KR20230063790A - Nanostructured lipid carriers containing remdesivir, preparation method thereof, and pharmaceutical composition comprising the same - Google Patents

Abstract

The present invention includes remdesivir, a solid lipid, a liquid lipid, and a surfactant, and includes remdesivir, and the solid lipid and liquid lipid are mixed in the center, and the surfactant is in the form of particles forming the outside. As related, it has the effect of improving the poor solubility of remdesivir and increasing its bioavailability.

Description

Nanostructured lipid carriers containing remdesivir, preparation method thereof, and pharmaceutical composition comprising the same}

The present invention relates to a nanolipid carrier containing remdesivir, a method for preparing the same, and a pharmaceutical composition comprising the nanolipid carrier.

Remdesivir is a 2-ethylbutyl (2S)-2-[[[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f derivative from GS-441524). ][1,2,4]trizin-7-yl)-5-cyano-3,4-dihydroxyoxolan-2-yl]methoxy-phenoxyphosphoryl]amino]propanoate (2 -ethylbutyl (2S)-2-[[[(2R,3S,4R,5R)-5-(4-aminopyrrolo[2,1-f][1,2,4]triazin-7-yl)-5- cyano-3,4-dihydroxyoxolan-2-yl] methoxy-phenoxyphosphoryl] amino] propanoate), and is a compound having the structure of Formula 1 below.

Remdesivir, marketed under the trade name Veklury, is a prodrug of an adenosine triphosphate analog with potential antiviral activity against a variety of RNA viruses. Upon administration, the prodrug remdesivir is metabolized to its active form, GS-441524. GS-441524 is a drug that inhibits the action of viral RNA-dependent RNA polymerase (RdRp), resulting in termination of RNA transcription and reduced viral RNA production. It is mainly used in the form of an intravenous injection.

However, remdesivir has a logP partition coefficient of 2.01, which is close to hydrophobicity and is poorly soluble in water. Therefore, it is necessary to improve the physical properties of these active ingredients due to their low bioavailability.

The commercially available lyophilized powder of remdesivir for intravenous injection (Veklury inj) uses a cyclodextrin derivative such as sulphobutyl ether β-cyclodextrin sodium as an excipient to Although the problem of poor solubility was solved, the increase in bioavailability was not maximized, and cyclodextrin is a cyclic oligosaccharide molecule that is mainly excreted by the kidney, so there is a problem that accumulation in the kidney can cause renal and hepatic impairment, resulting in renal impairment. And patients suffering from hepatic impairment have the inconvenience of limited administration or periodic monitoring.

As a prior patent technology, Chinese Patent Publication No. 112843073 A discloses the use of remdesivir used in the manufacture of drugs for the treatment of bovine parainfluenza virus type 3 (BPIV-3) infections, while remdesivir and at least one Discloses a composition consisting of other non-drug ingredients of, and Korean Patent Registration 10-1822348 B1 discloses a method for treating Filoviridae infection, particularly Ebola virus infection and Marburg virus infection, and chemical formulas and formulas of compounds used for treatment The chemical formula of the prodrug is described. However, these prior patents do not disclose a method for improving bioavailability by improving the physical properties of remdesivir.

Korean Registered Patent No. 10-2236174 B1 discloses a nano-lipid carrier containing econazole, solid lipid, liquid lipid and surfactant to improve skin permeability and enable continuous release of econazole, an active substance, and utilize it to prepare a nano-lipid carrier. Although the production of a film-forming pharmaceutical composition that is topically applied to the skin is disclosed, an ingredient composition suitable for improving the low solubility and bioavailability of remdesivir, an active ingredient of the present invention, or a nanoparticle containing the same Lipid carriers are not suggested.

As a result of research on improving the poor solubility and bioavailability of remdesivir, the present inventors developed a formulation in which the active ingredient remdesivir was applied to a nanolipid carrier, and this formulation solubilized remdesivir. The present invention was completed by confirming that the bioavailability of remdesivir could be improved by making it exist in a stable emulsion state.

Korean Registered Patent No. 10-1822348 B1 (Method for treating filoviridae virus infection, published on January 19, 2018) Chinese Patent Publication 112843073 A (Application of Remdesivir in preparation of anti-bovine parainfluenza virus type 3 medicine, published on May 28, 2021) Korean Registered Patent No. 10-2236174 B1 (Nano-lipid carrier containing econazole and pharmaceutical composition for topical film formation containing the same, published on March 30, 2021)

An object of the present invention is to provide nanostructured lipid carriers capable of improving bioavailability by improving the poor solubility of remdesivir and pharmaceutical compositions containing the same.

