WO2002064115A1

WO2002064115A1 - Formulation for delivery of insulin and preparation method thereof

Info

Publication number: WO2002064115A1
Application number: PCT/KR2002/000205
Authority: WO
Inventors: Seo-Young Jeong; Ick-Chan Kwon; Hesson Chung
Original assignee: Korea Institute Of Science And Technology
Priority date: 2001-02-13
Filing date: 2002-02-08
Publication date: 2002-08-22
Also published as: KR20020066776A; KR100508695B1

Abstract

The present invention relates to a liquid insulin formulation and the preparation method thereof. More particularly, the present invention relates to a liquid and powder formulations comprising a monoglyceride, an emulsifier, organic solvent, insulin and acidic aqueous solution. The present invention relates to the liquid and powder insulin formulations with a higher encapsulation efficiency of insulin, higher bioavailability upon oral administration with higher patient compliance and the preparation method thereof.

Description

FORMULATION FOR DELIVERY OF INSULIN AND PREPARATION

METHOD THEREOF

TECHNICAL FIELD

The present invention relates to an insulin formulation and the preparation method thereof. More particularly, the present invention relates to liquid and powder insulin formulations for administration comprising a monoglyceride, an emulsifier, organic solvents, insulin and an acidic aqueous solution and the preparation method thereof. The formulations of the present invention have high encapsulation efficiency of insulin and high bioavailability. Since it is possible to administer orally the formulations of the present invention to achieve the effect of lowering blood glucose levels, the formulations of the present invention would provide a higher patient compliance.

BACKGROUND OF THE INVENTION

Lipid-based drug delivery systems are widely used since the raw materials are usually biocompatible and sub-micron sized particulate systems can be easily formulated.

Generally, emulsions and liposomes are two of the most widely used lipid-based drug delivery systems. In particular, the emulsions are heterogeneous mixture of oil and water by the use of emulsifiers. The oil-in- water type emulsions, composed of oil components dispersed in water, are widely used in solubilizing water-insoluble pharmaceutical compounds. Liposome formulations consist of spherical lipid vesicles with lipid bilayers, and water-insoluble pharmaceutical compounds are enclosed within the lipid bilayer.

U.S. Pat. No. 5,531 ,925 discloses Cubosome^®, another type of formulation using lipid as a solubilization medium, which was first developed by Swedish scientists in early 1990s. Cubosome^® is prepared by dispersing the hydrated lipid cubic phase in water using an emulsifier. The interior of Cubosome^® comprises cubic phase wherein lipid and water components constitute continuous but separate three-dimensional channels, and there exists an interface between lipid headgroup and water. Therefore, Cubosome^® could be advantageous over the conventional emulsion type or liposome type formulations, which only allow solubilization of hydrophobic and hydrophilic pharmaceutical compounds, respectively, in that they can solubilize amphiphilic pharmaceutical compounds as well as hydrophobic and hydrophilic pharmaceutical compounds.

Cubosome^® can be formed by first forming a very viscous liquid cubic phase by adding water and an emulsifier to monoglyceride, and then by dispersing the mixture in water. Cubosome^® has average particle size of as large as several micrometers in diameter with the aid of emulsifiers. Since it is preferential to have submicron-sized particles for the solubilization of pharmaceutical compounds, it is also possible to obtain submicron-sized particles by applying mechanical forces such as homogenization and microfluidization

Preparing submicron-sized Cubosome particles by means of a mechanical force, however, may result in physicochemical instability of the enclosed materials or the constituting ingredients of the formulations due to high energy and high temperature accompanied with the mechanical process. The formulation process may also incur hydrolysis and oxidization of constituting ingredients because the enclosed materials or the constituting ingredients of the formulations may be mixed with air during the microfluidization process. Moreover, the dispersed Cubosome prepared by the microfluidization process may experience the instability of the dispersion system after a prolonged storage and subsequently result in phase separation due to aggregation of particles. Although Cubosome type formulations have advantageous properties as described above over the conventional type of formulations, they also have disadvantages that they cannot encapsulate the thermally labile pharmaceutical compounds. Also, the physical and chemical stability of the formulation need to be improved greatly to provide a successful drug delivery system.

Peptide and proteins are physiologically active compounds can be used as therapeutics with specific functions when compared to the molecules with smaller molecular weights.

Proteins, however, cannot be orally administered since they are digested into amino acids by proteases inside the gastro-intestinal tract and loses the physiological activity. The clearance rate of pharmaceutical proteins is also high in the blood stream when injected, causing great difficulties in administering the protein drugs.

To solve the above-mentioned problems, a variety of drug delivery systems are being developed. Cyclosporin is the only peptide drug that can be administered orally. Cyclosporin is commercially available as pre- concentrates of Cremophor emulsion which can spontaneously form microemulsion upon dispersion in water (U.S. Pat. No. 5,438,072). There are no other oral peptide or protein formulations in the market, however, excepting the oral cyclosporin formulation. The main difficulty arises from the fact that peptide or protein is hard to be encapsulated inside a carrier, can be easily denatured and loses the pharmaceutical activity upon the storage even if it can be encapsulated in the carrier.

Insulin is a drug that must be administered to the insulin dependent diabetes mellitus (IDDM) patients. Currently, insulin is administered mainly to the patients by subcutaneous injection.

Successful oral insulin formulation will bring a revolutionary advancement in the treatment of IDDM. To this end, many pharmaceutical companies and research groups have endeavored to make an oral insulin formulation with high bioavailability. Due to the limitations in the absorption of insulin in the gastrointestinal tract, however, it is only the beginning stage of the development. Therapeutic peptides including insulin degrades fast by the protease in the intestine resulting in low absorption rate and physiological activity.

