JPH0219092B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a polypeptide preparation for nasal administration containing a physiologically active polypeptide and a cyclodextrin. It has been known that pharmaceutical compounds that are highly hydrophilic and have a low oil-water partitioning ratio are not absorbed or are extremely difficult to absorb from the digestive tract. Physiologically active polypeptides are generally highly hydrophilic and have a low oil-water partitioning ratio, and are also subject to hydrolysis by enzymes within or on the walls of the gastrointestinal tract, making absorption from the gastrointestinal tract extremely difficult. Therefore, in order to expect sufficient medicinal efficacy, administration of these physiologically active polypeptides has been limited to injections. However, administration by injection is limited to specialists and is painful to the recipient, so a formulation that is simpler and easier to apply is desired, especially during continuous administration. On the other hand, cyclodextrin is a cyclic oligosaccharide and as a monomolecular host molecule with a hydrophobic cavity in the molecule, it forms inclusion compounds with fat-soluble drugs, increasing the solubility of drugs, stabilizing them, and promoting biological utilization. It is an extremely safe compound that is well known to exhibit effects such as improving bioavailability and reducing bitterness [Pharmaceutical Journal Vol. 101, No. 857
Page (1981)]. However, cyclodextrins do not form packaging compounds with water-soluble drugs such as physiologically active polypeptides, and to date, they have not been studied as mucosal absorption enhancers for these drugs. In order to effectively exert the pharmacological effects of physiologically active polypeptides that are poorly absorbed in the gastrointestinal tract, the present inventors have conducted intensive research on formulations that improve bioavailability. It has been found that the absorption of the polypeptide is significantly increased when administered nasally by the concomitant use of cyclodextrin, which has almost no interaction.Based on this, further research has led to the completion of the present invention. The present invention is a nasal preparation containing a physiologically active polypeptide and a cyclodextrin. Examples of the physiologically active polypeptide used in the present invention include those that constitute two or more peptides. Examples of the polypeptide include those having strong hydrophilicity and a low oil-water partitioning ratio. More specifically, the oil-water partition ratio between octanol and water is approximately 0.1.
These include: The polypeptide preferably has a molecular weight of about 200 to 60,000. Specific examples of the physiologically active polypeptide include L-pyroglutamyl-L-histidyl-L-prolinamide (thyrotropin releasing hormone; hereinafter abbreviated as "TRH") or salts thereof. , especially tartrate (JP-A-Sho
50-121273) or the formula () [In the formula, A represents hydrogen, alkyl, aralkyl, alkoxyalkyl, hydroxyalkyl or alkoxy. R is

【formula】

[expression] or

[Formula] and X represents -CH ₂ -, -CH ₂ CH ₂ - or -S-. Each of R and the other constituent amino acid residues may be in the L form, D form, or racemic form. ] or a salt thereof (see JP-A-52-116465). In addition, in this specification, among the compounds represented by the above formula (), the following formula The compound is called "DN-1417". Furthermore, the polypeptide is luteinizing hormone-releasing hormone (hereinafter abbreviated as "LH-RH") or a homologue having a similar effect thereto, which has the formula () (Pyr) Glu-R ₁ −Trp−Ser−R ₂ −R ₃ −R ₄ −
Arg−Pro−R ₅ () [R ₁ is His, Tyr, Trp or p-NH ₂ −Phe,
R ₂ is Tyr or Phe, R ₃ is Gly or a D-type amino acid residue, R ₄ is Leu, Ile or Nle, R ₅ is Gly-
It represents NH- _R6 ( _R6 is H or a lower alkyl group with or without a hydroxyl group) or NH- _R6 ( _R6 has the same meaning as above). [U.S. Pat. No. 3,853,837,
4008209, 3972859, British Patent No. 1423083,
Proceedings of the National Academy of Sciences
National Academy of Sciences of the