Another object of the present invention is to provide a method for preparing a remdesivir-containing nanolipid carrier.

The present invention relates to a nano-lipid carrier containing remdesivir, a solid lipid, a liquid lipid and a surfactant as an active ingredient.

In the nanolipid carrier, the solid lipid is preferably glycerol monostearate, the liquid lipid is preferably caprylic / caprylic acid glyceride, and the surfactant is preferably polyoxyethylene stearate .

The nano-lipid carrier may be in the form of particles containing remdesivir and having a central portion in which a solid lipid and a liquid lipid are mixed, and a surfactant forming the exterior.

Remdesivir included in the nanolipid carrier may be amorphous.

The nanolipid carrier may contain 1 to 5% by weight of active ingredient, 35 to 58% by weight of solid lipid, 0 to 25% by weight of liquid lipid, and 23 to 45% by weight of surfactant based on the total weight of nanolipid carrier.

According to one aspect, the present invention is a method for producing a nanolipid carrier,

(i) preparing a lipid mixture by dissolving and mixing remdesivir, solid lipid, and liquid lipid by heating to 75 to 85 ° C;

(ii) preparing a surfactant solution by heating a solution in which a surfactant is dissolved in water to the same temperature as the lipid mixture; and

(iii) preparing a nano-lipid carrier by dispersing the lipid mixture of step (i) in the surfactant solution of step (ii);

The present invention, according to one aspect, relates to an antiviral pharmaceutical composition comprising the nano-lipid carrier. The virus may be a coronavirus or a filovirus, and the virus may be severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory It may be selected from the group consisting of MERS-CoV, Ebola virus, and Marburg virus.

The pharmaceutical composition may be formulated as an injection.

Hereinafter, the present invention will be described in more detail.

In the present invention, a nanolipid carrier is a nanoparticle comprising remdesivir as an active ingredient and composed of a solid lipid, a liquid lipid, and a surfactant, and refers to a composition that improves the bioavailability of the active ingredient to increase the efficacy of the active ingredient.

The nanolipid carrier is preferably a mixture of 1 to 5% by weight of active ingredient, 35 to 58% by weight of solid lipid, 0 to 25% by weight of liquid lipid and 23 to 45% by weight of surfactant based on the total weight, and active ingredient 1 ~ 3% by weight, 37-56% by weight of solid lipid, 1-22% by weight of liquid lipid and 25-43% by weight of surfactant are more preferably mixed, 1.5-2.5% by weight of active ingredient, 38-43% by weight of solid lipid %, it is most preferred that 13 to 20% by weight of liquid lipid and 36 to 42% by weight of surfactant are mixed.

In the present invention, solid lipids are lipids or waxes having a melting point of 60° C. or higher, and pharmaceutically acceptable solid lipids having sufficient solubility for the active ingredient remdesivir may be used. The solid lipid is preferably glycerol monostearate.

If the content of the solid lipid is less than 35% by weight based on the total weight of the nanolipid carrier, encapsulation of remdesivir may be difficult and the drug may leak into the aqueous phase, and if it exceeds 58% by weight, the lipid is homogeneously dispersed in the aqueous phase If this is not done, the size of the particles may increase and sedimentation may occur. Therefore, the solid lipid is preferably used at 35 to 58% by weight based on the total weight of the nanolipid carrier, more preferably at 37 to 56% by weight, and most preferably at 38 to 43% by weight. do.

In the present invention, the liquid lipid is mixed with the molten solid lipid to help incorporate the drug into the solid lipid, and a pharmaceutically acceptable liquid lipid having high solubility for the active ingredient, remdesivir, can be used. The liquid lipid is preferably caprylic/caprylic acid glyceride (Capmul MCM).

When the content of the liquid lipid exceeds 25% by weight based on the total weight of the nanolipid carrier, the particle size of the nanolipid carrier increases and it becomes difficult to homogeneously disperse in the aqueous phase. Therefore, the liquid lipid is preferably used in an amount of 0 to 25% by weight based on the total weight of the nanolipid carrier, more preferably in an amount of 1 to 22% by weight, and most preferably in an amount of 13 to 20% by weight .

