Medicinal compositions for nasal absorption

专利摘要:
The present invention relates to a nasal absorption pharmaceutical composition, and when administered intranasally (administration), the present invention relates to a nasal absorption pharmaceutical composition having an excellent absorption rate of a physiologically active polypeptide contained as an active ingredient in vivo. More specifically, in the pharmaceutical composition, a bioactive polypeptide having an isoelectric point of 7 or less is a water-insoluble or poorly water-soluble polyvalent metal compound carrier, for example, a divalent or higher metal compound such as an aluminum compound, a calcium compound, a magnesium compound, silicon The compound, the iron compound or the zinc compound is uniformly dispersed or embedded by using an additive capable of dispersing or embedding the polypeptide on the surface of the carrier.
公开号:KR20040058324A
申请号:KR10-2004-7007961
申请日:2002-11-26
公开日:2004-07-03
发明作者:미나미따께요시하루；쯔까다요시오；가나이야스시；야나가와아끼라
申请人:다이이찌 산토리 파마 가부시키가이샤；
IPC主号:

专利说明:

Nasal absorption pharmaceutical composition {MEDICINAL COMPOSITIONS FOR NASAL ABSORPTION}
[2] Bioactive polypeptides are high molecular weight compounds that exhibit a variety of specific pharmacological activities and are very useful compounds that have been used in various medical fields. For example, peptide hormones derived from glucagon-like peptide I (hereinafter referred to as GLP-1), glucagon precursor (proglucagon) are known (Bell et al., Nature, 304 , 368, 1983). Proglucagon, found in mammals, is a precursor protein of 160 amino acids and is produced in pancreatic islets (islets of Langerhans) A-cells and intestinal L-cells. In the pancreas, proglucagon is processed as a glucagon and a major proglucagon fragment by a processing enzyme, whereas in the intestine proglucagon is treated in a different way, resulting in glycentin, GLP-1 and glucagon-like peptides. -2 (hereinafter referred to as GLP-2). [Mojsovr et al., J. Biol. Chem., 261 , 11880, 1986].
[3] Peptides (corresponding to GLP-1) formed from 72-108 amino acids in proglucagon exhibit activity of promoting insulin secretion. In addition, GLP-1 (7-37) formed by removing the first six N-terminal amino acids from GLP-1, and GLP-1 resulting as a result of amidation of GLP-1 (7-37) at position 36 (7-36) is the most effective insulin secretagogue among known insulin secretagogues [Mojsovr et al., J. Clin. Inves., 79 , 616, 1987]. It also has the activity of inhibiting glucagon secretion. GLP-1 is known to be secreted from the intestinal L-cells into the blood circulation in the form of GLP-1 (7-36) NH ₂ [Gutniak et al., N. Engl. J. Med., 326 , 1316, 1993].
[4] GLP-1 (7-36) NH ₂ is immediately secreted from intestinal L-cells in response to stimulation of food intake and acts on the pancreas to promote insulin secretion. At the same time, it acts to reduce glucagon secretion, to increase mRNA expression in insulin-secreting cells, to reduce glucose gluconeogenesis in the liver, and to inhibit the activity of the gastrointestinal tract. This means an important role in vivo of GLP-1 including GLP-1 (7-36) NH ₂ as an incretin (insulin secretion stimulant) made to meet the requirements of energy metabolism in the human body.
[5] The peptides having the physiological activity described above have been found to be used as a medicament for the treatment of diabetes. Specifically, GLP-1 (7-36) NH ₂ may be administered before meals to inhibit postprandial elevations of blood glucose levels, thus acting as an effective treatment for patients with type II diabetes. Sulfonyl urea agents with similar activities that promote insulin secretion are at risk of excessively low blood sugar levels, because they are active regardless of blood sugar levels. When administered over a long period of time, the medicament can also make the insulin-producing cells less active. Conversely, GLP-1 (7-36) NH _{2 results} in excessively low blood glucose because the activity of GLP-1 (7-36) NH ₂ that promotes insulin secretion is regulated by an antagonistic mechanism that reflects blood glucose levels. I never do that. In addition, GLP-1 (7-36) NH ₂ stimulates insulin-producing cells. Thus, there is a clear contrast between GLP-1 (7-36) NH ₂ and sulfonyl urea agents currently being used clinically as diabetic agents.
[6] GLP-1 (7-36) NH, including inhibition of glucose synthesis by the liver, activation of insulin-producing cells, promotion of sugar uptake by muscles, inhibition of gastrointestinal tract activity, and suppression of appetite through the central nervous system _In addition to the activity of correcting post-prandial blood sugar levels, the various activities of ₂ may be achieved by prolonged administration of GLP-1 (7-36) NH ₂ to normalize and activate the integrity of systemic glucose metabolism, It has the expectation that one of the main factors can suppress obesity.
[7] Other peptides with incretin activity (ie, promotion of insulin secretion) similar to GLP-1 (7-36) NH ₂ include exendin-4, which is a Gila monster. It was separated from the structure and its structure was determined. The peptide is less degraded than GLP-1 (7-36) NH ₂ in plasma and thus can maintain its activity to promote insulin secretion for a long time. As in GLP-1 (7-36) NH ₂ , exendin-4 is known to induce differentiation / neogenesis of β-cells.
[8] Since the eighth position of GLP-1 (7-37) NH ₂ and GLP-1 (7-36) NH ₂ is cleaved by dipeptidyl peptidase IV (DPP-IV) present in vivo, Ala at the eighth position Derivatives substituted by amino acids that are not readily cleaved, such as Val, ie [Val ⁸ ] -GLP-1 (7-37), and fatty acids are used to delay the apparent plasma half-life by controlling dissolution rate. Derivatives modified with GLP-1, namely [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37), are expected to have the same effect. On the other hand, recent studies have shown that GLP-1 (9-37) cleaved at the eighth position has the same effect on reducing blood glucose levels [Deacon et al., Am. J. Physiol. Endocrinol. Matb. 282 , 873-879, 2002. Due to this finding, the conversion of amino acid residues of GLP-1 (7-36) NH ₂ at the eighth position, and the modification by fatty acids of GLP-1 (7-36) NH ₂ , are advantageous in delaying the apparent plasma half-life. You need to check the point.
[9] Gastric inhibitory polypeptide (GIP, hereinafter), a peptide that stimulates glucose-dependent insulin secretion, distinguishes it from GLP-1 and exendin-4 in that, in addition to insulin secretion promoting activity, the peptide may promote glucagon secretion. do.
[10] Among the peptide incretins, GLP-1 (7-36) NH ₂ has a common amino acid sequence in mammals and is considered an ideal drug for diabetes. However, GLP-1 (7-36) NH ₂ is hardly absorbed from the gastrointestinal tract due to its nature as a peptide. This is a significant obstacle to the development of these peptides as diabetic agents.
[11] As is known, in addition to oral administration, the peptides can be administered transdermally by subcutaneous injection. However, long-term subcutaneous injections must be controlled under the physician's observations and, given the burden of hospitalization and pain at the injection site, injection is not a suitable route for long-term administration of diabetes treatment agents. If the peptide is administered after each meal, subcutaneous injection given three times daily is clearly not a viable method of administering the peptide, although it is possible to effectively correct hyperglycemia levels. In addition, self-regulating insulin injections administered to type I diabetic patients as well as type II diabetic patients cannot be used in combination with self-regulating type GLP-1 injection formulations.
[12] In order to solve the problems disclosed above, attempts have been made to allow GLP-1 (7-36) NH ₂ to be absorbed through the oral mucosa in the form of a patch preparation [Gutniak et al. , Diabetes care , 20 , 1874, 1997 ]. However, certain specific dosage forms involve the use of absorption enhancers, such as sodium taurocholate (very stimulating in the type of bile acids). As a result, irritation is inevitable and the mucous membranes can be destroyed, making the route of administration unsuitable for long term administration.
[13] Thus, to date, practical non-injection of GLP-1 (7-36) NH ₂ and other peptide incretin administration, which is safe, achieves high bioavailability and is suitable for frequent drug delivery. ) no method. Development of such a method is expected.
[14] Unlike low molecular weight compounds, bioactive polypeptides are not effectively administered by routes of administration other than injection. The main cause is that bioactive polypeptides are easily digested by the digestive enzymes present in the stomach and small intestine and the large intestine, or in the absorbing epithelium of these organs, nasal and lungs, and the polypeptides, due to their high molecular weight, have a typical transport route. It is not transported through. For this reason, nasal peptide preparations for nasal absorption have recently been proposed as a practical non-injection method of peptide administration. Usually, the nasal peptide preparation is administered by spraying the peptide solution into the nasal cavity using a nebulizer in the presence of an absorption enhancer. One problem with this method is that, among the various known bioactive polypeptides, certain peptides, including GLP-1 (7-36) NH ₂ , having an isoelectric point (pI) in the acidic or neutral range, become unstable in acidic or neutral solutions. Will be.
[15] For example, in the observation by the inventors, when administered to rats and other animals nasally, the solution of GLP-1 (7-36) NH ₂ is absorbed to some extent, whereas the solution The peptide becomes insoluble when stored for 10 hours (see Reference Example below). Thus, although the formulation is in the form of dissolving the peptide each time it is used, the solution formulation is not suitable for use as a pharmaceutical composition.
[16] Likewise, glucagon and insulin have an isoelectric point in the acidic or neutral pH range and are known to not dissolve or crystallize in acidic or neutral solutions. Many of these peptides are known to not dissolve or crystallize in acidic or neutral solutions because they have an isoelectric point in the acidic or neutral pH range. Thus, it is practically impossible to administer the peptide nasally in the form of an acidic or neutral solution formulation.
[17] On the other hand, acidic bioactive polypeptides dissolve very well in alkaline (basic) solutions. However, when an acidic bioactive polypeptide is exposed to a basic solution, the acidic bioactive polypeptide is not only susceptible to degradation, such as hydrolysis (even in an acidic environment), but also tends to racemize. As a result, the above chemical stability is reduced. Both acidic and basic bioactive polypeptides can undergo this side reaction. For example, casein (pI = about 4.6), a basic bioactive polypeptide, is known that its amino acid residues of aspartic acid, phenylalanine, glutamic acid and alanine become unstable as racemization proceeds in basic solutions [Friedman et al., Et al.J. Food Sci., 47 , 760-764, 1982]. Organic acids include a variety of substances, including acetic acid and butyric acid, each being a biological compound and long chain carboxylic acids such as octanoic acid and decanoic acid (both nutrients). Many of these organic acids can be used as additives in pharmaceutical compositions. On the other hand, many organic bases such as serotonin and dopamine are known to exhibit pharmacological activity. Alkaline metals such as sodium are in many cases unsuitable for use as additives in pharmaceutical compositions because they make it difficult to control the pH of the composition and tend to form salts with acidic peptides, thus affecting the properties of the peptides. Because it is crazy. For this reason, given the range of chemical stability and selection of additive components, it is not desirable to provide acidic bioactive polypeptides in the form of alkaline solutions.