United States of America) Volume 78 No. 650-6512
(1981)]. In the above formula (), the D-type amino acid residue represented by _R3 includes, for example, α-D-amino acids having up to 9 carbon atoms (e.g., D-Leu, Ile, Nle,
Val, Nval, Abu, Phe, Phg, Ser, Thr,
Examples include Met, Ala, Trp, α-Aibu, etc., and they may have an appropriate protecting group (eg, t-butyl, t-butoxy, t-butoxycarbonyl, etc.). Of course, acid salts and metal complex compounds of peptide (2) can also be used in the same way as peptide (2). Regarding the amino acids, peptides, protecting groups, etc. in the polypeptide represented by formula (), when abbreviations are used, IUPAC-IUB Commission on
The abbreviations are based on BiologiCal Nomenclature or the abbreviations commonly used in the field, and when an amino acid may have optical isomers, the L form is indicated unless otherwise specified. In addition, in this specification, in the above formula (), R ₁ =His, R ₂ =Tyr, R ₃ =D-Leu, R ₄ =
The polypeptide in which Leu, _R5 = _NHCH2 - _CH3 is referred to as "TAP-144." Furthermore, the polypeptides include, for example, insulin, somatostatin, growth hormone,
prolactin, adrenocorticotropic hormone (ACTH), melanocyte stimulating hormone (MSH),
Thyroid stimulating hormone (TSH), luteinizing hormone (LH), follicle stimulating hormone (FSH), vasopressin, vasopressin derivatives {see Desmopressin [Journal of the Japanese Endocrine Society, Vol. 54, No. 5, pp. 676-691 (1978)]} , oxytocin, calcitonin,
Parathyroid hormone, glucagon, gastrin, secretin, pancreozymine, cholecystokinin, angiotensin, human placental lactogen,
Human chorionic gonadotropin (HCG), enkephalin, enkephalin derivatives [U.S. Pat.
4277394, European Patent Application Publication No. 31567], endorphin, interferon (α type, β type, γ type), urokinase, kallikrein, thymopoietin, thymosin, motilin,
Examples include deinorufin, bombesin, neurotensin, caerulein, pradeikinin, supstans P, kyotorfuin, nerve growth factor, and the like. Examples of the cyclodextrin used in the present invention include various cyclodextrin derivatives obtained by hydrolyzing starch with acid or amylase. As the cyclodextrin, for example, α
(degree of polymerization 6), β (degree of polymerization 7), and γ (degree of polymerization 8) [Pharmacia Vo1.16, No.1
(1980), Pharmaceutical Journal Vol. 101 (10), 857-873 (1981)
,
See Special Publication No. 53-31223]. Examples of the cyclodextrin derivative include tri-O-methylcyclodextrin [chemical pharmaceutical bulletin (Che-
micical & Pharmaceutical Bulletin) Volume 28 1552
- p. 1558 (see 1980), Triaminocyclodextrin [Angewandte Chemie: International Edition
in English), Volume 19, pp. 344-362 (1980)
reference. ] etc. As the cyclodextrin used in the present invention, α-cyclodextrin is particularly preferred. The nasal preparation of the present invention can be manufactured according to a method known per se. For example, small amounts of pH regulators, preservatives or thickeners (such as natural gums, cellulose derivatives, acrylic acid polymers, vinyl polymers, etc.) or excipients are added. The polypeptide-containing preparation for nasal administration of the present invention is formed into a solid, liquid or semi-solid form. In the case of a solid, the above-mentioned components may be mixed to form a simple powder composition, or a freeze-dried product may be used, preferably having a particle size of about 20 to 250 microns.
In the case of a liquid, an aqueous solution, aqueous suspension, or oil suspension is preferable. If it is semi-solid, a water-based or oil-based gel or softener is preferable. The proportion of each component in the preparation is, in the case of solid form, the polypeptide in the preparation about 0.005 to 50 W/V%, more preferably about 0.01 to 30 W/V%, and the cyclodextrin about 2 to 99.995%. W/V%, more preferably about 5 to 99.99 W/V%. When in liquid or semi-solid form, the amount of polypeptide in the formulation is about 0.01 to 50 W/V%, more preferably about 0.05 to 40 W/V%, and the amount of cyclodextrin is about 0.5 to 50 W/V%. /V%, more preferably about 1 to 30 W/V%. Solid preparations can be produced by methods known per se.
