HK1096295B - Nanoparticle compositions of water-soluble drugs for oral administration and preparation methods thereof - Google Patents

Description

Orally administered water-soluble drug nanoparticle composition and preparation method thereof

Technical Field

The present invention relates to an orally administered nanoparticle composition comprising a water-soluble drug having improved gastrointestinal absorption rate, and a method for preparing the same. In particular, the present invention relates to an orally administered nanoparticle composition having an improved entrapment rate of a water-soluble drug in nanoparticles composed of lipids and polymers and being stable to lipase, wherein the nanoparticles are prepared by combining the water-soluble drug with a counter ionic substance and adding the lipids, the polymers and an emulsifier, and a method for preparing the same.

Background

Water-soluble drugs and protein drugs including physiologically active agents have low stability in the gastrointestinal tract and low permeability to the intestinal wall, and thus these drugs are generally used in intravenous injection. However, intravenous injection is inconvenient for most patients in daily life, so many efforts have been made to prepare water-soluble drugs into oral administration preparations.

Once orally administered, water-soluble drugs are chemically degraded by the action of pH and digestive enzymes, and their stability in the gastrointestinal tract is reduced. Therefore, most or a part of these drugs administered cannot exert their effects, and their pharmacological effects cannot be sufficiently expressed in oral administration. In particular, when these drugs are degraded or degraded with changes in pH, they lose their efficacy by the action of gastrointestinal enzymes and have very low bioavailability. To solve these problems, studies have been made in the preparation of water-soluble drug formulations in which these drugs are not exposed to external chemical environments, such as pH or digestive enzymes, by being entrapped in lipids or polymers having high affinity for biological membranes.

For example, oral formulations of water-soluble drugs using w/o or w/o/w emulsifiers or liposomes are known in the art. However, they have disadvantages of insufficient drug loading rate and low stability.

Us patent 6,004,534 discloses targeting of these water-soluble drugs as vaccines or allergens to specific tissues by the use of targeting liposomes. However, there is a problem that the entrapment rate of the drug in the liposome is low, about 35%.

U.S. Pat. No. 6,191,105 discloses the preparation of a w/o microemulsion formulation of insulin polymers.

However, once inside the body, the w/o emulsion structure can be usually split by phase transition, so that the drug dissolved in the aqueous phase cannot be protected by the oil phase, but is directly exposed to the body.

U.S. Pat. No. 6,277,413 discloses the preparation of w/o/w emulsions wherein a water soluble drug is added to the inner aqueous phase. However, in this patent, the prepared emulsion has a very large particle size of 10 to 20 μm, and the drug entrapped in the inner aqueous phase may be released to the outer aqueous phase during the preparation process thereof, so that the drug entrapment rate of the emulsion is low.

Korean patent publication No. 2002-. However, the carrier is composed of only lipids, and thus is degradable by lipase in vivo.

Journal of controlled Release No. 69, p 283 & 295 (2000) discloses microspheres of PLGA containing insulin with a drug loading of less than 50% and a particle size of greater than 5 microns. It is reported that 100 nanometer size particles absorb 15-250 times more in the intestinal tract than micron size particles, which are not normally found on the epithelial cell surface of the intestine [ pharm. Res.13(12) p1838-1845(1996) ]. Therefore, PLGA microspheres of 5 μm size have a low insulin entrapment rate and are less effective at delivering insulin than nanoparticles.

In addition, U.S. Pat. No. 5,962,024 discloses dissolving a drug at pH 6.5 or higher by preparing granules or coating the granules with an enteric polymer such as hydroxypropylmethylcellulose acetate succinate or methylmethacrylate copolymer. However, microspheres formed of enteric polymers alone have a drawback in that it cannot stabilize a drug unstable in the gastrointestinal tract because the polymer is dissolved in the intestinal tract, and the drug entrapped in the microspheres is exposed to the intestinal tract.

On the other hand, since water-soluble drugs have low affinity with carriers composed of lipophilic substances or polymers, only a small portion of them should be entrapped in the carriers, and thus removal of their charges should decrease their water solubility, thus increasing their entrapment rate in the carriers.

WO 94/08599 discloses the preparation of complexes in which insulin is ionically bound to sodium lauryl sulfate. The preparation method comprises dissolving insulin-sodium dodecyl sulfate complex in organic solvent, and using the solution as lung preparation or suppository. However, once orally administered, the drug is directly exposed to the gastrointestinal tract and therefore does not remain stable.

In addition, U.S. Pat. No. 5,858,410 discloses nano-particulated water-insoluble drugs by milling and using a microfluidizer, but has a drawback that it is not suitable for water-soluble drugs unstable in vivo, such as protein drugs, because these drugs are directly exposed to the in vivo environment.

From the above, for oral administration of water-soluble drugs (including protein drugs) that are unstable in vivo, nanoparticles that are stable to gastrointestinal lipase should be designed, and the water-soluble drugs should be efficiently encapsulated in drug carriers. The technical requirements for achieving these objects are as follows.

First, in order to keep the drug entrapped in the nanoparticles stable, without degradation in the gastrointestinal tract, the lipid nanoparticles should contain an appropriate amount of polymer to improve their stability in vivo.

Secondly, the entrapment rate of the water-soluble drug in the lipophilic carrier is increased by preparing a complex of the water-soluble drug and a counter-ion substance and selecting a lipid/polymer system having similar affinity, by modifying the water-soluble drug to have affinity with the lipophilic carrier.

Third, the particle size of the lipid/polymer nanoparticles containing a complex of a water-soluble drug and a counter-ionic substance should be minimized. In addition, the nanoparticles should be prepared so that these drugs are entrapped therein so that they are not exposed to the external environment and can be absorbed into the body while retaining their maximum activity.

DISCLOSURE OF THE INVENTION

The present inventors have conducted repeated and extensive studies to solve the above-mentioned problems and to develop a drug carrier that meets the above-mentioned technical requirements. As a result, the present inventors have found that an orally administrable nanoparticle composition is prepared from a water-soluble drug-counter ion substance complex and a lipid, a polymer, and the like in a certain ratio so that the particle size thereof is 500nm or less, which can improve drug entrapment rate and resistance to in vivo degrading enzymes, and thus have completed the present invention.

