HK1138197A - A biodegradable microsphere composition suitable for the controlled release of glucose controlling peptide and formulation thereof - Google Patents

Description

Biodegradable microsphere composition suitable for controlled release of glucose control peptide and preparation thereof

Technical Field

The present invention relates to a biodegradable polymeric microsphere comprising a biodegradable polymeric carrier having encapsulated therein a glucose-regulating peptide, said microsphere being capable of releasing the glucose-regulating peptide in a controlled manner; the invention also relates to a preparation method of the microsphere.

Background

After oral administration, most protein drugs and peptide drugs lose their active structures in the acidic environment of the stomach or under the degradation action of enzymes. In addition, their absorption rate through the gastric or intestinal mucosa is very low. For these reasons, protein drugs or peptide drugs are usually administered by a non-oral route, i.e., by injection. Since most of the non-orally administered protein drugs or peptide drugs have a short half-life in vivo and low bioavailability, the non-oral administration of the protein drugs or peptide drugs must be repeated. In addition, in many cases, the administration process may last for a long period of time, e.g., several months. In order to avoid such problems, active studies have been made on sustained-Release preparations, resulting in the use of biodegradable polymer carriers in which protein-based drugs or peptide-based drugs are encapsulated, which polymer carriers can Release the protein-based drugs or peptide-based drugs therefrom during their biodegradation (Heller, J. et al, Controlled Release of water-soluble drugs from bioorganic drugs, Biomaterials, 4, 262-266, 1983; Langer, R., New methods of drug delivery, Science, 249, 1527-1533, 1990; Langer, R., chem. Eng. Commun, 6, 1-48, 1980; Langer, R.S. and Peppas, N.A., Biomaterials, 2, 201, 1981; Helm. Eng. Comun, 6, 1-48, 1980; Langer, R.S. and Peppas, N.A. Biomaterials, 2, 201, 1981; Heller, J. control, C. J. 1980; Tail, J. 155, J. Release, J. 4, J. 1984, R.S. 180).

Currently, aliphatic polyesters used as polymeric carriers for protein or peptide drugs are approved by the FDA for their biocompatibility. They are widely used as carriers in drug delivery or surgical suturing. Specific examples of the aliphatic polyester include: poly-L-lactic acid, polyglycolic acid, D-lactic-glycolic acid copolymer, L-lactic-glycolic acid copolymer, D, L-lactic-glycolic acid copolymer (hereinafter referred to as "PLGA"), polycaprolactone, polypentanolide, polyhydroxybutyrate, and polyhydroxyvalerate (Peppas, l.b., int.j.pharm., 116, 1-9, 1995).

With the recent development of high molecular weight peptides or proteins as novel therapeutics, various attempts have been made to release them from polymeric carriers in a controlled manner. However, dosage forms comprising polyester microspheres encapsulating a protein drug suffer from the following drawbacks: initial burst effect (initial burst release effect), uncontrolled release rate over time due to various factors, or incomplete release of encapsulated drug.

For example, model protein drugs such as bovine serum albumin, lysozyme, etc. are released in large amounts at the beginning, but show a final release rate of about 50% (Crotts, G. and Park, T.G., J.Control.Release, 44, 123-. As for the microspheres using the aliphatic polyester carrier in which recombinant human growth hormone is encapsulated, the amount of drug released at the initial stage is 30% to 50%, but the amount of drug remaining in the microspheres is 40% to 60% (Yan, C. et al, J.Control.Release, 32, 231. minus 241, 1994; Kim, H.K. and Park, T.G., Biotechnol.Bioeng., 65, 659. minus 667, 1999).

The initial burst effect of the drug is attributed to the fact that the protein drug aggregated or absorbed on the surface or pores of the microspheres is released by rapid diffusion at the initial stage.

During the preparation of microspheres, protein drugs may denature at the interface between water and organic solvents and thus form irreversible aggregates leading to unstable release. To avoid interfacial induced denaturation of protein drugs, the use of surfactants (e.g., nonionic surfactants, Tween, Pluronic F68, Brij 35, etc.) and stabilizers (e.g., mannitol, gelatin, trehalose, carboxymethylcellulose, etc.) or anhydrous organic solvents (Gombotz, W.R.; Healy, M., Brown, L., U.S. Pat. No.5019400) in the preparation of microspheres has been reported.

To solve the problems of uncontrollable drug release rate over time and incomplete release of the encapsulated drug, many recent studies have been associated with alternative methods for preparing sustained-release drug microspheres, which include encapsulating a drug in the form of a mixture of two or more polymers having different degradation rates in a predetermined ratio (Ravivarapu, H.B.; Burton, K.; Deluca, P.P., Eur J Pharm Biopharm, 50(2), 263-270, 2000; Korean patent application No. 1998-2140062) or mixing two or more polymer microspheres having different degradation rates, in which a drug is encapsulated, respectively, in a predetermined ratio (U.S. Pat. No.4897268), thereby controlling both initial and sustained release of the drug from the microspheres. Then, in the microspheres prepared by the conventional method, the products of degradation from the polymers having high degradation rates (for example, lactic acid and glycolic acid) lower the pH, thereby promoting the degradation of the polymers having low degradation rates, resulting in a great difference in the release rate of the drugs respectively encapsulated in the respective polymers from the calculated release rate. Further, it is disadvantageous from the viewpoint of manufacturing process and economy to prepare two or more kinds of microspheres for one dosage form (Korean patent application No. 2000-0036178).