For example, the polypeptide dissolved in a small amount of water is gradually added to a mixture of cyclodextrin and, if necessary, excipients added to a mixer and kneaded. Thereafter, this is dried at an appropriate temperature under vacuum, and the dried product is pulverized to obtain a solid preparation. Alternatively, add water to the mixed powder of the polypeptide and cyclodextrin, and if necessary, excipients to completely dissolve them, then dehydrate and dry by freeze-drying or spray-drying, and crush the dried product appropriately to obtain a solid preparation. obtain. Examples of such excipients include glucose, mannitol, inositrol, steamed sugar, lactose, fructose, starch, cornstarch, microcrystalline cellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, and the like. The liquid preparation can be produced by a method known per se. For example, an aqueous solution for nasal administration can be prepared by dissolving, suspending, or emulsifying the polypeptide and cyclodextrin in water, a buffer, or an aqueous solution. In addition, an oily suspension for nasal administration can be produced by suspending or emulsifying the polypeptide and cyclodextrin in an oily base. As the buffer solution, for example, Sorensen buffer solution [Ergeb.
Physiol. 12 , 393 (1912)], Clark-Lubs buffer [J.Bact. 2 , (1), 109,
191 (1917)] Macll-vaine buffer [J.Biol, Chem. 49 , 183 (1921)], Michaelis buffer [Die
Wasserstoffionenkomzentration, p.186
(1914)], Kolthoff buffer [Bio-
chem.Z, 179 , 410 (1926)].
Examples of oily bases include sesame oil, olive oil,
Examples include corn oil, soybean oil, cottonseed oil, peanut oil, lanolin, petrolatum, paraffin, isopar, silicone oil, and fatty acids having 6 to 30 carbon atoms or their glycerin or alcohol esters, and these may be used alone. You may also use a mixture of two or more types. As a method for producing a semi-solid preparation, an aqueous or oil-based gelatin or ointment can be produced by a method known per se. For example, for an aqueous gel for nasal administration, an aqueous solution or suspension of cyclodextrin is prepared, and if necessary, a PH adjusting agent, preservative, etc. are added to this. This solution is divided into two parts, an aqueous gel base is dissolved or dispersed in one part, and a stable gel is formed by heating or cooling appropriately. An aqueous gel can be prepared by dissolving the polypeptide in the other solution and uniformly mixing the two. Adjustment of the PH in the above can be done by using acids, bases,
This can be done by adding a buffer or the like during the manufacturing process of the preparation. Examples of acids used to adjust pH include inorganic acids (e.g., hydrochloric acid, boric acid, phosphoric acid, carbonic acid, bicarbonate, etc.), amino acids, or organic acids (e.g., monocarboxylic acid, oxycarboxylic acid, polycarboxylic acid). Examples of the base include sodium hydroxide, potassium hydroxide, sodium bicarbonate, and sodium carbonate, and examples of the buffer include those similar to the above-mentioned buffers. Examples of the aqueous gel base include natural gums (e.g., gum tragacanth, gum acacia, gum karaya, Irish moss, guaiac gum, xanthan gum, locust bean gum, etc.), cellulose derivatives (e.g., methylcellulose, carboxymethylcellulose, etc.), and acrylic acid polymers. (e.g., polyacrylic acid, polymethacrylic acid, etc.) Vinyl polymers (e.g., polyvinylpyrrolidone, polyvinyl alcohol, polyvinyl methyl ether, carboxypolymethylene, etc.), synthetic polysaccharides (e.g., polysucrose, polyglucose, polylactose) etc.), starch, dextrin, pectin, sodium alginate, etc. These bases can be used as a mixture of two or more types as appropriate. An oily ointment for nasal administration can be produced by uniformly dispersing cyclodextrin and the polypeptide in a heat-molten oily base and cooling the mixture while stirring. Examples of the oily base include those similar to those mentioned above. A preservative may be added to the preparation for nasal administration, and examples of the preservative include paraoxybenzoic acid esters, phenolic substances such as phenol and cresol, and alcohols such as chlorobutanol, phenitylethyl alcohol, and propylene glycol. Inverse soaps such as benzalkonium chloride and benzethonium chloride, acids such as benzoic acid, sorbic acid, dehydroacetic acid, sulfurous acid and salts thereof, and sodium hydrogensulfite or salts thereof can be used. In the case of a solid preparation, the