The object of the present invention is to provide an orally administrable nanoparticle composition and a method for preparing the same, in which the drug entrapment rate and the resistance to lipase are improved by a complex containing a water-soluble drug and a counter ion substance, thereby improving the in vivo oral absorption.

Brief description of the drawings

Figure 1 shows the stability of an orally administrable nanoparticle composition of the invention comprising insulin as the active ingredient against pancreatin (protease/lipase) in experiment 2.

-. solid: example 1

- ■ -: comparative example 1

-. solidup-: comparative example 2

-X-: comparative example 3

- * -: example 1a

Figure 2 shows the relative blood glucose concentrations following enteral application of an orally administrable nanoparticle composition of the invention comprising insulin as the active ingredient in experiment 3(2) for the treatment of type I diabetes in rats.

-. solid: reference group (buffer containing insulin, pH7.4, insulin dose 3IU/kg)

- ■ -: example 1 (insulin dose 3IU/kg)

FIG. 3 shows the relative blood glucose concentrations following enteral application of an orally administrable nanoparticle composition of the invention comprising insulin as the active ingredient in experiment 3(3)1) for the treatment of type I diabetes in rats.

-. solid: reference group 1 (buffer containing insulin, pH7.4, insulin dose 20IU/kg)

- ■ -: example 1 (insulin dose 20IU/kg)

-. solidup-: comparative example 1 (insulin dose 20IU/kg)

-X-: example 1a (insulin dose 20IU/kg)

FIG. 4 shows the relative blood glucose concentrations following enteral application of an orally administrable nanoparticle composition of the invention comprising insulin as the active ingredient in experiment 3(3)1) for the treatment of type I diabetes in rats.

-. solid: example 1 (insulin dose 20IU/kg)

- ■ -: reference group 2 (insulin injection, subcutaneous injection, insulin dose 0.2IU/kg)

FIG. 5 shows the insulin concentration after enteral application of the orally administrable nanoparticle composition of the present invention comprising insulin as an active ingredient in experiment 3(3)2) for the treatment of type I diabetes in rats.

-. solid: control group (oral nanoparticle composition without insulin)

- ■ -: example 1 (insulin dose 20IU/kg)

-. solidup-: comparative example 1 (insulin dose 20IU/kg)

-X-: example 1a (insulin dose 20IU/kg)

FIG. 6 shows the insulin concentration after enteral application of the orally administrable nanoparticle composition of the present invention comprising insulin as an active ingredient in experiment 3(3)2) for the treatment of type I diabetes in rats.

-. solid: example 1 (insulin dose 20IU/kg)

- ■ -: reference group (insulin injection, subcutaneous injection, insulin dosage 0.2IU/kg)

Figure 7 shows the ceftriaxone concentration after intraduodenal application of the orally administrable nanoparticle composition of the invention comprising ceftriaxone as the active ingredient given to normal rats in experiment 4(1) 1.

-. solid: example 6 (ceftriaxone dosage 40mg/kg)

- ■ -: example 7 (ceftriaxone dosage 40mg/kg)

-. solidup-: comparative example 5 (ceftriaxone dosage 40mg/kg)

-X-: reference group 1 (aqueous solution containing ceftriaxone, ceftriaxone dosage 40mg/kg)

Fig. 8 shows the concentration of ceftriaxone after intraduodenal application of the orally administrable nanoparticle composition of the present invention, which contains ceftriaxone as the active ingredient given to normal rats in experiment 4 (1).

-. solid: example 6 (ceftriaxone dosage 40mg/kg)

- ■ -: example 7 (ceftriaxone dosage 40mg/kg)

-. solidup-: reference group 2 (ceftriaxone injection, intravenous injection, ceftriaxone dosage 20mg/kg)

Best mode for carrying out the invention

The present invention provides an orally administrable composition containing nanoparticles having a particle size of 500nm or less, the composition comprising: 0.1 to 30% by weight of a complex of a water-soluble drug and a counter-ionic substance, in which complex the charged water-soluble drug is bound to the counter-ionic substance; 0.5-80% by weight of lipids; 0.5 to 80% by weight of a polymer; and 1-80% by weight of an emulsifier, wherein the weight ratio of lipid to polymer is 1: 0.05-3. The weight ratio of the lipid to the polymer is preferably 1: 0.2-1.

In addition, the present invention provides a method for preparing the orally administrable nanoparticle composition of the present invention, comprising the steps of: (a) ionizing and combining the charged water-soluble drug and the counter ion substance to form a complex of the water-soluble drug and the counter ion substance; (b) adding a lipid, a polymer and a solubilizer to the resulting complex and dissolving the whole mixture, and then introducing the resulting solution into an aqueous solution containing an emulsifier to obtain a uniform liquid phase; (c) removing the solubilizer from the mixture obtained in step (b); and (d) optionally, minimizing particle size using a microfluidizer.

According to the present invention, a water-soluble drug is entrapped in such a nanoparticle composition at an entrapment rate of 70% or more, and the entrapment rate can be maintained at 80% or more in the presence of a pancreatin solution.

In addition, the present invention provides a method for preparing the orally administrable nanoparticle composition of the present invention, comprising the steps of: (a) ionizing and combining the charged water-soluble drug and the counter ion substance to form a complex of the water-soluble drug and the counter ion substance; (b) adding a lipid and a solubilizer to the resulting complex and dissolving the entire mixture, and then introducing the resulting solution into an aqueous solution containing an emulsifier and a polymer to obtain a homogeneous liquid phase; (c) removing the solubilizer from the mixture obtained in step (b); and (d) optionally, minimizing particle size using a microfluidizer.

The present invention will be described in detail below.

The orally administered nanoparticle composition of the present invention comprises: a complex of a water-soluble drug and a counter-ionic substance, in an amount of 0.1 to 30% by weight, in which the charged water-soluble drug is ionically bound to the counter-ionic substance; lipid, 0.5-80% (by weight); 0.5 to 80% by weight of a polymer; and an emulsifier in an amount of 1-80% by weight, wherein the weight ratio of the lipid to the polymer is 1: 0.05-3, and the composition further comprises nanoparticles having a particle size of 500nm or less. The weight ratio of the lipid to the polymer is preferably 1: 0.2-1.

