JP2002069000A - Oral medicine for treating diabetes mellitus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a therapeutic agent containing insulin and suitable for oral administration. SOLUTION: An oral medicine containing a hybrid type liposome containing insulin is used. The hybrid type liposome is prepared by using a phospholipid and a micelle-based surfactant. An l-α-diacylphosphatidylcholine, an l-α- diacylphosphatidylglycerol, sphingomyelin, or the like, is exemplified as the phospholipid. A polyoxyethylenesorbitan fatty acid ester, a sorbitan fatty acid ester, an alkyl glycoside, an alkyl thioglycoside, a cerebroside, an alkylmaltopyranoside, a fructofuranosylalkylglocopyranoside, or the like, is exemplified as the micelle-based surfactant.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to an oral preparation for treating diabetes. More particularly, the present invention relates to orally administered insulin preparations for treating diabetes.

[0002]

BACKGROUND OF THE INVENTION Diabetes is defined as a disease in which there is a persistently high blood sugar level. It is known that if such a condition continues, serious complications such as retinopathy leading to blindness, renal failure requiring artificial dialysis, or neurosis may occur. Above all, in those classified as insulin-dependent diabetes, insulin production and secretion are not performed at all, and eventually a coma occurs. Therefore, administration of insulin is indispensable for survival. Recent research is elucidating the genes responsible for diabetes,
Diabetes has been shown to be caused not by a single gene but by multiple genes. Therefore, it is believed that the fundamental therapy by gene therapy is well established.

[0003] Since insulin is a protein, there is a problem that, when administered orally, it is decomposed by digestive enzymes before it is absorbed in the digestive tract. Therefore, a subcutaneous injection by a diabetic patient itself is generally performed for the treatment, but since the duration of action is not long, the injection must be performed every day, which involves physical and mental pain. For these reasons, it has been desired to develop a method of administering insulin that enables oral administration. Various methods for oral administration of insulin, such as chemical modification of insulin molecules and the use of drug carriers such as liposomes, have been considered.However, those that have been found to have actually been effective have been reported. Absent.

[0004]

SUMMARY OF THE INVENTION It is an object of the present invention to provide an insulin preparation suitable for oral administration.
More specifically, an object of the present invention is to provide an insulin preparation for oral administration that protects insulin from digestion fluid digestion and has a long-lasting effect.

Means for Solving the Problems The present inventors have been able to produce various hybrid liposomes,
It has already been reported that hybrid liposomes themselves or hybrid liposomes containing fat-soluble anticancer agents have a cancer cell-suppressing effect. The invention has been completed. That is, the present invention is an oral therapeutic agent for diabetes, comprising a hybrid liposome containing insulin. As used herein, the term "liposome" is used in the ordinary sense used in this field, and is formed by dispersing a phospholipid, which is a component of a biological membrane, in water at a relatively low concentration. Means ER. The “hybrid liposome” refers to a liposome containing one or more phospholipids, one or more surfactants, and, if necessary, other components.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION In the present invention, an insulin-containing hybrid liposome is used. The hybrid liposomes used in the present invention can be made from one or more phospholipids, one or more surfactants and insulin. Phospholipids that can be used in the hybrid liposome of the present invention include, for example,
L-α-diacylphosphatidylcholine, L-α-diacylphosphatidylglycerol, L-α-diacylphosphatidylserine, L-α-diacylphosphatidylethanolamine, sphingomyelin, L-α-diacylphosphatidyl acid, L-α-diacylphosphatidylinositol And cardiolipin. All of these phospholipids are physiologically compatible phospholipids. In these phospholipids, the two acyl groups may be the same or different as long as a stable hybrid liposome is formed.
To 22 are preferred. Of these, L-α
-Diacylphosphatidylcholine, particularly preferred is L-α-dimyristoylphosphatidylcholine (DMPC) having the following structural formula:

[0006]