method for administering the nasal preparation of the present invention is to set a powder-filled capsule in a special spray device equipped with a needle and pierce the needle into the upper and lower portions of the capsule. Examples include making tiny holes, then blowing air in with a rubber bulb to squirt the powder into the nasal cavity. In the case of liquid formulations, methods include placing the formulation in a nasal dropper, spray bottle, or similar container suitable for intranasal application of such liquid formulations, and administering the formulation dropwise or by squirting into the nasal cavity. In the case of semi-solid preparations, the preparation is filled into a tube and when administered, an applicator is attached to the mouth of the tube and administered directly into the nasal cavity, or a fixed amount of the preparation is taken into the nasal cavity using an intranasal insert and administered into the nasal cavity. Examples include. The amount of polypeptide administered varies depending on the type or disease condition, but the amount of the preparation per dose is approximately 5 mg to 100 mg for solid preparations, approximately 0.05 ml to 0.5 ml for liquid preparations, and approximately 0.05 ml to 0.5 ml for semi-solid preparations. For formulations approx.
A suitable range is 50mg to 500mg. The present invention has the following features. 1. High bioavailability can be obtained by administering physiologically active polypeptides with poor gastrointestinal absorption through routes other than injection. 2. Physiologically active polypeptides can be easily administered without pain during administration. 3. If continuous multiple administration is required, patients can easily administer the drug themselves and can perform home therapy. 4. Cyclodextrin used as an absorption enhancer is tasteless and odorless, has little toxicity, and has almost no mucous membrane irritation, so it is possible to produce extremely safe preparations even when administered multiple times. Hereinafter, the present invention will be explained in more detail with reference to experimental examples and examples. In addition, below, percentage (%) representing concentration represents weight/volume percentage (W/V%). Experimental Example 1 SD male rats weighing approximately 250 g (more than 3 rats in each group) were fasted for 16 hours and were anesthetized with pentobarbital.
of Pharmaceutics) Vol. 7, p. 317 (1981), after performing the surgery for nasal administration, administer insulin in a volume of 0.1 ml/kg using a micropipette directly from the external nostril. It was administered intranasally, and blood was collected from the tail vein over time to measure blood sugar levels. In addition, as an insulin administration solution, porcine insulin 10U or 20U (approximately 0.2mg or 0.8mg),
0 α-, β- or γ-cyclodextrin
mg~10mg [equivalent to 0~10%] in PH7.4 isometric buffer
A solution dissolved in 0.1 ml was used. However, in the case of β-cyclodextrin, the saturation solubility is approximately 1.8%.
Therefore, when the concentration was higher than that, it was administered as a suspension. As a control, blood glucose levels were measured in the same manner after intravenous administration of insulin. The results are shown in Table-1. As shown in Table-1, α
The addition of -, β-, or γ-cyclodextrin resulted in a significant drop in blood sugar compared to the case without the addition, indicating that insulin was effectively absorbed through the nasal mucosa.

[Table] Experimental Example 2 An amount equivalent to 2 mg/Kg of ¹⁴ C-DN-1417 and 5 mg (equivalent to 5%) of α-cyclodextrin were dissolved in 0.1 ml of physiological saline, and the same procedure as shown in Example 1 was carried out. Inject 0.1ml into the rat nasal cavity with a micropipette using the method described in
was administered, blood was collected from the tail vein over time, and the total radioactivity in blood sugar was measured to determine the blood concentration. As a comparative experiment, similar experiments were conducted with subcutaneous administration of the same dose and nasal administration without addition of α-cyclodextrin. The results are shown in Table 2, and it is clear that nasal administration of the formulation of the present invention significantly increases the absorption of peptides, and the bioavailability for subcutaneous administration ranges from about 10% to about 50%. %, an increase of five times.