The complex of the water-soluble drug and the counter-ionic substance is obtained by reacting the charged drug with the counter-ionic substance. The water-soluble drug used as the active ingredient may be a drug charged in an aqueous solution, preferably a drug charged in water, selected from peptide/protein drugs such as insulin, erythropoietin, calcitonin, growth hormone, interferon, somatostatin and the like; heparin; cephalosporin antibiotics; alendronate sodium; etidronate sodium; pamidronate sodium, and the like.

The negatively charged substance that can be ionically bound to the positively charged drug may preferably be selected from sodium salts of C8-18 fatty acids such as sodium oleate, sodium lauryl sulfate, sodium caproate, sodium laurate, and the like; sodium salts of bile acids; sodium alginate; and sodium carboxymethyl cellulose. The positively charged substance that can be ionically bound to the negatively charged drug may preferably be selected from quaternary ammonium compounds, such as carnitine salts, benzalkonium chloride, cetyltrimethylammonium bromide, and the like.

The content of the complex of the water-soluble drug and the counter ion substance is preferably 0.1 to 30% by weight based on the total weight of the nanoparticle composition.

When a water-soluble drug forms a complex with a counter-ionic substance, the initial charge of the water-soluble drug is removed or weakened, thus increasing its affinity with the lipophilic carrier. As a result, the water-soluble drug can be more entrapped in the lipophilic carrier. As shown in Table 4 of experiment 1 below, the drug loading of the composition of the present invention is high.

The molar ratio of the water-soluble drug to the counter ion substance in the complex of the water-soluble drug and the counter ion substance can be adjusted according to the ion number of the water-soluble drug, and is preferably 1: 0.1-20, more preferably 1: 3-10.

The lipid may preferably be selected from one or more of the following fatty alcohols: such as monoglycerides, diglycerides, propylene glycol esters of fatty acids (Capriol et al), glycerol esters of fatty acids (GELucier et al), cetostearyl alcohol, cetyl alcohol, etc., and the lipid content is preferably 0.5 to 80% by weight, more preferably 0.5 to 30% by weight, based on the total weight of the nanoparticle composition.

The polymer may preferably be selected from one or more of the following: enteric polymers such as methacrylic acid copolymer, hydroxypropylmethylcellulose phthalate, hydroxypropylmethylcellulose acetate succinate, cellulose acetate phthalate and the like; shellac, chitosan, hydroxypropylmethylcellulose and its derivatives, ethyl cellulose, methyl cellulose, polyvinyl alcohol, sodium alginate and carbomer, and the content of the polymer may preferably be 0.5 to 80% by weight, more preferably 0.5 to 30% by weight, based on the total weight of the nanoparticle composition. In particular, if enteric polymers are used, drugs (e.g., proteins) that are unstable in gastric acid are not decomposed, and they are selectively absorbed by the small intestine or the large intestine depending on the composition of the enteric polymers.

The polymer is insoluble in water or becomes viscous when dissolved in water, and thus may be slowly degraded when added to lipid nanoparticles, thereby controlling the dissolution rate of the water-soluble drug entrapped in the nanoparticles. In addition, the polymer is wrapped around the surface of the lipid nanoparticle and is inserted in the middle of the lipid molecule, thus making the nanoparticle enzyme resistant. That is, the addition of the polymer prevents the degradation of lipid, which is a structural component in the nanoparticle, by the action of lipase, thus maintaining the structure of the nanoparticle and also significantly improving the chemical stability of the water-soluble drug entrapped in the nanoparticle.

When the polymer is added to the lipid nanoparticle composition, the size of the lipid nanoparticle may be changed according to the content of the polymer, and the ratio of the lipid to the polymer should be appropriately adjusted to obtain nanoparticles of 500nm or less.

The emulsifier is preferably selected from one or more of the following: polyoxyethylene polyoxypropylene copolymer (trade name: Poloxamer)^TM) Polyethylene glycol alkyl ether (trade name: bris-^TM) Polyoxyethylene castor oil (trade name: tween (Green)^TM) Polyoxyethylene sorbitan fatty acid ester (trade name: span^TM) And transesterification products of natural vegetable oil triglycerides and polyalkylene polyols (trade name: labrafil^TM、Labrasol^TM) Glycerin fatty acid ester (trade name: plurol^TMoleique), vitamin E polyethylene glycol succinate (vitamin E polyethylene glycol succinate), lecithin, sodium lauryl sulfate, and bile acid and its derivatives, and the content of the emulsifier may preferably be 1-80% by weight based on the total weight of the nanoparticle composition. More preferably, the emulsifier is present in an amount of 30 to 80% by weight. When the prepared lipid/polymer nanoparticles are dispersed in an aqueous solution, the emulsifier stabilizes the dispersion.

The solubilizer may preferably be selected from one or more of the following: c_1-8Alcohols, dimethyl sulfoxide, methylene chloride, toluene, propylene glycol, polyethylene glycol and 12-hydroxystearate (trade name: Solutol)^TM). This solubilizer is eliminated while the nanoparticles are prepared, and thus the content of the solubilizer in the final nanoparticle composition is preferably 50% by weight or less. In addition, if the solubilizer alone is not solubleA complex, polymer, lipid, etc. of the water-soluble drug and the counter ion substance is dissolved or dispersed, and the above-mentioned emulsifier may be added thereto to obtain a uniform solution.

In addition, the composition may contain conventional oral absorption enhancers, preferably selected from p-glycoprotein inhibitors, carnitine salts, chelating agents, etc., in an amount of 0-20% by weight.

Alternatively, the compositions of the present invention may be freeze-dried, preferably containing 0.1 to 30% by weight of the cryoprotectant, and may be prepared as a powder and then prepared into conventional oral dosage forms, such as capsules or tablets. The cryoprotectant may preferably be a saccharide such as glucose, mannitol, sorbitol, trehalose; amino acids, such as arginine; proteins such as albumin, and the like.

The orally administrable nanoparticle compositions of the present invention are prepared by a method comprising the steps of: (a) ionically binding a charged water-soluble drug with a counter-ionic species to form a complex of the water-soluble drug and the counter-ionic species; (b) adding a lipid, a polymer and a solubilizer to the resulting complex to dissolve the entire mixture, and then adding the resulting solution to an aqueous solution containing an emulsifier to obtain a uniform liquid phase; (c) removing the solubilizer from the mixture obtained in step (b); and (d) optionally, minimizing particle size using a microfluidizer.