Embedded image

The surfactant which can be used for the hybrid liposome used in the present invention is a surfactant capable of forming micelles, and any of natural and synthetic substances can be used as long as they are physiologically compatible. Can be used. As surfactants capable of forming such micelles, non-sugar micelle surfactants and sugar micelle surfactants can be used. Among them, it is preferable to use a non-sugar micelle type surfactant. When a sugar micelle surfactant is used, it is preferable to use it together with a non-sugar micelle surfactant.
Here, the “sugar micelle surfactant” refers to a surfactant having a sugar skeleton among surfactants that form micelles, and the “non-sugar micelle surfactant” refers to a surfactant that forms micelles It does not have a sugar skeleton. Among the non-sugar micelle surfactants, non-ionic surfactants are preferred, and particularly surfactants usually sold under the name `` Tween '', for example, polyoxyethylene sorbitan such as Tween 20 to Tween 80. Fatty acid esters, or surfactants usually sold under the name "Span", for example, sorbitan fatty acid esters such as Span 20 to Span 80, or alkyl or aryl poly represented by the following general formula (I) Oxyethylene glycol

[0008]

Embedded image (I) (wherein n = 10 to 25, m = 12 to 20, Z = C _{6 to} C ₁₂ (preferably C _{8 to} C ₁₂ ) alkyl group) is preferred. These non-sugar micelle surfactants may be used alone or in combination of two or more. More specifically,
Polyoxyethylene sorbitan monolaurate (particularly Tween 20) and in the general formula (I), m is 12 to
Alkyl polyoxyethylene glycol having 14 and n is 10 to 23 is preferred. Particularly preferred are polyoxyethylene glycols wherein m is 12 or 14 and n is 10 or 12.

Examples of the sugar micelle surfactant include alkyl glucosides represented by the following general formula (II) and general formula (II)
An alkylthioglucoside represented by the formula (I), represented by the following formula (I
V) or a monosaccharide such as glucocerebroside whose sugar moiety is glucose,
Examples of alkylmaltopyranoside represented by the following general formula (V), fructofuranosylalkylglucopyranoside represented by the general formula (VI), alkyllactopyranoside represented by the general formula (VII), and the like. Polysaccharides such as sugar, amylose, inulin and the like can be mentioned. These sugars may be either α-type or β-type.

[0010]

Embedded image (II) (wherein n = 7 to 17, and --- means α-type or β-type)

[0011]

Embedded image (III) (wherein, n = 7 to 17, and --- means α-type or β-type.)

[0012]

Embedded image (IV) (where n = 7 to 17)

[0013]

Embedded image (V) (wherein n = 7 to 17, and --- means α-type or β-type.)

Embedded image (VI) (where n = 7 to 17)

Embedded image (VII) (wherein n = 7 to 17, and --- means α-type or β-type)

Among these sugar micelle surfactants, fructofuranosylalkylglucopyranoside represented by the general formula (VI), particularly β-D-fructofuranosyl-α-D-glucopyranoside, is preferred. The alkyl group may be branched or unbranched as long as it can form micelles, and the number of carbon atoms is generally preferably 8 to 18, more preferably 8 to 16, even more preferably 8 in any sugar micelle surfactant. ~ 10. Particularly preferred is β-D-fructofuranosyl-α-D
-Glucopyranoside monodecanoate (DeFG) (general formula (V
In I), n = 8) and β-D-fructofuranosyl-α-D-
It is glucopyranoside monododecanoate (DoFG) (n = 10 in the general formula (VI)). As optional components that can be used for preparing the liposome, cholesterol for decreasing the fluidity in the membrane, a charged substance for preventing aggregation of the liposomes, and the like can be used. These optional components can be used in amounts that do not impair the effects of the present invention.