To overcome these problems, a variety of methods have been developed to protect insulin in the gastrointestinal tract. For instance, insulin was encapsulated inside liposomes or polymeric microspheres or administered together with protease inhibitors to prohibit the degradation of insulin. The oral bioavailability could not be enhanced much, however, since the carriers such as liposomes have a relatively low encapsulation efficiency, and the insulin can also degrade during the encapsulation process.

Also, other proteins beside insulin in the intestine cannot be digested causing adverse side effects if the protease inhibitors are administered simultaneously with insulin. There is a method for using an absorption enhancer of insulin in order to increase the absorption of insulin in the intestine, but the enhancer can cause severe side effects. Insulin was administered as a partially unfolded or molten globule (MG) state to gain access to the pore of an integral membrane transporter. The development of oral insulin formulation is still at the beginning stage, and commercial products are yet to come on the market.

The present inventors have developed a solubilizing composition for insoluble drugs comprising a monoglyceride, an emulsifier and an organic solvent (Korea patent application 2000-12465) to overcome the problems of poor oral bioavailability.

The above solubilizing composition solubilizes various compounds, especially insoluble and amphiphilic substances effectively. This liquid formulation can be easily dispersed in water, by shaking or vortexing, to form a dispersion of particles of less than 500 nm. The above solubilizing composition can encapsulate most of the drugs efficiently even though the encapsulating efficiency depends on many factors including the molecular weight or hydrophobicity of the drugs. Insulin, however, cannot be solubilized in the above solubilizing composition since it does not mix with any of the components and forms precipitations.

There is a great need for a successful oral insulin formulation that can encapsulate insulin effectively since the conventional Cubosome^® has a stability problem, and the solubilizing composition of Korea patent application 2000-12465 cannot solubilize insulin.

SUMMARY OF THE INVENTION

After intensive studies to solve the above problems, the inventors of the present invention finally succeeded in preparing a homogeneous liquid formulation comprising monoglycerides, emulsifiers, organic solvents, insulin and acidic aqueous solution. This liquid formulation according to the present invention can be easily dispersed in water, without using a harsh physical force, to form a dispersion of particles of less than 500 nm. Moreover, the encapsulation efficiency of insulin is high after the dispersion process. Therefore, it is the object of the present invention to provide an oral insulin formulation and the preparation method thereof. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the relative blood glucose levels in normal male rats after injecting the liquid insulin formulation through tail vein; — •— ; the dispersion of the liquid formulation which does not contain insulin

— O— ; Insulin solution in phosphate buffered saline (Insulin concentration 3 U/kg) and

— ▼ — ; the dispersion of the liquid insulin formulation of the present invention (Insulin concentration 3 U/kg)

Fig. 2 is a graph showing the relative blood glucose levels in diabetic male rats after oral administration of the dispersion of the liquid insulin formulation of the present invention; — •— ; the dispersion of the liquid formulation which does not contain insulin,

— O — ; the dispersion of the liquid insulin formulation of the present invention (Insulin concentration 100 U/kg),

— — ; the dispersion of the liquid insulin formulation of the present invention (Insulin concentration 50 U/kg) and

— V — ; the dispersion of the liquid insulin formulation of the present invention (Insulin concentration 30 U/kg).

Fig. 3 is a graph showing the relative blood glucose levels in normal male rats after oral administration of the dispersion of the liquid insulin formulation of the present invention;

— • — ; the dispersion of the liquid formulation which does not contain insulin, — O — ; the dispersion of the liquid insulin formulation of the present invention (Insulin concentration 100 U/kg),

Fig. 4 is a graph showing the relative blood glucose levels in normal male rats after oral administration of the dispersion of the liquid insulin formulation of the present invention; — • — ; the dispersion of the liquid formulation which does not contain insulin,

— O — ; the dispersion of the liquid insulin formulation of the present invention (Insulin concentration 30 U/kg),

— ▼ — ; the dispersion of the liquid insulin formulation of the present invention containing oleic acid (Insulin concentration 30 U/kg).

Fig. 5 is a graph showing the relative blood glucose levels in normal male rats after injecting the dispersion of the liquid insulin formulation of the present invention into duodenum; — • — ; the dispersion of the liquid formulation which does not contain insulin,

— O — ; Insulin solution in phosphate buffered saline (Insulin concentration 100 U/kg), — — ; a mixture of the dispersion of the liquid formulation which does not contain insulin and insulin solution in phosphate buffered saline (Insulin concentration 100 U/kg),

— V — ; the dispersion of the liquid insulin formulation in phosphate buffered solution (Insulin concentration 100 U/kg) and — ■ — ; the dispersion of the liquid insulin formulation in bile salt solution (Insulin concentration 100 U/kg).

Figure 6 is a graph showing the in vitro release rate of the liquid insulin formulation; — • — ; insulin dissolved in phosphate buffer solution,

— O — ; Cubosome® containing insulin prepared by the conventional technology,

— — ; the dispersion of the liquid insulin formulation of the present invention in water, and — V — ; the dispersion of the liquid insulin formulation of the present invention in sodium deoxycholate solution

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a liquid insulin formulation for oral administration comprising 0.01 ~20% by weight of insulin as an active ingredient, 9~90% by weight of at least one monoglyceride, 0.01-80 % by weight of at least one emulsifier, 0.01-10 % by weight of acidic aqueous solution and 1-90 % by weight of an organic solvent.