[18] As disclosed above, it is not desirable to provide the acidic bioactive polypeptide in the form of an acidic or neutral solution or in the form of an alkaline solution. Thus, acidic bioactive polypeptides are not suitable for use in nasal solution formulations.
[19] For certain types of bioactive polypeptides, such as insulin, calcitonin, parathyroid hormone (PTH), human growth hormone (HGH) and hypothalamic hormone (LH-RH), non-absorbable powder formulations may be used as the solution- It has been proposed as an alternative to nasal preparations. Many compounds have been investigated for their ability as carriers for such powder formulations for non-administration, whereby different powder compositions using several different carriers have been proposed to date for nasal administration of the bioactive polypeptide.
[20] Through the course of the study, it has been found that substances which are insoluble in water or poorly in water but soluble in water under acidic conditions can be used as very effective carriers for non-administered powder formulations of bioactive polypeptides. For example, as a carrier, polyvalent metal compounds such as hydroxyapatite and calcium carbonate [Japanese Patent Laid-Open No. 8-27031], substances having the ability to regenerate or protect mucous membranes, especially gastric mucosa [Japanese Patent Laid-Open No. Pseudo 9-255586], or a pharmaceutical composition for non-administration using a powder of grain [Japanese Patent Laid-Open No. 2000-239187] is proposed.
[21] However, powder formulations prepared by dispersing and adsorbing GLP-1 (7-36) NH ₂ on a carrier such as the polyvalent metal compound, when administered to animals at a costly rate of about 4% in dogs and GLP-1 (7-36) NH ₂ bioavailability of 15% or less in monkeys. Thus, the nasal absorption of the formulation is less than satisfactory.
[22] Bioactive polypeptides, such as GLP-1 (7-36) NH ₂ , having isoelectric points in the acidic or neutral pH range, have low solubility in the acidic or neutral pH range and tend to aggregate even when dissolved in solution. Such polypeptides, when administered orally in the form of a solution, as well as when administered nasally in the form of a powder formulation, do not obtain sufficient bioavailability. Thus, an effective non-injection route for the peptide administration has not yet been established.
[23] A non-administration composition consisting of a cyclic peptide and a polyvalent metal composition carrier is disclosed in WO 01/52894 A2. It has also been described that absorption enhancers such as rice flour and starch may be added, wherein the particle size of the enhancer is preferably 250 μm or less, more preferably 20 to 180 μm. However, there is no disclosure of a method of improving bioavailability by adding an enhancer having a particle size substantially the same as the carrier to a bioactive acid polypeptide having an isoelectric point of 7 or less.
[24] Accordingly, it is an object of the present invention to provide a pharmaceutical composition that enables non-administration of a bioactive polypeptide having an isoelectric point in the acidic or neutral pH range. Such polypeptides, when administered orally or through other non-injection routes of administration, have poor bioavailability, have low solubility in the acidic or neutral pH range, and tend to aggregate when dissolved in solution. The pharmaceutical composition is safe and does not cause irritation and has high bioavailability.
[25] In an effort to find a method for achieving the above object, the present inventors investigated the performance of the peptide as a component of an oral composition for possible additives. First, it was investigated whether starch could contribute to the stabilization of the peptide as an additive.
[26] Starch, which is rich in nutrients in cereals, is a substance that can be stably used as an additive in nasal absorption compositions. Starch consisted of amylose (consisting of glucose units linked by α-1,4 bonds to form a straight chain) and amylopectin (branched including α-1,6 bonds).
[27] To prepare a nasal absorption pharmaceutical composition, a multi-metallic compound is used as a carrier in combination with various different types of starch (containing amylose and amylopectin in various proportions) as additives. The nasal absorption rate was investigated for each pharmaceutical composition. The effect of the particle absorption of starch as an additive in the pharmaceutical composition was also investigated.
[28] As a result, the inventors have found that administration of the following powder nasal preparations into the nasal passages can achieve improved nasal absorption and thus provide effective clinical treatment. The composition comprises acidic bioactive polypeptides such as GLP-1 (7-36) NH ₂ , including rice flour (Domyo-ji powder), corn starch, potato starch, and their pregelatinized or partially gelatinized starches (respectively, Under the aid of additives such as silver amylose and amylopectin in specific proportions, powder or crystalline polyvalent metal compound carriers (e.g., divalent or higher valent metals) that are water insoluble or poorly water soluble and have an average particle size Compounds, such as calcium compounds), by uniformly dispersing and embedding on the surface.
[29] In addition, the present inventors have found that when a polyvalent metal compound (average particle size of 100 µm or less) such as calcium carbonate is used as the water insoluble starch as an additive and a carrier, the water insoluble starch having a smaller particle size than the carrier is GLP-1 ( 7-36) it has been found to exhibit a noticeable absorption promoting effect on acidic peptides such as NH ₂ . The present invention has been completed based on the above findings.
[1] The present invention relates to a nasal absorption pharmaceutical composition, and more particularly, to a nasal absorption comprising a bioactive acid polypeptide having an isoelectric point of 7 or less as an active ingredient, and an additive for improving the bioavailability of the polypeptide. It relates to a pharmaceutical composition for.
[35] 1 is a view showing a change in the plasma concentration of GLP-1 (7-36) NH ₂ after subcutaneous administration in the second embodiment.
[36] FIG. 2 is a diagram showing changes over time of plasma concentrations of GLP-1 (7-36) NH ₂ against nasal administration of a composition without additives in Example 2. FIG.
[37] FIG. 3 is a diagram showing changes over time of plasma concentrations of GLP-1 (7-36) NH ₂ against nasal administration of a composition without additives, using sucralate as an additive in Example 2. FIG.
[38] FIG. 4 is a diagram showing changes over time of plasma concentrations of GLP-1 (7-36) NH ₂ against nasal administration of an additive-containing composition in Example 2. FIG.
[39] Best Mode for Carrying Out the Invention
[40] As disclosed above, the present invention allows for the non-administration of bioactive polypeptides having an isoelectric point of 7 or less, which have a high bioavailability, exhibit low solubility in the acidic or neutral pH range and tend to aggregate when dissolved in solution, Provide a pharmaceutical composition. Such polypeptides have poor bioavailability and are not suitable for oral or other non-injection administration. In a preferred detailed embodiment, the present invention relates to GLP-1 derivatives such as GLP-1, GLP-1 amide, GLP-1 (7-36) NH ₂ , GLP-1 (9-36) NH ₂ , GLP-1 (9-37), GLP-1 (7-37), [Val ⁸ ] -GLP-1 (7-36) NH ₂ , [Val ⁸ ] -GLP-1 (7-37), [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37), and GLP-2, exendin-3, exendin-4, glucagon, gastric inhibitory peptide ( Provided is a nasal absorption pharmaceutical composition containing GIP) or insulin.
[41] The high bioavailability of the nonabsorbable pharmaceutical composition of the present invention is due to the bioactive polypeptide dispersed and embedded on the carrier surface in a stable and uniform manner with the aid of an additive.
[42] When using water-insoluble or poorly water-soluble beta- or partially gelatinized starch as an additive, the use of rice starch or corn starch with a small average particle size can be achieved by expanding the superficial area and improving elution. Absorption can be improved. Therefore, additives having an average particle size of 1 µm to 20 µm are preferably used in the present invention to improve the absorption.
[43] Accordingly, the additive used in the present invention may be any additive capable of dispersing and embedding the bioactive polypeptide on the carrier surface in a stable and uniform manner. For example, starch containing amylopectin and amylose independently or in a specific ratio may be used as the additive. Starches obtained from rice, corn, and the like, are "nonglutinous rice" -type "glucose rice" -types containing amylopectin and amylose in a ratio of about 7: 3 to 8: 2. It is generally classified as starch. Specifically, examples of the additives used in the present invention include the following: rice flour, rice starch, corn starch, potato starch, beta-starch such as rice beta-starch (non-rice type), rice beta-starch (glucose rice type) ), Corn beta-starch (rice type), corn beta-starch (glucorice type), and potato beta-starch (rice type); Luxury rice starch (non-rice type), luxury rice starch (glutinous rice type), luxury corn starch (non-rice type), luxury corn starch (glutinous rice type), luxury potato starch (non-rice type), luxury wheat starch (non-rice type), and these Part of the luxury starch.
[44] Although poorly water soluble, starch can be gelled by heating with water and loosen its crystal structure. Both fully gelled starch (stabilized starch, or alpha-starch) and partially gelatinized starch can be used as additives of the present invention.
[45] Rice flour is made by grinding the albumen of rice seeds, which remain after the shells and embryos of the rice grains have been removed. Rice flour is rich in starch and commonly used in food and pharmaceutical additives. In the present invention, unheated rice flour consisting of beta-starch is preferred over heat-treated rice flour containing gelatinized starch (alpha-starch) or partially gelatinized starch, but heat-treated rice flour may be used. An example of preferred rice flour is Domyo-ji powder containing fine rice starch. In addition to corn starch consisting of beta-starch, partially gelatinized or gelatinized starch (alpha-starch) corn starch may be used in the present invention.
[46] Mixtures of the starch can also be used as additives for the non-composition of the present invention.
[47] In addition, oligosaccharides, carboxyvinyl polymers, povidones, hydroxypropylcellulose (HPC), xanthan gum, pectin, sodium alginate, gum arabic powder, and gelatin can be used as additions in the present invention.
[48] The bioactive polypeptide used in the nonabsorbable composition of the present invention has an isoelectric point (pI) of 7 or less. Such polypeptides exhibit low solubility in the acidic or neutral pH range and tend to aggregate even when dissolved in solution.
