RU2670091C1 - Method for delivering nanoparticles intended for the transport of drugs to the brain of mammals through the blood-brain barrier - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to experimental medicine, namely to method for non-invasive delivery of nanoparticles to the mammalian brain, comprising the following steps: preparation of tungsten monocarbide or vanadium monocarbide powder in the form of nanoparticles with a size of 15 to 60 nm, preparation of a mixture of said powder in the form of nanoparticles with a liquid carrier, endotracheal administration of the resulting mixture in an amount of 20 to 32 mg per kilogram of mammal weight.

EFFECT: invention provides a high degree of targeted delivery to the brain of drugs through the blood-brain barrier and obtaining a pronounced and persistent clinical effect due to the new technology developed by the authors, which involves the introduction of nanoparticles into the body through the air-lung barrier of the lungs, in which they are captured by erythrocytes, transported through the circulatory system and overcome the blood-brain barrier.

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Description

The method of delivery of nanoparticles intended for the transport of drugs to the brain of mammals through the blood-brain barrier

The invention relates to the field of experimental medicine, methods for the targeted delivery of nanoparticles intended for the transport of medicinal substances to the brain. It can also be used in other fields: nanobiotechnology, experimental pharmacology, nanopharmacology.

The problem of drug delivery to the brain is relevant for the treatment of a number of socially significant diseases, for example: cerebrovascular, neurodegenerative brain lesions, oncological, neuroinfectious diseases, etc.

It is known that in order to achieve the desired effect of the introduced medicinal substance, it is necessary to ensure its delivery in optimal concentration to the target area of the body, which is achieved by using various routes of administration to the body.

However, there is the problem of introducing drugs into the body and delivering them to the brain (GM). The internal environment of the GM is separated from the circulatory system by the blood-brain barrier (BBB), which performs a protective function and prevents the penetration into the brain of some foreign substances that enter the bloodstream.

Thus, the effectiveness of the effect on drugs on GM can be improved by searching for new ways and methods of their targeted (directed) delivery, allowing them to penetrate the BBB.

Several methods for delivering drugs to the brain are known.

A known method of treating metastatic brain lesions (RU 2238731, 04/14/2000), including intrathecal administration of methotrexate in an amount of 1.2-2.5 mg per kg of patient weight in combination with cytosar in an amount of 3-5 mg per kg of weight and hydrocortisone the amount of 125 mg with preliminary brain dehydration and intravenous administration of cerebrolysin. From the second day after the introduction of chemotherapy, 1-2 sessions of plasmapheresis are carried out. The drugs are administered three times with an interval of 7 days, then 1 time per month three times.

The known method of intrathecal drug administration has significant disadvantages:

- trauma, which can lead to damage to the spinal cord;

- the risk of infection of the cerebrospinal fluid, which is fraught with serious complications

- when using the indicated method of administration and delivery of drugs, they cannot overcome the blood-brain barrier (BBB) or do not overcome it sufficiently to provide a therapeutic effect. This is due to the properties of the barrier that prevents the penetration into the brain of some foreign substances that enter the bloodstream, which can be attributed to the vast majority of complex compounds that do not have an active transport mechanism developed during phylogenesis.

A known method of treating cancer patients with metastatic brain damage (RU 2290974, 04/11/2005), including endoliquor therapy. After surgical removal of a brain tumor, in the early postoperative period, the subarachnoid space of the spinal cord is catheterized at the level of L4-L5, an endolumbar catheter is placed. 5 ml of cerebrospinal fluid are taken. It is mixed with methotrexate in a dose of 5 mg and a suspension of hydrocortisone in a dose of 50 mg. The mixture is incubated for 30 minutes at 37 ° C. Then, a jet is introduced through the endolumbar catheter into the subarachnoid space. Within one course of treatment, 5 such infusions are made with an interval of 3 days. A total of 3 described courses are carried out in combination with 5 cycles of adjuvant systemic chemotherapy with the introduction of cytostatics on autologous blood.