In the above step (a), the complex of the water-soluble drug and the counter ion substance is prepared by dissolving the drug in water or a suitable buffer to obtain an aqueous solution containing the drug, and then reacting the counter ion substance with the drug solution.

The optimum ratio of the water-soluble drug and the counter ion substance is adjusted in the range of 1: 0.1-20 in molar ratio according to the ion number of the drug, and is determined by analyzing the unreacted drug in the above reaction. The resulting complex of the water-soluble drug and the counter-ionic substance is preferably washed 3 or more times with water to remove the unreacted drug.

In particular, in preparing complexes of proteinaceous drugs and counter-ionic substances, pH adjusters may be used to impart a charge to the proteinaceous drug. The pH regulator can be selected from acidifying agents such as hydrochloric acid, phosphoric acid, carbonic acid, citric acid, etc.; alkalinizing agents such as sodium hydroxide, sodium/potassium monohydrogen phosphate, sodium/potassium dihydrogen phosphate, sodium citrate, and the like; or a buffer consisting of a mixture thereof, and the amount of the pH adjustor can be 0.1 to 10 times that of the drug.

In the above-mentioned steps (b) to (c), the complex of the water-soluble drug and the counter ion substance and the lipid are dissolved in the solubilizing agent, then, the solution is added to the aqueous solution containing the polymer, the mixture is stirred, and then, the solubilizing agent is removed therefrom. In this step, in order to increase the solubility of the polymer, an acid, a base and an emulsification aid, in an amount of 0.5 to 50% by weight, may be added thereto, and the mixture may be heated, preferably to 30 to 70 ℃.

In the above step (c), if the solubilizer is highly volatile, it can be removed by injecting nitrogen gas or using a vacuum evaporator. If it is not highly volatile, it can be removed by dialysis.

In the above step (d), the solution is preferably cyclized 3 to 10 times at 100MPa using a microfluidizer. The particle size of the nanoparticles can be further reduced by heating if desired. The heating temperature is preferably 30 to 70 ℃.

Another method for preparing an orally administrable composition of the invention comprises the steps of: (a) ionically binding a charged water-soluble drug with a counter-ionic species to form a complex of the water-soluble drug and the counter-ionic species; (b) adding a lipid and a solubilizing agent thereto to dissolve the entire mixture, and then adding the resulting solution to an aqueous solution containing a polymer and an emulsifier to obtain a uniform liquid phase; (c) removing the solubilizer from the mixture obtained in step (b); and (d) optionally, minimizing particle size using a microfluidizer.

The resulting nanoparticle composition is liquid and, if desired, can be freeze-dried with the addition of a cryoprotectant to obtain a powder. The cryoprotectant is used to prevent denaturation of the components of the composition during lyophilization. The cryoprotectant is preferably used in an amount of 0.1 to 30% by weight based on the total weight of the nanoparticle composition. This cryoprotectant may preferably be a sugar such as lactose, mannitol, trehalose, and the like; amino acids, such as arginine; proteins such as albumin, and the like.

The liquid and powder compositions obtained as above can be easily dispersed in water by simple physical mixing, for example, by shaking by hand, and the particle size of the dispersion formed is 500nm or less, which is smaller than that of the previous orally administered formulation, depending on the characteristics of the emulsifier or drug used. Preferably, the orally administered nanoparticle composition has a particle size of 20-300 nm.

Sealed at room temperature or lower, the nanoparticle powder composition of the present invention can be stably stored for a long period of time, and water can be added thereto before use to prepare a dispersion solution thereof.

In addition, the nanoparticle composition of the present invention can be prepared into a solution, suspension, capsule, tablet, etc. by adding other absorption enhancers, and in case of a solid preparation, its oral bioavailability can be further improved by a conventional preparation technique such as enteric coating, etc.

The present invention will be described in more detail below with reference to examples, but they should not be construed as imposing any limitation on the scope of the present invention.

Examples

Example 1: preparation of orally administrable nanoparticle compositions containing insulin

(1) Preparation of complexes of insulin and counter-ion substances

50mg of insulin (Sigma) was dissolved in 20ml of citrate buffer pH3 and docusate sodium (Aldrich) dissolved in 80ml of citrate buffer pH3 was reacted therewith in molar ratios of 1: 3, 1: 6 and 1: 9 with 1 mole of insulin to obtain a complex of insulin and a counter ion substance. Upon completion of this reaction, the supernatant was analyzed for insulin according to the insulin analysis method described in the U.S. pharmacopoeia to obtain the results shown in table 1 below.

Table 1: reaction ratio of insulin to counter ion substance complex

Insulin: molar ratio of docusate sodium	Residual reactant in supernatant
		1∶31∶61∶9	20.7％0％0％

As shown in Table 1 above, most of the insulin forms a complex with the counter ion substance when the molar ratio of insulin to docusate sodium is 1: 6 or more. Thereafter, the complexes prepared with insulin and counter-ion species in a 1: 6 molar ratio were washed 3 times each with citrate buffer at pH3 and water, and left to be used in the following step.

(2) Preparation of orally administrable nanoparticle compositions containing insulin

15mg of insulin-docusate sodium complex and 60mg of glycerol monooleate (TCI) were dissolved in 10ml of ethanol. This solution was added to 20ml of a solution containing 20mg of chitosan (Korean chitosan) and 200mg of Poloxamer407^TM(BASF) in water, the whole mixture was stirred to obtain a homogeneous solution, from which ethanol was then removed by injecting nitrogen. In thatWherein 1% citric acid is added as an additive for improving the solubility of chitosan. Using a microfluidizer, Emulsiflex-C5^TM(Avestin), cyclizing the lipid/polymer nanoparticle 10 times at room temperature under 100MPa to obtain an orally administrable nanoparticle composition containing insulin.

(3) Determination of particle size and distribution

200. mu.l of the resulting liquid composition was dispersed in 1ml of distilled water, followed by photonic spectroscopy (ELS-8000)^TM0tsuka electronics) the particle size and dispersion of the composition was determined. As a result, the average particle diameter of the liquid composition of example 1 was 83.0nm, and the dispersion degree was 0.300.