The insulin contained in the insulin-containing hybrid liposome of the present invention may be an intact molecule or any modified molecule as long as it has the physiological action of insulin. In addition, intact molecules and modified molecules can be used in combination.
Here, “has a physiological action of insulin” means that it has an action of binding to an insulin receptor and lowering a blood glucose concentration. The term "alteration" includes deletion, addition, substitution, and chemical modification of an amino acid in an insulin molecule. In particular, a modification that improves the lipid solubility of insulin and improves the efficiency of incorporation into liposomes, and / or a modification that protects against degradation by digestive enzymes is preferred. Such modifications include the acylation of insulin. Acylation improves the efficiency of insulin uptake into the hybrid liposome and protects it from the degradative action of digestive enzymes due to steric hindrance. Such insulin acylation is described, for example, in Hashimoto (M. Hashimoto et al., Pharm. Re.
s. 6, 171 (1989)). According to this method, a monoacylated derivative and a diacylated derivative of insulin can be obtained as a mixture. If necessary, each derivative can be separated and used using HPLC according to, for example, the method of Nakazawa et al. (Nakazawa et al., Pharmaceutical Journal 106, 398 (1986)).
Insulin that can be used in the present invention may be one directly extracted from a biological tissue or the like, or may be recombinant insulin, regardless of its biological origin. Particularly when recombinant insulin is used, modification at the primary structure level is easy.

In the hybrid liposome of the present invention,
Phospholipids, non-sugar micellar surfactants, insulin and
And the amount of sugar micelle surfactant used contains insulin
Optional as long as liposomes can be formed
But the phospholipid concentration is 1 × 10^-Five~ 5 × 10
^-3M, micelle-based surfactant concentration of 1 × 10^-6~ 1 × 10
^-3M, make the insulin concentration 10-30 U / mL
Is appropriate. In addition, phospholipids and micelle surfactants
Although the ratio of use can be arbitrary,
The activator is preferably set to 50:50 to 98: 2 (mass ratio).
More preferably, the ratio is 30 to 95: 5. The insulin of the present invention
DMPC: Tw
It is particularly preferred to use een20 = 9: 1 (molar ratio). I
Ratio of insulin to phospholipid or surfactant
Can be arbitrarily selected, but its solubility in the buffer used
Limits its concentration. Insulin of the present invention
Of hybrid liposomes containing
The composition of the surfactant and the surfactant
Selected so that posomes are stable in gastric or intestinal fluid
Preferably. Specifically, disintegration of solid preparation for internal use
First and second liquids commonly used for testing (Japanese Pharmacy
Is preferably stable for at least about 2 hours in
No. Stability measures changes over time in liposome membrane size
Can be evaluated by Film size is dynamic light
It can be measured by the scattering method, and the calculation formula for that
And the measuring device is well known to those skilled in the art.

The insulin-containing hybrid liposome of the present invention is prepared by mixing the above-mentioned phospholipid, non-sugar micelle type surfactant and insulin in an appropriate amount and ratio as described above in a buffered aqueous solution. It can be manufactured by sonication at a temperature of, for example, about 45 ° C. for a relatively short time. When using a sugar micelle-based surfactant, after performing the above-described ultrasonic treatment, adding a sugar micelle-based surfactant to the solution, and further performing ultrasonic treatment under the same conditions. Can be prepared. The buffer aqueous solution can be arbitrarily selected within a range in which insulin can be dissolved and a hybrid liposome is formed.
For example, it is preferable to use a phosphate buffer having a pH in the range of 7.0 to 8.0. For example, PBS (phosphate buffered saline) or the like can be used.

The insulin-containing hybrid liposome preparation of the present invention is orally administered to a diabetic patient.
The dose and the number of doses can be selected in consideration of the patient's condition, general health condition, age, lifestyle, and the like. The insulin-containing hybrid liposome preparation of the present invention generally uses, for example, 200 to 400 U / day, preferably 200 to 350 U / day, more preferably insulin 200 / kg / kg of patient weight as a total dose per day. An equivalent of ~ 300 U / day is used. Also, the insulin concentration in the administered formulation is 1
It is administered at 0-30 U / ml, preferably 10-20 U / ml, more preferably 10-15 U / ml. The insulin-containing hybrid liposome preparation of the present invention is generally prepared from water or other physiologically acceptable solution, for example, PBS.
The dosage form is provided in the form of a solution in a phosphate buffer having a pH in the range of 7.0 to 8.0, and can be provided in a single-dose ampule or a sealed container such as a capsule. It may be provided as.