The present invention also relates to a method of preparing the liquid insulin formulation for oral administration comprising the steps of;

1) dissolving at least one emulsifier in at least one organic solvent; 2) adjusting pH to an acidic pH by adding a small amount of acidic aqueous solution to said liquid in step (1);

3) adding insulin powder as an active ingredient to said liquid in step (2) and solubilizing insulin completely by stirring;

4) preparing a viscous composition by adding at least one monoglyceride to said liquid in step (3); and

5) removing the volatile organic solvent from said viscous liquid in step

(4). The present invention also relates to a method of preparing the liquid insulin formulation for oral administration comprising the steps of; 1) dissolving at least one emulsifier in at least one organic solvent;

2) dissolving insulin powder as an active ingredient in acidic aqueous solution;

3) mixing said liquid in step (1) and liquid in step (2) to prepare a clear liquid; 4) preparing a viscous composition by adding at least one monoglyceride to said liquid in step (3); and

5) removing the volatile organic solvent from said viscous liquid in step (4). The present invention relates to a powder insulin formulation manufactured by lyophilization of the dispersion of the liquid insulin formulation by adding 0~30% (w/v) of a cryoprotectant.

The present invention also relates to a method of preparing the powder insulin formulation for oral administration, wherein said method comprises the steps of: 1) dispersing the liquid insulin formulation according to the present invention in water to prepare the dispersion; and

2) lyophilizing the dispersion in step (1) in the presence of cryoprotectant to prepare the powder insulin formulation.

A liquid Insulin formulation for peroral administration according to the present invention comprises 0.01 ~ 20 % by weight of insulin as an active ingredient, 9 — 90 % by weight of at least one monoglyceride compound,

0.01-80 % by weight of at least one emulsifier, 0.01 ~ 10 % by weight of acidic aqueous solution and 1 - 90 % by weight of organic solvent.

A liquid Insulin formulation according to the present invention can additionally comprise 0 - 5 % of additives.

Insulin used as an active ingredient is preferably selected from a group consisting of a bovine pancreatic insulin, a human insulin, a human recombinant insulin, a porcine pancreatic insulin, an arg-insuϊm, an insulin- biotin, an insulin-FITC, LysPro (Eli Lilly) and a polyethylene glycol-insulin, (PEG-insulin), and the content of insulin in the total amount of the liquid formulation is preferably 0.01 - 20 % by weight.

The above monoglycerides are preferably selected from a group consisting of one or more saturated or an unsaturated monoglycerides having 10 - 22 carbon atoms in the hydrocarbon chain, and the content of monoglycerides in the total amount of the liquid formulation is preferably 9 - 90 % by weight. Monoglycerides is selected preferably from a group of consisting of monoolein, monopalmitolein, monomyristolein, monoelaidin, and monoerucin, and more preferably monoolein. The emulsifier is preferably selected from a group consisting of a phospholipid, a sphingolipid, a non-ionic surfactant, an anionic surfactant, a cationic surfactant or bile acid, and the content of emulsifiers in the total amount of the liquid formulation is preferably 0.01 - 80 % by weight

The phospholipid used as the above emulsifier is preferably selected from the group consisting of a phosphatidylcholine (PC) and its derivative, a phosphatidylethanolamine (PE) and its derivative, a phosphatidylserine (PS) and its derivative and a polymeric lipid wherein a hydrophilic polymer is conjugated to the lipid headgroup.

The sphingolipid used as the above emulsifier is preferably selected from the group consisting of a ceramide, a cerebroside and a sphingomyelin.

The non-ionic surfactant used as the above emulsifier is preferably selected from the group consisting of a poloxamer (Pluronic: polyoxyethylene- polyoxypropylene copolymer), a sorbitan ester (Span), a polyoxyethylene sorbitan (Tween) and a polyoxyethylene ether (Brij). The anionic surfactant used as the above emulsifier is preferably selected from the group consisting of a phosphatidylserine (PS) and its derivative, a phosphatidic acid (PA) and its derivative, and sodium dodecyl sulfate (SDS). The cationic surfactant used as the above emulsifier is selected from the group consisting of 1,2- dioleyl-3-trimethylammonium propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), N-[1-(1 ,2-dioleyloxy)propyl]- N,N,N-thmethylammonium chloride (DOTMA), 1 ,2-dioleyl-3- ethylphosphocholine (DOEPC) and 3β-[N-[(N',N'- dimethylamino)ethan]carbamoyl]cholesterol (DC-Choi).

The bile acid used as the above emulsifier is selected from the group consisting of cholic acid, its salt and derivatives; deoxycholic acid, its salt and derivatives; ursodeoxycholic acid, its salt and derivatives; chenocholic acid, its salt and derivatives; and lithocholic acid, its salt and derivatives. It is preferable to use hydrochloric acid aqueous solution, phosphoric acid aqueous solution, carbonic acid aqueous solution and their mixtures as the acidic aqueous solutions, and he content of the acidic aqueous solution in the total amount of the liquid formulation is preferably 0.01 ~ 10 % by weight. If a neutral or basic aqueous solution is used instead of acidic aqueous solution, insulin could aggregate and is not solubilized in the liquid formulation.

It is preferable that the organic solvent is selected from the group consisting of alcohol, ethyleneglycol, propylene glycol, polyethyleneglycol, dimethylsulfoxide, and the mixture of these solvents, and the content of the organic solvent in the total amount of the liquid formulation is preferably 1 ~ 90 % by weight.

Also, it is possible to include 0 ~ 5 % by weight of other additives such as fatty acids, fatty acid esters and fatty acid alcohols in the above liquid insulin formulation. The method of preparing the liquid insulin formulation for oral administration comprises the steps of:

1) dissolving at least one emulsifier in at least one organic solvent;

2) adjusting pH to an acidic pH by adding a small amount of acidic aqueous solution to said liquid in step (1); 3) adding insulin powder as an active ingredient to said liquid in step

(2) and solubilizing insulin completely by stirring;

5) removing the volatile organic solvent from said viscous liquid in step (4).

