CN104857014A - Application of 2-hydroxypropyl-beta-cyclodextrin to the preparation of drug for treatment of X-linked adrenoleukodystrophy - Google Patents

Abstract

The invention discloses the application of 2-hydroxypropyl-beta-cyclodextrin (HPCD) in the preparation of medicine for treating X-linked adrenoleukodystrophy (X-ALD), belonging to the field of biomedicine. The present invention injects 2-hydroxypropyl-β-cyclodextrin into wild-type and X-ALD model mice (ABCD1 gene knockout mice), and slices cells, adrenal gland and cerebellum tissue of the cells injected with HPCD Filipin staining was performed to measure plasma total cholesterol, very long-chain fatty acids, body weight, and behavioral tests. It was found that HPCD can reduce the cholesterol levels of plasma, cerebellum, and adrenal glands to within the normal range, and reduce the level of VLCFA. Reduced behavioral abnormalities caused by neurodegeneration in X-ALD mice. Based on the results, it is shown that HPCD can treat X-ALD, and it can be used to prepare medicines for treating X-ALD.

Description

Application of 2-hydroxypropyl-β-cyclodextrin in preparation of medicine for treating X-linked adrenoleukodystrophy

technical field

The invention belongs to the field of biomedicine, and specifically relates to the application of 2-hydroxypropyl-β-cyclodextrin in the preparation of medicines for treating X-linked adrenoleukodystrophy.

Background technique

X-linked adrenoleukodystrophy (X-ALD) is an X-linked recessive genetic disorder of lipid metabolism. At present, it is believed that because peroxisomes in the cells are hindered in the oxidation of very long-chain fatty acids (VLCFA), mainly C23-C30 fatty acids, especially C26, resulting in a large amount of VLCFA in blood, brain white matter, adrenal cortex and other organs and tissues. Aggregates, causing demyelination of the central nervous system and atrophy or dysplasia of the adrenal cortex. About 0.5-1 person per 100,000 people has the disease, of which 95% are males and 5% are heterozygous females. The X-ALD pathogenic gene ABCD1 (Sarde et al., 1994) encodes the peroxisome membrane protein ALDP (adrenoleukodystrophy protein), which contains 745 amino acids. ALDP combines with three other proteins located on the peroxisome membrane to form a dimer, which can transport saturated VLCFA to the peroxisome for oxidation. Due to the mutation of ABCD1 gene, the ALDP function is abnormal, which hinders the oxidation of VLCFA and causes the accumulation of VLCFA. The accumulation of VLCFA in the nervous system can destroy the stability of myelin sheath and the normal formation of myelin sheath, while the accumulation in adrenal cortical cells will reduce the function of ACTH receptors on the surface of adrenal cortical cell membranes, inhibit the synthesis of intracellular steroids, and cause adrenal Decreased function.

At present, there is no effective X-ALD disease treatment plan, and the current treatment methods include adrenocortical hormone replacement therapy and diet therapy. When adrenal insufficiency occurs, hormone replacement therapy can be used. Giving steroid hormones can significantly improve the endocrine state of the patient, but cannot improve the symptoms of the nervous system; a low-fat diet combined with Lorenzo's oil (trierucic acid glyceric acid: trioleic acid glyceride = 4:1) may have a certain effect on reducing the level of VLCFA in plasma in some patients, but it cannot prevent the demyelination of the brain; gene therapy has great prospects but is not feasible in the near future; hematopoietic stem cell transplantation can effectively treat patients with brain type in the early stage of the disease, However, it is not suitable for patients with brain type in the rapidly progressive stage of the disease; the use and efficacy of drug-induced gene therapy such as lovastatin are still at the experimental level.

Lorenzo's oil, which is currently the most widely used treatment for X-ALD, can reduce the level of VLCFA in the plasma of some patients, but it cannot improve the brain lesions and behavioral abnormalities caused by X-ALD. Another small molecule 4-PBA used in the experiment was well tolerated in patients, but it could not reduce the level of VLCFA in the body.