[49] Examples of preferred such bioactive polypeptides are shown below with their respective isoelectric points:
[50] GLP-1 (pI = 5.05); GLP-1 amide (pI = 5.47); GLP-1 (7-36) NH ₂ (pI = 6.76); GLP-1 (7-37) (pI = 5.53); GLP-1 (9-36) NH ₂ (pI = 4.68); GLP-1 (9-37) (pI = 4.87); [Val ⁸ ] -GLP-1 (7-36) NH ₂ (pI = 6.76); [Val ⁸ ] -GLP-1 (7-37) (pI = 5.53); [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37) (pI = 4.57); GLP-2 (pI = 4.45); Exendin-3 (pI = 4.96); Exendin-4 (pI = 4.96); Glucagon (pI = 6.75); And gastric inhibitory peptide (GIP) (pI = 6.92); Insulin (pI = 5.39).
[51] Other bioactive polypeptides can also be used which can be administered nasally, examples of which are below (pI means isoelectric point, MW means molecular weight, and all compounds are exendin-3 and exendin). From humans, except -4):
[52] Calcitonin (pI: 6.72, MW: 3420.88), catacalcin (pI: 5.26, MW: 2436.62), cholecystokinin-12 (pI: 3.93, MW: 1535.71), cholecystokinin-8 (pI: 3.56, MW: 1064.20), cortico Tropin-Lipotropin precursor (pI: 5.22, MW: 8469.32), corticotropin-like intermediate peptide (pI: 3.91, MW: 2309.51), reportropin-β (pI: 6.17, MW: 9805.94), report Lopin-γ (pI: 4.66, MW: 6074.57), melanotropin-β (pI: 5.57, MW: 2204.40), corticoriberine (pI: 5.09, MW: 4758.49), endothelin-1 (pI: 4.54, MW: 2495.94), endothelin-2 (pI: 4.54, MW: 2550.9), endothelin-3 (pI: 5.38, MW: 2647.09), galanin message-associated peptide (pI: 4.49, MW : 6671.52), gastrin-71 (pI: 5.17, MW: 8066.88), gastrin-34 (pI: 4.25, MW: 3867.26), gastrin-17 (pI: 3.40, MW: 2116.24), gastric inhibitory polypeptide (pI: 6.92 , MW: 4983.59), glycentin-related polypeptide (pI: 4.13, MW: 3384.50), glucagon (pI: 6.75, MW: 3482.79), glucagon-like peptide-1 (pI : 5.05, MW: 4167.02), glucagon-like peptide-1 amide (pI: 5.47, MW: 4111.50), glucagon-like peptide-1 (7-36) amide (pI: 6.76, MW: 3297.68), glucagon-like Peptide-1 (7-37) (pI: 5.53, MW: 3355.71), [Val ⁸ ] -glucagon-like peptide-1 (7-36) amide (pI: 6.76, MW: 3326.74), [Val ⁸ ]- Glucagon-like peptide-1 (7-37) (pI: 5.53, MW: 3383.87), [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37 ) (pI: 4.57, MW: 3751.2), GLP-1 (9-36) NH ₂ (pI: 4.68, MW: 2933.2), GLP-1 (9-37) (pI: 4.87, MW: 3090.4), glucagon -Like peptide-2 (pI: 4.21, MW: 3922.35), exendin-3 (pI: 4.96, MW: 4202.63), exendin-4 (pI: 4.96, MW: 4186.60), insulin β-chain (pI: 6.90, MW: 3429.96), insulin α-chain (pI: 3.79, MW: 2383), insulin (pI: 5.39, MW: 5807.6), progonadoriberine-I (pI: 5.63, MW: 7893.83), gondori Berine-II (pI: 6.92, MW: 1254.33), gonadoriberin-related peptide-I (pI: 4.67, MW: 6370.11), neuromedin C (pI: 6.92, MW: 1121.28), insulin-like group Vaginal (INSL) A-chain (pI: 6.36, MW: 3542.16), motilin-related peptide E (pI: 4.72, MW: 7433.47), leucine-enkephalin (pI: 5.52, MW: 555.63), methionine-enkephalin ( pi: 5.52, MW: 573.66), leumorphine (pI: 6.21, MW: 3351.68), oxytocin (pI: 5.51, MW: 1010.19), neuropisin-1 (pI: 4.94, MW: 9600.88), neuropisin-2 (pI: 5.05, MW: 9787.07), copeptin (pI: 4.11, MW: 4021.46), neuromedin B (pI: 6.74, MW: 1133.29), neuromedin N (pI: 5.52, MW: 617.79), neuropeptide Y (pI: 6.76, MW: 4272.72), neuropeptide AF (pI: 6.05, MW: 1979.18), PACAP-related peptide (pI: 5.38, MW: 4800.32), pancreatic hormone (pI: 6.26, MW: 4182.74), Pancreatic 20-icosapeptide (pI: 6.01, MW: 2235.44), peptide YY (pI: 6.77, MW: 4310.80), tyroriberine (pI: 6.74, MW: 380.40), neuroquinine A (pI: 6.74, MW: 1134.32), Eurocortin (pI: 5.58, MW: 4697.29), Eurotensin II (pI: 4.37, MW: 1390.59), Intestinal peptide (PHM-27) (pI: 6.75, MW: 2986.43) , And enteric peptide 42 (pI: 6.76, MW: 4552.1 8).
[53] In addition to those indicated above, the compositions of the present invention may be any bioactive peptide that can be administered nasally.
[54] In the present invention, the carrier supporting the physiologically active polypeptide together with the additive includes a carrier which is water insoluble or poorly water soluble. For example, a polyvalent metal compound having two or more valences selected from aluminum compounds, calcium compounds, magnesium compounds, silicon compounds, iron compounds, or zinc compounds can be used.
[55] Specifically, examples of each type of the polyvalent metal compound are as follows:
[56] As said aluminum compound, dry aluminum hydroxide gel, aluminum chloride hydroxide, synthetic aluminum silicate, hard aluminum oxide, colloidal hydrous aluminum silicate, aluminum magnesium hydroxide, aluminum hydroxide, aluminum hydroxide gel, aluminum sulfate, dihydroxy aluminum Acetates, aluminum stearate, natural aluminum silicate, aluminum monostearate, and potassium aluminum sulfate.
[57] Examples of the calcium compound include apatite, hydroxyapatite, calcium carbonate, calcium disodium edetate, calcium chloride, calcium citrate, calcium glycerophosphate, calcium gluconate, calcium silicate, calcium oxide, calcium hydroxide, calcium stearate, 3 Primary calcium phosphate, calcium lactate, calcium pantothenate, calcium oleate, calcium palmitate, calcium D-pantothenate, calcium alginate, anhydrous calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium acetate, calcium saccharate, calcium sulfate, phosphate Calcium hydrogen, calcium para-aminosalicylate, and biologically calcified compounds.
[58] Examples of the magnesium compound include magnesium L-aspartate, magnesium chloride, magnesium gluconate, magnesium aluminosilicate, magnesium silicate, magnesium oxide, magnesium hydroxide, magnesium stearate, magnesium carbonate, magnesium aluminate, magnesium sulfate, and magnesium silicate. Sodium magnesium, and synthetic magnesium silicate.
[59] The silicon compound includes hydrous silicon dioxide, hard silicic anhydride, synthetic hydrotalcite, diatomaceous earth, and silicon dioxide. Iron sulfate is contained as said iron compound. The zinc compound includes zinc chloride, zinc stearate, zinc oxide and zinc sulfate.
[60] The polyvalent metal compounds can be used individually or as a mixture of two or more compounds. Among the polyvalent metal compounds, calcium compounds such as phosphate hydroxide, calcium carbonate or calcium lactate provide good results. If the average particle size of the polyvalent metal compound is too large, the spraying performance of the compound is lowered and the particles precipitate quickly. Conversely, if the average particle size is too small, the particles cannot stay in the nasal cavity and are sucked into the bronchus and lungs. Thus, it is preferred that the polyvalent metal compound has an average particle size of 10 to 100 μm, more preferably 20 to 60 μm, whereby the metal compound can remain effectively in the nasal cavity.
[61] The amount of the bioactive polypeptide in the composition of the present invention, which provides an effective dosage of the polypeptide, depends on a number of factors (type of active substance selected, type of disease to be treated, desired frequency of administration, age, weight, symptoms of the patient). Severity, route of administration, desired effect, and other factors), but, for example, in the case of GLP-1 (7-36) amide, the composition of the present invention may have It is preferred to administer it nasally at a dose capable of delivering GLP-1 (7-36) amide.
[62] Specifically, the effective dosage of the physiologically active polypeptide is determined by the carrier (eg, the polyvalent metal compound including the calcium compound, aluminum compound, magnesium compound, silicon compound, iron compound and zinc compound) and the additive and dry -Mixed. The carrier, which is water insoluble or poorly water soluble, is provided in the form of powder or crystals and has an average particle size of 250 μm or less, preferably 100 μm or less, and more preferably 20 to 60 μm. Alternatively, the components can each be wet-mixed in water or in an organic solvent such as ethanol and then dried. In this manner, the bioactive polypeptide is uniformly dispersed and embedded on the surface of the carrier to provide a nonabsorbable pharmaceutical composition of the present invention.
[63] The nonabsorbable pharmaceutical composition of the present invention may suitably contain a carrier which is commonly used in the formulation of a medicament: lubricants, DPP-IV inhibitors, excipients, thickeners, sustainers, stabilizers, antioxidants , Binders, disintegrators, humectants, colorants, fragrances, fragrances, suspending agents, emulsifiers, solubilizers, buffers, isotizing agents, surfactants, soothing agents, and other functional ingredients.
[64] Such lubricants include calcium stearate, magnesium stearate, aluminum stearate, stearic acid and talc.
[65] As such stabilizers, quaternary ammonium salts such as benzalkonium chloride, benzogenium chloride, cetylpyridinium chloride, polyoxyethylene sorbitan monooleate (Tween 80) and sorbitan mono Sorbitan fatty acid esters such as oleate (Span 80).
[66] When an acidic bioactive polypeptide such as GLP-1 is used, which is susceptible to degradation by dipeptidyl peptidase IV (DPP-IV), the pharmaceutical composition preferably contains a DPP-IV inhibitor.
[67] Examples of DPP-IV inhibitors include dopamine A, baccitracin and isoleucine thiazolidide. The amount of the DPP-IV inhibitor added may vary depending on the inhibitory activity of each inhibitor, but it may be added to the pharmaceutical composition in an amount of about 1 to 10,000 times the weight of the bioactive polypeptide or active ingredient.