Also it is possible to exchange some of the steps in the above method of preparing liquid insulin formulation according to the present invention. For example, insulin powder can be added in step (2) instead of step (3) as follows; 1) dissolving at least one emulsifier in at least one organic solvent;

5) removing the volatile organic solvent from said viscous liquid in step (4). As shown above, it is possible to obtain the same formulation even if the order and the process is changed slightly in each step of the preparation method of the liquid insulin formulation for oral administration according to the present invention.

Also, the present invention provides a powder insulin formulation manufactured by lyophilization of the dispersion of the above liquid insulin formulation by adding 0-30% (w/v) of a cryoprotectant.

The preparation method of the above powder formulation comprises the steps of:

1) dispersing the liquid insulin formulation according to the present invention in water to prepare the dispersion; and

2) lyophilizing said dispersion in step (1) in the presence of cryoprotectant to prepare the powder insulin formulation.

A cryoprotectant may be used to prevent the morphological changes of the particles in the dispersion of the above formulation during lyophilization, and it is preferable to add it less than 30% (w/v) to the liquid formulation.

Preferred Examples of a cryoprotectant include sugars such as mannitol or trehalose, amino acids such as arginine, and proteins such as albumin.

The liquid and powder insulin formulation of the present invention can be easily dispersed in water mediated by such a minimal mechanical aid as a simply shaking with hands. The size of particles is approximately 200 nm in general and can become as large as 500 nm depending on the property of a given pharmaceutical compounds or an emulsifier. Moreover, the constituting ingredients of the particles and pharmaceutical compound in the particles are not degraded since a strong mechanical force is not required in generating the particles.

The formulations according to the present invention can be stored at room temperature in a sealed state for a long period of time without undergoing phase separation or the change in properties of the formulations. When a long-term storage is required, the powder formulation is desirable because it does not contact with an organic solvent or moisture.

Moreover, the formulations of the present invention can be used as an insulin delivery system since the loading efficiency of insulin is as high a 50 ~ 100% with the simple preparation protocol. When applying these formulations in drug delivery system, it is preferred to use various administration routes including oral administration, buccal administration, mucosal administration, nasal administration, intra peritoneal administration, subcutaneous injection, intra muscular injection, transdermal administration and intravenous injection, and more preferably an oral administration. This invention is explained in more detail based on the following

Examples but they should not be construed as limiting the scope of this invention.

Example 1. Preparation of Liquid ϊnsuV Formulation 1 1-1. Preparation of the Liquid Insulin Formulation

In the mixed solvents of 280 mg anhydrous ethanol and 280 mg propylene glycol, 20 mg Pluronic F-127 was dissolved. The mixture was heated up to 40 °C to accelerate the dissolution when necessary. Eight microliters of 1 N hydrochloric acid solution was added to the mixture to adjust the pH to 3 - 4, and 8 mg (1 mg s 28.3 U) of insulin powder was added and stirred. After insulin solubilize completely, 100 mg of monoolein was mixed and dissolved totally. Ethanol in the formulation was evaporated completely by purging with oxygen-free nitrogen gas or decompressing to prepare the liquid insulin formulation.

1-2. Property Analysis of thus prepared Liquid Formulation

© The size and the polydispersity of the emulsion particles were measured after diluting 10 μl of thus obtained liquid formulation by adding 3 ml of phosphate buffered saline (PBS) at pH 7.4. The size the polydispersity of the emulsion particles were measured by Photon Correlation spectroscopy (QELS method) using Malvern Zetasizer (Malvern Instruments Limited, England), and the polydispersity is a variance of the particle size in a logarithmic scale of a log-normal distribution function of the particle size. The average particle size and polydispersity were obtained by measuring a given formulation three times (Orr, Encyclopedia of emulsion technology, 1, 369-404, 1985). This method was used in measuring the particle size and the polydispersity throughout the following Examples.

The above liquid formulations were dispersed well in water, and the average particle size and polydispersity were about 378.3 nm and 0.377, respectively.

(2) The size and polydispersity of the emulsion particles were measured by the above method after diluting the emulsion by mixing 3 ml of 0.01 M sodium deoxycholate with 10 μL of thus obtained liquid formulation.

The above liquid formulations were dispersed well in water, and the average particle size and polydispersity were about 365.1 nm and 0.431 , respectively.

(3) Cubosome® encapsulating insulin according to in US patent 5,531 ,925 was prepared for comparison. A very viscous cubic phase was prepared by mixing 1) a mixture of 100 mg monoolein and 20 mg pluronic F- 127 in one syringe and 2) an aqueous solution of 8 mg of 8 mg insulin in 120 mg of PBE (pH 7,4) in another syringe by connecting the two syringes with a three-way stopcock and by pushing the syringes back-and-forth. The formed cubic phase was viewed under a polarized light microscope. In the cubic phase containing insulin, 1 ml of PBS (pH 7.4) was added and mixed roughly. The mixture was microfluidized at 80 °C and cooled slowly to form a dispersion containing Cubosome®. The average particle size and polydispersity measured as above were about 148.5 nm and 0.178, respectively. If the mixture was not microfluidized, the cubic phase containing insulin did not disperse in PBS. When vortexed, particles bigger than a few millimeters in diameter were observed. Example 2. Preparation of Liquid Insulin Formulation 2 2-1. Preparation of the Liquid Insulin Formulation

A liquid insulin formulation was obtained using the same procedure as described in Example 1 except that 2 mg of insulin was added instead of 8 mg of insulin.