As a preparation technique of the microspheres, a phase separation method (U.S. Pat. No.4673595, korean patent application No.2007 & 0031304), a spray drying method (korean patent application No.2003 & 0023130), and an organic solvent evaporation method (U.S. Pat. No.4389330) are well known. In the phase separation method, a methylene chloride solvent is used together with silicone oil, heptene and ethanol, but they are all removed, and thus it is disadvantageous from an economical point of view. As for the spray-drying method, since it is required to spray-dry the peptide drug or the protein drug together with an organic solvent at a high temperature (e.g., 60 ℃ or more), denaturation of the peptide drug or the protein drug may be caused. For these reasons, the organic solvent evaporation method is most widely used in the preparation of peptide drugs or protein drugs. In this method, one of the most important factors in the art is the encapsulation efficiency (korean patent application No. 2003-.

Therefore, there is a need for a method for preparing microspheres that meets the following requirements: the initial burst effect is not generated, and the medicine release is not complete; so that the medicine is subjected to zero-order release; simple and convenient operation and is economically favorable; and ensures that the encapsulated drug has high encapsulation efficiency and high stability.

Glucose-regulating peptides belong to a group of peptides with therapeutic potential for the treatment of insulin-dependent diabetes, gestational diabetes or non-insulin-dependent diabetes, obesity and lipid metabolism disorders (U.S. Pat. No. 6506724). Examples of glucose-regulating peptides include: exendin-3, Exendin-4 and their homologues and agonists; and glucagon, glucagon-like peptides (e.g., GLP-1, GLP-2) and their homologs and agonists (Korean patent application No. 2006-7015029).

Exendin-4 isolated from the salivary secretions of Exendin mexicana (Heloderma horridum) or Exendin hula (Heloderma suspecum) is a physiologically active peptide consisting of 39 amino acid residues. Exendin-4 stimulates secretion of insulin from islet beta cells, decreases elevated glucagon secretion and causes a decrease in appetite and is therefore useful in the treatment of diabetes and obesity (Eng.J. et al, 1990; Raufman, J.P., 1992; Goeke, R., 1993; Thorens, B., 1993).

In order to effectively treat and prevent diabetes, microspheres for sustained release of exendin-4 have been studied (korean patent application No. 2006-. However, conventional methods are complex and inefficient, as evidenced by the following examples: the large amount of organic solvents used and removed during the phase separation process, degradation of peptides due to the use of high energy during the sonication process, and the use of a variety of excipients including stabilizers (e.g., sugars) and release enhancers (e.g., inorganic acids and salts).

Disclosure of Invention

It is therefore an object of the present invention to provide microspheres that meet the following requirements: the initial burst effect is not generated, and the medicine release is not complete; no matter the stable release time is long, the medicine is subjected to zero-order release; the preparation is simple, convenient and easy, and is economically favorable; and ensures that the encapsulated drug has high encapsulation efficiency and high stability. It is also an object of the present invention to provide a method for preparing said microspheres which does not use organic solvents and high energy treatments (such as ultrasonication) nor release promoters, and which is simple and easy to perform.

To achieve the above object, the present invention provides biodegradable polymeric microspheres comprising a biodegradable polymeric carrier having glucose-regulating peptides encapsulated therein, said microspheres being capable of releasing the glucose-regulating peptides in a controlled manner.

Similarly, the invention also provides a preparation method of the biodegradable polymer microsphere.

In addition to being simple and economically advantageous in its preparation process and ensuring high encapsulation efficiency and high stability of the encapsulated drug, the microspheres of the present invention are shown to enable zero-order release of the drug (e.g., exendin-4) and thus allow stable release of the drug therefrom in vitro and in vivo over a period of three to four weeks, with neither initial burst effect nor incomplete release of the drug.

Drawings

FIG. 1 is a graph showing an in vitro release pattern of microspheres prepared by the drug dispersion method in example 1 of the present invention.

FIG. 2 is a graph showing in vitro release patterns of microspheres prepared in examples 4-8 of the present invention.

Figure 3 shows a graph of the in vitro release pattern of microspheres prepared in comparative example 1 and comparative example 2.

FIG. 4 is a graph showing the in vivo release pattern of microspheres prepared in example 1-1.

FIG. 5 is a chromatogram obtained by reverse phase high performance liquid chromatography (RP-HPLC) analysis of exendin-4 obtained from the microspheres prepared in example 1-1.

Detailed Description

The present invention will be described in detail below.