The disadvantages of the method are trauma, which can lead to brain damage, as well as the likelihood of infection of the cerebrospinal fluid, which is fraught with serious complications.

A known method of intravenous administration of drugs for the treatment of cancer, including the brain, through combination therapy that provides an anticancer effect when administered to a person affected by cancer, effective amounts of (SP-4-3) - (cis-ammindichloro- [2-methylpyridine] platinum (II) (ZD0473) or its prodrug and anticancer funds not based on platinum selected from the group consisting of taxane, camptothecin, gemcitabine, capecitabine, 5-fluorouracil, vinorelbine, topotecan, anthracycline, irinotecan, etoposide, vinblastine, pemetrexed, vindesine, cyclophosphamide, ifosfamide 2011 g.).

However, this method is invasive and traumatic, there is a risk of infection of the body. Air bubbles can enter the vascular bed along with the drug and cause embolism. The method is not effective enough, which is associated with low bioavailability of drugs due to the need to overcome the BBB.

Known methods of oral administration of drugs for the treatment of diseases or disorders of the central nervous system, for example, "Pharmaceutical composition having nootropic activity, and method for its preparation" (RU 2240783, 07.17.2003).

The disadvantages of the oral route of administration are:

- primary and indirect effects of drugs on the mucous membranes of the oral cavity and esophagus, for example, a direct irritant effect on the mucous membranes;

- direct and side effects on the mucous membrane of the gastrointestinal tract (GIT), for example: stimulation of the release of hydrochloric acid, which can be a complicating factor for patients with a hyperacid form of gastritis; inhibition of the production of protective mucus; slowing down the process of physiological regeneration of the epithelium of the mucous membrane;

- inactivation of drugs with enzymes of the gastrointestinal tract;

- slow and incomplete absorption of drugs in the digestive tract;

- the effect of food and other substances on absorption;

- partial inactivation of drugs in the liver;

- deposition of drugs in the liver, which, due to an increase in their concentration in the liver tissue above the maximum allowable concentrations, can lead to hepatotoxic effects;

- possible death of macrophages in the submucosal gastrointestinal tract leading to a decrease in immunity;

- Drugs under the influence of blood plasma enzymes can form new compounds and have undesirable side effects on other organs.

The factors listed above reduce the bioavailability of drugs, increase the risk of side effects and, as a result, reduce the effectiveness of its use.

A known method for the delivery of drugs or diagnostic tools to the central nervous system through the blood-brain barrier of mammals [patent US 6117454]. Medicines are included in polybutyl cyanoacrylate nanoparticles (NPs) obtained and coated with an appropriate surfactant or adsorbed onto their surface and delivered to the brain without the need to change the structure of the drug. Surfactants allow complexes with drug-filled polybutyl cyanoacrylate NPs to overcome the blood-brain barrier and targeted delivery of drugs to the central nervous system.

The disadvantage of the proposed method is the high probability of toxic side effects caused by surfactants used to cover polybutyl cyanoacrylate NPs.

As the closest analogue adopted "Method of obtaining a targeted drug delivery system for the introduction of a pharmacologically active substance into the central nervous system of mammals through the blood-brain barrier" (patent RU 2423104; 2011)

A targeted drug delivery system contains poly (DL-lactide) and / or poly (DL-lactide-soglycolide) nanoparticles (NPs), a pharmacologically active substance adsorbed and / or incorporated into nanoparticles, and contains TPGS (D-β-tocopherol polyethylene glycol 1000 succinate) or has a coating of the surfactant pluronic 188, which is deposited on nanoparticles filled with a drug substance.

A system containing nanoparticles with a medicinal substance is administered orally or by intravenous injection so that it can enter the bloodstream, due to which the drug reaches the blood-brain barrier (BBB), overcomes it and enters the central nervous system.

The disadvantages of the method for intravenous administration of nanoparticles with drugs are:

- invasiveness, because the route of administration is traumatic;

- the possibility of infection of the body; air bubbles that can cause embolism can enter the bloodstream along with the medicine.