EXAMPLES Materials In the following examples, L-α-dimyristoyl phosphatidylcholine (DMPC) was a commercial product (Nippon Oil & Fat) used as it was. As the polyoxyethylene sorbitan monolaurate (Tween 20), a commercially available product (Nacalai Tesque) was used as it was. As other reagents, commercial grade reagents were used as they were. As insulin, a commercial product (Sigma) extracted from calf pancreas was used as it was.

Embodiment 1 FIG. Preparation of Insulin-Containing Hybrid Liposomes (1) Preparation of Insulin Solution After dissolving insulin in 0.5 ml of 0.01 M hydrochloric acid, 4.5 ml of a phosphate buffer aqueous solution was added to obtain an insulin solution. (2) Preparation of hybrid type liposome 16.9 mg of L-α-dimyristolyphosphatidylcholine (DMPC) and Tween 20 in 5 mL of a phosphate buffer aqueous solution
3.4 mg of the mixture was mixed and subjected to ultrasonic irradiation (Yamamoto BRANSONIC MODEL B2210: 90W) at 45 ° C under a nitrogen atmosphere for 1 minute / ml to prepare DMPC / 10mol% Tween20 hybrid liposome. After sonication, after confirming that a uniform solution was obtained, the solution containing the hybrid liposome was sterilized by filtration with a 0.45 μm filter to obtain a test solution. When insulin was contained in the hybrid liposome, treatment was performed under the same conditions as above, but an insulin solution having a predetermined concentration was used instead of the phosphate buffer aqueous solution.

Embodiment 2 FIG. Stability evaluation of hybrid liposomes in artificial gastric juice and artificial intestinal fluid First and second liquids used in artificial gastric juice and artificial intestinal juice for the disintegration test of solid preparations for internal use Using the manual, 349 (1986)), the stability test of the hybrid liposome prepared in Example 1 was performed. To 0.1 g of sodium chloride was added 1.2 ml of 1M hydrochloric acid, the total volume was made up to 50 ml with ion-exchanged water, and an artificial gastric juice (first solution) was prepared so that the pH became about 1.4 at 37 ° C. Also, after dissolving 0.544 g of potassium dihydrogen phosphate in a small amount of ion-exchanged water, 5.9 ml of a 0.2 M sodium hydroxide aqueous solution was added, and the total amount was made up to 50 ml with ion-exchanged water.
An artificial intestinal fluid (second fluid) was prepared so that the pH became about 6.8.

Using these artificial gastric juice and artificial intestinal juice, the hybrid liposome was diluted 10-fold, and the change over time in the membrane size was measured. The membrane size of the hybrid liposome was measured by a dynamic light scattering method using a submicron sizer (BROKKHAVEN BI-90). He-Ne as light source
Using a laser beam of 632.8 nm at an output of 35 mW, the scattering angle
Measurement was performed at 90 °, and from the obtained diffusion coefficient (D), equation (1) (Stok
The diameter d _hy of the membrane was determined according to the es-Einstein equation). d _hy = kT / 3πηD (1) where k is the Boltzmann constant, T is the absolute temperature, and η is the viscosity of the solvent. The value of d _hy was measured over a period of 0 to 2.0 hours for hybrid liposomes incubated at 37 ° C. after dilution with artificial gastric juice or artificial intestinal fluid, and the results were recorded (Table 1). As a control, the stability of the hybrid liposomes similarly diluted with an aqueous phosphate buffer solution was also measured (Table 2).