The present invention relates to biodegradable polymeric microspheres for controlled release of glucose-regulating peptides, comprising a biodegradable polymeric carrier in which the glucose-regulating peptides are encapsulated.

Examples of glucose-regulating peptides suitable for use in the present invention include: natural, recombinant or synthetic exendin-3, exendin-4 and their homologues and agonists; glucagon, glucagon-like peptides (e.g., GLP-1, GLP-2), and their homologs and agonists. Among them, synthetic exendin-3, exendin-4, and their homologs and agonists are preferable. Most preferred is synthetic exendin-4.

The content of glucose-regulating peptide in the microspheres varies with the route of administration, dosage and protein properties.

Materials suitable for use as biodegradable polymeric carriers are biodegradable polyester polymers. When used as a scaffold for microspheres (scaffold) and containing glucose-regulating peptides therein, the biodegradable polyester polymer gradually degrades to release the glucose-regulating peptides. Examples of biodegradable polyester polymers include, but are not limited to: poly L-lactic acid, polyglycolic acid, D-lactic acid-glycolic acid copolymer, L-lactic acid-glycolic acid copolymer, D, L-lactic acid-glycolic acid copolymer, polycaprolactone, polypentanolide, polyhydroxybutyrate and polyhydroxyvalerate. Any particular limitation is not imposed on the use of the biodegradable polyester polymer in the present invention as long as it is frequently used in the art. The polymer is preferably selected from the group consisting of: poly-L-lactic acid, poly-D-lactic acid-co-glycolic acid, poly-L-lactic acid-co-glycolic acid, poly-D, L-lactic acid-co-glycolic acid, PLGA, and combinations thereof. More preferably, D, L-lactic-glycolic acid copolymer (PLGA) is used by itself or in combination with poly-L-lactic acid.

In addition, the present invention relates to a method for preparing biodegradable polymer microspheres for controlled release of glucose-regulating peptides.

The preparation method of the biodegradable polymer microsphere comprises the following steps:

adding an organic solvent to the polymer to obtain a polymer solution (step 1);

dispersing glucose regulating peptide in the polymer solution of step 1 to obtain dispersion (dispersion), and then adding alcohol or a mixture of alcohol and organic acid to the dispersion to obtain drug-dispersed solution (step 2); and

microspheres are formed from the drug-dispersed solution described in step 2 (step 3).

A detailed description of the method will be given step by step.

First, step 1 is to prepare a polymer solution.

In step 1, the polymer is dissolved in an organic solvent. The polymer is biodegradable and can be used as a carrier, preferably a biodegradable polyester polymer. Any volatile organic solvent may be used without particular limitation as long as the biodegradable polymer carrier has high solubility therein and can be easily removed by evaporation. In the present invention, the organic solvent functions not only as a solubilizer for dissolving the polymer but also as a dispersant for uniformly dispersing the glucose-regulating peptide in the polymer solution.

Examples of organic solvents suitable for use in the present invention include: dichloromethane, ethyl acetate, chloroform, acetone, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethyl acetate, methyl ethyl ketone, acetonitrile and combinations thereof, with dichloromethane, ethyl acetate and chloroform being preferred, and dichloromethane being most preferred.

Then, step 2 is to prepare a drug-dispersed solution.

In step 2, glucose-regulating peptides are dispersed in the polymer solution. The glucose-regulating peptide is as described above. Synthetic exendin-4 is preferably added to obtain a drug dispersion (adrug dispersion). In the pharmaceutical dispersion, the ratio (w/w) of glucose regulating peptide to the polymer is selected from a range sufficient to solubilize the glucose regulating peptide.

Then, the alcohol is dissolved in the drug dispersion alone, or a combination of the alcohol and the organic acid is dissolved in the drug dispersion. The alcohol and organic acid act as solubilizers capable of dissolving the polymer and glucose-regulating peptide. A stabilizer or a surfactant may be further added.

In the process of the present invention, it is important to carry out the preparation of the pharmaceutical dispersion in the following order: adding an organic solvent, adding a glucose-regulating peptide, adding an alcohol or a mixture of an alcohol and an organic acid to the polymer. When the order of addition is changed, i.e., after adding the organic solvent and the alcohol or the mixture of the alcohol and the organic acid to the polymer, the glucose-regulating peptide is dissolved; or when a solution of glucose-regulating peptide in alcohol or a mixture of alcohol and organic acid is added to the polymer solution, the resulting microspheres may exhibit an incomplete release pattern.

Useful alcohols in the present invention are methanol, ethanol, isopropanol and butanol, with methanol being preferred due to its high solubility for the biodegradable polymer carrier and glucose-regulating peptide. The alcohol used to dissolve the drug dispersion is preferably used in as small an amount as possible, but must be sufficient to dissolve the drug dispersion. The amount may be determined depending on the kind of the alcohol. In the case of methanol, the ratio of the drug dispersion to the alcohol (v/v) is preferably in the range of 1: 1 to 6: 1, more preferably 3: 1 to 4: 1, to completely dissolve the drug dispersion.