In the present invention, when the solid and liquid lipid mixture is dispersed in the aqueous phase, the hydrophobic group of the surfactant is attached to the lipid phase and the hydrophilic group contacts the aqueous phase so that it is dispersed in the aqueous phase in a stable state. It prevents precipitation of the generated nanolipid carrier and maintains small and homogeneous particles. The surfactant is preferably polyoxyethylene stearate (Myrj52).

In the present invention, the use of less than 23% by weight of the surfactant based on the total weight of the nanolipid carrier does not lower the surface tension of the lipid and aqueous phase, so that the nanolipid carrier cannot be stably dispersed in the aqueous phase, and aggregation may occur, resulting in precipitation. The use of surfactant in excess of 45% by weight increases the hydrophilicity of remdesivir, an active ingredient, so that remdesivir in the nanolipid carrier cannot be encapsulated and escapes into the external aqueous phase, thereby lowering the encapsulation rate. Therefore, the surfactant is preferably used in an amount of 23 to 45% by weight based on the total weight of the nanolipid carrier, more preferably in an amount of 25 to 43% by weight, and most preferably in an amount of 36 to 42% by weight. do.

Nanostructured lipid carriers according to the present invention were confirmed to be superior in drug release control, drug delivery efficiency, and drug stabilization effect compared to other nanotechnology drug delivery systems while solving the problem of remdesivir's poor solubility.

According to one aspect, the present invention relates to an antiviral pharmaceutical composition comprising the remdesivir-containing nanolipid carrier.

The present invention has antiviral activity against various RNA viruses through a mechanism of terminating RNA transcription by inhibiting the action of RNA-dependent RNA polymerase (RdRp) of the virus. Preferably, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) belonging to the Coronaviridae family as an RNA virus, severe acute respiratory syndrome coronavirus (Severe acute respiratory syndrome coronavirus) , SARS-CoV), coronaviruses such as Middle East respiratory syndrome coronavirus (MERS-CoV); and filoviruses such as Ebola virus and Marburg virus belonging to the Filoviridae family. There are five known types of Ebolavirus: Zaire ebolavirus, Sudan ebolavirus, Tai Forest ebolavirus, Bundibugyo ebolavirus, and Reston ebolavirus. there is.

The pharmaceutical composition of the present invention can be administered in various oral and parenteral dosage forms during clinical administration, and when formulated, diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants, etc. manufactured using

Solid preparations for oral administration include tablets, patients, powders, granules, capsules, troches, etc., and these solid preparations contain at least one or more excipients such as starch, calcium carbonate, It is prepared by mixing sucrose or lactose or gelatin. In addition to simple excipients, lubricants such as magnesium styrate and talc are also used. Liquid preparations for oral administration include suspensions, solutions for oral administration, emulsions, or syrups. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, and preservatives may be included. can

Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspension solutions, emulsions, freeze-dried formulations, suppositories, and the like. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspending agents. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerol, gelatin, and the like may be used.

As a specific example, the pharmaceutical composition of the present invention may be formulated in the form of an injection for parenteral administration by intravenous, intramuscular, subcutaneous, intraperitoneal, intrauterine intrathecal or intracerebrovascular injection. The injection may be formulated in a lyophilized form as well as a liquid form, and sterile water, physiological saline, or an isotonic solution containing glucose, mannitol, sodium chloride, or the like may be used as the injection. In addition, the injection may further appropriately contain pharmaceutically acceptable ingredients such as isotonic agents, suspending agents, solubilizing agents, pH adjusting agents, buffering agents, analgesic agents, preservatives, antioxidants and the like, if necessary.

In addition, in the pharmaceutical composition of the present invention, the effective dosage of remdesivir for the human body may vary depending on the patient's age, weight, sex, administration form, health condition, and disease degree, and is generally about 0.001-100 mg/kg/day, preferably 0.01-50 mg/kg/day. For example, based on an adult patient weighing 70 kg, the daily dose may be in the range of 1-2000 mg, preferably 5-1000 mg, at regular intervals according to the judgment of the doctor or pharmacist. It can also be administered once or several times a day in divided doses.

The remdesivir-containing nanolipid carrier of the present invention has the effect of improving the solubility of the active ingredient remdesivir in water and improving the bioavailability to increase the efficacy. As the efficacy increases, it is also possible to lower the amount of the active ingredient at the time of manufacture, and there is an effect of reducing side effects due to the dose according to the minimization of the dose of the active ingredient.