2-2. Property Analysis of thus prepared Liquid Formulation

The average particle size and polydispersity of the dispersion of the liquid insulin formulation determined as described in Example 1-2 were 259.3 nm and 0.174, respectively, in PBS at pH 7.4 and 250.3 nm and 0.351 , respectively, in 0.01 M sodium deoxycholate aqueous solution.

Example 3. Preparation of Liquid Insulin Formulation 3 according to the Change in the amount of a Solvent 3-1. Preparation of the Liquid Insulin Formulation

A liquid insulin formulation was obtained using the same procedure as described in Example 1 except that 280 mg of anhydrous ethanol was added instead of a solvent mixture of 280 mg of anhydrous ethanol and 280 mg of propyleneglycol.

3-2. Property Analysis of thus prepared Liquid Formulation

The above liquid formulations were dispersed well in water, and the average particle size and polydispersity of the dispersion of the liquid insulin formulation were determined as described in Example 1-2 were 188.9 nm and 0.393, respectively, in PBS at pH 7.4.

Example 4. Preparation of Liquid Insulin Formulation 4 4-1. Preparation of the Liquid Insulin Formulation A liquid insulin formulation was obtained using the same procedure as described in Example 1 except that solvent mixture of 180 mg of anhydrous ethanol and 180 mg of propyleneglycol was added instead of a solvent mixture of 280 mg of anhydrous ethanol and 280 mg of propyleneglycol.

4-2. Property Analysis of thus prepared Liquid Formulation

The average particle size and polydispersity of the dispersion of the liquid insulin formulation were determined as described in Example 1-2 were 307.6 nm and 0.093, respectively, in PBS at pH 7.4.

Example 5. Preparation of Liquid Insulin Formulation 5 5-1. Preparation of the Liquid Insulin Formulation

A liquid insulin formulation was obtained using the same procedure as described in Example 1 except that solvent mixture of 120 mg of anhydrous ethanol and 120 mg of propyleneglycol was added instead of a solvent mixture of 280 mg of anhydrous ethanol and 280 mg of propyleneglycol.

5-2. Property Analysis of thus prepared Liquid Formulation

The average particle size and polydispersity of the dispersion of the liquid insulin formulation were determined as described in Example 1-2 were 252.9 nm and 0.135, respectively, in PBS at pH 7.4.

Example 6. Preparation of Liquid Insulin Formulation 6 6-1. Preparation of the Liquid Insulin Formulation A liquid insulin formulation was obtained using the same procedure as described in Example 1 except that 280 mg of PEG₄₀₀ was added instead of 280 mg of propyleneglycol.

6-2. Property Analysis of thus prepared Liquid Formulation The average particle size and polydispersity of the dispersion of the liquid insulin formulation were determined as described in Example 1-2 were 239.8 nm and 0.223, respectively, in PBS at pH 7.4.

Example 7. Preparation of Liquid Insulin Formulation 7 7-1. Preparation of the Liquid Insulin Formulation

A liquid insulin formulation was obtained using the same procedure as described in Example 4 except that 180 mg of PEG₄₀₀ was added instead of 180 mg of propyleneglycol.

7-2. Property Analysis of thus prepared Liquid Formulation

The average particle size and polydispersity of the dispersion of the liquid insulin formulation determined as described in Example 1-2 were 222.8 nm and 0.278, respectively, in PBS at pH 7.4. Example 8. Preparation of Liquid Insulin Formulation 8 8-1. Preparation of the Liquid Insulin Formulation

A liquid insulin formulation was obtained using the same procedure as described in Example 5 except that 120 mg of PEG₄₀₀ was added instead of 120 mg of propyleneglycol.

8-2. Property Analysis of thus prepared Liquid Formulation

The average particle size and polydispersity of the dispersion of the liquid insulin formulation determined as described in Example 1-2 were 229.8 nm and 0.278, respectively, in PBS at pH 7.4.

Example 9. Preparation of Liquid Insulin Formulation 9 9-1. Preparation of the Liquid Insulin Formulation

In the mixed solvents of 280 mg anhydrous ethanol and 280 mg propylene glycol, 20 mg Pluronic F-127 was dissolved. The mixture was heated up to 40 °C to accelerate the dissolution when necessary. Eight milligrams (1 mg s 28.3 U) of insulin powder was dissolved in 10 μl of PBS whose pH was adjusted to 3 - 4 by adding 1N hydrochloric acid solution. After the above solvent solution and insulin aqueous solution were mixed completely, 100 mg of monoolein was mixed and dissolved totally. Ethanol in the formulation was evaporated completely by purging with oxygen-free nitrogen gas or decompressing to prepare the liquid insulin formulation.

9-2. Property Analysis of thus prepared Liquid Formulation The average particle size and polydispersity of the dispersion of the liquid insulin formulation determined as described in Example 1-2 were 268.6 nm and 0.216, respectively, in PBS at pH 7.4.

Example 10. Preparation of Liquid Insulin Formulation containing Absorption Enhancer 10-1. Preparation of the Liquid Insulin Formulation

A liquid insulin formulation was obtained using the same procedure as described in Example 1-1 except that 2 mg of oleic acid was additionally included in the liquid formulation as an absorption enhancer.

10-2. Property Analysis of thus prepared Liquid Formulation

The average particle size and polydispersity of the dispersion of the liquid insulin formulation were determined as described in Example 1-2 were 276.1 nm and 0.334, respectively, in PBS at pH 7.4.

Example 11. Preparation of Powder Insulin Formulation

In 0.1 ml of the liquid insulin formulation obtained in Example 1 , 1 ml of 5 % trehalose aqueous solution was added and solubilized completely by shaking. The mixture was lyophilized to prepare a powder insulin formulation. The average particle size and polydispersity of the dispersion of the powder insulin formulation were determined after dispersing approximately 3 mg of the above powder insulin formulation in 3 ml of water and 0.01 M sodium deoxycholate, respectively, as described in Example 1-2.