[Table] Experimental Example 3 100 μg of TAP-144 and 5 mg of α-cyclodextrin were dissolved in 0.1 ml of physiological saline, and an amount equivalent to 0.1 ml/Kg was intranasally administered to rats in the same manner as in Example 1. (The dose of TAP-144 corresponds to 100 μg/Kg). Blood was collected from the tail vein over time, and TAP-144 in the serum was quantified by radioimmunoassay. As a comparative experiment, the same dosage was administered subcutaneously or a preparation without α-cyclodextrin was administered nasally. The results are shown in Table 3. When the composition of the present invention is administered nasally, the absorption of the peptide is significantly increased, and the bioavailability compared to subcutaneous administration is approximately
It has become clear that this will increase by 3.5 times from 20% to approximately 70%.

[Table] Example 1 5000 U (approximately 200 mg) of porcine insulin was dissolved in 8 ml of isotropic phosphate buffer of pH 7.4, and 500 mg of α-cyclodextrin and 20 mg of chlorobutanol were added and dissolved completely, followed by physiological saline. A solution of 10 ml was made with water. Pour this into a nasal spray container, about 0.1ml per dose.
Administer by injection. Example 2 200 mg of DN-1417, 200 mg of mannitol and 200 mg of β-cyclodextrin were dissolved in 40 ml of purified water and freeze-dried. The obtained dried product is crushed and about 20
It was made into a powder with a particle size of ~250 microns. Among them
30 mg was filled into a No. 4 hard gelatin capsule. For administration, this capsule is placed in a special spray tool equipped with a needle to make a hole in the capsule and a rubber bulb to send air, then holes are made at both ends of the capsule, and the rubber bulbs are pressed to send the air to the destination. Administer the powder into the nasal cavity from the end. Example 3 Methylparaben 0.12%, propylparaben 0.01
1 g of α-cyclodextrin and 2 g of TAP-144 were dissolved in 16 ml of PH7.4 isotropic buffer, and methylcellulose (Metrose) was added to this.
Add 200 mg of 90SH4000 (manufactured by Shin-Etsu Chemical Co., Ltd.) and stir well to make a uniform viscous solution.
It was set to 20g. Fill 100 mg of this into a nasal applicator and administer into the nasal cavity. Example 4 In natural LH-RH [general formula ()], R ₁
= His, R ₂ = Tyr, R ₃ = Gly, R ₄ = Leu, R ₅ =
500 mg of peptide Gly-NH ₂ and 1 g of α-cyclodextrin were placed in a mortar, 1 g of lanolin dissolved by heating was added, and the mixture was thoroughly mixed and dispersed. Next, under stirring Miglyol 812 [Dynamit Novel (Dyna
- Mit Nobel, West Germany] was gradually added to the mixture to give a total weight of 10 g to form an oil-based suspension. Place this in a container with a dropper and administer 0.1g directly into the nasal cavity each time. Example 5 50 mg of α-cyclodextrin and 100,000 U of α-interferon (interferon derived from human leukocytes) were dissolved in 1 ml of physiological saline to form a solution. Place this in a nasal dropper and administer 0.1ml into the nasal cavity. Example 6 2 mg of desmopressin and 1 g of γ-cyclodextrin were dissolved in 10 ml of physiological saline, and 100 mg of methylcellulose was added to make a viscous liquid. Of this
Take 0.2ml with an applicator and administer directly into the nasal cavity. Example 7 1 g of enkephalin and 3 g of α-cyclodextrin were dissolved in physiological saline to form a solution. Place this in a spray container and spray 0.2ml into the nasal cavity each time.

Claims (1)

[Claims] 1. A nasal preparation containing a physiologically active polypeptide and cyclodextrin.