p=0.047), the BSA group was 3.39±1.47umol/L, and the difference was not statistically significant compared with the PBS group (p>0.05). After 2h, the concentration of 3-IAA in the three groups of dialysate gradually became platform, and the concentration of 3-IAA in the dialysate side of the liposome group was always higher than that in the PBS group (p<0.05). The 3-IAA concentration on the dialysate side of the BSA group was statistically significant (5.52 ± 1.27 vs. 4.21 ± 0.59 umol/L, p = 0.031) until the end of dialysis compared with the PBS group.

5) Changes in blood side BSA concentration during in vitro closed circulation

As shown in Table 2, during the extracorporeal circulation, the concentration of BSA on the blood side of the three groups decreased slightly within 30 min before dialysis, but did not reach statistical significance. After 30 min, there was no significant change in the BSA concentration on the blood side of the three groups.

Table 2 Changes in BSA concentration on the blood side (B side) of each group during cardiopulmonary bypass

The above examples are intended to illustrate the disclosed embodiments of the invention and are not to be construed as limiting. In addition, various modifications of the present invention, as well as variations of the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope of the invention. While the invention has been described in detail with reference to the preferred embodiments embodiments In fact, various modifications to the invention as apparent to those skilled in the art are intended to be included within the scope of the invention.

Claims

A use of a liposome for the preparation of a pharmaceutical preparation for the clearance of protein-bound toxins having a diameter of from 50 to 500 nm.
The use according to claim 1, characterized in that the protein-bound toxin is a protein-bound toxin in a uremic patient.
The use according to claim 1, characterized in that the liposome has a diameter of from 100 to 200 nm.
The use according to claim 1, characterized in that the pharmaceutical preparation refers to a preparation used in any of the following processes: interstitial hemodialysis, peritoneal dialysis, continuous renal replacement therapy.
The use according to claim 1, characterized in that the only active ingredient or the main active ingredient of the scavenging protein-binding toxin in the pharmaceutical preparation is a liposome.
The use according to claim 1, characterized in that the liposome is added in a concentration of from 30 to 50 g/l when used.
A dialysate characterized in that the dialysate contains liposomes.
The dialysate according to claim 7, wherein the concentration of the liposome in the dialysate is 30 to 50 g/l.
The dialysate according to claim 7, wherein the liposome has a diameter of 100 to 200 nm.
The dialysate according to claim 7, wherein the dialysate is any one of interstitial hemodialysis dialysate, peritoneal dialysate or continuous renal replacement therapy dialysate.