[68] In the preparation of the composition of the present invention, the amount of the bioactive polypeptide is preferably in the range of 0.005 to 50%, more preferably in the range of 0.01 to 20%, even more, assuming that the weight of the preparation is 100%. It is preferably selected in the range of 0.1 to 10.0%. The amount of carrier in the composition of the present invention may be any amount suitable for clinical use, assuming that the weight of the formulation is 100%, for example, in the range of 70 to 99.995%, preferably 80 to 99.99% Range, even more preferably in the range of 90 to 99.9%. By using an amount of the carrier within the above range, even better nasal absorption can be achieved. The amount of the additive in the composition of the present invention is, for example, assuming that the weight of the formulation is 100%, in the range of 0.005 to 50%, preferably in the range of 0.01 to 20%, even more preferably from 0.05% to It is in the range of 10.0%.
[69] The non-absorbable pharmaceutical composition of the present invention can be obtained by mixing the polyvalent metal compound carrier, the bioactive polypeptide, and the additive which are water insoluble or poorly water soluble. In one embodiment, GLP-1 (7-36) NH ₂ powder provided as peptide component is thoroughly mixed with corn starch. The mixture is then introduced into the vessel and calcium carbonate is slowly added with a small amount of purified water to form a slurry. The slurry is dried in a desiccator under reduced pressure. The dried product is filtered through a sieve and, if desired, an appropriate amount of calcium stearate can be mixed together. The above provides a pharmaceutical composition of the present invention.
[70] In addition, the pharmaceutical composition for non-absorption of the present invention can be obtained by first forming a slurry by corn starch and calcium carbonate added with a small amount of purified water. The mixture is then introduced into a vessel, to which GLP-1 (7-36) NH ₂ powder is slowly added and kneaded with water containing benzalkonium chloride. After drying and filtering the mixture, an appropriate amount of calcium stearate is mixed together. The above also provides a pharmaceutical composition of the present invention.
[71] An appropriate amount of the obtained nonabsorbable pharmaceutical composition is filled into a capsule consisting of hydroxypropylmethylcellulose (HPMC), starch or gelatin, and the capsule is suitably packaged, preferably in a sealed manner. Preferred sealed packages are a combination of a blister package and an aluminum package. If desired, a desiccant may be introduced into the aluminum bag. The entire process is preferably performed at a humidity of 60% or less.
[30] Accordingly, the present invention provides a non-absorbable pharmaceutical composition comprising a bioactive acid polypeptide having an isoelectric point of 7 or less, a water insoluble or poorly water soluble carrier, and an additive for dispersing and embedding the polypeptide on the surface of the carrier. .
[31] In addition, the present invention comprises a physiologically active polypeptide having an isoelectric point of 7 or less, that is, a neutral or acidic pH range, a polyvalent metal compound carrier, and an additive for dispersing and embedding the polypeptide on the carrier surface. To provide a composition.
[32] Specific embodiments of the non-absorbable pharmaceutical composition of the present invention contain an effective dose of the bioactive polypeptide having an isoelectric point within the neutral or acidic pH range, together with an additive having an average particle size of 1 μm to 20 μm, The polypeptide is uniformly dispersed and embedded on the surface of a powder or crystalline polyvalent metal compound carrier having an average particle size of 100 μm or less.
[33] Another specific embodiment of the non-absorbable pharmaceutical composition of the present invention contains a peptide incretin, a polyvalent metal compound carrier, and more particularly, the present invention, finely divided or crystallized having an average particle size of 100 μm or less Together with a polyvalent metal compound in form, a composition comprising a carrier that is water insoluble or poorly water soluble, and an additive having an average particle size of 1 μm to 20 μm for dispersing and embedding the peptide incretin on the surface of the carrier.
[34] The average particle size of the additive is the average of the water insoluble or poorly water soluble starch composition on the surface of the carrier when the formulation is prepared according to the method of the present invention using starch composed of gelatinized starch or gelatinized starch. Means particle size.
[72] The invention is disclosed in more detail by way of examples, but the examples are not intended to limit the scope of the invention in any case.
[73] Unless otherwise specified, the test methods and equipment described below are used in the Examples.
[74] The main abbreviations used in the present invention have the following meanings:
[75] Fmoc; Fluorenylmethoxycarbonyl
[76] Boc; tert-butoxycarbonyl
[77] Trt; Trityl
[78] Pmc; Pentamethylchromansulfonyl
[79] DCC; Dicyclohexylcarbodiimide
[80] HOBt; N-hydroxybenzotriazole
[81] TFA; Trifluoroacetic acid
[82] DIPEA; Diisopropylethylamine
[83] DMF; Dimethylformamide
[84] NMP; N-methylpyrrolidone
[85] TFE; Trifluoroethanol
[86] 1. Peptide Analysis by HPLC
[87] The following equipment and conditions are used to perform reversed-phase-HPLC for the purpose of peptide analysis in the determination and stability of peptide content in the formulations:
[88] Instrument: SHIMADZU LC-9A System
[89] Column: YMC-PROTEIN-RP (4.6mmφx 150mm)
[90] Column temperature: 40 ℃
[91] Eluent: Acetonitrile in 0.1% trifluoroacetic acid (linearly changes the concentration of acetonitrile at 10 minute intervals)
[92] Flow rate: 1mL / min
[93] Detection: UV (214nm)
[94] Loaded volume: 50 μl
[95] 2. Mass Spectrometry
[96] The mass of the peptide was measured using the following equipment and conditions:
[97] Instrument: Finnigan MAT TSQMS
[98] Ion Source: ESI
[99] Ion Detection Mode: Positive
[100] Spray voltage: 4.5kV
[101] Capillary Temperature: 250 ℃
[102] Mobile phase: 0.2% acetic acid / methanol mixture (1: 1)
[103] Flow rate: 0.2mL / min
[104] Scan Range: m / z 550 to 850
[105] 3. Amino Acid Sequencing
[106] The following instrument was used to analyze the amino acid sequence of the peptide:
[107] Instrument: Perkin Elmer 477A Sequencer
[108] 4. Analysis of Amino Acid Composition
[109] The following instrument was used to analyze the amino acid composition in the peptide:
[110] Instrument: Hitachi L-8500 Amino Acid Analyzer
[111] 5. Storage of samples (stability test)
[112] Samples were stored in incubators under the following temperature conditions:
[113] Instrument: Nagano Science LH-30-14
[114] Temperature setting: 40 ± 2 ℃, 25 ± 1 ℃
[115] 6. Lyophilization
[116] Instrument: LABCONCO Corp., using FZ-6.
[117] 7. Determination of Concentration in Plasma
[118] After administration of the pharmaceutical composition, the concentration of GLP-1 (7-36) NH ₂ in plasma was measured by radioimmunoassay (RIA) or enzyme immunoassay (ELISA).
[119] 7-1. Radioimmunoassay (RIA)
[120] Rabbits were sensitized using a combination adjuvant of GLP-1 (7-36) NH ₂ and bovine tyroglobulin to obtain antiserum (IgG fraction). Plasma was introduced in vitro with anti-GLP-1 (7-36) NH ₂ -rabbit antibody obtained from the antiserum and the mixture was left at 4 ° C. overnight. Then ¹²⁵ I-GLP-1 (7-36) NH ₂ was added and the mixture was left at 4 ° C. overnight. Then anti-rabbit IgG goat serum was added and the mixture was left at 4 ° C. for 1 hour. The obtained sample was centrifuged and the radioactivity of the precipitate (gamma-ray) was measured by a gamma ray meter.
[121] 7-2. Enzyme Immunoassay (ELISA)
[122] Anti-GLP-1 (7-36) NH ₂ antibody (rabbit polyclonal antibody) was immobilized on a 96-well plate. The serum was added to the plate and allowed to react for 2 hours. After washing the plate, add anti-GLP-1 (7-36) NH ₂ antibody (mouse polyclonal antibody) labeled with horseradish peroxidase and allow the reaction to proceed for 1 hour at room temperature. It was left. After washing the plate, tetramethylbenzidine was added for reaction. Absorbance was measured at 450 nm.
[123] 8. Calculation of isoelectric point (pI) of bioactive polypeptide
[124] An ExPASy (Expert Protein Analysis System) Molecular Biology Server, Swiss Institute of Bioinformatics was used. For the calculation of C-terminal amides, one of the Asp or Glu residues in the sequence was replaced with Asn or Gln.
[125] 9. Synthesis of Peptides (Incretin Peptides)
[126] Preserved peptide resin was synthesized according to ABI standard FastFmoc (0.25 mmol) using a 433A synthesizer (ABI corporation).
[127] 10. Particle size distribution (measuring particle size of calcium carbonate and starch)
[128] Instrument: A laser diffractometer (RODOS SR) from SYMPATEC HELOS & RODOS Corp. was used.
[129] Reference Example 1: GLP-1 (7-36) NH ₂Manufacture
[130] An expression plasmid pG97ompPR was prepared for expressing a fusion protein consisting of an E. coli β-galactosidase derivative (β-gal 97), a linker of 25 amino acids in length and GLP-1 (7-37). Publication No. WO99 / 38984. The linker region of the expressed fusion protein comprises a cleavage motif of the ompT protease (Arg-Arg) and a cleavage motif of Kex2 protease (Pro-Arg) and is cleaved by the protease at each cleavage site.
[131] To obtain the fusion protein, pG97ompPR was introduced into E. coli strains derived from the W3110 strain. The transformant obtained was cultured in a growth medium containing yeast extract, inorganic salts and glucose in a 300L incubator. The cells were incubated until the concentration of bacteria reached OD660 = 180. The obtained culture solution was treated in a high pressure homogenizer to destroy cell bodies and then centrifuged to obtain an inclusion body. The precipitate containing the inclusion body was redispersed in deionized water and centrifuged and then redispersed in deionized water to give a condensate (about 30 L) of inclusion body with an OD660 of 1000.