X-ALD is generally considered to be a lipid metabolism disease caused by the accumulation of VLCFA in tissues. The present invention finds that HPCD acts on X-ALD mice, not only reducing the cholesterol levels of plasma, cerebellum, and adrenal glands to within the normal range, but also reducing the level of VLCFA, and alleviating the behavioral changes caused by neurodegeneration in X-ALD mice abnormal.

Contents of the invention

The object of the present invention is to provide an application of 2-hydroxypropyl-β-cyclodextrin (HPCD) in the preparation of medicine for treating X-linked adrenoleukodystrophy (X-ALD).

The object of the present invention is achieved through the following technical solutions:

The present invention injects HPCD into wild-type (WT) and X-ALD model mice (ABCD1 gene knockout mice, ABCD1 ^-/- mice), 6-month-old to 7-month-old mice are injected 4 times a week, in Cell Filipin staining, Filipin staining of adrenal gland and cerebellar tissue sections, and measurement of plasma total cholesterol and very long-chain fatty acids were performed at the age of 7 months; injections were performed 5 times a month from the age of 7 months to 12 months, and body weight was measured at the age of 12 months ; Mice were injected 8 times a month after 12 months of age, and behavioral tests were performed at 20 months of age. It was found that HPCD can reduce the cholesterol levels of plasma, cerebellum, and adrenal glands to within the normal range, and reduce the level of VLCFA, alleviating the behavioral abnormalities caused by neurodegeneration in X-ALD mice. Based on the results, it is shown that HPCD can treat X-ALD, and it can be used to prepare medicines for treating X-ALD.

The invention has the following beneficial effects: compared with the existing therapeutic drugs Lorenzo's oil and 4-PBA, HPCD not only reduces the VLCFA level in X-ALD mice, but also alleviates the abnormal behavior symptoms of X-ALD mice. HPCD will be a new and effective compound for treating X-ALD disease.

Description of drawings

Figure 1 is a diagram of the procedure for HPCD injection of X-ALD mice.

Figure 2 is a confocal micrograph of Filipin staining of tail tip fibroblasts of 7-month-old WT and ABCD1 ^-/- mice.

Fig. 3 is a fluorescence microscope image of Filipin staining of tissue sections of adrenal gland and cerebellum of 7-month-old WT and ABCD1 ^-/- mice.

Fig. 4 is a graph showing the statistical results of plasma total cholesterol levels in 7-month-old WT and ABCD1 ^-/- mice; NS: no significant difference; *: significant difference, P<0.05.

Figure 5 is a graph of the statistical results of VLCFA content in the adrenal gland or brain tissue of 7-month-old WT and ABCD1 ^-/- mice; NS: no significant difference; *: significant difference, P<0.05; **: significant difference, P <0.01.

Figure 6 is the body weight chart of 12-month-old WT and ABCD1 ^-/- mice; NS: no significant difference; *: significant difference, P<0.05; **: significant difference, P<0.01.

Figure 7 is a graph of the statistical results of the 20-month-old WT and ABCD1 ^-/- mice rotarod experiments; NS: no significant difference; *: significant difference, P<0.05; **: significant difference, P<0.01.

Fig. 8 is a graph showing the statistical results of the number of forelimbs off the ground in the open field test between 20-month-old WT and ABCD1 ^-/- mice; NS: no significant difference; **: significant difference, P<0.01.

Figure 9 is a graph of the statistical results of the total distance moved by the mice in 15 minutes in the open field test of 20-month-old WT and ABCD1 ^-/- mice; NS: no significant difference; *: significant difference, P<0.05; **: yes Significant difference, P<0.01.

Fig. 10 shows the trajectories of the mice in the open field test of 20-month-old WT and ABCD1 ^-/- mice within 15 minutes.

Detailed ways

Below in conjunction with embodiment the present invention is described in further detail, but should not be interpreted as the restriction of the present invention, under the situation of not departing from the spirit and essence of the present invention, the modification or replacement that the method, step or condition of the present invention is done all belong to the present invention range. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

Embodiment 1 HPCD injects X-ALD mouse procedure

Materials used in this example: 2-hydroxypropyl-β-cyclodextrin (2-hydroxypropyl-β-cyclodextrin) HPCD was purchased from Cyclodextrin Technologies Development, Inc., and ABCD1 knockout mice were purchased from Jackson Laboratory.

