WO2018126522A1

WO2018126522A1 - Injectable intraocular sustained-release antiviral drug and manufacturing method and application thereof

Info

Publication number: WO2018126522A1
Application number: PCT/CN2017/074975
Authority: WO
Inventors: 陶勇; 黄延宾; 孙玉玲; 曹庆辰
Original assignee: 首都医科大学附属北京朝阳医院; 清华大学
Priority date: 2017-01-05
Filing date: 2017-02-27
Publication date: 2018-07-12
Also published as: CN108276440B; CN108276440A

Abstract

An injectable intraocular sustained-release antiviral drug and a manufacturing method and application thereof. The antiviral drug is an insoluble phosphonoformate salt microcrystal. The insoluble phosphonoformate salt is a multivalent metal salt of phosphonoformic acid. The manufacturing method comprises: reacting a monovalent metal salt of phosphonoformic acid with a soluble multivalent metal salt in water; and obtaining a system containing the insoluble phosphonoformate salt microcrystal. An insoluble phosphonoformate salt microcrystal sustained release system is designed and developed to meet special needs of a sustained and controlled release intraocular injection. The nominal drug loading of the system is higher than 100%. The insoluble phosphonoformate salt microcrystal sustained-release antiviral can be released sustainably and stably in a vitreous chamber of a rabbit eye. The vitreous concentration of phosphonoformate continuously remains higher than an effective therapeutic concentration of sodium phosphonoformate. The antiviral drug has a sustained release period of up to 12 weeks and does not induce significant toxic or inflammatory reactions. The insoluble phosphonoformate salt can be used as an antiviral drug suitable for sustained release intravitreal injection technology.

Description

Antiviral sustained-release drug for intraocular injection and preparation method and application thereof

Technical field

The invention belongs to the field of medicines, and particularly relates to an anti-virus sustained-release medicine which can be used for intraocular injection, a preparation method and application thereof.

Background technique

Viral retinal disease is an ocular lesion with a high risk of visual impairment and blindness, and is diverse in many diseases, including cytomegalovirus retinitis, acute retinal necrosis, and progressive outer retinopathy. The incidence of viral retinal diseases has increased in recent years due to the combination of an aging population, the spread of AIDS, the use of new immunosuppressants, unhealthy lifestyle habits, and increased stress.

Intravitreal injection of antiviral drugs is an important way to treat viral retinal diseases clinically. Injectable drugs include sodium foscarnet and ganciclovir. The half-life of sodium foscarnet in the eye is very short, and after 12 hours, the concentration of sodium foscarnet in the eye drops by more than half. After a single injection of sodium foscarnet, the effective drug concentration in the eye does not exceed 3 days. Therefore, clinically, patients with viral retinal diseases often require frequent intraocular drug injections, which greatly increases the economic burden on patients and families, and also makes the overall therapeutic effect of viral retinal diseases poor. In 1996, the FDA approved a sustained release device for ganciclovir, but the device has the disadvantage of requiring re-surgery after the drug is released. However, there have been no reports on the sustained release of sodium foscarnet.

The development of antiviral drugs with sustained release effect in the eye will undoubtedly reduce the frequency and side effects of intravitreal injections and improve the therapeutic effect; it has broad prospects in terms of medical value, social benefits and market applications.

However, there are many special requirements for intravitreal injection: First, the upper limit of the injection volume is 0.1 mL, and considering that the solid content of the injection suspension cannot be too high, which requires that the sustained release system must have a high drug loading amount. However, the dosage of sodium foscarnet is much higher than that of ganciclovir. The long-term sustained release in the eye requires a specially designed system with a drug loading of nearly 100%, which is technically challenging. Second, intravitreal injection The needle model is 27G or finer, and its inner diameter is only 210μm, which requires the particles of the sustained release system to have suitable size and is not easy to aggregate. Third, the intraocular environment requires strict sterile materials, and the materials used for injection must pass through. Strictly disinfected. These requirements make The conventional degradable microsphere sustained-release system cannot be applied, and a new system needs to be invented to solve the on-the-spot demand for sustained release of sodium foscarnet.

Invention disclosure

One of the objects of the present invention is to provide a phosphonic acid insoluble salt microcrystal.

In the phosphoric acid insoluble salt microcrystal provided by the present invention, the phosphoric acid insoluble salt is a polyvalent metal salt of foscarnet, and the polyvalent metal may be selected from at least one of calcium, magnesium, zinc and aluminum. .

The phosphonium insoluble salt microcrystals have a diameter ranging from 0.1 to 100 μm, preferably from 1 to 50 μm.

The foscarnet poorly soluble salt microcrystals may comprise bound water.