[132] To 3.9 L of the inclusion condensate, 1 L of 1 M Tris-HCl without pH control was added along with 10 L of 8 M urea. Deionized water was then added to bring the final volume to 20 liters. The N-terminus of 5% hydrochloric acid was used to adjust the pH of the obtained solution to 6.5 and the solution was maintained at 37 ° C. for 2 hours to allow the E. coli ompT protease present in the inclusion body to act on the fusion protein. GLP-1 (7-37) with 13 amino acids added to was cleaved off (hereinafter designated RHHGP [G]). After the reaction was completed, urea powder was added to the reaction mixture to 7M concentration and the pH was adjusted to 8.0 with 5N NaOH. The reaction mixture was then filtered-pressed to give 30 L of supernatant. The supernatant was packed on SP-Sepharose Big Beads column (140mmID x 160mm, Amersham Pharmacia Biotechnology) equilibrated with 5M urea / 20mM Tris-HCl (pH8.0) /0.1% Tween 80 solution, followed by 0.2M NaCl Washed with a / 20 mM Tris-HCl (pH 8.0) /0.1% Tween 80 solution. RHHGP [G] was then eluted with 0.5M NaCl / 20 mM Tris-HCl (pH8.0) /0.1% Tween 80 solution, resulting in a fraction containing about 100 g of RHHGP [G] (about 20 L ) Was obtained.
[133] Purified water (UF water) was used to adjust the resulting RHHGP [G] fraction to 5.0 mg / mL. To this solution was added 20 mM sodium acetate (pH 5.2), 5.0 μM copper sulfate, 0.5 g / L-ascorbic acid, 1 mg / L catalase, 0.1% Tween 80, and 1500 units / mL amidation enzyme. Subsequently, the reaction was allowed to proceed at 32 ° C. for 80 minutes, while oxygen was bubbled into the solution to maintain the dissolved oxygen concentration at 100%. As a result, the C-terminus of RHHGP [G] was amidated to form the amide form (RHHGP-1). To the solution, Tris-HCl (pH8.0), Tween 80, calcium chloride and Kex2 protease were added so that the final concentrations were 20 mM, 0.1%, 1 mM and 8,000 U / mL, respectively, and the reaction was 2.5 hours at 32 ° C. It was left to proceed.
[134] 13 amino acids were removed from the N-terminus of RHHGP-1 to form free GLP-1 (7-36) NH ₂ . About 10 L portion (3.4 g / L) of the solution was diluted (to 26 L) with 0.3% Tween 80/20 mM MBriton Robinson (hereinafter referred to as BR) buffer (pH4.5), 20 mM NaCl / 0.3% Filled onto a cation-exchange chromatography column (90 mm ID × 400 mm, MacroPrep High-S, Biorad) equilibrated with Tween 80/20 mM BR buffer (pH 4.5). After washing with the same solution as above, GLP-1 (7-36) NH ₂ was dissolved in Solution A {20 mM BR buffer (pH 6.0) / 20 mM NaCl / 0.3% Tween 80} and Solution B {20 mM BR buffer (pH 7. 5) / 20 mM NaCl / 0.3% Tween 80} (during elution, a linear gradient is formed by linearly changing the proportion of solution B in the eluent from 50% to 100%).
[135] The obtained fractions with a purity of at least 98% were diluted with water to a GLP-1 (7-36) NH ₂ concentration of 6 mg / mL and equilibrated with 20 mM sodium acetate (pH4.5) (90 mmID). x 240 mm) (Waters). After washing with 10% acetonitrile solution containing 20 mM sodium acetate (pH4.5) and 0.2% acetic acid, GLP-1 (7-36) NH ₂ was added with 30% acetonitrile solution containing 2% acetic acid. Elution was used to give a solution (2.5 L) containing 27 g GLP-1 (7-36) NH ₂ . Using an evaporator, acetonitrile was removed from the eluate and injected with water to adjust the concentration of GLP-1 (7-36) NH ₂ to 10 mg / mL. The solution was then freeze-dried in RL-903BS freeze-dryer (Kyowa Vacuum Engineering Co., Ltd.) to yield 22g freeze-dried GLP-1 (7-36) NH ₂ product.
[136] The obtained product was confirmed to be GLP-1 (7-36) NH ₂ by having the following molecular weight and amino acid composition. ESI-MS: 3297.4 (Theory: 3297.68). Leu standard amino acid composition after hydrolysis with 6N hydrochloric acid: Asp 1.0; (1), Thr; 2.0 (2), Ser; 2.7 (3), Glu; 4.0 (4), Gly; 3.0 (3), Ala; 4.1 (4), Val; 2.0 (2), Ile; 1.0 (1), Leu; 2.0, Tyr; 1.0 (1), Phe; 2.1 (2), Lys; 2.0 (2), His; 1.0 (1), Arg; 1.0 (1).
[137] Reference Example 2: GLP-1 (7-36) NH using rats ₂Nasal absorption test of pharmaceutical composition solution
[138] About 10 mg of GLP-1 (7-36) NH ₂ obtained in Reference Example 1, 180 mg of sucrose, 8 mg of citric anhydride and 0.2 mg of benzalkonium chloride were dissolved in 2 mL water, and the concentration of 5 mg / mL was measured by reverse phase HPLC. A sample solution of was formed. Seven to nine week old male SD rats (Crj: CD, Charles River Japan Inc.) weighing about 250 g were metal cages in a 12 hour day / night cycle while maintaining a temperature of 22 ± 5 ° C. and a humidity of 30 to 70%. Stored within. The rats were freely fed solids and tap water and fasted for 24 hours prior to testing (3 animals per group).
[139] For nasal absorption, a cannula was introduced into the femoral artery under anesthesia using pentobarbital, and 5 μl of the sample solution was administered into the left nasal cavity using a precision pipette (about 100 μg / kg). Blood was collected and centrifuged into tubes containing anticoagulants and enzyme inhibitors at 0, 5, 10, 15, 20, 30, 60 and 90 minutes after administration to obtain plasma. GLP-1 (7-36) NH ₂ concentration in the plasma was measured by RIA using anti-GLP-1 (7-36) NH ₂ antibody. For subcutaneous administration, a sample solution for subcutaneous administration (about 15 μg / mL) was administered subcutaneously in the back of the rat using a syringe at a dose of 1 mL / kg. GLP-1 (7-36) NH ₂ concentration in plasma was measured in the same manner as in the above nasal administration. The results are shown in Table 1 below.
[140]
[141] As can be seen from the above results, the bioavailability was 11.2 ± 6.5% (mean ± SD, n = 3), indicating that GLP-1 (7-36) NH ₂ was effectively absorbed from the nasal mucosa.
[142] Reference Example 3: GLP-1 (7-36) NH ₂Stability of Solution of Pharmaceutical Composition
[143] The GLP-1 (7-36) NH ₂ sample solution prepared in Reference Example 2 was stored at 25 ° C and 40 ° C.
[144] The results are shown in Table 2 below. As can be seen from the results, formation of small particles was observed after one week at each temperature condition.
[145]
[146] The results of Reference Example 2 show that a solution of the pharmaceutical composition containing GLP-1 (7-36) NH ₂ has the ability to be nasal absorbed in rats, but if the solution has a pH of about 2.7, its physicochemical stability is Low. Therefore, when the said pharmaceutical composition solution has such a pH, it is not preferable.
[147] Reference Example 4: GLP-1 (7-36) NH ₂Stability of solution
[148] 1,000 mL Solution A was prepared by adding distilled water to a mixture of 3.92 g phosphoric acid, 2.40 g acetic acid, 14.91 g potassium chloride and 2.47 g boric acid, and 1,000 mL Solution B was prepared by adding water to 8.0 g sodium hydroxide. The solution B was added dropwise to the solution A so that each pH value was pH2.0, pH3.0, pH4.0, pH5.0, pH6.0, pH7.0, and pH9.0 buffer solution {Britton Robinson ( BR) buffer} 100 mL were formed.
[149] Meanwhile, about 300 mg of GLP-1 (7-36) NH ₂ prepared in Reference Example 1 was dissolved in 30 mL distilled water. The solution (10 mg / mL) was divided into 2.0 mL aliquots and the BR buffer and 0.1M hydrochloric acid were separately added to the aliquots to form a 20 mL sample solution. The sample was then stored in the incubator at 40 ° C. for 1, 4 and 7 days to observe the appearance of the sample and to determine the residual ratio of GLP-1 (7-36) NH ₂ . The results are shown in Table 3 below.
[150]
[151] As shown in Table 3 above, for samples of pH1.2, pH2.0, pH3.0, pH6.0, and pH7.0, after 1 day, for samples of pH 5.0, after 4 days, and pH4. After 7 days for a sample of 0, a precipitate formed. No precipitate was formed in the sample at pH 9.0, but the residual ratio of GLP-1 (7-36) NH ₂ was reduced to 75.6% after 7 days. Thus, GLP-1 (7-36) NH ₂ exhibited a relatively low stability in acidic, neutral or basic solutions, and thus appeared not suitable for use in the pharmaceutical composition solution.
[152] In view of the above, as an alternative to the pharmaceutical composition solution, the absorption rate of the powder formulation of the non-administration composition was tested.
[153] Example 1 Absorption Rate Test of Nonadministered Powder Formulations Using Dogs
[154] The non-administration composition was prepared in the following Preparation Examples 1 to 9 in a mixture of GLP-1 (7-36) NH ₂ obtained in Reference Example 1 and a calcium carbonate fine powder having an average particle size of 50 μm provided as a carrier. Prepared by adding the different additives disclosed.
[155] As a control, one non-administration composition without an additive was prepared.
[156] Three beagle dogs each weighing about 10 kg were used, and the non-administration pharmaceutical composition disclosed in Preparation Examples 1 to 9 was administered nasally using a powder sprayer. The concentration of GLP-1 (7-36) NH ₂ in plasma was measured at 0, 5, 10, 20, 30, 45, 60, 90 and 120 minutes after administration. As a control, a fine powder of GLP-1 (7-36) NH ₂ without additives with calcium carbonate carrier was administered. The concentration of GLP-1 (7-36) NH ₂ in plasma was measured by RIA using anti-GLP-1 (7-36) NH ₂ antibody. Bioavailability was calculated by comparing the lower area of the curve (AUC) after nasal administration with AUC after subcutaneous administration of GLP-1 (7-36) NH ₂ in saline. The results are shown in Table 4 below.
[157]
[158] As can be seen from the above results, although the bioavailability of the pharmaceutical composition without the additive was about 4%, the administration of the pharmaceutical composition of the present invention improved the absorption of GLP-1 (7-36) NH ₂ to about 6% to 14 An increased bioavailability of% was achieved.