Comparative example 1

A liquid composition was prepared in the same manner as in example 1, except that insulin was used instead of the insulin-counterion substance complex, and neither polymer nor microfluidizer was used. The particle diameter and the degree of dispersion were measured by the method of example 1, and as a result, the average particle diameter was 117.4nm and the degree of dispersion was 0.251.

Comparative example 2

A liquid composition was prepared in the same manner as in example 1, except that insulin was used instead of the insulin-counterion substance complex, and a microfluidizer was not used. The particle size and the degree of dispersion were measured by the method of example 1, and as a result, the average particle size was 241.8nm and the degree of dispersion was 0.090.

Comparative example 3

15mg of the insulin-counterion complex obtained according to example 1 and 60mg of glycerol monooleate were sonicated at 50 ℃ for 1 hour, then 20ml containing 20mg of chitosan and 200mg of Poloxamer407 were added^TM1% aqueous citrate solution and they were again sonicated for 30 minutes. This mixture was then cyclized 10 times at 100MPa at room temperature using a microfluidizer to obtain a liquid composition containing an insulin complex. Particle size measurement according to the method of example 1And a dispersion degree, as a result, the average particle diameter was 211.6nm, and the dispersion degree was 0.213.

Example 1a

A liquid composition was prepared in the same manner as in example 1, except that no microfluidizer was used. The particle diameter and the degree of dispersion were measured in accordance with the method of example 1, and the average particle diameter was 234.3nm, and the degree of dispersion was 0.291.

Example 2: preparation of orally administrable nanoparticle powder formulations containing insulin

In solidifying the orally administrable nanoparticle liquid preparation obtained in example 1, the increase in the nanoparticle size was minimized by adding 5% mannitol to the orally administrable nanoparticle liquid preparation, and then the liquid preparation was solidified under various freeze-drying conditions and dispersed in water to determine the particle size. The results are shown in Table 2 below.

TABLE 2

Conditions of lyophilization		Before freezing out (nm)	After freeze-drying (nm)	Increase rate of particle size before and after lyophilization
					Condition 1	Room temperature, 48 hours	68.1	339.8	5.0
Condition 2	0 ℃ C: 12 hours → 0 ℃: 8 hours → 5 ℃: 24 hours	82.5	188.5	2.3
					Condition 3	-25 ℃ C: 24 hours → 0 ℃: 24 hours	118.1	196.4	1.7

As can be seen from Table 2 above, to minimize the variation in particle size, freeze-drying at < 0 ℃ is preferred.

Mannitol was added to the orally administrable nanoparticle liquid formulation at 0.5%, 1%, 2%, 4% and 5%, respectively, and then the mixture was cured at-25 ℃ for 6 hours, followed by another 6 hours at 20 ℃. The obtained solid was dispersed in water, and then the particle size was measured according to the method of example 1. The results obtained are shown in Table 3 below.

TABLE 3

Mannitol	-25 ℃, 6 hours → 20 ℃, 6 hours
			Particle size	Increase rate of particle size before and after lyophilization
	0.5％	394.1nm			3.4
1％			335.1nm	2.8
	2％	321.6nm			2.7

4％	217.3nm	1.8
			5％	196.4nm	1.7

As can be seen from the results of the measurement, when the concentration of the cryoprotectant was about 5%, the variation in particle size was minimized.

Example 3: preparation of an orally administrable nanoparticle preparation containing insulin 2

In example 1, sodium lauryl sulfate (Sigma) was used in place of docusate sodium in a ratio of 1: 7 to obtain a complex of insulin with a counter ion substance. The complex of insulin with a counter ion substance, glycerol monooleate and polyethylene glycol 2000 were weighed in an amount of 15mg, 45mg and 90mg, respectively, and dissolved in 10ml of ethanol. An orally administrable nanoparticle composition containing insulin was prepared according to the same method.

The particle size and the degree of dispersion were measured by the method of example 1, and as a result, the average particle size was 121nm and the degree of dispersion was 0.314.

Example 4: preparation of an insulin-containing nanoparticle composition for oral administration 3

An orally administrable nanoparticle composition containing insulin was prepared in the same manner as in example 1, except that Labrafac was used^TM(Gattefosse) instead of glycerol monooleate. The particle diameter and the degree of dispersion were measured in accordance with the method of example 1, and as a result, the average particle diameter was 129.4nm and the degree of dispersion was 0.227.

Example 5: preparation of an insulin-containing nanoparticle composition for oral administration 4

An orally administrable nanoparticle composition containing insulin was prepared in the same manner as in example 1, except that 60mg of Eudragit was used^TM(Rohm) and Eudragits^TM(Rohm) 1: 1 mixture instead of chitosan. The particle size and the degree of dispersion were measured in accordance with the method of example 1, and the average particle size was 194.2nm, and the degree of dispersion was 0.128.

Comparative example 4

This liquid preparation was prepared in the same manner as in example 5, except that neither glycerol monooleate nor microfluidizer was used. The particle diameter and the degree of dispersion were measured in the same manner as in example 1, and as a result, the average particle diameter was 350.9nm, and the degree of dispersion was 0.009.

Example 6: preparation of orally administrable nanoparticles containing ceftriaxone

(1) Preparation of ceftriaxone and counter ion substance complex

100mg of ceftriaxone (Hawon fine chemical) was dissolved in 20ml of distilled water, benzalkonium chloride (Sigma) was weighed at a 1: 2 molar ratio to ceftriaxone, and dissolved in distilled water, followed by reaction at 4 ℃. The supernatant was collected and analyzed for ceftriaxone according to the methods described in the united states pharmacopeia for ceftriaxone. As a result, it was confirmed that 99.9% of ceftriaxone formed a complex with the counter ion substance.

(2) Preparation of oral liquid preparation containing ceftriaxone

120mg of a complex of ceftriaxone and a counter-ionic substance, 60mmg of glycerol monooleate and 30mg of Eudragit L^TMAnd Eudragit S^TMThe 1: 1 mixture was dissolved in 15ml ethanol. This solution was added to 30ml of a solution containing 375mg Labrasol^TM(Gattefose) and 300mg Poloxamer407^TMThe whole mixture was stirred to obtain a uniform solution. An orally administrable nanoparticulate formulation containing ceftriaxone was prepared according to the method of example 1. The particle size and degree of dispersion were measured by the method of example 1, and the average particle size was 199.0nm, and the degree of dispersion was 0.135.