In addition, any organic acid may be used without particular limitation as long as it can dissolve the polymer carrier and the glucose-regulating peptide. Examples of organic acids suitable for use in the present invention include: oxalic acid, oxaloacetic acid, fumaric acid, malic acid, succinic acid, acetic acid, butyric acid, palmitic acid, tartaric acid, ascorbic acid, uric acid, sulfonic acid, sulfinic acid, formic acid, citric acid, isocitric acid, alpha-ketoglutaric acid, succinic acid, and nucleic acids, preferably acetic acid, formic acid, and combinations thereof. The amount of the organic acid is determined according to the kind of the organic acid, similarly to the alcohol.

No particular limitation is imposed on the additive as long as it is capable of dissolving the drug dispersion and is soluble in the solvent for the drug dispersion. For example, polyethylene glycol (SolutolHS-15) can be used^TM、TPGS^TM、Gelucire^TM) Oils (Labrafil)^TM、Labrasol^TMMediumChain trigyceride (medium chain triglycerides)^TM) Proteins (lectins), surfactants (N-methylpyrrolidone, polyvinylpyrrolidone, Tween)^TM、Span^TM、Cremophor^TMPoloxamers, and methods of use^TM、Brij^TM、Sunsoft 818H^TM) And hydroxypropyl methylcellulose. Its concentration in the solubilizer ranges from 0.01% to 15% (w/v), preferably from 0.1% to 12.5% (w/v).

Finally, step 3 is the formation of microspheres from the drug-dispersed solution described in step 2.

The formation of microspheres can be achieved by dispersing the drug-dispersed solution in an aqueous solution containing an emulsifier or by using a spray dryer.

When the drug-dispersed solution is dispersed in an aqueous solution containing an emulsifier, a stirrer and a homogenizer are used to form microspheres, and the microspheres are then dried. The emulsifier useful in the present invention may be a lipophilic emulsifier dispersible in an organic solvent or a hydrophilic emulsifier dispersible in an aqueous solvent. Examples of hydrophilic emulsifiers include: tween, Triton, Brij, polyvinylpyrrolidone and polyvinyl alcohol, preferably polyvinyl alcohol. The emulsifiers used may be saturated or unsaturated in organic solvents. The organic solvent is preferably dichloromethane, ethyl acetate or chloroform, most preferably dichloromethane. The concentration of the emulsifier in the aqueous solution ranges from 0.01% to 5.0% (w/v), preferably from 0.5% to 2% (w/v).

In this step, drying may be performed by freeze-drying or vacuum-drying. The microspheres obtained can be collected by centrifugation during freeze-drying or by vacuum filtration during vacuum drying before final drying.

The microspheres prepared according to the process are of the oil-in-water (O/W) type and have an average size ranging from 5 μm to 70 μm, and preferably from 10 μm to 30 μm, which is suitable for injection. The particle size can be set to various values by controlling the volume ratio of the oil phase, i.e., the volume ratio of the drug-dispersed solution to the emulsifier-dissolved aqueous phase.

In the case of spray drying, the preparation of microspheres can be simply carried out by spraying the drug-dispersed solution from a spray dryer. To improve the production efficiency, the temperature of the spray dryer was set to 115 ℃ to 125 ℃ at the time of injection, and to 80 ℃ to 90 ℃ at the time of ejection. The spray dried microspheres may thereafter be subjected to additional drying processes such as freeze drying or vacuum drying to remove residual solvent therefrom.

Further, the biodegradable polymeric microspheres of the present invention can be prepared by a method comprising the steps of:

adding an organic solvent to the polymer to obtain a polymer solution (step 1);

emulsifying the polymer solution in the step 1 by using a glucose-regulated peptide aqueous solution containing a surfactant to obtain primary emulsion (primary emulsion) (step 2'); and

microspheres are formed from the primary emulsion described in step 2 '(step 3').

In step 1, the polymer is dissolved in an organic solvent. The polymer is biodegradable and can be used as a carrier, preferably a biodegradable polyester polymer. Any volatile solvent may be used without particular limitation as long as it has high solubility to the biodegradable polymer carrier and can be easily removed by evaporation. In the present invention, the organic solvent functions not only as a solubilizer for dissolving the polymer but also as a dispersant for uniformly dispersing the glucose-regulating peptide in the polymer solution.

Examples of organic solvents suitable for use in the present invention include: dichloromethane, ethyl acetate, chloroform, acetone, dimethyl sulfoxide, dimethylformamide, N-methylpyrrolidone, dioxane, tetrahydrofuran, ethyl acetate, methyl ethyl ketone, acetonitrile and combinations thereof, with dichloromethane, ethyl acetate and chloroform being preferred, and dichloromethane being most preferred.

In step 2', an aqueous solution of glucose-regulating peptide containing a surfactant is added to the polymer solution, followed by emulsification with a stirrer or homogenizer to obtain a primary emulsion. Synthetic exendin-4 can be used as a preferred glucose-regulating peptide. Adding glucose regulating peptide aqueous solution containing surfactant into polymer solution to form double emulsion (double emulsion) microsphere of water-in-oil-in-water (W/O/W) type.