Figure 1 shows the composition of remdesivir (RDS), glycerol monostearate (GMS), polyoxyethylene stearate (Myrj 52), Example 5, but remdesivir-free nanolipid carrier (Blank-NLC), implementation It is a differential scanning calorimetry graph of the remdesivir-containing nanolipid carrier (RDS-NLC) of Example 5 and the physical mixture of additives used.
Figure 2 is a transmission electron microscopy (TEM) photograph confirming the particle formation of the remdesivir-containing nanolipid carrier of Example 5.
Figure 3 shows the composition of remdesivir (RDS), glycerol monostearate (GMS), polyoxyethylene stearate (Myrj 52), Example 5, but remdesivir-free nanolipid carrier (Blank-NLC), implementation It is an X-ray diffraction analysis graph of the remdesivir-containing nanolipid carrier (RDS-NLC) of Example 5 and the physical mixture of additives used.
Figure 4 shows the composition of remdesivir (RDS), glycerol monostearate (GMS), polyoxyethylene stearate (Myrj 52), Example 5, but remdesivir-free nanolipid carrier (Blank-NLC), implementation It is an infrared spectroscopy (surface reflection) graph of the remdesivir-containing nanolipid carrier (RDS-NLC) of Example 5 and the physical mixture of additives used.
5(A) and 5(B) are results of cytotoxicity evaluation of Example 5 (RDS-NLC) and remdesivir (RDS), respectively.
6(A) is a graph showing in vitro dissolution evaluation of Example 5 (RDS-NLC) and Comparative Example 1 (RDS Suspension), and FIG. 6(B) is a graph showing a portion of FIG. 6(A).
Figure 7 shows the results of evaluating the efficacy of Example 5 (RDS-NLC) and remdesivir (RDS) in coronavirus-infected cells, measuring the viability of coronavirus-infected cells (Fig. 7 (A)), and plaque assay results. (FIG. 7(B)) and RT-qPCR analysis results (FIG. 7(C)) are shown.
8 is a chromatogram of remdesivir at the highest concentration.
9 is a linearity test graph of a standard product for performing LC-MS/MS analysis using remdesivir.
10 is a graph comparing pharmacokinetic parameters after intravenous injection of Example 5 (RDS-NLC) and Comparative Example 1 (remdesivir suspension, RDS) to rats.

Hereinafter, preferred embodiments of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the disclosure herein is provided so that it will be thorough and complete, and will fully convey the spirit of the invention to those skilled in the art.

< Experimental example One. of nanolipid carrier manufacturing>

1-1. Example 1 to Example 5's nano lipid carrier , and comparative example manufacture of 1

Nanolipid carriers of Examples 1 to 5 were prepared according to the composition shown in Table 1 below by using remdesivir as a pharmacologically active ingredient and varying the dosage of solid lipid and liquid lipid. That is, for the preparation of Examples 1 to 5, first, remdesivir, glyceryl monostearate, and caprylic/caprylic acid glyceride (Capmul MCM) were accurately weighed, mixed, and melted at 80°C. Then, polyoxyethylene stearate (Myrj 52) was accurately weighed and a solution dissolved in distilled water was added thereto, followed by homogenization using a disperser for 2 minutes. Then, a nano-lipid carrier was prepared by homogenizing with a probe-type ultrasonicator for 5 minutes. In addition, for the preparation of Comparative Example 1, sulfobutyl ether-beta-cyclodextrin sodium was accurately weighed, added to distilled water, and then dissolved. In addition, after accurately weighing and adding remdesivir, Comparative Example 1 was prepared by homogenizing using an ultrasonic grinder.

ingredient Example comparative example One 2 3 4 5 One Content (mg) Remdesivir 5 5 5 5 5 5 Glyceryl Monostearate 189 168 147 126 105 - Caprylic/Caprylic Glycerides 81 72 63 54 45 - polyoxyethylene stearate 100 100 100 100 100 - Sulfobutylether-beta-cyclodextrin sodium - - - - - 40 gross weight 375 345 315 285 255 45

1-2. Example 1 to Example With a grain size of 5 polydispersity Analysis

The particle size and polydispersity of the nanolipid carriers of Examples 1 to 5 prepared as described above were evaluated. Particle size and polydispersity were analyzed using an electrophoretic laser scattering analyzer (ELS-Z, Otsuka Electronics, Osaka, Japan). As a result of the experiment, as shown in Table 2, in the case of Examples 1 to 5, the average particle size was 200 nm or less, and the polydispersity representing the homogeneity of the particle size showed a suitable value of 0.3 or less.