The foscarnet poorly soluble salt microcrystals provided by the present invention are prepared according to the method comprising the following steps:

The monovalent metal salt of the foscarnet and the soluble salt of the polyvalent metal are reacted in water to obtain a system containing the micron crystal of the phosphonium insoluble salt.

In the above method, the monovalent metal salt of foscarnet may specifically be sodium foscarnet or potassium phosphonate.

The molar ratio of the monovalent metal salt of foscarnet to the soluble salt of the polyvalent metal is from 1:1 to 1:10.

The reaction time is from 0.1 to 24 hours, specifically 1 hour.

The reaction is carried out with stirring.

The monovalent metal salt of foscarnet is added as an aqueous solution; the soluble salt of the polyvalent metal is also added as an aqueous solution.

The aqueous solution of the monovalent metal salt of foscarnet has a concentration of 0.1 to 500 mg/mL, specifically 1 to 10 mg/mL.

The concentration of the aqueous solution of the soluble salt of the polyvalent metal is from 0.1 to 500 mg/mL, specifically from 1 to 10 mg/mL.

The method further includes the operation of isolating the phosphonic acid insoluble salt microcrystals from the obtained system containing the phosphonic acid insoluble salt microcrystals. The operation is: the system containing the phosphonic acid insoluble salt microcrystals is allowed to stand, filtered, the solid is collected, washed, and the suspension is lyophilized to obtain a phosphonic acid insoluble salt microcrystal.

The use of the above-mentioned phosphonium insoluble salt microcrystals for the preparation of a medicament for preventing/treating viral retinal diseases is also within the scope of the present invention.

Another object of the present invention is to provide an antiviral drug having a sustained release effect which can be injected intraocularly.

The antiviral drug having a sustained release effect which can be administered intraocularly according to the present invention is the above-mentioned phosphonic acid insoluble salt microcrystal or a suspension containing the above-mentioned phosphonium insoluble salt microcrystal.

The invention aims at the special requirement of intraocular injection and controlled release, and designs and develops a phosphonium insoluble salt microcrystalline sustained release system, which can be loaded with a nominal amount of more than 100% relative to the sodium foscarnet. The sustained release of the drug in the vitreous cavity is achieved by preparing an injectable phosphofuric acid insoluble salt microcrystal using its dissolution equilibrium.

Animal experiments show that the phosphonium insoluble salt microcrystalline sustained-release drug prepared by the invention can be stably and sustainedly released in the vitreous cavity of the rabbit eye, and the concentration of the phosphonate root in the vitreous cavity is always maintained above the effective therapeutic concentration of the sodium foscarnet. The sustained release cycle lasted for 12 weeks, and the health of the rabbit eye was good after 12 weeks.

Therefore, it has been proved by animal experiments that the foscarnet insoluble salt microcrystalline sustained-release drug prepared by the invention can well solve the problem that the sodium foscarnet needs frequent injections, and the insoluble salt of the foscarnet should be used as a glass. Injectable slow release techniques for antiviral drugs in body cavity are used.

DRAWINGS

1 is a flow chart showing the preparation of a phosphonium insoluble salt microcrystalline sustained-release drug prepared by the present invention.

2 is a scanning electron micrograph of a phosphonium insoluble salt microcrystalline sustained-release drug prepared according to the present invention.

Figure 3 is an X-ray diffraction pattern of the foscarnet insoluble salt microcrystalline sustained release drug and the raw material sodium foscarnet prepared according to the present invention.

Fig. 4 is a view showing the presence of a phosphonium insoluble salt microcrystalline sustained-release drug prepared by the present invention in rabbit eyes and the health condition of a rabbit eye in an animal experiment.

Figure 5 is a scanning electron micrograph of a foscarnet insoluble salt microcrystalline sustained release drug taken from rabbit eyes in an animal experiment.

Figure 6 is a graph showing changes in the concentration of foscarnet in the vitreous and aqueous humor of rabbit eyes in an animal experiment.

The best way to implement the invention

The experimental methods in the following examples are conventional methods unless otherwise specified.

The invention will now be described by way of specific examples, but the invention is not limited thereto.

The experimental methods used in the following examples are conventional methods unless otherwise specified; the reagents, materials and the like used in the following examples are commercially available unless otherwise specified.

Example 1. Preparation of Phosphonic Acid Insoluble Salt Microcrystals

According to the preparation scheme shown in Fig. 1, a phosphonic acid insoluble salt microcrystal was prepared.

Prepare 2mg/mL sodium phosphonate sodium solution and 2mg/mL calcium chloride solution, pour and mix the two solutions in a volume ratio of 1:1 under magnetic stirring. After mixing, continue stirring for 1 hour; stop stirring and let stand for 10 minutes. The supernatant was slowly poured off; the sample was washed 3 times with pure water; the suspension was lyophilized to obtain product a.