In step 2', the aqueous glucose-regulating peptide solution may contain any surfactant as long as the glucose-regulating peptide can be dissolved in the aqueous solution. Examples of surfactants useful in the present invention include: tween, Triton, Brij, polyvinylpyrrolidone and polyvinyl alcohol.

The formation of microspheres can be achieved by: dispersing the primary emulsion in the step 2' in an aqueous solution containing an emulsifier; stirring with a stirrer or homogenizer, and drying. The emulsifier useful in the present invention may be a lipophilic emulsifier dispersible in an organic solvent or a hydrophilic emulsifier dispersible in an aqueous solvent. Examples of hydrophilic emulsifiers include: tween, Triton, Brij, polyvinylpyrrolidone and polyvinyl alcohol, preferably polyvinyl alcohol. The emulsifiers used may be saturated or unsaturated in organic solvents. The organic solvent is preferably dichloromethane, ethyl acetate or chloroform, most preferably dichloromethane. The concentration of the emulsifier in the aqueous solution ranges from 0.01% to 5.0% (w/v), preferably from 0.5% to 2% (w/v).

In this step, drying may be performed by means of freeze drying or vacuum drying. The microspheres obtained can be collected by centrifugation during freeze-drying or by vacuum filtration during vacuum drying before final drying.

Microspheres prepared according to the present invention are useful agents for releasing exendin-4 in a controlled manner, which have the following advantages: the initial burst effect is not generated, and the medicine release is not complete; maintaining zero-order release of exendin-4; the encapsulated exendin-4 has high encapsulation efficiency and high stability due to the simple preparation method; and stably releasing exendin-4 therefrom in vitro and in vivo for a period of three or four weeks.

Examples

The invention will be better understood by reference to the following examples which are given for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Example 1: preparation of microspheres (oil-in-water O/W type) according to the type and mixing ratio of the polymer Emulsion)

300mg of polymer (Boehringer Ingelheim) were completely dissolved in dichloromethane. 9mg of exendin-4(American Peptide) was dispersed in the polymer solution to obtain an exendin-4 dispersion. The polymer used is a single polymer product or a mixture of two different polymer products in various mixing ratios as shown in table 1. To each of the drug dispersions having different polymer species and mixing ratio, a predetermined amount of methanol (alcohol to drug dispersion in a volume ratio of 1: 4) was added to obtain a drug-dispersed solution. 10ml of each drug-dispersed solution was taken, and 250ml of a 1% (w/v) aqueous polyvinyl alcohol solution saturated with methylene chloride was used, which was emulsified by a stirrer or a homogenizer to form microspheres. The microspheres were cured while stirring at room temperature and atmospheric pressure for several hours to slowly evaporate the dichloromethane into the air. After centrifugation, the microspheres thus collected were washed with distilled water and frozen at-70 ℃ and then freeze-dried at room temperature and a pressure of 50mTorr for 3 days using an adVantage freeze dryer (VirTis, NY, U.S. A) to give oil-in-water (O/W) type microspheres capable of releasing exendin-4 in a controlled manner.

TABLE 1

Example 2: preparation of microspheres according to the ratio of alcohol to drug dispersion (oil-in-water O/W type) Emulsion)

300mg of polymer (RG502H, Boehringer Ingelheim) were completely dissolved in dichloromethane. 9mg of exendin-4(American Peptide) was dispersed in the polymer solution to obtain an exendin-4 dispersion. To the drug dispersion was added a predetermined amount of methanol (alcohol to drug dispersion in a volume ratio of 1: 1 to 1: 7, as shown in Table 2) to obtain a drug-dispersed solution. It was emulsified and dried in the same manner as in example 1 to obtain microspheres.

TABLE 2

As shown in table 2, when the volume ratio of the drug dispersion to methanol was 7 or more, a solution could not be formed.

Example 3: preparation of microspheres from a drug-dispersed solution containing additives (oil-in-water) O/W type emulsion)

Microspheres were prepared in the same manner as in example 1-1, except that various additives in an amount of 0.1% or 12.5% by volume of the solution were mixed with the drug-dispersed solution. The additives and their volume percentages mixed with the solution are summarized in table 3.

TABLE 3

As shown in table 3, various additives having a wide range of concentrations can be mixed with the drug-dispersed solution.

Example 4: preparation of microspheres from an aqueous emulsifier solution not saturated with organic solvent (oil-in-water) O/W type emulsion)

Microspheres were prepared in the same manner as in example 1-1, except that the drug-dispersed solution was added to 250ml of a 1% aqueous solution (w/v) of polyvinyl alcohol not saturated with methylene chloride, and emulsified with a stirrer or homogenizer.

Example 5: preparation of microspheres with different particle sizes (oil in water O/W type emulsions)

Microspheres were prepared in the same manner as in example 1-1, except that the volume ratio of a 1% aqueous solution (w/v) of polyvinyl alcohol saturated with methylene chloride to the drug-dispersed solution (i.e., the volume ratio of the aqueous phase to the oil phase) was set as shown in table 4 below.