Example Average particle size (nm) polydispersity D50% (nm) Example 1 184.5 0.222 197.8 Example 2 152.6 0.201 161.7 Example 3 139.2 0.213 146.6 Example 4 133.6 0.213 144.2 Example 5 117.0 0.198 124.2

The D50% means a particle size corresponding to 50% of the volume measured in the particle size distribution determined by laser diffraction particle size distribution measurement, and Examples 1 to 5 all showed values of 200 nm or less.

In addition, the properties of the nano-lipid carrier were greatly affected by the amount of solid lipid and liquid lipid. In the case of Example 1, the nanolipid carrier having the largest average particle size of 184.5 nm was obtained, whereas in Example 5, the nanolipid carrier having the smallest average particle size of 117.0 nm was formed.

< Experimental example 2. Optimized Remdesivir nano lipid carrier evaluation>

The remdesivir nanolipid carrier of Example 5 was prepared under conditions optimized through Experimental Example 1. That is, 5 mg of remdesivir was added to 105 mg of solid lipid glycerol monostearate and 45 mg of liquid lipid Capmul MCM, and then heated to 80 ° C to make a transparent solution. After dissolving 100 mg of Myrj 52 in 10 mL of purified water, the mixture was heated to 80°C. After adding 10 mL of a heated surfactant solution to the mixture of lipid and drug, it was homogenized with a probe-type sonicator for 5 minutes to prepare a nanolipid carrier. The prepared nanolipid carrier was lyophilized and powdered.

The properties of this formulation were evaluated in the following experimental examples.

2-1. Differential scanning calorimetry (Differential scanning calorimetry, DSC )

Remdesivir raw material (RDS), glycerol monostearate (GMS), Mryj 52, nano-lipid carrier (Blank-NLC) having the composition of Example 5 but not containing remdesivir, containing the remdesivir of Example 5 Differential scanning calorimetry was performed on the physical mixture of all raw materials used except for the nanolipid carrier (RDS-NLC) and capmul MCM.

As confirmed in the experimental results of FIG. 1, the endothermic peak was confirmed at around 150° C. corresponding to the melting point of the remdesivir raw material. In the case of glycerol monostearate and Myrj 52, each endothermic peak was observed, but in the nanolipid carrier of Example 5, it was confirmed that the endothermic peak was reduced due to amorphous or structural change. It was confirmed that the endothermic peak of remdesivir also disappeared due to amorphous form or combination with other excipients after preparation of the nanolipid carrier. Even in the case of the physical mixture of raw materials used, the endothermic peak of remdesivir disappeared, but the peaks of glycerol monostearate and Myrj 52 could be confirmed. Based on this result, it can be expected that the endothermic peak disappeared due to encapsulation of remdesivir and excipients in the nanolipid carrier during the preparation or mixing process of the nanolipid carrier, or binding between each excipient and change in crystal form.

2-2. nano lipid carrier check formation

The formation of the nano-lipid carrier of Example 5 prepared according to the present invention was confirmed by a transmission electron microscope (Transmission electron microscope, Tecnai G2 F30, FEI company, Oregon, USA). As a result, as shown in FIG. 2, it was confirmed that the remdesivir-containing nanolipid carrier of Example 5 formed spherical particles having a particle size of 150 nm or less, particularly 100-130 nm.

2-3. of nanolipid carrier X-ray diffraction analysis evaluation

X-ray diffraction analysis of the nanolipid carrier of Example 5 prepared according to the present invention was analyzed with an X-ray diffractometer (Multipurpose X-ray Diffractometer, D8 ADVANCE, Bruker axs, Massachusetts, USA). As a result, when compared with the crystalline structure of remdesivir, as shown in FIG. 3, in the remdesivir-containing nanolipid carrier of Example 5, it was confirmed that remdesivir was changed to an amorphous form.

