KR20020032795A - Dry Liposome Compositions Containing Water-soluble, Peptide-based Drugs, and Preparation Method Thereof - Google Patents

Abstract

PURPOSE: Provided are dry liposome composition containing water-soluble, peptide-based drugs and its preparation method. Particularly, water-soluble peptide or water-soluble drugs are formulated into solid liposome to transfer drugs stably. CONSTITUTION: The manufacturing method of dry liposome composition comprises the steps of; mixing water-soluble peptide drugs with oil and surfactant to W/O emulsion type of water-soluble peptide drugs; dispersing multi-porous powdery carrier into the W/O emulsion then including it; removing oil from the surface then evaporating water to manufacture dry carrier which includes water-soluble peptide drugs; and coating the dry carrier with mixed a mixed solution of phospholipid and organic solvent.

Description

Dry Liposome Compositions Containing Water-soluble, Peptide-based Drugs, and Preparation Method Thereof}

The present invention relates to a method of formulating a water soluble peptide drug into stable targeting liposomes. More specifically, the present invention relates to a novel method of developing stable drug delivery transporters by preparing water soluble peptides or generic water soluble drugs into solid liposomes. The method of the present invention is widely applicable to general water-soluble drugs, and improves the stability and in vivo kinetic parameters of the drug to enable various formulation designs. In addition, the methods of the present invention can improve the bioavailability and targeting of water-soluble drugs.

Water-soluble peptide drugs with biological activity are generally unstable for enzymes in the gastrointestinal tract and have very low bioavailability, which is a great difficulty in treating systemic action. Therefore, studies on stabilizing the drug and improving the efficacy of the agent have been carried out for these unstable water-soluble peptide drug through various methods.

Conventional methods of stabilizing unstable water-soluble peptide drugs include preparing microparticles using polymers or preparing liposomes using lipids. However, there have been problems with drug stability in high temperature and liquid phases in the preparation of such formulations of labile water soluble peptide drugs. In addition, the conventional stabilized manufacturing methods have to be selected and used according to the characteristics of the individual drugs, there are a number of problems to apply a wide range for the general unstable peptide drugs.

Specifically, deamidation reaction occurs in asparaginyl residues and glutamine residues in the structure of the peptide drug which is unstable in the conventional methods of using high temperature underwater, or methionine (Met), cysteine (Cys), histidine ( His), tryptophan (Trp), tyrosine (Tyr) residues may be oxidized to decompose the drug and reduce its efficacy.

On the other hand, the conventional method for preparing a dry liposome formulation can be applied only to poorly soluble drugs, it was not possible to enclose a water-soluble drug. This is because poorly soluble drugs are well soluble in organic solvents containing phospholipids, while water-soluble drugs are not soluble in phospholipids or organic solvents. Therefore, there is a need for a microemulsion system with excellent dispersibility for uniformly encapsulating a water-soluble drug in a sorbitol carrier. The present invention provides such a microemulsion having excellent dispersibility.

The present invention solves the above-mentioned problems, and provides a water-soluble peptide-containing dry liposome composition and a method for preparing the same, which can increase the bioavailability by delaying the loss of the water-soluble peptide drug in the body, as well as excellent physical and chemical stability. do.

Therefore, the novel production method of the present invention improves the above-mentioned problems by stabilizing and formulating an unstable water soluble peptide drug, and can be widely applied to a general unstable water soluble peptide drug, as well as oral, transdermal, intravenous, etc. Various formulation designs are possible for the administration of. By the method of the present invention, a composition excellent in stability and efficacy can be obtained.

1 is a graph comparing the loading efficiency of drugs according to the type of surfactant in the preparation of rhEGF dry liposomes.

Figure 2 is a graph comparing the drug loading efficiency of mannitol and sorbitol as a carrier in the preparation of rhEGF dry liposomes.

Figure 3 is a graph comparing the sealing efficiency when using liquid paraffin, soybean oil, ethyl oleate, sesame oil as the oil in the preparation of rhEGF dry liposomes.

Figure 4 is a graph showing the effect of the oil volume on the loading efficiency during the preparation of rhEGF dry liposomes.

5 is a graph showing the effect of rhEGF drug concentration on the loading efficiency.

6 is an analytical diagram showing particle size when rhEGF dry liposomes are dispersed in water.

7 is a photograph of rhEGF dry liposomes observed under an electron microscope (7a: dry sorbitol surface, 7b: rhEGF dry liposome surface, and 7c: particles when rhEGF dry liposomes are dispersed in water)

8 is a graph showing blood concentration patterns after intravenous administration of rhEGF dry liposomes to rats.