According to the program shown in Figure 1, HPCD was injected into the back of the neck of wild-type (WT) and X-ALD model mice (ABCD1 knockout mice, ABCD1 ^-/- mice), and the injection dose was 4000 mg HPCD per kg mouse body weight , specifically as follows: prepare 20% HPCD (W/V) with 0.9% normal saline, inject 20% HPCD 4 times a week to mice aged 6 months to 7 months, inject the same volume of normal saline into the control group, Carry out Filipin staining of cells, Filipin staining of adrenal gland and cerebellar tissue sections, and measurement of plasma total cholesterol and very long-chain fatty acids; inject 5 times a month from the age of 7 months to 12 months, and measure body weight at the age of 12 months; mice 12 After 1 month of age, inject 8 times a month, and conduct behavioral examination at 20 months of age.

Example 2 HPCD alleviates intracellular cholesterol accumulation in X-ALD mice

Materials used in this example: Filipin was purchased from Sigma.

In order to detect whether HPCD can improve the symptoms of cholesterol accumulation at the cellular level in X-ALD mice, 7-month-old WT and ABCD1 ^-/- mice injected with normal saline and HPCD were taken in Example 1, and their tail tip fibroblasts were isolated and cultured. Cells were observed by Filipin staining.

Filipin staining: the isolated and cultured tail tip fibroblasts were fixed with 4% paraformaldehyde at room temperature for 30 min; washed twice with PBS; diluted ethanol-dissolved Filipin mother solution (5 mg/mL) with PBS containing 10% fetal bovine serum (FBS) to The final concentration was 50 μg/mL, and incubated at room temperature in the dark for 30 minutes; washed three times with PBS and twice with deionized water; sealed, dried overnight, observed under a confocal laser microscope, and stored at -20°C.

The results of Filipin staining are shown in Figure 2. Compared with WT mouse cells, there is a large amount of cholesterol accumulation in ABCD1 ^-/- mouse cells injected with normal saline, while in ABCD1 ^-/- mouse cells injected with HPCD, cholesterol accumulation phenomenon is alleviated.

Example 3 HPCD alleviates tissue cholesterol accumulation in X-ALD mice, reduces plasma total cholesterol and very long-chain fatty acids (VLCFA)

To test whether HPCD can improve the symptoms of cholesterol accumulation at the tissue level in X-ALD mice, and reduce plasma total cholesterol and very long-chain fatty acids (VLCFA). Take the 7-month-old WT and ABCD1 ^-/- mice injected with normal saline and HPCD respectively in Example 1, separate the adrenal gland and cerebellum, perform frozen sections and Filipin staining, and measure plasma total cholesterol and very long-chain fatty acids.

(1) Staining of tissue sections: cut open the thorax of the mouse after anesthesia, insert a 20mL syringe connected to the scalp needle through the left ventricle, cut a small mouth at the right atrial appendage with scissors, push in 20mL of normal saline, and quickly change to 20mL after pushing 4% paraformaldehyde; take out the adrenal gland and cerebellum, and fix them in 4% paraformaldehyde overnight; soak the fixed tissues in 30% sucrose (dissolved in PBS) overnight; embed with OCT The tissue was stored at -80°C; the OCT-embedded tissue was fixed on a cryostat with a slice thickness of 20 μm, the OCT attached to the tissue slice was washed away with PBS, and the slice was soaked in filipin solution containing 100 μg/mL for 4 hours; The tissue sections were washed 3 times with PBS, and once with deionized water; fixed and observed with a fluorescence microscope. Frozen tissue sections were dried and stored at -20°C.

The results are shown in Figure 3. Compared with WT mouse cells, the adrenal cortex and cerebellum of ABCD1 ^-/- mice injected with normal saline had a large amount of cholesterol accumulation, while the adrenal cortex and cerebellum of ABCD1 ^-/- mice injected with HPCD In the process, the accumulation of cholesterol is relieved.