2-4. of nanolipid carrier Infrared spectroscopy (surface reflection) evaluation

In order to confirm the IR surface reflection of Example 5 prepared according to the present invention, it was analyzed using an infrared spectrometer (ALPHA-P, Bruker, Massachusetts, USA). As a result, as shown in Figure 4, it was possible to confirm the structure, and it was confirmed that there was no interaction between the active ingredient remdesivir and other additives and that it was well encapsulated in the nanolipid carrier.

2-5. Remdesivir contain of nanolipid carrier Cytotoxicity test

A cytotoxicity test was performed to confirm the toxicity of the remdesivir-containing nanolipid carrier of Example 5 prepared according to the present invention to renal cells (Vero cells).

<Cell culture>

Vero cells were cultured in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum and 1% penicillin/streptomycin (100 unit/mL units) at 37°C and 5% CO ₂ .

<Cytotoxicity test>

One day before the test, 1 X 10 ⁴ Vero cells were suspended and cultured in DMEM containing 5% fetal bovine serum in each well of a 96-well plate. Remdesivir-containing nanolipid carrier was diluted 3-fold from 11 μM to 5.1 nM and treated with vero cells prepared one day before, and remdesivir was used as a control. After 72 hours, the culture medium was removed, CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega) reagent was treated for 3 hours, and OD (optical density) was measured using SynergyTM H1 multi microplate reader (Biotek) equipment, The viability of the sample for each concentration was calculated by converting the viability of untreated cells to 100% using the measured values.

The cytotoxicity of the remdesivir-containing nanolipid carrier was 7 μM, and the toxicity of remdesivir used as a control was not observed within the experimental concentration range. Through this, it was confirmed that the remdesivir-containing nanolipid carrier had more than 1.6 times toxicity compared to remdesivir. The cytotoxicity test results are shown in FIG. 5 .

2-6. Remdesivir contain of nanolipid carrier In vitro dissolution evaluation

In order to evaluate the in vitro dissolution of the remdesivir-containing nanolipid carrier of Example 5 prepared according to the present invention, the test was conducted together with Comparative Example 1. A dose corresponding to 2 mg of remdesivir was placed in a dialysis bag and evaluated while stirring at 150 rpm in 50 ml of a pH 7.4 PBS solution. Sampling was performed at 0.09, 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24, and 48 hours after the dialysis bag was injected. As a result, as shown in Figure 6, the nanolipid carrier of Example 5 in a PBS solution mixed with 1% by weight of Tween80 at pH 7.4 shows a dissolution profile with a controllable rate while the final dissolution rate is similar to that of Comparative Example 1. Confirmed.

2-7. Remdesivir contain of nanolipid carrier Evaluation of efficacy in coronavirus-infected cells

To evaluate the efficacy of the remdesivir-containing nanolipid carrier of Example 5 according to the present invention in cells infected with SARS-CoV-2, a coronavirus that causes coronavirus disease 2019 (COVID-19) For this, it was carried out as follows.

(1) Test for evaluating the viability of coronavirus-infected cells

One day before, kidney cells (Vero cells) were cultured in each well of a 96-well plate at a number of 1 X 10 ⁴ . Remdesivir-containing nanolipid carrier (Example 5, RDS-NLC) diluted 3-fold from 100 μM to 5 nM was treated with cells one hour before virus infection, and then infected with SARS-CoV-2 at 0.1 MOI, as a control A solution of remdesivir dissolved in DMSO was diluted and used.

After incubation for 72 hours after infection, the culture medium was removed, treated with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS, Promega) reagent for 3 hours, and cell viability was measured using SynergyTM H1 multi microplate reader (Biotek) equipment. and calculated the observed value of antiviral efficacy using the measured value.

As shown in FIG. 7 (A), the EC ₅₀ of the remdesivir-containing nanolipid carrier in coronavirus (SARS-CoV-2)-infected cells was 0.16 μM, and the control, remdesivir-containing coronavirus (SARS-CoV-2) 2) The EC ₅₀ in the infected cells was 1.6 μM, confirming that the drug efficacy of the remdesivir-containing nanolipid carrier was 10 times higher.