The disadvantages of the oral method of administering a complex of nanoparticles and drugs are due to the features of nanoscale objects, which, being easily absorbed and moving through tissue and cell barriers of the body’s barrier systems, cause a change in the distribution of NPs, which consists in increasing the concentration of NPs in biological targets and in organs are such targets that causes their defeat and, as a result, the development of side effects.

Of great importance is the group of pharmacokinetic factors determined by the physicochemical processes of the interaction of NPs with the body's barrier systems. With the oral administration of NPs and drugs, the total thickness of the barrier structures is several tens of microns, including: intestinal epithelium in the gastrointestinal tract - 22-26 microns, liver barrier systems 30-40 microns, blood-brain barrier 0.5-0.7 microns. Direct and indirect side effects are possible along the entire path of translocation of NPs through barrier systems.

The specificity of the barrier systems of the liver is in the structure of sinusoidal capillaries. The basement membrane of the vessels on which endotheliocytes are located is not continuous, but discontinuous, and, therefore, NPs and drugs that enter the bloodstream can directly contact the liver cells, penetrate and accumulate in the liver cells, causing cytotoxic effects.

An important factor affecting the interaction of NPs and the body is the localization of NPs in the genome. The authors found that the severity of the genotoxic effect is determined by the localization of NP relative to certain sections of interphase chromatin and the nuclear membrane of a eukaryotic cell. For example, the localization of NPs in the intermembrane gap of a nuclear membrane can cause necrosis, accompanied by the destruction of the nuclear membrane. In the case of localization of metal-containing nano-objects in interphase chromatin near the nucleolus, their effect is accompanied by activation of the genome.

The likelihood of side effects on other organs that are not the target also depends on the length of the route of transfer of NPs through the circulatory system. With the oral method of administration, after passing through the liver, the NPs enter the circulatory system and, under the influence of hematodynamic forces, move along the circulatory system: vena cava, large circle of blood circulation, lungs, small circle of blood circulation, carotid artery. The length of the route of hematodynamic transfer of LF from the gastrointestinal tract to the central nervous system is approximately 70 cm.

Studies by the authors have shown other possible side effects of exposure to NPs, for example, unplanned reactions of substances released under the mucosa with NPs. The products of these reactions may be toxic.

The objective of the invention is to provide a non-invasive, easy-to-perform and effective method for the delivery of nanoparticles intended for the transport of drugs to the mammalian brain through the BBB, which reduces the likelihood of complications and ensures a high degree of targeted drug delivery.

The essence of the invention lies in the fact that the proposed method for non-invasive delivery of nanoparticles to the brain of mammals, comprising the following steps:

preparation of tungsten monocarbide powder or vanadium monocarbide in the form of nanoparticles with a size of from 15 to 60 nm,

preparing a mixture of the specified powder in the form of nanoparticles with a liquid carrier, for example, physiological saline, at the rate of 5 mg of nanoparticles per 0.5 ml of carrier,

endotracheal administration of the resulting mixture in an amount of from 20 to 32 mg per kilogram of mammal weight.

At the same time, nanoparticles are also intended for transporting a drug substance.

The technical results of the invention are:

- ensuring a high degree of targeted delivery of drugs to the brain through the blood-brain barrier and obtaining a pronounced and lasting clinical effect due to the new technology developed by the authors, which involves the introduction of NPs into the body through the airborne lung barrier, in which they are captured by red blood cells, transferred through the circulatory system and overcome the blood-brain barrier.

- targeted delivery of nanoparticles to the brain according to the proposed method significantly reduces the complications that are possible with their invasive or minimally invasive methods of introduction into the body, in which complications are possible: bleeding, the development of an infectious and inflammatory process, etc., which may adversely affect the results of treatment

- the method allows to avoid side effects characteristic of, for example, the oral route of administration, significantly reducing the load on the liver and other organs of the gastrointestinal tract

The key point of the method is the passage of NPs through the airborne barrier (AGB) of the lungs due to the endotracheal route of administration.