Comparative example 5

A liquid preparation was prepared in the same manner as in example 6, except that ceftriaxone was used in place of the ceftriaxone-counterion substance complex, and neither polymer nor microfluidizer was used. The particle diameter and the degree of dispersion were measured by the method of example 1, and the average particle diameter was 149.4nm, and the degree of dispersion was 0.124.

Example 7: preparation of orally administrable nanoparticle preparation containing ceftriaxone

240mg of ceftriaxone-counterion complex and 1200mg of Capryol^TM(Gattefose) was dissolved in 15ml ethanol. This solution was added to 30ml of a solution containing 240mg of Labrasol^TM30mg of chitosan and 300mg of Poloxamer407^TMThe whole mixture was stirred in the aqueous solution of (1). Then, according to the method of example 1, orally administrable nanoparticle preparation containing ceftriaxone was prepared. 300mg of citric acid was added as an additive to increase the solubility of chitosan. The particle size and degree of dispersion were measured by the method of example 1, and as a result, the average particle size was 290.1nm and the degree of dispersion was 0.197.

Experiment 1: determination of drug Loading Rate

500. mu.l of the liquid formulations prepared in example 1 and examples 1a to 7 above, and comparative examples 1 to 5 were dispersed in 500. mu.l of distilled water. Then, this dispersion was added to Centricon YM-30^TM(fractionation MW: 30,000, Millipore) and centrifuged at 1500g for 60 minutes. The drug in the above separated filtrate was analyzed to calculate the drug loading rate in the nanoparticles. The results obtained are shown in Table 4 below.

Table 4: drug loading rates in orally administrable nanoparticle formulations

	Drug loading (%)
		Example 1	99.8
Example 1a	95.7
		Example 2	98.7
Example 3	97.2
		Example 4	90.3
Example 5	91.3
		Example 6	90.5
Example 7	92.0
		Comparative example 1	72.4
Comparative example 2	64.8
		Comparative example 3	70.7
Comparative example 4	92.6
		Comparative example 5	40.8

Experiment 2: evaluation of enzymatic degradability-degradation of pancreatic enzymes by proteases/lipases

Lipase exists in the body, and the nanoparticle composed of lipid should be stable to lipase so that insulin entrapped in the lipid nanoparticle is not exposed to the outside and so that it is not degraded by protease. The preparations of examples and comparative examples were tested for stability against in vivo enzymes, which degrade proteins and lipids, such as pancreatin.

5ml of the liquid formulations prepared in examples 1, 1a and 3 to 5 above, and comparative examples 1 to 4 were added to 15ml of a buffer solution containing 0.0067% pancreatin (USP grade) and having a pH of 7.4. The reaction was carried out at 37 ℃ for 0, 1, 3 and 5 hours, and the remaining amount of insulin was measured, and the results are shown in Table 5 below.

Table 5: stability of orally administrable nanoparticulate formulations containing insulin to pancreatin

	Residual insulin (%)
						0 hour	1 hour	3 hours	5 hours
Example 1	100.0	95.8	93.6	91.6
					Example 1a	100.0	100.0	97.5	93.5
Example 3	100.0	100.0	97.5	93.5
					Example 4	100.0	99.1	96.8	93.4
Example 5	100.0	92.7	92.0	91.3
					Comparative example 1	100.0	9.1	0.8	0
Comparative example 2	100.0	87.5	82.6	82.1
					Comparative example 3	100.0	91.6	69.2	57.2
Comparative example 4	100.0	51.2	14.9	14.5

As shown in tables 4 and 5 above, the formulations of examples 1 to 5 containing insulin-counterion complex and lipid/polymer have higher drug loading and enzyme stability. In contrast, the formulations of comparative example 1 containing only lipids and comparative example 4 containing only polymers were extremely low in enzyme stability, i.e. the stability of insulin to pancreatin after 5 hours was even less than 20%.

In particular, the nanoparticle formulations of comparative examples 1 and2, which were prepared from insulin, but not from an insulin-counterion complex, had lower drug loading rates. As shown in table 5, the stability of the formulation of comparative example 2 containing a polymer to the enzyme was higher than that of the formulation of comparative example 1 containing no polymer.

The preparation of comparative example 3, which was prepared using only a microfluidizer in preparing nanoparticle formulations, had a lower entrapment rate of insulin-counterion complex and was less stable to enzymes than the preparation of example 1, which was prepared by first using such a solubilizer, which is ethanol, and then using a microfluidizer. The nanoparticle formulation of example 1a prepared using a solubilizing agent without using a microfluidizer has a higher drug loading rate and enzyme stability similar to that of the formulation of example 1.

As described above, nanoparticle formulations prepared from insulin rather than insulin-counterion complexes have lower drug loading rates, while nanoparticle formulations composed of lipids and polymers have higher stability to enzymes. In addition, it is not sufficient to use only a microfluidizer, and the nanoparticles preliminarily formed using a solubilizer are necessary to obtain a nanoparticle preparation having a high drug loading rate and stability to enzymes.

Therefore, the nanoparticles of the present invention prepared by forming a drug-counter ion complex and entrapping the complex in a carrier composed of lipid and polymer have been demonstrated to have not only excellent drug entrapment rate but also excellent stability against lipase and protease.

Experiment 3: evaluation of animal efficacy 1

Animal experiments were performed using the orally administrable nanoparticle liquid formulation prepared in example 1.

(1) Induce diabetes

Normal male Spraque Dawley rats weighing about 180-220g were injected intraperitoneally with 45mg/kg streptozotocin (Sigma)2 times with 2 days in between to generate type I diabetic rats. After 1 week, the rats were fasted for 12 hours, then blood was collected, and blood glucose was measured. As a result, rats having blood glucose of 300mg/dl or more, which were used in the following experiments, were considered to be type I diabetic rats. Using Glucotrand2^TM(Roche) blood glucose was measured.

(2) Determination of the physiological Activity of insulin in an orally administrable Nanoparticulate liquid formulation

2ml of the nanoparticle liquid formulation prepared in example 1 was dispersed in 2ml of physiological saline and intramuscular injected into type I diabetic rats at 3IU/kg of insulin.