(2) plaque assay

A plaque assay was performed to measure the number of infectious viruses released. One day before, kidney cells (Vero cells) were cultured in each well of a 6-well plate at a number of 1 X 10 ⁶ . Remdesivir nanolipid carrier was treated with 0, 0.1, 0.3, and 1 μM, and control remdesivir was treated with 0, 1, 3, and 10 μM, and SARS-CoV-2 was infected with 1 MOI. After 24 hours, the obtained supernatant was diluted 10 times, inoculated into each well of a 48-well plate at a number of 2 X 10 ⁵ the day before, treated with cultured kidney cells (Vero cells) for one hour, and then used as an overlay medium. Incubated for 72 hours. After culturing for 72 hours, the culture medium was removed, stained with crystal violet, and the number of plaques was calculated.

As shown in FIG. 7 (B), the number of infectious viruses released from coronavirus (SARS-CoV-2) infected cells treated with 0.1, 0.3, and 1.0 μM of remdesivir-containing nanolipid carrier was measured by plaque assay (plauqe). As a result of measurement by assay), it was not detected at all at 1.0 μM, and reduced virus discharge was observed at 0.3 μM and 0.1 μM compared to the virus group. The number of infectious viruses shed from coronavirus (SARS-CoV-2)-infected cells treated with 1, 3, and 10 μM of control remdesivir was not detected at 10 μM, but decreased at 3 μM and 1 μM By observing the release, it was observed that the remdesivir-containing nanolipid carrier suppressed the release of the infectious virus at a lower concentration than the control remdesivir.

(3) RT-qPCR (Reverse-transcription quantitative polymerase chain reaction) analysis

To measure the expression level of the virus-derived Spike gene produced in coronavirus (SARS-CoV-2) infected cells, RT-qPCR was performed as follows. One day before, kidney cells (Vero cells) were cultured in each well of a 6-well plate at a number of 1 X 10 ⁶ . Remdesivir nanolipid carrier was treated with 0, 0.1, 0.3, and 1 μM, and control remdesivir was treated with 0, 1, 3, and 10 μM, and SARS-CoV-2 was infected with 1 MOI. RNA was extracted from each cell obtained after 24 hours, and RT-PCR was performed using the virus spike gene (S gene) and β-actin as a control using SuperScript III Platinum SYBR Green One-Step RT-PCR kit (Invitrogen, USA). qPCR was performed.

The results of RT-qPCR analysis are shown in FIG. 7(C). In coronavirus (SARS-CoV-2) infected cells treated with 0.1, 0.3, and 1.0 μM of remdesivir nanolipid carrier, the spike gene was not expressed at all at 1 μM and was expressed at a very slight level at 0.3 μM, At 0.1 μM, the spike gene was expressed at a slightly reduced level compared to the virus-only treatment. As a control, in coronavirus (SARS-CoV-2) infected cells treated with 1, 3, and 10 μM of remdesivir, spike gene was not expressed at all at 10 μM and 3 μM, and spikes at a slightly reduced level at 1 μM Genes were expressed, and it was confirmed that the remdesivir nanolipid carrier inhibited the production of viral genes at a lower concentration than the control remdesivir.

As such, it was confirmed that the remdesivir-containing nanolipid carrier according to the present invention has an effect of exhibiting excellent antiviral activity even at a low concentration by improving the poor solubility of remdesivir.

2-8. Remdesivir contain of nanolipid carrier pharmacokinetic evaluation

To evaluate the pharmacokinetics of the remdesivir-containing nanolipid carrier according to the present invention, the test was conducted as follows. As experimental animals, 6 rats were used per experimental group and control group, and they were bred in cages under the same conditions for 4 days or more while supplying a certain amount of conventional solid feed and water. After the rats were fasted for more than 12 hours, they were used in the experiment, and they were allowed to drink water freely during fasting.

In the experimental group, the remdesivir-containing nanolipid carrier of Example 5 of the present invention was lyophilized, concentrated to a concentration of 2 mg/mL, suspended in physiological saline, and injected. In the control group, the remdesivir suspension of Comparative Example 1 was injected. Desivir was prepared at a concentration of 2 mg/mL in an aqueous solution of sulfobutyl ether-beta-cyclodextrin sodium and injected. All groups were intravenously injected with a dose equivalent to 10 mg/kg of remdesivir.

<Pharmacokinetic parameter measurement results>

About 0.5 mL of blood was obtained through the orbital vein every 0.09, 0.25, 0.5, 1, 2, 4, 6, 8, and 12 hours after intravenous administration to rats. The obtained sample was centrifuged at 15,000 rpm for 10 minutes to obtain plasma, and then stored at -20°C until HPLC-MS/MS analysis. Table 3 and Table 4 below are the conditions for analyzing GS-441524, an active metabolite of remdesivir, the active ingredient. In addition, for partial validation of the following analysis conditions, the results of repeated analysis of the standard material three times are shown in FIGS. 8 and 9.