[159] Preparation Example 1 Preparation of a Pharmaceutical Composition Containing Domyo-ji Powder (1.0%)
[160] A peptide component, an GLP-1 (7-36) wherein the amount of GLP-1 (7-36) NH ₂ powder for the NH ₂ 10mg (about 12mg) was mixed with 31mg Domyo-ji powder. The powder mixture was introduced into a beaker and slowly added 2.92 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (29 mg) corresponding to 1.0% of the total weight was mixed together to give 2.83 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 690 μg of GLP-1 (7-36) NH ₂ (about 226 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[161] Preparation Example 2 Preparation of Pharmaceutical Composition Containing Corn Starch (0.1%)
[162] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was determined by 3.5 mg of corn starch ( Japanese pharmacopoeia : average particle size = 13.3 μm). The powder mixture was introduced into a beaker and slowly added 2.96 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (28 mg) corresponding to 1.0% of the total weight was mixed together to give 2.75 g of a powder sample. An amount of the powder sample (about 297 mg) containing 1,000 μg of GLP-1 (7-36) NH ₂ was filled into a # 2 capsule to prepare a nasal absorption pharmaceutical composition.
[163] Preparation Example 3 Preparation of a Pharmaceutical Composition Containing Corn Starch (1.0%)
[164] A peptide component, of the GLP-1 (7-36) GLP- 1 (7-36) NH _2, the amount of powder that corresponds to the NH ₂ of the 10mg (about 12mg) 32mg of corn starch (Japanese pharmacopoeia: average particle size = 13.3 Μm). The powder mixture was introduced into a beaker and slowly added 2.93 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (29 mg) corresponding to 1.0% of the total weight was mixed together to give 2.89 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 678 μg of GLP-1 (7-36) NH ₂ (about 213 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[165] Preparation Example 4 Preparation of a Pharmaceutical Composition Containing Rice-Type Luxury Potato Starch (0.1%)
[166] As a peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was converted to 3.2 mg of rice-type gelatinized potato starch (AMYCOL HF, NIPPON STARCH CHEMICAL Co., Ltd.). The powder mixture was introduced into a beaker and slowly added 2.96 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (28 mg) corresponding to 1.0% of the total weight was mixed together to give 2.75 g of a powder sample. An amount of the powder sample (about 297 mg) containing about 1,000 μg of GLP-1 (7-36) NH _{2 was} filled into a # 2 capsule to prepare a nasal absorption pharmaceutical composition.
[167] Preparation Example 5 Preparation of a Pharmaceutical Composition Containing Non-rice-Type Luxury Potato Starch (1.0%)
[168] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was converted to 30 mg of rice-type gelatinized potato starch (AMYCOL HF, NIPPON STARCH CHEMICAL Co., Ltd.). The powder mixture was introduced into a beaker and slowly added 2.92 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and the dried product was passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (27 mg) corresponding to 1.0% of the total weight was mixed together to give 2.77 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 661 μg of GLP-1 (7-36) NH ₂ (about 217 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[169] Preparation Example 6 Preparation of Pharmaceutical Composition Containing Povidone (1.0%)
[170] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was compared with 32 mg of povidone K30 (The Japanese Standards of Pharmaceutical Additives). Mixed. The powder mixture was introduced into a beaker and slowly added 2.93 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and the dried product was passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (29 mg) corresponding to 1.0% of the total weight was mixed together to give 2.84 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 642 μg of GLP-1 (7-36) NH ₂ (about 211 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[171] Preparation Example 7 Preparation of Pharmaceutical Composition Containing Pectin (1.0%)
[172] As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was mixed with 31 mg of pectin (USP). The powder mixture was introduced into a beaker and slowly added 2.93 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and the dried product was passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (29 mg) corresponding to 1.0% of the total weight was mixed together to give 2.88 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 644 μg of GLP-1 (7-36) NH ₂ (about 210 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[173] Preparation Example 8 Preparation of a Pharmaceutical Composition Containing Non-rice-Type Luxury Corn Starch (1.0%)
[174] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was converted to 31 mg of rice-type deluxe corn starch (PCS, ASAHI KASEI Co., Ltd.). The powder mixture was introduced into a beaker and slowly added 2.92 g of calcium carbonate (average particle size = 51.9 μm). After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (28 mg) corresponding to 1.0% of the total weight was mixed together to give 2.88 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 646 μg of GLP-1 (7-36) NH ₂ (about 211 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[175] Preparation Example 9 Preparation of Pharmaceutical Composition Without Additives
[176] As the peptide component, an amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30 mg of GLP-1 (7-36) NH ₂ was introduced into the beaker and 8.88 g of calcium carbonate (average particles Size = 51.9 μm) was added slowly. After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a cold-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (87 mg) corresponding to 1.0% of the total weight was mixed together to give 8.76 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 937 μg of GLP-1 (7-36) NH ₂ (about 300 mg) was # Filled into 2 capsules to prepare a nasal absorption pharmaceutical composition.
[177] Example 2 Absorption Rate Test of Non-administered Powder Preparation Using Cynomolgus Monkeys
[178] GLP-1 (7-36) NH ₂ obtained in Reference Example 1 was used as a physiologically active polypeptide, and calcium carbonate or sucrose sulfate aluminum salt (sucralate: sucralfate) having an average particle size of 51.9 μm was used as a carrier. . According to the procedure disclosed in Preparation Examples 10 to 18, different nasal absorption pharmaceutical compositions containing different additives carried by a carrier were prepared. The dose of GLP-1 (7-36) NH ₂ was adjusted so that about 100 μg / animal of GLP-1 (7-36) NH ₂ was administered at one time. Each additive was added in the pharmaceutical composition in an amount of 0.5%, 1.0%, 10% and 50% by weight of the formulation. Two formulations without additives, one containing calcium carbonate and GLP-1 (7-36) NH ₂ , and the other containing sucralate and GLP-1 (7-36) NH ₂ as controls. Was prepared.
[179] Using a nasal nebulizer (manufactured by UNISIA JECS Co., Ltd.), the compositions of Preparations 10-18 were individually administered to Chinese monkeys (each weighing about 3 kg) into their nasal passages, 0, 5 after administration. The concentration of GLP-1 (7-36) NH ₂ in plasma at 10, 20, 30, 45, 60, 90 and 120 minutes was measured by RIA and in part by ELISA. Bioavailability was measured by comparing AUC after nasal administration with AUC after subcutaneous administration.
[180] To Figures 1, 2, and 3 is a saline solution of GLP-1 (7-36) each GLP-1 (7-36) was administered into the two nasal jehyeongreul with no additives and after subcutaneous administration of NH ₂ NH ₂ The plasma concentration-time curve of is shown. FIG. 4 shows plasma concentrations of GLP-1 (7-36) NH ₂ after intranasal administration of each non-formulation containing 1% Domyo-ji powder, 0.5% corn starch, or 0.5% non-rice-type luxury potato starch. Represent a time curve.
[181] The assessment of plasma GLP-1 (7-36) NH ₂ concentration, measured by RIA and partially ELISA, is shown in Tables 5 and 6, respectively.
[182]
[183]
[184] As apparent from Tables 5 and 6, each of the two formulations without additives exhibited low GLP-1 (7-36) NH ₂ nasal absorption of about 0.3 to 3.1%, while each of the pharmaceutical compositions of the present invention Significantly promoted GLP-1 (7-36) NH ₂ uptake by nasal mucosa.
[185] Preparation Example 10 Preparation of Pharmaceutical Composition Without Additives
[186] As the peptide component, an amount of GLP-1 (7-36) NH ₂ powder (approximately 36 mg) corresponding to 30 mg of GLP-1 (7-36) NH ₂ was introduced into the beaker and 8.87 g of calcium carbonate (average particle size = 51.9 μm) was added slowly. After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (84 mg) corresponding to 1.0% of the total weight was mixed together to give 8.20 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 100 μg of GLP-1 (7-36) NH ₂ (about 30 mg) was # 2. Filled in capsules to prepare a nasal absorption pharmaceutical composition.
[187] Preparation Example 11 Preparation of Pharmaceutical Composition Without Additives
[188] As the peptide component, an amount of GLP-1 (7-36) NH ₂ powder (approximately 36 mg) corresponding to 30 mg of GLP-1 (7-36) NH ₂ was introduced into the beaker and slowly added 8.87 g of sucralate. It was. After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (84 mg) corresponding to 1.0% of the total weight was mixed together to give 8.03 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 100 μg of GLP-1 (7-36) NH ₂ (about 30 mg) was # 2. Filled in capsules to prepare a nasal absorption pharmaceutical composition.
[189] Preparation Example 12 Preparation of Pharmaceutical Composition Containing Domyo-ji Powder (1.0%)
[190] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30 mg of GLP-1 (7-36) NH ₂ was mixed with 89 mg of Domyo-ji powder. 8.78 g calcium carbonate was slowly added to the powder mixture. After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the filtered product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (84 mg) corresponding to 1.0% of the total weight was mixed together to give 8.11 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 100 μg of GLP-1 (7-36) NH ₂ (about 30 mg) was # 2. Filled in capsules to prepare a nasal absorption pharmaceutical composition.
[191] Preparation Example 13 Preparation of Pharmaceutical Composition Containing Corn Starch (0.5%)
[192] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 36 mg) corresponding to 30 mg of GLP-1 (7-36) NH ₂ was determined by 45 mg of corn starch ( Japanese pharmacopoeia : average particle size = 13.3. Μm). The powder mixture was introduced into a beaker and 8.83 g of calcium carbonate (average particle size = 50 μm) was added slowly. After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (88 mg) corresponding to 1.0% of the total weight was mixed together to give 8.46 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reverse phase HPLC, and the amount of the powder sample containing about 98 μg of GLP-1 (7-36) NH ₂ (about 30 mg) was # 2. Filled in capsules to prepare a nasal absorption pharmaceutical composition.
[193] Preparation 14 Preparation of Pharmaceutical Composition Containing Non-rice-Type Luxury Potato Starch (0.5%)
[194] As a peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 36 mg) corresponding to 30 mg of GLP-1 (7-36) NH ₂ was converted to 45 mg of rice-type deluxe potato starch (AMYCOL HF, NIPPON STARCH CHEMICAL Co., Ltd.). The powder mixture was introduced into a beaker and 8.83 g calcium carbonate (average particle size = 51.9 μm) was added slowly. After the mixture was thoroughly mixed, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (84 mg) corresponding to 1.0% of the total weight was mixed together to give 8.47 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reverse phase HPLC, and the amount of the powder sample containing about 101 μg of GLP-1 (7-36) NH ₂ (about 30 mg) was # 2. Filled in capsules to prepare a nasal absorption pharmaceutical composition.