According to the above method, by adjusting the concentration of the sodium phosphonate solution and the calcium chloride solution, or by adjusting the mixing volume ratio of the sodium phosphonate solution to the calcium chloride solution, different sizes of the foscarnet insoluble salt microcrystalline sustained release drug can be prepared.

According to the above preparation method, a 6 mg/mL sodium phosphonate sodium aqueous solution and a 6 mg/mL calcium chloride solution were prepared, and the two solutions were mixed and reacted in a volume ratio of 1:1 to obtain a product b.

According to the above preparation method, 8 mg/mL aqueous solution of sodium foscarate and 8 mg/mL of calcium chloride solution were prepared, and the two solutions were mixed and reacted in a volume ratio of 1:1 to obtain product c.

Example 2, Characterization of in vitro performance of foscarnet insoluble salt microcrystals

Scanning electron microscopy characterization: The lyophilized powder samples a, b, and c of the foscarnet insoluble salt microcrystals prepared in Example 1 were uniformly sprinkled on the sample stage to which the conductive paste was adhered, and the excess sample on the surface was blown off. Scanning electron microscope observation. The experimental results are shown in Figure 2.

Fig. 2 (a), (b) and (c) are scanning electron micrographs of the prepared micronized crystals of foscarnet insoluble salts, a, b and c, respectively, and the average sizes thereof are estimated to be 10 μm, 60 μm and 100 μm, respectively. The crystals are grown in the radial direction from the center to form a micron-sized spherical crystal.

Characterization by plasma mass spectrometry: Weigh 50mg of foscarnet insoluble salt microcrystal freeze-dried powder and sodium foscarnet standard, respectively add 0.1M dilute hydrochloric acid until all dissolved, analyze by plasma mass spectrometer, main working parameters cooling gas flow rate: 13.88 L/min, auxiliary gas flow rate: 0.795 L/min, carrier gas flow rate: 0.952 L/min. The experimental results confirmed that the ratio of phospho-methyl and calcium ions in the product was close to 2:3.

X-ray diffraction characterization: phosphonic acid insoluble salt microcrystal freeze-dried powder and sodium foscarnet standard They were respectively ground with a mortar, sieved with a 100 mesh sieve, and characterized by an X-ray diffractometer. The test conditions were: 2 ° 2 θ / min, step size 0.02 ° 2θ, using Cu Kα ray. The experimental results are shown in Figure 3.

The above results indicate that the size of the phosphonium insoluble salt microcrystalline sustained release drug can be controlled by adjusting the initial concentration of the sodium foscarnet solution and the calcium chloride solution and the mixing volume ratio. By X-ray diffraction pattern analysis (see Figure 3), the crystallinity of the sample is close to 100%, and its crystal structure is different from that of the raw material sodium foscarnet. Determination of the content of Ca, Na and P elements foscarnet insoluble salts extended release pharmaceutical micron crystals utilizing plasma mass spectrometry (ICP-MS), whereby molecular structure was confirmed foscarnet insoluble salts of Ca ₃ (CO ₅ P) ₂ or write Ca ₃ (pfa) ₂ .

Table 1 is the equilibrium solubility value of the foscarnet insoluble salt microcrystals (product a) prepared in Example 1 under different conditions.

The above results indicate that the micron crystal of the foscarnet insoluble salt microcrystal can be prepared by directly mixing the sodium phosphonate solution with the calcium chloride solution, and the insoluble intravitate microcrystal can satisfy the intraocular injection (the particle size is below 100 microns). And have the basic requirements of anti-viral drugs with sustained release effect.

Example 3: Intraocular sustained release experiment of micron crystals of foscarnet insoluble salt microcrystals

Thirteen New Zealand white rabbits were selected and injected into both eyes with sustained release drugs.

The animal experiment steps are as follows:

1. Formulation of injection suspension: 180 mg of foscarnet insoluble salt sustained-release drug (product a) is dissolved in 1.8 mL of 1% CMV solution (carboxymethylcellulose), and the drug and CMV solution are released before vortex mixing. All should be UV-irradiated for 60 minutes for anti-virus sterilization.

2. Intravenous pentobarbital anesthesia for New Zealand white rabbits.

3, using a 29G insulin needle to intravitreal injection of foscarnet insoluble salt / CMV suspension into the rabbit's eyes, after the injection, it was observed that there was white matter in the vitreous cavity, confirming the injection was successful, no liquid spillover (See Figure 4d).