HPLC-MS/MS analysis of GS-441524 column Kinetex ^® 2.6 μm XB-C18 100Å column
(50 × 2.1 mm, 2.6 μm; Phenomenex, California, USA) mobile phase 5 mM ammonium acetate aqueous solution: methanol injection volume 2 μL flow rate 0.4 mL/min m/z 291.2 → 200.2 Column temperature (℃) 40 collision energy (eV) 19 Gas temperature (℃) 290 Gas flow rate (L/min) 11 Nebulizer pressure (psi) 40

time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL/min) 0.00 100 0 0.400 0.30 100 0 2.30 40 60 2.40 30 70 3.30 30 70 3.32 100 0 4.30 100 0

The main pharmacokinetic parameters of the plasma remdesivir concentration versus time profile of the remdesivir-containing nanolipid carrier of Example 5 are shown in Table 5 and FIG. 10 below.

Pharmacokinetic parameters after intravenous injection in red Pharmacokinetic parameters control group
(Comparative Example 1) experimental group
(Example 5 of the present invention) T _1/2 lz ₍ hr) 4.04 ± 2.92 3.07 ± 1.31 T _max (hr) 0.33 ± 0.13 0.22 ± 0.07 C _max (ng/mL) 46.32 ± 8.68 104.53 ± 17.68*** AUC _{0 -24} (hr*ng/mL) 111.91 ± 16.89 234.77 ± 33.79*** V/F (L/kg) 452.78 ± 277.88 192.12 ± 102.10 Cl/F (L/hr/kg) 81.83 ± 54.56 41.99 ± 5.62*** ***P<0.001

As confirmed in Table 5 and FIG. 10, AUC _0-24 of Example 5 of the present invention increased by about 210% and Cmax also increased by 226% compared to _Comparative Example 1, confirming that the bioavailability was improved.

As described above, the remdesivir-containing nanolipid carrier of the present invention can reduce the dose of the active ingredient by maximizing the increase in bioavailability while solving the problem of remdesivir's poor solubility, and can cause renal and hepatic disorders due to renal accumulation. Since cyclodextrin is not used, side effects can be reduced.

Claims (9)

A nanolipid carrier comprising remdesivir as an active ingredient, glycerol monostearate as a solid lipid, caprylic/caprylic acid glyceride as a liquid lipid, and polyoxyethylene stearate as a surfactant. The nano-lipid carrier according to claim 1, wherein the nano-lipid carrier comprises remdesivir in the center where solid lipid and liquid lipid are mixed, and a surfactant is formed on the outside. The nano-lipid carrier according to claim 1, wherein the remdesivir contained in the nano-lipid carrier is amorphous. The method of claim 1, wherein the nano-lipid carrier comprises 1 to 5% by weight of active ingredient, 35 to 58% by weight of solid lipid, 0 to 25% by weight of liquid lipid and 23 to 45% by weight of surfactant based on the total weight of nanolipid carrier. Nano-lipid carrier, characterized in that it comprises. A method for producing the nanolipid carrier according to any one of claims 1 to 4,
(i) preparing a lipid mixture by dissolving and mixing remdesivir, solid lipid, and liquid lipid by heating to 75 to 85 ° C;
(ii) preparing a surfactant solution by heating a solution in which a surfactant is dissolved in water to the same temperature as the lipid mixture; and
(iii) preparing a nano-lipid carrier by dispersing the lipid mixture of step (i) in the surfactant solution of step (ii);
Characterized in that it comprises a, method for producing a nano-lipid carrier. An antiviral pharmaceutical composition comprising the nano-lipid carrier according to any one of claims 1 to 4. The antiviral pharmaceutical composition according to claim 6, wherein the virus is a coronavirus or a filovirus. The method of claim 6, wherein the virus is severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), severe acute respiratory syndrome coronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus (MERS-CoV), Ebola virus ( Ebola virus), and Marburg virus (Marburg virus) characterized in that selected from the group consisting of a pharmaceutical composition for antiviral. The antiviral pharmaceutical composition according to claim 6, wherein the pharmaceutical composition is formulated as an injection.

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