[195] Preparation Example 15 Preparation of a Pharmaceutical Composition (Powder Mixture) Containing Hydroxy-propylcellulose (HPC, 10%)
[196] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was ground with 300 mg of HPC ( Japanese Pharmacopoeia ) to prepare the powder mixture. Obtained. The powder mixture was similarly ground until homogeneous with 2.69 g calcium carbonate (average particle size = 51.9 μm). The mixture was then passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (28 mg) corresponding to 1.0% of the total weight was mixed together to give 2.76 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reversed phase HPLC, and the amount of the powder sample containing about 100 μg of GLP-1 (7-36) NH ₂ (about 30 mg) was # 2. Filled in capsules to prepare a nasal absorption pharmaceutical composition.
[197] Preparation Example 16: Preparation of a pharmaceutical composition (mixture kneaded with water) containing HPC (10%)
[198] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was ground with 300 mg of HPC ( Japanese Pharmacopoeia ) to prepare the powder mixture. Obtained. The powder mixture was similarly ground until homogeneous with 2.69 g calcium carbonate (average particle size = 51.9 μm). Purified water was then added to the mixture to make the mixture a paste. The paste was mixed thoroughly and dried overnight in a desiccator under reduced pressure. The dry mixture was passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (28 mg) corresponding to 1.0% of the total weight was mixed together to give 2.70 g of a powder sample. The GLP-1 (7-36) NH ₂ content of the powder sample was measured, and about 30 mg of the powder sample containing 100 μg of GLP-1 (7-36) NH ₂ was filled into a # 2 capsule. The composition was prepared.
[199] Preparation 17: Preparation of pharmaceutical composition (mixture kneaded with ethanol) containing HPC (10%)
[200] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ was ground with 300 mg of HPC ( Japanese Pharmacopoeia ) to prepare the powder mixture. Obtained. The powder mixture was similarly ground until homogeneous with 2.69 g calcium carbonate (average particle size = 51.9 μm). Ethanol was then added to the mixture to make the mixture a paste. The paste was thoroughly kneaded and dried overnight in a desiccator under reduced pressure. The dry mixture was passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (28 mg) corresponding to 1.0% of the total weight was mixed together to give 2.73 g of a powder sample. The GLP-1 (7-36) NH ₂ content of the powder sample was measured, and about 30 mg of the powder sample containing 100 μg of GLP-1 (7-36) NH ₂ was filled into a # 2 capsule to prevent nasal absorption. The composition was prepared.
[201] Preparation 18: Preparation of pharmaceutical composition containing HPC (50%)
[202] As a peptide component, the powder mixture by grinding with an amount of GLP-1 (7-36) NH ₂ powder (about 12 mg) corresponding to 10 mg of GLP-1 (7-36) NH ₂ together with 1.5 g of HPC ( Japanese Pharmacopoeia ) Obtained. The powder mixture was similarly ground until homogeneous with 1.49 g calcium carbonate (average particle size = 51.9 μm). Ethanol was then added to the mixture to make the mixture a paste. The paste was thoroughly kneaded and dried overnight in a desiccator under reduced pressure. The powder obtained above was passed through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (27 mg) corresponding to 1.0% of the total weight was mixed together to give 2.54 g of a powder sample. The GLP-1 (7-36) NH ₂ content in the powder sample was measured by reverse phase HPLC, and about 30 mg of the powder sample containing 100 μg of GLP-1 (7-36) NH ₂ was filled into a # 2 capsule. A nasal absorption pharmaceutical composition was prepared.
[203] Example 3: Absorption rate test of the powder formulation for non-administration using Chinese monkey (Test-2)
[204] In this example, by administering a composition containing a mixture of partially gelatinized starch, hydroxypropylcellulose-SSL (HPC-SSL), partially gelatinized starch / HPC-SSL or corn starch (in amounts of 1.0 to 10%) as an additive The rate of absorption of GLP-1 (7-36) NH ₂ was investigated.
[205] Using a nasal spray (manufactured by UNISIA JECS Co., Ltd.), the compositions in Preparations 19-27 below were individually administered to Chinese monkeys (2-4 kg each weight) into their nasal passages and GLP-1 in plasma (7-36) NH ₂ concentration was measured by RIA at 0, 5, 10, 15, 30, 60, 90 and 120 minutes after administration. Bioavailability was measured by comparing AUC after nasal administration with AUC after subcutaneous administration.
[206] The results are shown in Table 7 below. As clearly shown in Table 7, formulations containing corn starch in a range of 1.0 to 10% show an effective non-absorption promoting effect of GLP-1 (7-36) NH ₂ compared to formulations without additives. It was. In addition, formulations containing partially gelatinized corn starch (partially water insoluble) also showed effective nasal absorption of GLP-1 (7-36) NH ₂ . By using poorly water-soluble HPC-SSL as an additive in the pharmaceutical composition, a certain effect of promoting absorption of GLP-1 (7-36) NH ₂ was observed. Although no absorption enhancement effect was observed when using HPC-SSL and partially gelatinized starch in combination, the nasal absorption of GLP-1 (7-36) NH ₂ was improved compared to the formulation without additives.
[207]
[208] Preparation Example 19 Preparation of a Pharmaceutical Composition Without Additives Containing Benzalkonium Chloride (0.01%)
[209] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (approximately 36 mg) corresponding to 30.6 mg of GLP-1 (7-36) NH ₂ was added to 2.93 g of calcium carbonate (average particle size = 53.6 μm). ) And slowly mixed. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 30 mg) corresponding to 1.0% of the total weight was mixed together to give 2.95 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and 30 mg of the powder sample containing 311 μg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[210] Preparation Example 20 Preparation of a Pharmaceutical Composition Containing Corn Starch (1.0%) and Benzalkonium Chloride (0.01%)
[211] About 2.91 g of calcium carbonate (average particle size: 53.6 μm) was mixed with about 29 mg of corn starch (average particle size: 13.3 μm). After complete mixing, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 29.9 mg of GLP-1 (7-36) NH ₂ was mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 29 mg) corresponding to 1.0% of the total weight was mixed together to give 2.88 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and 30 mg of the powder sample containing 312 μg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[212] Preparation Example 21 Preparation of a Pharmaceutical Composition Containing Corn Starch (5.0%) and Benzalkonium Chloride (0.01%)
[213] About 2.78 g of calcium carbonate (average particle size: 53.6 μm) was mixed with about 150 mg of corn starch (average particle size: 13.3 μm). After complete mixing, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 35 mg) corresponding to 29.6 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 30 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.89 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and 30 mg of the powder sample containing 307 µg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[214] Preparation Example 22 Preparation of a Pharmaceutical Composition Containing Corn Starch (10.0%) and Benzalkonium Chloride (0.01%)
[215] About 2.63 g of calcium carbonate (average particle size: 53.6 μm) was mixed with about 301 mg of corn starch (average particle size: 13.3 μm). After complete mixing, purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30.6 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 27 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.69 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reverse phase HPLC, and 30 mg of the powder sample containing 341 μg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[216] Preparation Example 23 Preparation of a Pharmaceutical Composition Containing HPC-SSL (0.1%) and Benzalkonium Chloride (0.01%)
[217] To about 2.93 g of calcium carbonate (average particle size: 53.6 μm) was added a solution containing about 3 mg of HPC-SSL, followed by further addition of purified water and kneading the mixture. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30.4 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 30 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.94 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reverse phase HPLC, and 30 mg of the powder sample containing 310 μg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[218] Preparation Example 24 Preparation of a Pharmaceutical Composition Containing HPC-SSL (0.5%) and Benzalkonium Chloride (0.01%)
[219] To about 2.92 g of calcium carbonate (average particle size: 53.6 mu m), a solution containing about 15 mg of HPC-SSL was added, followed by further addition of purified water and kneading the mixture. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30.5 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 30 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.93 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and 30 mg of the powder sample containing 312 μg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[220] Preparation 25: Preparation of pharmaceutical composition containing HPC-SSL (1.0%) and benzalkonium chloride (0.01%)
[221] After adding a solution containing about 30 mg of HPC-SSL to about 2.90 g of calcium carbonate (average particle size: 53.6 mu m), additional purified water was added and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30.6 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. In the sieved mixture, an amount of calcium stearate (about 28 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.85 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reverse phase HPLC, and 30 mg of the powder sample containing 322 µg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[222] Preparation Example 26 Preparation of a Pharmaceutical Composition Containing Partly Luxury Corn Starch (1.0%) and Benzalkonium Chloride (0.01%)
[223] To about 2.90 g of calcium carbonate (average particle size: 53.6 mu m), a solution containing about 30 mg of partially gelatinized corn starch was added, followed by further addition of purified water and kneading the mixture. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 37 mg) corresponding to 30.8 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 29 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.90 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reverse phase HPLC, and 30 mg of the powder sample containing 319 µg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[224] Preparation Example 27 Preparation of a Pharmaceutical Composition Containing Corn Starch (5.0%), HPC-SSL (0.1%) and Benzalkonium Chloride (0.01%)
[225] About 2.78 g of calcium carbonate (average particle size: 53.6 μm) was mixed with about 151 mg of corn starch (average particle size: 13.3 μm) and the mixture was thoroughly mixed. Then, a solution containing about 3 mg of HPC-SSL was added, and then purified water was further added to the mixture, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 36 mg) corresponding to 30.3 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 30 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.93 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reversed phase HPLC, and 30 mg of the powder sample containing 310 μg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[226] Example 4 Effect of Starch Addition on Nasal Absorption of Incretin Polypeptides
[227] In this example, using the effect of reducing blood glucose levels in rats as an indicator, it was investigated whether nasal absorption of acidic incretin hormone in addition to GLP-1 (7-36) NH ₂ was enhanced by starch addition.
[228] Male CD (SD) rats weighing about 300 g were anesthetized using pentobarbital. The front edge of the pipette tip is filled with 3 mg of the pharmaceutical composition (containing 100 μg of polypeptide) prepared by the preparation described below, the tip is placed in a syringe and the composition is air It was sprayed into the nasal cavity of the rat with 1 mL. Then, 5 minutes after nasal administration of the composition, 0.5 g / kg glucose was administered intravenously in the tail of the rat. Blood samples were taken from the aorta 15 minutes after nasal administration of the composition (10 minutes after glucose administration). Blood glucose levels were measured using a blood glucose level measuring instrument (FREESTYLE-KISSEI; Kissei Pharmaceutical Co., Ltd.).