After 4, 3 weeks, 6 weeks, and 12 weeks, 3 rabbits were sacrificed. For each rabbit eye, aqueous humor was taken for the test of foscarnet concentration, and the vitreous body was taken out (containing sustained-release drug), and the solid was centrifuged. The drug was separated from the vitreous solution, the solid drug was characterized by scanning electron microscopy, and the vitreous solution was tested for foscarnet concentration.

The foscarnet insoluble salt microcrystalline sustained-release drug prepared by the invention can stably and stably release in the vitreous cavity of the rabbit eye (see FIG. 4a-3, FIG. 4b-6, FIG. 4c-12), and has Good sustained release effect, the concentration of foscarnet in the vitreous cavity is always above 70μg/mL, higher than the IC _{50 of the} sodium foscarate = 54μg/mL, and the sustained release period is more than 12 weeks (see Figure 6). After 12 weeks, the rabbit eye was well sensitized, the lens was perfectly transparent (see Figure 4e), and there was still an incompletely dissolved phosphofuric acid insoluble salt microcrystalline sustained-release drug in the eye (see Figure 4f). The vitreous cavity remained clear and transparent, and the retina and choroid All were normal, without any bleeding or exudation. With the passage of time, the scanning electron microscopy morphology of sustained-release drugs in rabbit eyes is defective, and the original spherulite morphology cannot be maintained. The size of drug fragments is also gradually decreasing (see Figure 5), which also indicates that the drug is continuously slowing down. release. The above results indicate that the foscarnet insoluble salt microcrystalline sustained-release drug prepared by the present invention can fully meet the requirements of an intraocular injection and an antiviral drug having a sustained release effect.

Industrial application

The foscarnet insoluble salt microcrystalline sustained-release drug prepared by the invention can be stably and sustainedly released in the vitreous cavity of the rabbit eye, and the concentration of the phosphonate root in the vitreous cavity is maintained above the effective therapeutic concentration of the sodium foscarnet, and the sustained release period is long. After 12 weeks, the rabbit eyes were in good health after 12 weeks. Therefore, it has been proved by animal experiments that the foscarnet insoluble salt microcrystalline sustained-release drug prepared by the invention can well solve the problem that the sodium foscarnet needs frequent injections, and the insoluble salt of the foscarnet should be used as a glass. Injectable slow release techniques for antiviral drugs in body cavity are used.

Claims

A phosphonic acid insoluble salt microcrystal, wherein the phosphoric acid poorly soluble salt is a polyvalent metal salt of a foscarnet, and the polyvalent metal is at least one selected from the group consisting of calcium, magnesium, zinc, and aluminum.
The foscarnet insoluble salt microcrystal according to claim 1, wherein the phosphonic acid insoluble salt microcrystal has a diameter of from 0.1 to 100 μm.
The foscarnet poorly soluble salt microcrystal according to claim 1, wherein the phosphonic acid insoluble salt microcrystal contains crystal water.
A process for the preparation of the phosphonic acid insoluble salt microcrystals according to any one of claims 1 to 3, which comprises reacting a monovalent metal salt of a phosphonic acid with a soluble salt of a polyvalent metal in water to obtain a phosphorus-containing formic acid. A system of dissolved salt microcrystals.
The method according to claim 4, wherein in the method, the monovalent metal salt of foscarnet is sodium foscarnet or potassium phosphonate;

The molar ratio of the monovalent metal salt of foscarnet to the soluble salt of the polyvalent metal is 1:1 to 1:10;

The reaction time is 0.1-24 hours;

The reaction is carried out with stirring.
The method according to claim 4 or 5, wherein in the method, the monovalent metal salt of foscarnet is added as an aqueous solution; the soluble salt of the polyvalent metal is added as an aqueous solution;

The concentration of the aqueous solution of the monovalent metal salt of foscarnet is 0.1-500 mg/mL;

The concentration of the aqueous solution of the soluble salt of the polyvalent metal is from 0.1 to 500 mg/mL.
The method according to any one of claims 4-6, wherein the method further comprises the operation of separating the phosphonic acid insoluble salt microcrystals from the obtained system containing the phosphonic acid insoluble salt microcrystals,

The operation is: the system containing the phosphonic acid insoluble salt microcrystals is allowed to stand, filtered, the solid is collected, washed, and the suspension is lyophilized to obtain a phosphonic acid insoluble salt microcrystal.
Use of the foscarnet poorly soluble salt microcrystals according to any one of claims 1 to 3 for the preparation of a medicament for preventing/treating viral retinal diseases.
An intraocularly-injectable antiviral drug having a sustained release effect, which is a phosphonic acid poorly soluble salt microcrystal according to any one of claims 1 to 3, or comprising any one of claims 1-3 A suspension of the phosphonic acid insoluble salt microcrystals.