The present invention is explained in more detail below.

In the present invention, an aqueous solution of an unstable water soluble peptide drug is prepared in a W / O emulsion using an oil and a surfactant, the porous carrier is dispersed in the emulsion, and the water soluble peptide drug is encapsulated in the carrier and dried, and then it is phospholipid and an organic solvent. The present invention relates to a method for stabilization into granules having very good flowability by coating using a solution of. The granules thus prepared are shaken by adding water to form liposomes. This method of the present invention enables the production of a variety of targeted agents.

Representative of the labile water-soluble peptide drug that can be applied to the stabilizing production method of the present invention is recombinant human epidermal growth factor (rhEGF), transforming growth factor-α (TGF-α), platelet-derived growth factor (PDGF ), Water soluble peptides and protein drugs such as transforming growth factor-β (TGF-β), fibroblast growth factor (FGF), basic FGF and acidic FGF. In addition, a wide range of water soluble drugs as shown in Table 1 below may also be preferably stabilized and used by the present invention.

Water Soluble Peptide Drugs Applicable to the Present Invention Epithelial Growth Factor (EGF), rhEGF Transforming Growth Factor-α (TGF-α) Platelet-derived Growth Factor (PDGF) Transforming Growth Factor-β (TGF-β) Fibroblast Growth Factor (FGF) Adrenal Cortical Stimulating Hormone (ACTH Bacitracin Calcitonin (CT) Chorionic Gonadotropin (hCG) Growth Hormone (Somatotropin) Gluticidin (Grumicidin) Insulin LHH Free Hormone (LHRH) Oxytocin (OT) Somatostatin (SS) Thyroid Stimulating Hormone Free Hormone (TRH)

The porous powder carrier used in the present invention may have general conditions such as not causing toxicity in the body, not antigenic, and not accumulating in the body. In addition, the carrier must be water soluble so as to dissolve well in water, stable to impact during manufacturing, low solubility in organic solvents, applicable to the human body and suitable for the conditions according to the route of administration. Carriers that satisfy these conditions include sorbitol, sodium chloride, mannitol, lactose and the like. In particular, sorbitol is most suitable for the present invention because it has a porous structure and can accumulate a large amount of drug therebetween, and also prepare a formulation having various properties by coating a phospholipid on its surface.

The oils used in the primary encapsulation of the present invention are mineral oils such as liquid paraffin; Vegetable oils such as soybean oil, corn oil, castor oil, cottonseed oil, canola oil, sunflower seed oil, safflower oil, sesame oil, peanut oil; Vitamin oils such as tocopherol oil (VE); Ethyl oleate, caprylic / caprylic acid triglycerides, and the like. Liquid paraffin is particularly suitable oil for the present invention in terms of the encapsulation efficiency of the drug.

As the surfactant, an aqueous phase containing a drug is dispersed in an oil phase using a nonionic surfactant Span (Span 80) having a low HLB value to prepare an aqueous / oil phase (W / O) emulsion. Surfactants that can be used in the present invention include nonionic surfactants such as those shown in Table 2, for example, polysorbates (Tweens), sorbitan fatty acid esters (Spans), and polyoxyethylene alkyl ethers. (Brij), poloxamers (Pluronic), polyoxyl hydrogenated castor oil (HCO), polyethylene glycol esters (Labarfil, Labrafac; Gattefosse SA (France)), methyl glucoside derivatives (Glucate) Morphols (polyoxyethylated castor oil). In particular, the surfactant is selected from Tweens, Spans, poloxamers, and cremophors. Compared with Tweens and Spans as surfactants, Span 80 contains the most drugs (see FIG. 1).

Types of surfactants usable in the present invention Tween (polysorbate) Tween 20, Tween 40, Tween 60, Tween 80 Span (sorbitan fatty acid ester) Span 20, Span 40, Span 60, Span 80 Brij (polyoxyethylene alkyl ethers) Brij 72, Brij 76, Brij 78, Brij 700 Pluronic F-68, L-44, F-87, F-108, F-127 HCO (Polyoxyl Hydrogenated Castor Oil) HCO-60, HCO-50, HCO-40 Other Labrafil M 1944 CS Labrafil M 2125 CS Labrafac Hydro.wL 1219 Glucate DOCremophor RH 40 Cremophor EL

Phospholipids used in the formulations in the present invention may be used pharmaceutically acceptable phosphatidylcholine, phosphatidylglycerol and derivatives thereof. Such derivatives include dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), dimyristoylphosphatidylglycerol (DMPG), and disterraylphosphatidylethanolamine-N- Poly (ethyleneglycol) 2000 and its derivatives DSPE-PEG 1000, DSPE-PEG 2000, DSPE-PEG 5000, DSPE-PEG 10,000, and dioleoylphosphatidylcholine, dioleoylphosphatidylglycerol. In the present invention, after considering the maximum dose of the drug and its own properties to determine the suitable system and select and use the phospholipid.