The authors proceeded from the morphological structure of the lungs. The airborne barrier (AGB) is a tissue structure of relatively small thickness. The size of the alveolocyte in the thin part is 0.5-1.5 μm, two continuous basement membranes with a total thickness of 1-2 μm and the thickness of the vessel epithelium are 1-2 μm (Fig. 1). Thus, the total thickness of the barrier is about 3-6 microns, which is significantly less than the total thickness of the barrier systems (about 70 microns) when administered orally.

The authors calculated that the total length of the NP transfer route through the circulatory system from AHB to brain tissue is approximately 25 cm, which is significantly shorter than when administered orally (70 cm).

By reducing the route of hemodynamic transfer, the time of interaction between drugs and NPs with blood is reduced and, accordingly, the risk of NPs entering other organs decreases.

The authors found that NPs, having overcome AGB, are deposited in erythrocytes and transferred to them through the circulatory system. After that, NPs can go beyond the erythrocyte biomembranes and translocate through the BBB into the cytoplasm of brain cells and their nucleus (Fig. 2).

At the same time, the total cross section of vessels along the route of hematodynamic transfer of NPs through the circulatory system during endotracheal administration is greater than the total cross section of vessels along the transfer path when administered orally, which accordingly reduces the total concentration of NPs in the circulatory system and, thereby, reduces the risk of side complications

In addition, the area of the barrier layer of the epithelium in the lungs is significantly higher than in the gastrointestinal tract and liver and, therefore, the specific concentration of NPs at the same doses will be lower and the likelihood of side effects when interacting with NPs also decreases.

The method is as follows:

Tungsten monocarbide (WC) or vanadium monocarbide (VC) powders are prepared in the form of nanoparticles by heat treatment in a hydrogen medium of a multicomponent composition obtained in a plasma reactor at the IMET RAS experimental plasma chemical plant.

The Malvern Mastersizer 2000 laser diffraction particle size analyzer is used to determine the size of the nanoparticles. To prepare the samples, preliminary separation into fractions was carried out, including dispersion of the nanoparticle powder in a liquid (ethyl alcohol) by ultrasound followed by sedimentation of particles in a centrifuge. Metal-containing nanoparticles ranging in size from 15 to 60 nm are selected.

A mixture is prepared at the rate of 5 mg of nanoparticles per 0.5 ml of physiological saline in a volume of 2-3 ml. The mixture is stirred in a magnetic stirrer.

The finished mixture with the number of nanoparticles from 20 to 32 mg per kilogram of animal weight is placed in an endotracheal device, for example, in a syringe without a needle connected to an endotracheal tube. The animal is under general anesthesia, an endotracheal tube is inserted into the trachea. The mixture is introduced by smoothly pressing once on the syringe rod for 2-3 seconds.

After introduction, the nanoparticles cross the airborne barrier, enter the bloodstream and are transported through the circulatory system, reach the blood-brain barrier, overcome it and enter the GM.

The method is illustrated by the following examples.

Example 1

We studied the possibility of non-invasive administration for delivery to the brain via AHB and BBB of nanoparticles (NPs) of tungsten monocarbide (WC) 40 nm in size.

LF was introduced according to the proposed method to white outbred rats, males with an initial weight of 200-220 g, endotracheally in an amount of 20 mg per kilogram of weight and observation was established for 4 months.

The control group consisted of intact rats. Animals were kept in the same vivarium conditions. Number of animals 3.

After 1, 3, 8, 30, 60, 90, 120 days after endotracheal administration of NPs, biopsy samples of brain tissue and blood cells were studied. In anesthetized animals, a brain stem biopsy was performed, the biopsy samples were fixed in a buffer solution of osmium tetroxide, dehydrated in alcohols of increasing concentration and in absolute acetone, and poured into a mixture of epon and araldite. Semi-thin and ultrathin sections were prepared on an LKB-4800 ultratome. Semi-thin sections were stained with methylene blue (for review and detection of fat), Schiff's reagent according to the McManus method (for detection of glycogen) and examined under a light microscope.