TABLE 4

Example 6: preparation of microspheres by drying (oil in water O/W type emulsion)

Microspheres obtained in example 1-1 by gradually volatilizing dichloromethane by stirring at room temperature and atmospheric pressure for several hours and then solidifying were filtered through a vacuum filtration system, washed with distilled water, and then dehydrated for 3 days with an adVantage dryer (VirTis, NY, u.s.a) at room temperature and a pressure of 50mTorr before final drying.

Example 7: preparation of microspheres by spray drying (oil in water O/W type emulsion)

The drug-dispersed solution obtained in example 1-1 was not mixed with an aqueous emulsifier solution, but was injected into a spray dryer (Buchi Mini spray dryer, B-290) at a rate of 2.5ml per minute, and at the same time, was sprayed at a rate of 400Nl/h through a 0.7mm nozzle. The microspheres thus formed were dried in vacuum to obtain a microsphere formulation capable of stably releasing exendin-4. The temperature of the spray dryer was set to 120. + -. 2 ℃ at the time of injection, and to 85. + -. 2 ℃ at the time of discharge.

Example 8: preparation of microspheres Using an aqueous pharmaceutical solution (Water-in-oil-in-Water W/O/W type emulsion)

300mg of polymer (RG502H, Boehringer Ingelheim) were completely dissolved in dichloromethane. To the polymer solution was added an aqueous solution of exendin-4, which was formed by dissolving 9mg of exendin-4(American Peptide) in 0.3ml of a 0.5% aqueous solution (w/v) of polyvinyl alcohol, followed by stirring with a homogenizer to obtain a primary emulsion. 10ml of this primary emulsion was taken and emulsified by a stirrer or homogenizer using 250ml of a 1% aqueous solution (w/v) of polyvinyl alcohol saturated with methylene chloride to form microspheres. The microspheres were cured while stirring at room temperature and atmospheric pressure for several hours to slowly evaporate the dichloromethane into the air. After centrifugation, the microspheres thus collected were washed with distilled water and frozen at-70 ℃ and then freeze-dried at room temperature and a pressure of 50mTorr for 3 days using an adVantage freeze-dryer (VirTis, NY, U.S.A.) to give water-in-oil-in-water (W/O/W) type microspheres capable of releasing exendin-4 in a controlled manner.

Comparative example 1: without a drug dispersing stepPreparation of oil-in-water (O/W) type microspheres (1)

To a solution of 300mg of polymer (RG502H, Boehringer Ingelheim) in methylene chloride was added methanol in an amount corresponding to one-fourth of the volume of the methylene chloride solution to give a polymer/methylene chloride/methanol solution. Mixing the exendin-4 and the polymer/dichloromethane/methanol solution at a weight ratio of exendin-4 to polymer of 9: 300 to obtain a drug-dispersed solution without a drug-dispersing step. Microspheres were prepared from the drug-dispersed solution in the same manner as in example 1.

Comparative example 2: preparation of oil-in-water (O/W) type microspheres without drug Dispersion step (2)

Microspheres were prepared in the same manner as in example 1, except that a solution of 9mg of exendin-4 in 0.2ml of methanol was added to a solution of 300mg of polymer (RG502H, Boehringer Ingelheim) in 0.8ml of methylene chloride to give a drug-dispersed solution without the drug-dispersing step.

Experimental example 1: encapsulation rate of Exendin-4 in microspheres

30mg of each of the microspheres having exendin-4 encapsulated therein prepared in example 1 and examples 4-8 was taken, dissolved sufficiently in 0.5ml of DMSO in a polystyrene container, and 1.5ml of distilled water was added thereto, followed by stirring for 12 hours or more to extract exendin-4 into an aqueous phase. The extracted exendin-4 was quantitatively analyzed to calculate an encapsulation efficiency, which was expressed in a manner that the actual encapsulation amount was a percentage of the theoretical encapsulation amount.

The results of the calculations are summarized in table 5 below.

TABLE 5

Examples	Encapsulation efficiency (%)
		1-1	85
1-2	86
		1-3	88
1-4	81
		1-5	83
1-6	84
		1-7	80
4	84
		5-1	85
5-2	85
		5-3	84
6	85
		7	93
8	83

As shown in table 5, the encapsulation efficiency of the microspheres of the present invention was calculated to be 80% or more.

Experimental example 2: measurement of average particle size of microspheres

30mg of each of the microspheres prepared in example 1 and examples 4 to 8, in which exendin-4 was encapsulated, was dispersed in 1L of distilled water containing 0.02% (v/v) Tween20, and then the average particle size thereof was measured using a particle size analyzer. The measurement results are shown in table 6 below.