The above powder formulation was dispersed well in water, and the average particle size and polydispersity of the dispersion of the powder insulin formulation were 358.3 nm and 0.326, respectively, in water and 218.2 nm and 0.182, respectively, in 0.01 M sodium deoxycholate.

In Examples 1 - 11 , the liquid or powder insulin formulations according to the present invention formed dispersions containing smaller size than 380 nm in average confirming that that the liquid or powder insulin formulations according to the present invention forms sub-micron sized lipid particles with ease.

Experimental Example 1. Determination of Insulin Loading Efficiency

CD To determine the loading efficiency of insulin, 0.2 ml of the liquid insulin formulation obtained in Example 1 was dispersed in 0.8 ml of PBS (pH 7.4). The dispersion was diluted 20 times by adding PBS (pH 7.4), and 2 ml of the diluted dispersion was pippetted to Centricon (Millipore, MWCO 100,000) and centrifuged at 1000 g for 30 min. The filtered clear solution was gathered to determine the amount of unloaded insulin by BCA protein assay method. The amount of loaded insulin was calculated subsequently. The amount of loaded insulin in Cubosome® prepared in the Example 1-3 was also determined by using the same method for control. The loading efficiency of insulin inside the particles of the dispersion prepared from the liquid insulin dispersion of the present invention was 87 % whereas the loading efficiency of insulin inside the Cubosome® particles was 10 % indicating most of insulin molecules locates in the bulk aqueous phase. To determine the loading efficiency of insulin, 0.2 ml of the liquid insulin formulation obtained in Example 2 was dispersed in 0.8 ml of PBS (pH 7.4). The dispersion was filtered through a syringe filter with a pore size of 0.45 μm and subsequently through a syringe filter with a pore size of 0.2 μm. The amount of free insulin in the above filtrate was determined by Gel Permeation Chromatography (column : Ultrahydrogel 1000, Waters). The liquid formulation without insulin was also filtered as above and passed thorough the column to identify the peak of the dispersion itself as a control.

The free insulin was not observed in the filtrate obtained from the liquid insulin formulation of the above Example 2, and such result indicates that the loading efficiency of insulin inside the particles prepared from the liquid insulin formulation of the present invention is 100 %.

© To determine the loading efficiency of insulin, 0.2 ml of the liquid insulin formulation containing oleic acid obtained in Example 10 was dispersed in 0.8 ml of PBS (pH 7.4). The dispersion was diluted 20 times by adding PBS (pH 7.4), and 2 ml of the diluted dispersion was pippetted to Centricon (Millipore, MWCO 100,000) and centrifuged at 1000 g for 30 min. The filtered clear solution was gathered to determine the amount of unloaded insulin by BCA protein assay method. The amount of loaded insulin was calculated subsequently.

The loading efficiency of insulin inside the particles of the dispersion prepared from the liquid insulin dispersion containing oleic acid of the present invention was 50 %. Experimental Example 2. In vivo Oral administration

The dispersion of the liquid insulin formulation prepared in Example 1 was administered orally.

2-1. Determination of Insulin activity inside the Liquid Insulin

Formulation

A dispersion containing 28.3 U/ml insulin was prepared by dispersing 50 μl of the liquid insulin formulation in 1.0 ml of PBS (pH 7.4). The average particle size and polydispersity of the above dispersion were 345.8 nm and 0.341 , respectively, in PBS at pH 7.4. The baseline blood glucose level was determined in normal Wistar rats (male, 6~7 weeks old) fasted for 4 hours before administering insulin formulations and was used as an initial value. The dispersion of the liquid insulin formulation corresponding to 3 U/kg of insulin was administered via tail vein injection. The dispersion of the liquid formulation in PBS (pH 7.4) without insulin was also administered via tail vein injection for control.

As shown in Figure 1 , insulin retained the physiological activity inside the liquid insulin formulation when compared to the insulin solution for injection. The dispersion of the liquid insulin formulation of the present invention, therefore, was used for in vivo experiments.

2-2. Induction of Diabetes

Diabetes was induced by three consecutive intraperitoneal injections of 100 mg/kg of normal saline solution containing streptozotocin and alloxan monohydrate in 2-day intervals into 6-week old male Wister rats fasted for 8 - 10 hours. The rats were considered diabetic when the blood glucose level determined 1 week after the final injection was above 400 mg/dl at the non- fasting state and used to perform the in vivo experiments.

2-3. Oral Administration of Liquid Formulations A dispersion containing 28.3 U/ml insulin was prepared by dispersing 50 μl of the liquid insulin formulation in 1.0 ml of PBS (pH 7.4). The baseline blood glucose level was determined in normal Wistar rats (male, 6 — 7 weeks old) and diabetic rats fasted for 4 hours by withdrawing blood from the tail vein and was used as an initial value. Into the above animals, 0.8 ml of 3 % NaHCCyPBS was orally administered by using a gastric sonde to neutralized gastric acid. In about 30 minutes, the above dispersions having insulin concentrations of 30, 50 and 100 U/kg, respectively, were orally administered by using a gastric sonde. The dispersion of liquid formulation without insulin and the insulin solution in PBS were orally administered by using the same method for a control. One, 2, 3, 4 and 6h after the oral administration of the insulin formulations, blood samples were drawn from the tail vein using a 26 gauge needle under the light anesthesia to determine the glucose level. The relative blood glucose level was calculated by setting the initial blood glucose level to 100 % before the insulin treatment.