[0022]

[Table 1]

[0023]

[Table 2]

According to these measurement results, DMPC / 10 mol% T
Ween20 hybrid liposomes show a single size distribution of about 200 nm in phosphate buffered aqueous solution and form a stable membrane for more than 1 month, and furthermore, in artificial gastric juice and artificial intestinal juice, a particle size of 200 nm It was shown that a stable membrane of some degree was maintained for at least 2.0 hours.

Embodiment 3 FIG. Blood sugar level control by insulin-containing hybrid liposome The hybrid liposome prepared in Example 1 was used as an insulin drug carrier, and a therapeutic experiment was performed on a diabetic model rat. As a diabetic model rat, a rat three days after a single administration of streptozotocin (STZ) at 60 mg / kg was used. STZ-treated rats developed insulin-dependent diabetes mellitus by selectively necrotic pancreatic Langerhans islet β cells, which are insulin-producing cells, but they do not require insulin administration to survive. Easy and widely used. The insulin-containing hybrid liposome solution was administered to the stomach of the diabetic rat using a tube. The dose was 240 U of insulin per 1 kg of rat body weight, and the concentration of insulin in the solution was 15 U / ml. Glucose concentration in blood (blood sugar level)
Was measured hourly from 0 to 4 hours (Table 3).
For measurement of blood glucose concentration, a Fuji Drychem 3030 measuring device was used.

[0026]

[Table 3]

As shown in Table 3, one hour after administration of the insulin-containing hybrid liposome, the blood glucose level decreased to about 80% as compared to that before administration, and then gradually decreased to about 75% after 4 hours. % (Table 3). On the other hand, when the aqueous insulin solution was administered, the blood sugar level decreased only to about 90% before administration after 1 hour and decreased to about 80% after 2 hours, but thereafter the blood glucose level increased. At the beginning, it increased to about 95% after 4 hours, and continued to increase thereafter.

Embodiment 4 FIG. Evaluation of stability of acylated insulin (1) Acylation of insulin The method of Hashimoto et al. (M. Hshimoto et al., Pharm. Res., 6,
171 (1989)), acylated insulin was synthesized. An outline of the synthesis is shown below. Step 1: Synthesis of N-hydroxysuccinimide ester of fatty acid A solution of benzoic acid or caproic acid (50 mmol) in N-hydroxysuccinimide (NHS) (50 mmol) in ethyl acetate (1
50 ml). To this was added a solution of dicyclohexylcarbodiimide (DCC) (50 mmol) in ethyl acetate (15 ml),
Stirred overnight at 4 ° C. Dicyclohexylurea as a by-product was removed by suction filtration, and the filtrate was concentrated under reduced pressure. Recrystallization is performed using ethanol, and colorless needle crystals (Bz-O
su or Cap-Osu). Step 2: Synthesis of pMz-Insulin 10 ml of a mixed solution (1N NaOH: H ₂ O: DMF = 2: 3: 4) containing bovine insulin (200 mg) and p-methoxybenzyldicarbonylcarbonylazide) pMz-azide (152 μmol) Dissolved in
Stirred at room temperature for 2.5 hours. After the reaction, 50% acetic acid was added to the mixture, and the solvent was evaporated under reduced pressure. The residue was washed with anhydrous ether and 1% acetic acid, dissolved in 50% acetic acid, and lyophilized to obtain pMz-modified insulin.