TABLE 6

Examples	Average particle size (. mu.m)
		1-1	22
1-2	16
		1-3	18
1-4	16
		1-5	19
1-6	22
		1-7	21
4	26
		5-1	8
5-2	25
		5-3	65
6	21
		7	59
8	16

As shown in Table 6, microspheres prepared according to the present invention have an average particle size in the range of 8 μm to 65 μm, and have been small enough to be used with small-sized needles.

Experimental example 3: in vitro release of drugs from microspheres

Evaluation of exendin-4 in vitro release was performed on the microspheres prepared in examples and comparative examples under the following conditions.

30mg of microspheres in a polystyrene container were dispersed in 1.5ml of PBS (phosphate buffered saline, pH 7.4) containing 0.02% (v/v) Tween 20. During incubation (incubation) at 37 ℃, the dispersion was centrifuged to pellet the microspheres according to the incubation time. The supernatant was analyzed for the level of exendin-4 to determine the amount of exendin-4 released from the microspheres. The precipitated microspheres were redispersed in fresh PBS for subsequent analytical experiments. The amount (%) of exendin-4 released from the microspheres as a function of incubation time is plotted in FIGS. 1 to 3.

FIGS. 1 and 2 show the in vitro release of exendin-4 from microspheres prepared by the exendin dispersion procedure of examples 1 and 4-8 according to the present invention as a function of time; while FIG. 3 shows the in vitro release of exendin-4 from microspheres prepared in comparative examples 1 and 2, which did not include the exendin dispersion step, as a function of time.

As is apparent from the graphs of fig. 1 and 2, the microspheres prepared by the exendin dispersion step of the present invention neither had initial burst release (3% of exendin-4 released on the first day) nor incomplete release, and sustained zero-order release over 21 days.

In contrast, as shown in fig. 3, the microspheres prepared without the exendin dispersion step showed incomplete release, and only 83% (comparative example 1) and 49% (comparative example 2) of the drugs were released within 21 days.

Therefore, the microspheres containing exendin-4 prepared according to the present invention have neither initial burst release nor incomplete release, and continue zero-order release of exendin-4 for three weeks, so that they can be effectively used as sustained release agents for exendin-4.

Experimental example 4: pharmacokinetic evaluation of microspheres

The microspheres prepared in examples and comparative examples were evaluated for exendin-4 in vivo release and pharmacokinetic properties under the following conditions.

A predetermined amount (corresponding to 140. mu.g of exendin-4) of the microspheres prepared in examples 1 and 4-8 and comparative examples 1 and 2 was suspended in an aqueous solution containing 1.5% CMC, 0.5% Tween20 and 0.9% NaCl, and the suspension was injected subcutaneously into five Sprague-Dawley rats each at a dose of 0.2 ml. Blood samples were then taken from the rats at predetermined time intervals and the exendin-4 level was quantified by enzyme-linked immunosorbent assay (ELISA) to determine the release of exendin-4 from the microspheres. FIG. 4 is a graph showing the in vivo release pattern of exendin-4 from the microspheres prepared in example 1-1.

As is apparent from the release pattern of fig. 4, the microspheres prepared according to the present invention had neither initial burst nor incomplete release, and sustained zero-order release in the experimental animals over a period of 20 days as in the in vitro experiments.

In addition, the maximum blood concentration (C) of pharmacokinetic parameters was calculated from measurements of exendin-4 release using the WinNonlin program_max) And area under the curve (AUC) from 0 to 14 days_{0-14 days}). These parameters are summarized in table 7 below.

TABLE 7

Examples	Cmax(ng/ml)	AUC0-14 days(Tianxing ng/ml/mg/kg)
			1-1	1.92±0.30	11.85±0.30
1-2	2.78±0.69	9.29±0.57
			1-3	1.33±0.20	9.24±0.76
1-4	1.18±0.12	9.63±1.08
			1-5	1.04±0.13	8.98±0.62
1-6	2.26±0.37	9.92±0.81
			1-7	4.02±0.15	9.31±0.67
4	1.91±0.11	12.05±0.78
			5-1	3.13±0.31	9.74±1.08
5-2	1.31±0.11	12.01±0.11
			5-3	1.29±0.10	11.07±0.91
6	2.05±0.33	10.84±0.78
			7	1.67±0.27	8.31±0.27
8	3.28±0.42	9.40±0.58
			Comparative example 1	0.94±0.17	5.34±0.61
Comparative example 2	0.85±0.24	5.07±0.20

As shown in Table 7, the microspheres of examples 1-1 to 1-7, example 4, examples 5-1 to 5-3 and examples 6-8 had C calculated_maxAnd AUC values are higher than those of comparative example 1 and comparative example 2, indicating that the microspheres prepared according to the present invention are excellent exendin-4 controlled release agents.

Therefore, based on the data of fig. 4 and table 7, the biodegradable polymeric microspheres provided by the present invention not only have excellent encapsulation efficiency, but also stably and completely release exendin-4 without lag time within a period of 2-4 weeks.