As shown in Figure 2, when the dispersion of the liquid insulin formulation at a concentration of 100 U/kg of insulin was orally administered into the diabetic rats, the blood glucose level was lowered more than 80 % from the initial value in 6 hours. The blood glucose level was lowered more than 40 % in the group of rats orally administered with the dispersion of the liquid insulin formulation at a concentration of 30 or 50 U/kg of insulin. In contrast, the blood glucose level did not decrease in the group of rats orally administered with the dispersion of the liquid formulation without insulin. As shown in Figure 3, when the dispersion of the liquid insulin formulation at a concentration of 30 - 100 U/kg of insulin was orally administered into the normal rats, the blood glucose level was lowered 20 - 50 % from the initial value in 6 hours. In contrast, the blood glucose level did not decrease in the group of normal rats orally administered with the dispersion of the liquid formulation without insulin. In the group of normal or diabetic rats orally administered with insulin solution in PBS, the blood glucose level did not decrease from the initial value.

Experimental Example 3. In Vivo Oral administration 2

The dispersion of the liquid insulin formulation prepared in Example 10 was administered orally to normal rats by using the same experimental protocol in Experimental Example 2. For comparison, the dispersions of the liquid insulin formulation and the liquid formulation without insulin in PBS (pH 7.4) prepared in Example 1 were used.

As shown in Figure 4, when the dispersion of the liquid insulin formulation was orally administered into the normal rats, the blood glucose level was lowered 40 % from the initial value in 6 hours. In contrast, the blood glucose level decreased more than 75 % from the initial value in 2 hours when the dispersion of the liquid insulin formulation containing oleic acid was orally administered, indicating that the additional ingredient, oleic acid acted as an absorption enhancer of the oral insulin formulation of the present invention.

Experimental Example 4. In vivo Duodenal Administration

The dispersion of the liquid insulin formulation prepared in Example 2 was used for an animal experiment.

4-1. Determination of Insulin activity inside the Liquid Insulin Formulation

The insulin activity inside the liquid insulin formulation was determined by using the same method as in Experimental Example 2-1. After confirming that insulin activity was maintained, the dispersion of the liquid insulin formulation was used for in vivo experiments.

4-2. Preparation of the Dispersion

The dispersion with the insulin concentration of 1 mg (≡ 28.3U)/ ml was prepared by dispersion 200 μl of the liquid insulin formulation in Example 2 in 800 μl of PBS (pH 7.4) or 0.01 M sodium deoxycholate.

4-3. In vivo Duodenal Administration of the Dispersion

A group of male Whistar rats weighing 180—220 g were fasted for 16 hours. An anesthetic was prepared by mixing urethane and xylazine-HCI at concentrations of 1.0 g/ml and 12mg/ml, respectively, in PBS. The anesthetic was administered via intraperitoneal injection to the rat at 1.0 ml/kg. After ensuring that the rat is under anesthesia, the baseline blood glucose level was determined by withdrawing blood from the tail vein and was used as an initial value. The rat was transferred to an operating table, and the duodenum was exposed by surgically cutting the abdomen. Into the above animals, the dispersion of the liquid insulin formulation at an insulin dose of 100 U/kg was duodenally administered by using a 26-gauge needle. After suturing the incision site, blood samples were drawn from the tail vein using a 26-gauge needle, in 1 , 2, 3, 4 and 6h after the oral administration of the insulin formulations, and the blood glucose level of each sample was determined. The relative blood glucose level was calculated by setting the initial blood glucose level to 100 % before the insulin treatment.

As control groups, the insulin solution in PBS, the dispersion of the liquid formulation without insulin, the mixture of the dispersion of the liquid formulation without insulin and insulin solution were duodenally administered as above.

As shown in Figure 5, when the dispersion of the liquid insulin formulation in 0.01 M sodium deoxycholate was administered into the normal rats, the blood glucose level was lowered 20 % from the initial value. Such result indicates that sodium deoxycholate, which is a kind of bile salt, is a better dispersing medium than phosphate buffered saline in case of duodenal administration. Experimental Example 5. In Vitro Release Experiment of Insulin

After preparing the dispersion of 400 μl of the liquid insulin formulation in 1.6 ml of PBS (pH 7.4) as in Example 1 , the mixture was then added into a dialysis bag (MWCO = 300,000, Spectra/Por^R, Spectrum Medical Industries, Inc., USA), both ends were tied with a closure, and placed into a 50 ml conical tube containing 10 ml of phosphate buffered saline (PBS, pH = 7.4). The conical tube was put into a 37 °C shaking water bath for the release experiment. The PBS solution located outside the dialysis tube was collected at regular intervals to analyze concentration of insulin released from the dialysis bag by the BCA protein assay method.

As shown in Figure 6, more than 70 % of insulin remained inside the dialysis bag when the liquid insulin formulation of the present invention was dispersed in PBS or sodium deoxycholate whereas the insulin in Cubosome^® released completely. As shown in Experimental Examples 1 - 5, the dispersion of the liquid insulin formulation according to the present invention has a high encapsulation efficiency of insulin and can lower the blood glucose level effectively when injected or administered orally.

EFFECT OF THE INVENTION

As described above, the liquid insulin formulation according to the present invention can solubilize insulin stably and also generate homogeneous submicron sized particles when dispersed in water. Since the oral liquid insulin formulation according to the present invention can lower the blood glucose level effectively when injected or administered orally, it is suitable for use in insulin delivery system.

Claims

1. A liquid Insulin formulation comprising 0.01 — 20 % by weight of insulin as an active ingredient, 9 — 90 % by weight of at least one monoglyceride compound, 0.01-80 % by weight of at least one emulsifier, 0.01 - 10 % by weight of acidic aqueous solution and 1 - 90 % by weight of organic solvent.