Insulin diluted in pH7.4 buffer was intramuscularly administered to type I diabetic rats at 3IU/kg insulin as a reference group. Blood was taken from the tail vein at given time intervals.

As shown in FIG. 2, the orally administrable nanoparticle liquid formulation of the present invention was compared with the physiological activity of insulin in an insulin solution, confirming that the nanoparticle liquid formulation of the present invention can maintain the physiological activity of insulin intact. Therefore, the following animal experiments were conducted using the liquid formulation of the present invention.

(3) Gastrointestinal absorption test for orally administrable nanoparticle liquid formulations

1) Blood glucose determination

Type I diabetic rats were anesthetized with ether and the abdominal cavity was opened. The small intestine was removed and the liquid formulation containing insulin was applied 5cm above the top of the appendix at a dose of 20/kg. The incision was then sutured and the diabetic rat allowed free access to water.

The test groups used the formulations of example 1, comparative example 1 and example 1 a. The formulation of comparative example 1 is a nanoparticle formulation containing neither insulin-counterion complex nor polymer, so its drug loading rate and enzyme stability are lower than those of the formulation of example 1.

The formulation of example 1a had high drug loading and enzyme stability, but had a larger particle size than the formulation of example 1 because no microfluidizer was used. Insulin-containing phosphate buffer solution of pH7.4 was administered at a dose of 20IU/kg by the same route as in example 1, and used as reference group 1; insulin injections were administered subcutaneously at a dose of 0.2IU/kg, and they were used as reference group 2.

Blood was collected in the tail vein at 0, 0.5, 1, 1.5, 2, 3, 5 and 7 hours after administration, and blood glucose was measured. The blood glucose level was measured based on the initial value before administration as 100%.

As shown in fig. 3, in the case of administration of the formulation of example 1, blood glucose decreased by about 73%, but in the case of administration of the formulation of comparative example 1, blood glucose hardly decreased. In addition, the area of the hypoglycemic effect of the formulation of example 1a prepared by omitting the microfluidizer was about 70% of that of example 1.

As described above, the nanoparticle formulation in which insulin is merely loaded in a lipid carrier without an insulin-counterion complex shows little glucose-lowering activity in vivo. In addition, the reduction of particle size by microfluidization during the preparation of lipid nanoparticle formulations can increase the hypoglycemic activity by about 40%.

As shown in FIG. 4, the blood glucose lowering activity of the formulation of example 1 administered was very similar to that of 0.2IU/kg of insulin injected subcutaneously.

2) Measurement of blood concentration of insulin

Blood glucose was measured as described above. Experiments were performed using the formulations of example 1, comparative example 1 and example 1 a; the oral administration nanoparticle liquid preparation containing no insulin was used as a control group in the same manner as in example 1; 0.2IU/kg insulin injection was injected subcutaneously as a reference group.

Blood was collected from the tail vein at 0, 0.5, 1, 1.5, 2, 3 and 5 hours after administration, using an insulin-analyzing cartridge (Coat-a-count)^TMDiagnostic Products Corporation) to determine insulin blood concentration.

As shown in FIG. 5, the blood concentration of insulin in the control group without insulin was less than 20. mu.IU/ml, but the maximum blood concentration of insulin in the formulation of example 1 was about 170. mu.IU/ml, so that the rate of absorption of insulin was high. The comparative example 1 formulation, which only included insulin in a lipid carrier without the insulin-counterion complex, had only about 15 μ IU/ml of insulin absorption at maximum blood concentration. The formulation of example 1a, which did not pass through a microfluidizer, had an insulin absorption rate of about 70 μ IU/ml at maximum blood concentration. In addition, comparing the area of insulin increase (AUC) over time relative to the initial insulin concentration, the AUC increased by about 140% for the example 1 formulation compared to the example 1a formulation. This is similar to the result of comparing the area of blood concentration reduction by time.

It was confirmed that the inclusion of the insulin-counterion substance complex in the lipid carrier containing the polymer and the reduction of the particle size by the high-pressure homogenizer were effective for increasing the insulin absorption rate in type I diabetic rats.

As shown in FIG. 6, the blood insulin concentration profile of the formulation of example 1 was similar to 0.2IU/kg of insulin injected subcutaneously, which was used in early stage diabetic patients with 0.2IU/kg of insulin injected subcutaneously.

Experiment 4: evaluation of animal efficacy 2

Animal experiments were conducted using orally administrable nanoparticle-containing liquid formulations containing ceftriaxone prepared in examples 6 and 7, and comparative example 5.

(1) Gastrointestinal absorption test for orally administrable nanoparticle liquid formulations

1) Blood glucose determination

Normal rats (Sprague Dawley, male, approximately 200g in weight) were anesthetized with ether and the abdominal cavity was opened. The duodenum was removed and the liquid formulation of nanoparticles containing ceftriaxone was applied thereto at a dose of 40mg/kg ceftriaxone.

The formulations of example 6 and example 7, and comparative example 5 were used as the test group. Since the formulation of comparative example 5 contains neither ceftriaxone-counterion complex nor polymer, the drug loading rate of the formulation of comparative example 5 was lower than that of the formulation of example 6, and the dissolution of the drug could not be controlled.

Blood was collected from the tail vein at 0, 0.5, 1, 1.5, 2, 3 and 4 hours after administration and analyzed according to ceftriaxone assay in the united states pharmacopeia. Blood was centrifuged at 3000rpm for 10 minutes and pretreated by adding an equal amount of acetonitrile.

Ceftriaxone dissolved in water was administered by the same route as in example 6 at a dose of 40mg/kg, which was taken as reference group 1.

Ceftriaxone was administered intravenously in saline at a dose of 20mg/kg, as reference group 2.

As shown in fig. 7 and 8, the bioavailability was 22.8% and 35.1% in the case of administration of the formulations of examples 6 and 7, respectively, as compared to intravenous injection. In contrast, in reference group 1 to which the aqueous solution of ceftriaxone was administered, ceftriaxone was hardly absorbed in vivo, whereas the group to which the formulation of comparative example 5 was administered had a lower maximum blood concentration and a bioavailability of only about 12.4% compared to the group to which the formulation of example was administered, and ceftriaxone was entrapped only in the lipid in the formulation of comparative example 5. In the case of administration of the formulations of examples 6 and 7, the blood concentration of ceftriaxone decreased more slowly than that of the formulation of comparative example 5. In particular, the blood concentration of the drug remained high after 1.5 hours, while the initial drug concentration was lower than that of reference group 2, which was administered intravenously with ceftriaxone. From this result, it is considered that the composition of the present invention can reduce the side effects caused by high starting blood concentration and has a pharmacological effect for a longer period of time than that by intravenous injection.