Ultrathin sections were contrasted with lead uranyl acetate and citrate, after which they were examined under a MIKMED-5 light microscope and a JEM-7A electron microscope. To quantify the effect of NPs and their aggregates on Mammalia cells and tissue structures, computer morphodensitometry was used to determine the diameter of the equivalent circle of NP aggregates and its modification for in vivo study of blood cells, erythrocytes.

Example 2

We studied the possibility of non-invasive introduction of nanoparticles of vanadium monocarbide (VC) of sizes 15, 60, 1200 nm through AHB and BBB for their delivery to the brain.

LF was introduced according to the proposed method to white outbred rats, males with an initial weight of 200-220 g, endotracheally in an amount of 20 mg per kilogram of weight and observation was established for 4 months.

The control group consisted of intact rats. Animals were kept in the same vivarium conditions. The number of animals is 14.

In the time intervals of 1, 3, 8, 30, 60, 90, and 120 days, the animals were opened under anesthesia and the brain was taken from them to determine the content of NP in terms of the mass V / 1 g of the organ. The biopsy samples of tissues of the alveolar department of the lung, brain, and blood cells were studied.

Biopsies were fixed in a buffer solution of osmium tetroxide, dehydrated in alcohols of increasing concentration and in absolute acetone, and poured into a mixture of epon and araldite. Semi-thin and ultrathin sections were prepared on an LKB-4800 ultratome. Semi-thin sections were stained with methylene blue (for review and detection of fat), Schiff's reagent according to the McManus method (for detection of glycogen) and examined under a light microscope.

Ultrathin sections were contrasted with lead uranyl acetate and citrate, after which they were examined under a JEM-7A electron microscope (Japan) and a light microscope (MIKMED-5). To quantify the effect of NPs and their aggregates on Mammalia cells and tissue structures, computer morphodensitometry was used to determine the diameter of the equivalent circle of NP aggregates and its modification for intravital studies of red blood cells.

It was shown that NPs with a size of 15 nm penetrate through the AEG and BBB, accumulate in the brain at a concentration of 2.9 μg / ml on the 3rd day after administration, and the time of their 50% excretion is 36 days (Fig. 3a).

LFs with a size of 60 nm accumulate in the brain at a concentration of 2.75 μg / ml on the 3rd day after administration, and the time of their 50% elimination is 34 days (Fig. 3b).

NPs with a size of 1200 nm accumulate in the brain at a concentration of less than 0.1 μg / ml, which is close to the background values (Fig. 3c)

Thus, the studies of the proposed method for the introduction of low frequencies showed that:

- the non-invasiveness of the method is achieved by the introduction of LF endotracheally through the airborne barrier (AGB) of the lungs;

- LF transfer from AHB to the BBB is carried out by red blood cells through the circulatory system;

- the optimal NP size for their translocation to the brain during endotracheal administration through AHB is the range from 15 to 60 nm in the amount of NP from 20 to 32 mg per kilogram of weight.

Claims (7)

1. The method of non-invasive delivery of nanoparticles to the mammalian brain, comprising the following steps: preparation of tungsten monocarbide powder or vanadium monocarbide in the form of nanoparticles with a size of from 15 to 60 nm, preparing a mixture of the specified powder in the form of nanoparticles with a liquid carrier, endotracheal administration of the resulting mixture in an amount of from 20 to 32 mg per kilogram of mammal weight. 2. The method according to p. 1, characterized in that the nanoparticles are intended for the transport of a medicinal substance. 3. The method according to p. 1, characterized in that the mixture of the specified powder in the form of nanoparticles with a liquid carrier is prepared at the rate of 5 mg of nanoparticles per 0.5 ml of carrier. 4. The method according to p. 1, characterized in that the liquid carrier is physiological saline.

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