2. The liquid insulin formulation according to claim 1 , wherein said insulin is selected from the group consisting of a bovine pancreatic insulin, a human insulin, a human recombinant insulin, a porcine pancreatic insulin, an arg-insulin, an insulin-biotin, an insulin-FITC, a LysPro (Eli Lilly) and a polyethylene glycol-insulin, (PEG-insulin).

3. The liquid insulin formulation according to claim 1 , wherein said monoglyceride is selected from the group consisting of a saturated or an unsaturated monoglyceride having 10 - 22 carbon atoms in the hydrocarbon chain.

4. The liquid insulin formulation according to claim 3, wherein said monoglyceride compound is selected from the group consisting of monoolein, monopalmitolein, monomyristolein, monoelaidin, and monoerucin.

5. The liquid insulin formulation according to claim 1 , wherein said emulsifier is selected from the group consisting of a phospholipid, a sphingolipid, a non-ionic surfactant, an anionic surfactant, a cationic surfactant or bile acid.

6. The liquid insulin formulation according to claim 5, wherein said phospholipid is selected from the group consisting of a phosphatidylcholine (PC) and its derivative, a phosphatidylethanolamine (PE) and its derivative, a phosphatidylserine (PS) and its derivative and a polymeric lipid wherein a hydrophilic polymer is conjugated to the lipid headgroup.

7. The liquid insulin formulation according to claim 5, wherein said sphingolipid is selected from the group consisting of a ceramide, a cerebroside and a sphingomyelin.

8. The liquid insulin formulation according to claim 5, wherein said non- ionic surfactant is selected from the group consisting of a poloxamer (Pluronic: polyoxyethylene-polyoxypropylene copolymer), a sorbitan ester (sorbitan esters; Span), a polyoxyethylene sorbitan (Tween) and a polyoxyethylene ether (Brij).

9. The liquid insulin formulation according to claim 5, wherein said anionic surfactant is selected from the group consisting of a phosphatidylserine (PS) and its derivative, a phosphatidic acid (PA) and its derivative, and sodium dodecyl sulfate (SDS).

10. The liquid insulin formulation according to claim 5, wherein said cationic surfactant is selected from the group consisting of 1,2- dioleyl-3- trimethylammonium propane (DOTAP), dimethyldioctadecylammonium bromide (DDAB), N-[1-(1 ,2-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 1 ,2-dioleyl-3-ethylphosphocholine (DOEPC), and 3β- [N-[(N',N'-dimethylamino)ethan]carbamoyl]cholesterol (DC-Choi).

11. The liquid insulin formulation according to claim 5, wherein said bile acid is selected from the group consisting of cholic acid, its salt and derivatives; deoxycholic acid, its salt and derivatives; ursodeoxycholic acid, its salt and derivatives; chenocholic acid, its salt and derivatives; and lithocholic acid, its salt and derivatives.

12. The liquid insulin formulation according to claim 1 , wherein said acidic aqueous solution is selected from the group consisting of the hydrochloric acid aqueous solution, phosphoric acid aqueous solution, carbonic acid aqueous solution and their mixtures.

13. The liquid insulin formulation according to claim 1 , wherein said organic solvent is selected from the group consisting of alcohol, ethyleneglycol, propylene glycol, polyethyleneglycol, dimethylsulfoxide, and the mixture of these solvents.

14. The liquid insulin formulation according to claim 1 , wherein the formulation additionally comprises 0 - 5 % of additives selected from the group consisting of fatty acids, fatty acid esters and fatty acid alcohols.

15. The liquid insulin formulation according to claim 1 , wherein the administration route is selected from oral administration, buccal administration, mucosal administration, nasal administration, intra peritoneal administration, subcutaneous injection, intra muscular injection, transdermal administration and intravenous injection.

16. A method of preparing the liquid insulin formulation, wherein said method comprises the steps of: 1) dissolving at least one emulsifier in at least one organic solvent;

2) adjusting pH to an acidic pH by adding a small amount of acidic aqueous solution to said liquid in step (1);

3) adding insulin powder as an active ingredient to said liquid in step (2) and solubilizing insulin completely by stirring; 4) preparing a viscous composition by adding at least one monoglyceride to said liquid in step (3); and 5) removing the volatile organic solvent from said viscous liquid in step

(4).

17. A method of preparing the liquid insulin formulation, wherein said method comprises the steps of:

1) dissolving at least one emulsifier in at least one organic solvent;

3) mixing said liquid in step (1) and liquid in step (2) to prepare a clear liquid;

4) preparing a viscous composition by adding at least one monoglyceride to said liquid in step (3); and 5) removing the volatile organic solvent from said viscous liquid in step

(4).

18. A powder insulin formulation manufactured by lyophilization of the dispersion of the said liquid insulin formulation according to claim 1 by adding 0-30% (w/v) of a cryoprotectant.

19. The powder insulin formulation according to claim 18, wherein said cryoprotectant is selected from the group consisting of a sugar, an amino acid and a protein.

20. The powder insulin formulation according to claim 19, wherein said sugar is selected from mannitol or trehalose, said amino acid is arginine, and said protein is albumin.

21. The powder insulin formulation according to claim 18, wherein the administration route is selected from oral administration, buccal administration, mucosal administration, nasal administration, intra peritoneal administration, subcutaneous injection, intra muscular injection, transdermal administration and intravenous injection.

22. A method of preparing the powder insulin formulation, wherein said method comprises the steps of:

1) dispersing the liquid insulin formulation according to claim 1 in water to prepare the dispersion; and