As described above, the composition of the present invention has a high entrapment rate of the water-soluble drug in the nanoparticles, can protect the drug from the action of lipase or protease in vivo, and has a high absorption rate in the gastrointestinal tract, thus having a high blood concentration of the drug.

Industrial applications

As described above, the orally administrable nanoparticle composition of the present invention has a high water-soluble drug loading rate in nanoparticles, protects unstable drugs from gastrointestinal enzymes, and has a high absorption rate to gastrointestinal mucosa. Thus, for water-soluble drugs whose oral administration is limited due to charge problems, the compositions of the present invention are very useful as drug delivery systems in enhancing the bioavailability of these water-soluble drugs.

Claims (19)

1. A composition for oral administration comprising nanoparticles having a particle size of 500nm or less, the composition comprising:

0.1-30% by weight of a complex of a charged water-soluble drug and a counter-ionic species, in which complex the charged water-soluble drug is ionically bound to the counter-ionic species, wherein the counter-ionic species is selected from the group consisting of C_8-18Sodium salt of fatty acid, sodium lauryl sulfate, sodium salt of bile acid, sodium alginate, and anionic compound of sodium carboxymethylcellulose, or salt selected from carnitine, benzalkonium chloride, and decabromideCationic compounds of hexaalkyltrimethylammonium and mixtures thereof;

0.5-80% by weight of a lipid, wherein the lipid is selected from the group consisting of monoglycerides, diglycerides, propylene glycol esters of fatty acids, glycerol esters of fatty acids, stearyl alcohol, cetyl alcohol, and mixtures thereof;

from 0.5 to 80% by weight of a polymer, wherein the polymer is selected from the group consisting of methacrylic acid copolymer, hydroxypropyl methylcellulose phthalate, hydroxypropyl methylcellulose acetate succinate, cellulose acetate phthalate, shellac, chitosan, hydroxypropyl methylcellulose, ethylcellulose, methylcellulose, polyvinyl alcohol, sodium alginate, carbomer, and mixtures thereof; and

an emulsifier in an amount of 1-80% by weight,

wherein the weight ratio of the lipid to the polymer is 1: 0.05-3, the complex is entrapped in a carrier formed by the lipid and the polymer, and the polymer is entrapped on the surface of the lipid nanoparticle and is inserted between the lipid molecules.

2. The composition of claim 1, wherein 70% by weight or more of the water-soluble drug is entrapped in the nanoparticles.

3. The composition of claim 1, wherein 80% or more by weight of the water-soluble drug is retained within the nanoparticle composition in the presence of the pancreatin solution.

4. The composition of claim 1, wherein the water soluble drug is selected from the group consisting of insulin, erythropoietin, calcitonin, growth hormone, interferon, and somatostatin.

5. The composition of claim 1, wherein the water-soluble drug is a drug charged in water and selected from the group consisting of heparin, cephalosporins, alendronate sodium, etidronate sodium, and pamidronate sodium.

6. The composition of claim 1 wherein the sodium salt of a fatty acid is selected from the group consisting of sodium oleate, sodium caproate and sodium laurate.

7. The composition of claim 1, wherein the molar ratio of water-soluble drug to counter-ionic species is 1: 0.1-20.

8. The composition of claim 7, wherein the molar ratio of water-soluble drug to counter-ionic species is 1: 3-10.

9. The composition of claim 1, wherein the weight ratio of lipid to polymer is 1: 0.2-1.

10. The composition of claim 1, wherein the emulsifier is selected from the group consisting of polyoxyethylene polyoxypropylene copolymers, polyethylene glycol alkyl ethers, polyoxyethylene castor oils, polyoxyethylene sorbitan fatty acid esters, transesterification products of natural vegetable oil triglycerides and polyalkylene polyols, glycerol fatty acid esters, vitamin E polyethylene succinate, lecithin, sodium lauryl sulfate, bile acids, and mixtures thereof.

11. The composition of claim 1, further comprising 50% by weight or less of a solubilizing agent.

12. The composition of claim 11, wherein the solubilizing agent is selected from the group consisting of C_1-8Alcohols, dimethyl sulfoxide, methylene chloride, toluene, propylene glycol, polyethylene glycol and 12-hydroxystearate.

13. The composition of claim 1, further comprising 0.1 to 30% by weight of a cryoprotectant.

14. The composition of claim 13, wherein the cryoprotectant is selected from the group consisting of glucose, mannitol, sorbitol, trehalose, amino acids, albumin and mixtures thereof.

15. The composition of claim 1, wherein the nanoparticles have a particle size of 20-300 nm.

16. A process for the preparation of an orally administrable composition according to claim 1, which comprises the steps of:

(a) ionically binding a charged water-soluble drug with a counter-ionic species to form a complex of the water-soluble drug and the counter-ionic species;

(b1) adding a lipid, a polymer and a solubilizer to the complex obtained in step (a) and dissolving them, and then adding the resulting solution to an aqueous solution containing an emulsifier to obtain a homogeneous liquid phase; or

(b2) Adding a lipid and a solubilizer to the resulting complex and dissolving them, and then adding the resulting solution to an aqueous solution containing a polymer and an emulsifier to obtain a uniform liquid phase; and

(c) removing the solubilizer from the mixture obtained in step (b1) or (b 2).

17. The method of claim 16, further comprising the step of (d) minimizing particle size using a microfluidizer.

18. The method of claim 17, wherein the charged water-soluble drug is obtained in step (a) by treating the water-soluble drug with a pH adjusting agent to charge it.

19. The method of claim 18, wherein the pH adjusting agent is selected from the group consisting of hydrochloric acid, phosphoric acid, carbonic acid, citric acid, sodium hydroxide, sodium monohydrogen phosphate, potassium monohydrogen phosphate, sodium dihydrogen phosphate, potassium dihydrogen phosphate, sodium citrate, and mixtures thereof.