In the present invention, in consideration of economics and targeting, low-purity 60% phosphatidyl choline is used for oral administration, and 99% phosphatidyl choline or DSPE for continuous administration in the body during intravenous injection. -PEG 2000 is mixed.

Organic solvents that can be used for secondary coatings in the present invention dissolve phospholipids, but do not dissolve sorbitol and attached drugs, and must be volatile. As a solvent that satisfies these conditions, dichloromethane, acetone, chloroform, methanol, ethanol and a mixture of one or more thereof can be used. The organic solvent is selected differently depending on the drug, but mainly chloroform, methanol, ethanol and a mixture of one or more thereof are suitable.

Granules prepared according to the method of the present invention need to be shaded because the drugs and phospholipids are easily oxidized by light, and the stability of the drugs and liposomes is reduced by high temperature oxygen, so they are stored at 4 ° C. or avoided contact with oxygen. shall. Therefore, the prepared granules should be stored after being filled with nitrogen in a brown bottle.

Looking at the manufacturing process of the granules according to the present invention, first, W / B prepared by dispersing sorbitol (298-500 μm) using a rotary mixer as a manufacturing device and dispersing an aqueous phase containing a drug in an oil phase using a surfactant. Add to O emulsion and mix for 20 minutes at 250 rpm. The oil on the particle surface is washed 3-5 times with hexane and then the organic solvent is evaporated completely in a rotary evaporator to obtain a dry carrier with drug encapsulation. Carrier particles are used to make the carrier particles 298-500 μm, followed by a secondary coating as follows.

After the carrier is sufficiently dried in a rotary evaporator, an organic solvent containing a certain amount of phospholipid is injected using a production apparatus having a three-way connector with a stable glass wire. Finally, the organic solvent is added and dried, followed by drying in a vacuum descater for about 24 hours. To obtain granules of even particle size, the granules were dried and pulverized to collect 298-500 μm particles, filled with nitrogen, and stored in a brown bottle.

In addition, after recovering the organic solvent and the solution adsorbed on the carrier, the carrier is slowly stirred by using a glass rod to prevent the carriers from condensing to prepare granules. The granules thus prepared have very good fluidity.

Thus prepared granules can be stored in a dry state, unlike liquid liposomes can overcome the physicochemical instability of the liposomes, it is possible to obtain a homogeneous product with a relatively reproducible. In particular, industrial scale sterilization is also possible, which is more advantageous than conventional liposomes. Thus, it is possible to effectively stabilize general water soluble drugs, not limited drugs, by the method of the present invention.

As described above, the solid granules prepared by the present invention are stabilized systems of new water-soluble peptides which are widely used in pharmaceutical formulations, and when the obtained granules are added to water or body fluids, they become liposomes. Bioavailability can be significantly improved. In addition, these granules are advantageous for storage because they are made of powdery powder with good flowability. In addition, since it is possible to prepare a solid liposome of various drugs, it can be applied to various fields. That is, the granules obtained in the present invention can be prepared by liposome granules, liposome capsules, liposome tablets, soluble powder-type liposome injections, etc. in a conventional manner, and thus can be applied as an effective drug transport system through various formulation designs.

As described above, according to the method of the present invention, the unstable water-soluble peptide drug is encapsulated in a porous powdery carrier as a W / O emulsion to be prepared and stabilized in a solid granular state with good fluidity. Unlike, it is possible to produce a homogeneous product that can be stored in a dry state, and industrial scale sterilization treatment is very advantageous. The method of the present invention is particularly widely available for general water-soluble drugs, and can be used for various formulation designs, and can also improve the bioavailability of water-soluble drugs. Hereinafter, an Example demonstrates this invention.