Experimental example 5: stability analysis of Exendin-4 in microspheres

10mg of the exendin-4-containing microspheres prepared in example 1-1 were placed in a polystyrene container and dissolved well in 1ml of DMSO. The resulting solution was diluted five times with ammonium bicarbonate and then analyzed by reverse phase high performance liquid chromatography to observe the peak and retention time of exendin-4. The results are shown in FIG. 5.

FIG. 5 is a chromatogram of exendin-4 obtained from the microspheres prepared in example 1-1. As shown in the chromatogram, a single peak was observed and the retention time was the same as that of the control sample.

Claims (24)

1. A biodegradable microsphere capable of releasing a glucose-regulating peptide in a controlled manner, comprising a biodegradable polymeric carrier in which the glucose-regulating peptide is encapsulated.

2. The biodegradable microsphere of claim 1, wherein said glucose regulating peptide is selected from the group consisting of synthetic exendin-3, exendin-4, and homologs and agonists thereof.

3. The biodegradable microsphere of claim 2, wherein the glucose-regulating peptide is exendin-4.

4. The biodegradable microspheres of claim 1, wherein said biodegradable polymer is selected from the group consisting of: poly-L-lactic acid, D-lactic acid-glycolic acid copolymer, L-lactic acid-glycolic acid copolymer and D, L-lactic acid-glycolic acid copolymer.

5. The biodegradable microsphere of claim 4, wherein said biodegradable polymer is D, L-lactic acid-glycolic acid copolymer.

6. A method of making the biodegradable polymeric microsphere of claim 1, comprising:

step 1: adding an organic solvent to a polymer to obtain a polymer solution;

step 2: dispersing glucose regulating peptide in the polymer solution in the step 1 to obtain a drug dispersion, and then adding alcohol or a mixture of alcohol and organic acid into the drug dispersion to obtain a solution in which the drug is dispersed; and

and step 3: forming microspheres from the drug-dispersed solution described in step 2.

7. The method of claim 6, wherein the organic solvent is selected from the group consisting of dichloromethane, ethyl acetate and chloroform.

8. The method of claim 6, wherein the alcohol in step 2 is methanol.

9. The method of claim 6, wherein the alcohol of step 2 is used in a ratio of 1: 1 to 1: 6 of the alcohol to the polymer solution.

10. The method of claim 6, wherein the drug-dispersed solution of step 2 further comprises an additive.

11. The method of claim 10, wherein the additive is selected from the group consisting of: polyethylene glycol, Labrafil, Labrasol, medium chain triglycerides, lecithin, N-methyl pyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, Tween, Span, Cremophor, poloxamer, Brij, and Sunsoft 818H.

12. The method of claim 10, wherein the additive is present in an amount of 0.01% to 15% (w/v) by volume of the solution.

13. The method of claim 6, wherein the step 3 is performed by: dispersing the solution dispersed with the medicine in the step 2 into an aqueous solution containing an emulsifying agent; forming the microspheres with a stirrer or homogenizer; and drying the microspheres by freeze drying or vacuum drying.

14. The method of claim 13, wherein the emulsifier is selected from the group consisting of Triton, Brij, polyvinylpyrrolidone, and polyvinyl alcohol.

15. The method of claim 13, wherein the emulsifier is saturated in an organic solvent selected from the group consisting of dichloromethane, ethyl acetate, and chloroform.

16. The method of claim 6, wherein the step 3 is performed by spraying the solution dispersed with the drug in the step 2 with a spray dryer.

17. The method of claim 16, wherein the temperature of the spray dryer is set to 115 ℃ to 125 ℃ at the time of injection and to 80 ℃ to 90 ℃ at the time of ejection.

18. The method of claim 16, wherein said spray drying is followed by additional freeze drying or vacuum drying.

19. A method of making biodegradable polymeric microspheres comprising:

step 1: adding an organic solvent to a polymer to obtain a polymer solution;

step 2': emulsifying the polymer solution in the step 1 by using a glucose-regulated peptide aqueous solution containing a surfactant to obtain a primary emulsion; and

step 3': dispersing the primary emulsion in the step 2' into an aqueous solution containing an emulsifier, stirring by using a stirrer and a homogenizer to form the microspheres, and then drying the microspheres by freeze drying or vacuum drying.

20. The method of claim 19, wherein the organic solvent in step 1 is selected from the group consisting of dichloromethane, ethyl acetate and chloroform.

21. The method of claim 19, wherein the surfactant is selected from the group consisting of: polyvinyl alcohol, polyethylene glycol, Labrafil, Labrasol, medium chain triglycerides, lecithin, N-methylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, Tween, Span, Cremophor, poloxamer, Brij, and Sunsoft 818H.

22. The method of claim 19, wherein said emulsifier is selected from the group consisting of Triton, Brii, polyvinylpyrrolidone, and polyvinyl alcohol.

23. The method of claim 19, wherein the emulsifier is saturated in an organic solvent selected from the group consisting of dichloromethane, ethyl acetate, and chloroform.

24. Biodegradable polymeric microspheres for the controlled release of glucose regulating peptides prepared according to the method of any one of claims 6 to 23.