(2) Determination of total cholesterol and very long chain fatty acids:

Among them, the measurement method of plasma total cholesterol is: take 3 μL of mouse serum, and 300 μL containing cholesterol esterase (200U/L), cholesterol oxidase (100U/L), peroxidase (3KU/L), 4-amino The cholesterol determination reagent of antipyrine (0.3mmol/L) was reacted at 37°C for 5 minutes, the absorbance value at about 500nm was measured, and the cholesterol content was calculated by the standard curve.

The measurement method of very long-chain fatty acids is as follows: adrenal gland or brain tissue is homogenized with an organic solvent (chloroform:methanol=2:1, v/v), extracted and _N2 dried to prepare fatty acid methyl esters. Fatty acid methyl esters were dissolved in heptane after drying, separated by gas chromatography, and the concentration of fatty acids was identified and calculated from peak diagrams.

The results are shown in Figures 4 and 5: ABCD1 ^-/- mice injected with normal saline had significantly higher plasma total cholesterol and VLCFA than WT mice, but HPCD injection restored the plasma cholesterol of ABCD1 ^-/- mice to normal Level (Figure 4), improved the accumulation of VLCFA in its brain tissue and adrenal gland (Figure 5).

Embodiment 4HPCD reduces the body weight of X-ALD mice

The body weight of 12-month-old WT and ABCD1 ^-/- mice injected with saline and HPCD in Example 1 was taken. The results are shown in Figure 6: the body weight of ABCD1 ^-/- mice injected with normal saline was significantly higher than that of WT mice, while the body weight of ABCD1 ^-/- mice injected with HPCD returned to normal.

Embodiment 5HPCD alleviates behavioral abnormalities in X-ALD mice

In order to test whether HPCD can improve the behavioral abnormalities of X-ALD mice, 20-month-old WT and ABCD1 ^-/- mice injected with saline and HPCD in Example 1 were subjected to rotarod test and open field test respectively.

(1) Rotating rod experiment: Mice were trained two days before the test. On the day of the experiment, the mice were placed on the balance fatigue rotating rod device, and the device was started to rotate at an initial speed of 4 rpm, and the speed was gradually increased to 10 rpm within two minutes. When the speed of the rotating rod reaches 10 rpm, start recording the time for the mouse to fall from the rotating rod.

The rotarod test detects the time for mice to persist on a gradually accelerated horizontal rotarod without falling. The results are shown in Figure 7. The persistence time of ABCD1 ^-/- mice injected with normal saline on the rotarod decreased to less than that of WT. 19% of that of mice, while HPCD injection restored the persistence time of ABCD1 ^-/- mice to 87% of that of WT mice.

(2) Open field experiment: the mice were pre-placed in the experimental field before the experiment to adapt to the environment. On the day of the experiment, the mice were placed in a resin glass frame with a length, width, and width of 40×40 cm and a height of 40 cm, and a camera was placed above it to record the activities of the mice within 15 minutes. The Plexiglas bottom is divided into 9 equal squares. The camera device monitors the behavior of the mouse in the device, such as the trajectory, the distance of the movement, and the lifting of the upper limbs.

The statistical results of the number of forelimb lifts in mice are shown in Figure 8. The number of lifts of the forelimbs of ABCD1 ^-/- mice injected with normal saline was significantly reduced to 32% of that of WT mice, while HPCD injection made ABCD1 ^-/- mice The number of forelimb lifts recovered to 95% of that of WT mice. The statistical results of the total distance moved by the mice within 15 minutes are shown in Figure 9, and the movement trajectories within 15 minutes in the open field are shown in Figure 10. Similarly, the ABCD1 ^-/- mice injected with normal saline within 15 minutes The total distance moved was 32% of that of WT mice, whereas HPCD injection restored the total distance moved by ABCD1 ^−/− mice.

Claims (1)

1. Application of 2-hydroxypropyl-β-cyclodextrin in the preparation of medicine for treating X-linked adrenoleukodystrophy.