Example 1

RhEGF was used as the water soluble peptide drug. To encapsulate it, 0.5 g of sorbitol was first weighed as a porous powder carrier, and 12.5 ml of oil selected from liquid paraffin, ethyl oleate, soybean oil, castor oil, cottonseed oil, safflower oil, sesame oil, peanut oil, 0.05 ml of Span 80 and 0.1 ml of rhEGF. In a w / o emulsion containing (0.1 mg / ml) it was dispersed for 20 minutes at 250 rpm using a rotary mixer. Subsequently, washing with n-hexane three times to remove oil on the surface of the particles and then sufficiently removing the organic solvent in a rotary evaporator, yielding a dry carrier encapsulated with rhEGF. The carrier is used to make the carrier particles 298-500 μm, followed by secondary coating.

75 mg of DPPC and DSPE-PEG 2000 (molar ratio 5: 1) were dissolved in 7.5 ml of chloroform (10 mg / ml) and the respective carriers were repeatedly coated until they aggregated together.

The coating procedure was as follows: 5 g of the rhEGF containing sorbitol carrier obtained above was taken and placed in a flask of a rotary evaporator, followed by drying under reduced pressure for about 1 hour. Using a three-way linkage of a rotary evaporator, a chloroform solution in which the liposome components were dissolved was adjusted to be introduced at a predetermined amount, and the rhEGF containing sorbitol carrier was coated. The rotational speed of the rotary evaporator was maintained at 100 rpm, and when the sorbitol was completely dried and free flowing, the solution was added again and repeatedly coated. Sorbitol containing rhEGF and phospholipid was freeze-dried for 12 hours and then refrigerated at 4 ° C.

As shown in FIG. 3, the encapsulation efficiency in the case of using liquid paraffin, soybean oil, ethyl oleate, sesame oil, and castor oil as oil was the highest in the case of using liquid paraffin.

Example 2

The blue coloration of methylene blue on the dry liposome surface was confirmed as in Example 1 by replacing the water soluble drug with 2% methylene blue pigment in sorbitol of parenter particle size 298-500 μm. This confirmed that the general water-soluble drug can also be encapsulated in a carrier by the method of the present invention.

Example 3

Dry liposomes were prepared as in Example 1 using Tween 20 (HLB 16.7), Tween 80 (HLB 15.0), Span 20 (HLB 8.6), Span 80 (HLB 4.3) as surfactants. As a result of measuring the encapsulation efficiency of the thus prepared dry liposome, the surfactant having a large HLB value was smaller than the surfactant having a small HLB value. That is, when Span 80 with a low HLB value was used as the surfactant, the drug was most encapsulated (FIG. 1).

Example 4

It carried out as in Example 1 using mannitol instead of sorbitol as a carrier. The results showed that the drug loading efficiency of mannitol was lower than that of sorbitol (FIG. 2).

Experimental Example 1 . Measurement of encapsulation efficiency

The encapsulation efficiency of the prepared dry liposome was calculated from the actual encapsulation amount / theoretical encapsulation amount.

In Example 1, the influence on the filling efficiency at different oil volumes is shown in FIG. 4. As can be seen in FIG. 4, the larger the amount of oil, the smaller the encapsulation efficiency. When the amount of oil is 12.5 ml, the encapsulation efficiency is about 98%.

In addition, the effect on the loading efficiency by varying the loading amount of the drug in Example 1 is shown in FIG. The higher the amount of drug, the greater the encapsulation efficiency.

Experimental Example 2 . Measurement of Particle Size

Light scattering spectrophotometer: Nicomp 370, Particle Sizing System, Inc. (Santa, California, USA) was used to determine the particle size of liposomes formed by adding distilled water for injection to the rhEGF-containing dry liposome composition prepared in Example 1 and stirring. Barbara)).

The result is as follows (see Figure 6):

Particle size of the liposomes of Example 1: 166.3 ± 53.5 nm.

Experimental Example 3 . Observation under an electron microscope

To observe the surfaces of dry sorbitol and dry liposomes, they were coated with platinum using an ion coater and then observed by scanning electron microscopy (SEM) (FIGS. 7A and 7B). In addition, in order to observe the particle shape after dispersing the dry liposomes in water, the sample was placed on a carbon-coated grid and stained with 2% ammonium molybdate and observed by transmission electron microscopy (TEM) (FIG. 7C).

Looking at Figure 7a it can be seen that the surface of the sorbitol is porous, and looking at Figure 7b it can be seen that the surface of the sorbitol is coated with drugs and phospholipids. In addition, in Figure 7c it can be seen that the liposome particles are formed by dispersing the dry liposomes in distilled water for injection.