Step 3: Reaction of pMz-insulin with Bz-OSu or Cap-OSu The above-mentioned pMz-insulin was dissolved in DMF, and 50-fold molar amount of Bz-OSu or Cap-OSu was added thereto. Stirred at room temperature for 2.5 hours. Step 4: Recovery of acylated insulin After the above reaction, dimethylformamide (DMF) was evaporated under reduced pressure. Thereafter, the residue was treated with trifluoroacetic acid (TFA) in the presence of anisole for 60 minutes in an ice bath to remove the pMz group. After evaporating TFA at 30 ° C. under reduced pressure, anhydrous ether was added to the residue and the mixture was cooled, and the precipitated white crystals were collected by suction filtration. The obtained product was confirmed by the method of Nakazawa et al. (Hiro Nakazawa, Masayuki Nagase, Pharmaceutical Journal, 10
6, 398 (1986)). The measurement conditions were as follows: the column temperature was room temperature, the flow rate was 0.5 ml / min, and the detection wavelength was 25.
4 nm. The column is SHISEIDO UG120 PAC (4.6mmφx150
acetonitrile: TFA: distilled water = 500: 1: 4
99, 0.1 M NaCl was used as the mobile phase. Compared to unmodified insulin, two large peaks that eluted later were observed, corresponding to the mono- and diacyl-derivatives, respectively, confirming that the product was a mixture thereof. (2) Stability of acylated insulin to degrading enzymes Stability to various enzymes was examined using acylated insulin. Insulin and acylated insulin
For 20 U / ml, pepsin (0.005 g / ml), chymotrypsin (0.001 g / ml), carboxypeptidase (0.01 g / m
l), reacting with aminopeptidase (0.05 g / ml)
After time, the amount of residual insulin was measured. Table 4 shows the results.

[0030]

[Table 4]

As is clear from the table, unmodified insulin is degraded by about 50% after 3 hours by pepsin, chymotrypsin and carboxypeptidase, whereas acylated insulin is almost degraded by 3 hours. Did not.

Industrial Applicability According to the present invention, there is provided an oral therapeutic agent for diabetes capable of controlling blood glucose level for a long period of time lower than when an insulin aqueous solution is directly administered. The insulin-containing hybrid liposome preparation of the present invention can control a patient's blood glucose level to be low for at least 4 hours.

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI theme coat ゛ (Reference) A61K 47/34 A61P 3/10 A61P 3/10 A61K 37/26 (72) Inventor Kentaro Nakajima Kumamoto, Kumamoto 3-23-10 Hanazono F-term (reference) 4C076 AA19 BB01 CC21 CC41 DD08F DD08H DD09F DD09H DD15F DD15H DD63F DD63H DD69F DD69H EE23F EE23H FF36 FF68 4C084 AA03 BA44 DB34 MA05 MA24 MA52 NA10 ZC352

Claims (10)

[Claims] 1. An oral preparation for treating diabetes, comprising a hybrid liposome containing insulin. 2. The oral preparation for treating diabetes according to claim 1, wherein the insulin is modified. 3. The oral preparation for treating diabetes according to claim 2, wherein the insulin is acylated. 4. The oral preparation for treating diabetes according to claim 1, wherein the hybrid liposome is prepared from one or more phospholipids and one or more surfactants. 5. The method according to claim 1, wherein the phospholipid is L-α-diacylphosphatidylcholine, L-α-diacylphosphatidylglycerol, L-α-diacylphosphatidylserine, L-α-diacylphosphatidylethanolamine, sphingomyelin, L-α-diacyl. The oral preparation for treating diabetes according to any one of claims 1 to 4, wherein the oral preparation is selected from the group consisting of phosphatidic acid, L-α-diacylphosphatidylinositol and cardiolipin. 6. The oral preparation for treating diabetes according to claim 4, wherein the surfactant is a micellar surfactant. 7. The oral preparation for treating diabetes according to claim 6, wherein the micelle surfactant is a non-sugar micelle surfactant. 8. The oral preparation for treating diabetes according to claim 7, wherein the non-sugar micellar surfactant is selected from the group consisting of polyoxyethylene sorbitan fatty acid ester, sorbitan fatty acid ester and alkyl or aryl polyoxyethylene glycol. 9. The hybrid liposome according to claim 1, further comprising a sugar micelle surfactant.
Item 2. The oral preparation for treating diabetes according to item 1. 10. The sugar micelle surfactant is selected from the group consisting of alkylglucoside, alkylthioglucoside, cerebroside, alkylmaltopyranoside, fructofuranosylalkylglucopyranoside and alkyllactopyranoside. Oral preparation for treating diabetes.