Experimental Example 4 . Pharmacokinetic Studies

Pharmacokinetic experiments were conducted using 200-250 g of Sprague-Dawley rats. Rats were injected intravenously with 100 ng / kg of the rhEGF formulation dissolved in physiological saline and the composition of Example 1, and blood was injected at 0, 2, 4, 6, 8, 10, 15, 30, 60, 90, and 120 hours. Was collected. The rhEGF concentration in blood was measured by ELISA, and pharmacokinetic parameters were calculated and compared. The results are shown in Table 2 (see Figure 8).

AUC and T _1/2 of the rhEGF-containing dry liposome composition according to the present invention were increased by 1.38 and 3.35 times, respectively, compared to the solution formulation. Therefore, it was confirmed that the composition of the present invention exhibited higher bioavailability due to delayed loss of body compared to the solution formulation.

Body Pharmacokinetic Parameters Following Intravenous Administration of rhEGF Dry Liposomes to Rats Parameter AUC (ngmin / ml) T _1/2 (min) CL (ml / min / kg) MRT (min) Vdss (ml / kg) solution 686.34 9.26 0.0146 4.332 0.0633 Dry liposomes 9473.89 31.04 0.0106 6.106 0.0644 AUC: area under the curve of blood drug concentration, T _1/2 : half-life in vivo, CL: clearance, MRT: mean residence time, Vdss: volume of distribution

According to the method of the present invention, the unstable water-soluble peptide drug is prepared as a W / O emulsion and encapsulated in a porous powdery carrier to prepare and stabilize the solid granule with good fluidity, thereby solving the problems of the prior art, and unlike the conventional liposomes. It is possible to produce a homogeneous product that can be stored in a dry state, and is very advantageous, such as industrial scale sterilization treatment. The method of the present invention is particularly widely available for general water-soluble drugs, and can be used for various formulation designs, and can also improve the bioavailability of water-soluble drugs.

Claims (8)

Mixing an aqueous solution of a water soluble peptide drug with an oil and a surfactant to prepare a W / O emulsion of the water soluble peptide drug; Dispersing and encapsulating the porous powder carrier in the W / O emulsion, removing oil from the surface and evaporating water to prepare a dry carrier in which the water-soluble peptide drug is encapsulated; And Coating the dry carrier with a solution of phospholipids and organic solvents and drying the method. The method of claim 1, wherein the water soluble peptide drug is recombinant human epidermal growth factor (rhEGF), transforming growth factor-α (TGF-α), transforming growth factor-β (TGF-β), platelet derived growth Factor (PDGF), fibroblast growth factor (FGF), basic FGF and acidic FGF, corticosteroids (ACTH), bacitracin, calcitonin (CT), chorionic gonadotropin (hCG) With growth hormone (somatotropin), gumicidin S, insulin, luteinizing hormone free hormone (LHRH), oxytocin (OT), somatostatin (SS), thyrotropin free hormone (TRH) And selected from the group consisting of: The method of claim 1, wherein the carrier is selected from the group consisting of sorbitol, sodium chloride, glucose, and mannitol. The method of claim 1, wherein the oil used to prepare the W / O emulsion of the water soluble peptide drug is mineral oil such as liquid paraffin; Vegetable oils such as soybean oil, corn oil, castor oil, cottonseed oil, canola oil, sunflower seed oil, safflower oil, sesame oil, peanut oil; Vitamin oils such as tocopherol oil (VE); Ethyl oleate, caprylic / caprylic acid triglyceride. The method of claim 1, wherein the surfactant used to prepare the W / O emulsion of the water soluble peptide drug is a nonionic surfactant such as polysorbate (Tweens), sorbitan fatty acid esters (Spans), poly Oxyethylene alkyl ethers, poloxamers, polyoxyl hydrogenated castor oil, polyethylene glycol esters, methyl glucoside derivatives and cremophors. The method of claim 1, wherein the phospholipid used to coat the dry carrier is phosphatidylcholine, phosphatidylglycerol, dimyristoylphosphatidylcholine, dimyristoylphosphatidylglycerol, dioleoylphosphatidylcholine, dioleoylphosphadiylglycerol, disteroyl Phosphatidylethanolamine-N-poly (ethyleneglycol) (DSPE-PEG) and a method using one selected from the group consisting of cholesterol. The method according to claim 1, wherein the secondary coating uses one selected from chloroform, methanol, ethanol and a mixture of one or more thereof as an organic solvent. A formulation of a stabilized dry liposome of a water-soluble peptide drug prepared by the method of claim 1 in a formulation selected from liposome granules, liposome capsules, liposome tablets, external preparations of liposomes and injectable soluble powdered liposomes.