[229] The results are shown in Table 8 below. As can be seen from the results, the blood glucose level of the control group not administered the incretin hormone was 196 mg / dL (mean value of 3 rats). For incretin hormone containing compositions, blood glucose levels were lowered compared to the control. Furthermore, for incretin hormone-containing compositions, when a composition containing 5% corn starch was added, blood glucose levels were lowered compared to the case where 5% corn starch was not added.
[230] The results indicate that large amounts of incretin hormone can be absorbed with the pharmaceutical composition of the present invention, and the absorption rate of the incretin hormone can be improved by using starch as an additive to the composition.
[231]
[232] Reference Example 5 Synthesis of [Lys-26, ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37)
[233] Methylene chloride (30 mL) and 5 mg (6.9 mmol) of swelling Cl-Trt (2-Cl) resin, methylene chloride of Fmoc-Glu-OtBu (5.8 g, 13.7 mmol) and DIPEA (1.5 g, 11.8 mmol) The solution was added and the mixture was stirred slowly for 30 minutes. The resin was obtained by filtration and washed with methylene chloride, isopropanol and methylene chloride, respectively. A 20% piperidine / DMF (30 ml) mixed solution was then added to the resin obtained above and the mixture was stirred slowly for 20 minutes. The resin was obtained by filtration and washed with DMF, isopropanol and methylene chloride respectively and dried. The H-Glu (α-OtBu) -Trt (2-Cl) resin (6.1 g, 6.9 mmol) obtained above was added to a mixed solution of N-methyl pyrrolidone (NMP) / methylene chloride (1: 1, 30 mL). Suspension was added to the mixture and 3 equivalents of palmitic acid (5.3 g, 20.7 mmol), HOBt (2.8 g, 20.7 mmol) and water-soluble dicyclohexylcarbodiimide (DCC: 4.0 g, 20.7 mmol) were added. The mixture was stirred slowly overnight. The obtained resin was washed with methylene chloride, NMP and methylene chloride respectively and dried. To the obtained palmitoyl-Glu (α-OtBu) -Trt (2-Cl) resin (8 g, 6.9 mmol), a mixed solution of acetic acid / TFE / methylene chloride (1: 2: 7, 20 ml) was added, The mixture was stirred slowly for 20 minutes. The resin was filtered off and washed with 10 mL of TFE and the filtrate was concentrated. The obtained residue was treated with hexane to give a precipitate. The precipitate obtained was recrystallized from methylene chloride / hexane to give 2.0 g (yield: 66%) of glutamic acid derivative, palmitoyl-Glu (α-OtBu), which was introduced into the side chain of Lys at the 26 position. It was. ESI-MS: [M + H] 442.3; M + Na] 462.4 (Theoretical value: 441.3 [M]).
[234] Then Fmoc-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (Trt) -Phe-Thr (Trt) -Ser (tBu) -Asp (tBu) -Val-Ser (tBu) -Ser (tBu) ) -Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (4-methyltrityl) -Glu (OtBu) -Phe-Ile-Ala-Trp-Leu-Val -Arg (Pmc) -Gly-Arg (Pmc) -Gly-Wang resin was formulated by Fmoc method (0.25 mmol protocol) using an automated peptide synthesizer 433A (ABI Co., Ltd.). The obtained resin was treated for 30 minutes using a mixed solution of 1% TFA / 5% TIPS / methylene chloride (30 mL) to remove the 4-methyltrityl group from the side chain of Lys at position 26. The obtained resin was neutralized using a mixed solution of 5% DIPEA / methylene chloride, washed with methylene chloride and swollen using NMP (30 ml). The swelled resin was then reacted with palmitoyl-Glu (α-OtBu) (441 mg, 1 mmol) for 3 hours in the presence of HOBt (135 mg, 1 mmol) and DCC (260 mg, 1 mmol). The peptide resin obtained was treated with 20% piperidine to remove Fmoc and further treated with a mixed solution of 88% TFA / 2% TIPS / 5% water / 5% phenol (20 mL) for 1 hour. The resin was filtered off and the resin was washed with 10 mL of TFA and the combined filtrates were concentrated. The residue was treated with ether to give a precipitate (720 mg). 500 mg of the crude peptide was dissolved in saturated urea solution (200 mL) and 5 to a linear gradient of 13% to 60% acetonitrile / 0.1% TFA using C4 (YMC-pack, PROTEIN-RP, 2 cm x 25 cm). Purification by reverse phase HPLC at a flow rate of 10 mL / min. Fractions containing the desired product were obtained and lyophilized to yield 145 mg of the desired peptide. ESI-MS: 3751 (Theory: 3751.2). Leu standard amino acid composition after hydrolysis with 6N hydrochloric acid: Asx: 0.97 (1), Thr: 1.89 (2), Ser: 2.75 (3), Glx: 5.08 (5), Gly: 4.05 (4), Ala: 4.01 (4), Val: 1.93 (2), Ile: 0.99 (1), Leu: 2 , Tyr: 0.91 (1), Phe: 1.90 (2), Lys: 1.10 (1), His: 0.90 (1), Arg: 1.92 (2).
[235] Reference Example 6 Synthesis of GLP-1 (7-37)
[236] H-His (Trt) -Ala-Glu (OtBu) -Gly-Thr (Trt) -Phe-Thr (Trt) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu)- Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (Boc) -Glu (OtBu) -Phe-Ile-Ala-Trp-Leu-Val-Lys (Boc)- Gly-Arg (Pmc) -Wang resin (1.1 g) was used as the starting material Fmoc-Arg (Pmc) -Wang by Fmoc method (0.25 mmol protocol) using an automated peptide synthesizer 433A (ABI Co., Ltd.). It was made from resin. 600 mg of the obtained resin was treated with a mixed solution of 88% TFA / 2% TIPS / 5% water / 5% phenol (20 mL) for 1 hour. The resin was filtered to concentrate the filtrate and the residue was treated with ether to give a precipitate (380 mg). The crude peptide obtained was dissolved in purified water (20 mL) and 10 mL / min with a linear gradient of 9% to 68% acetonitrile / 0.1% TFA using C4 (YMC-pack, PROTEIN-RP, 2 cm × 25 cm). Purification by reverse phase HPLC at flow rate of. Fractions containing the desired product were obtained and lyophilized to give 53 mg of the desired peptide. ESI-MS: [M] 3355 (Theory: 3355.7). Leu standard amino acid composition after hydrolysis with 6N hydrochloric acid: Asx: 1.00 (1), Thr: 1.99 (2), Ser: 2.79 (3), Glx: 4.10 (4), Gly: 4.01 (4), Ala: 4.02 (4), Val: 1.92 (2), Ile: 0.98 (1), Leu: 2 , Tyr: 0.92 (1), Phe: 1.92 (2), Lys: 2.18 (2), His: 0.96 (1), Arg: 0.94 (1).
[237] Reference Example 7: [Val ⁸ Synthesis of GLP-1 (7-37)
[238] H-His (Trt) -Val-Glu (OtBu) -Gly-Thr (Trt) -Phe-Thr (Trt) -Ser (tBu) -Asp (OtBu) -Val-Ser (tBu) -Ser (tBu)- Tyr (tBu) -Leu-Glu (OtBu) -Gly-Gln (Trt) -Ala-Ala-Lys (Boc) -Glu (OtBu) -Phe-Ile-Ala-Trp-Leu-Val-Lys (Boc)- Gly-Arg (Pmc) -Wang resin (1.4 g) was used as the starting material Fmoc-Arg (Pmc) -Wang by Fmoc method (0.25 mmol protocol) using an automated peptide synthesizer 433A (ABI Co., Ltd.). It was made from resin. 0.7 g of the obtained resin was treated with a mixed solution of 88% TFA / 2% TIPS / 5% water / 5% phenol (20 mL) for 1 hour. The resin was filtered to concentrate the filtrate and the residue was treated with ether to give a precipitate (327 mg). The crude peptide obtained was dissolved in purified water (50 mL) and 10 mL / min with a linear gradient of 5% to 72% acetonitrile / 0.1% TFA using C4 (YMC-pack, PROTEIN-RP, 2 cm x 25 cm). Purification by reverse phase HPLC at flow rate of. Fractions containing the desired product were obtained and lyophilized to yield 75 mg of the desired peptide. ESI-MS: [M] 3383 (Theory: 3383.8). Leu standard amino acid composition after hydrolysis with 6N hydrochloric acid: Asx: 0.99 (1), Thr: 1.98 (2), Ser: 2.80 (3), Glx: 4.10 (4), Gly: 4.01 (4), Ala: 3.03 (3), Val: 2.86 (3), Ile: 0.98 (1), Leu: 2 , Tyr: 0.92 (1), Phe: 1.92 (2), Lys: 2.18 (2), His: 0.92 (1), Arg: 0.94 (1).
[239] Reference Example 8: Synthesis of Exendin-4
[240] H-His (Trt) -Gly-Glu (OtBu) -Gly-Thr (Trt) -Phe-Thr (Trt) -Ser (tBu) -Asp (OtBu) -Leu-Ser (tBu) -Lys (Boc)- Gln (Trt) -Met-Glu ((OtBu) -Glu (OtBu) -Ala-Val-Arg (Pmc) -Leu-Phe-Ile-Glu (OtBu) -Trp-Lue-Lys (Boc) -Asn (Trt ) -Gly-Gly-Pro-Ser (tBu) -Ser (tBu) -Gly-Ala-Pro-Pro-Pro-Ser (tBu) -Wang resin (1.9 g), Fmoc method (0.25 mmol protocol) It was formulated from the starting material, Fmoc-Ser (tBu) -Wang resin, 1.9 g of the obtained resin with a mixed solution of 88% TFA / 2% TIPS / 5% water / 5% phenol (20 mL) for 1 hour. The resin was filtered and the filtrate was concentrated and the residue was treated with ether to give a precipitate (870 mg) The crude peptide obtained was dissolved in purified water (80 mL) and C4 (YMC-pack). , PROTEIN-RP, 2 cm × 25 cm), was purified by reverse phase HPLC for 60 minutes at a flow rate of 10 mL / min with a linear gradient of 18% to 72% acetonitrile / 0.1% TFA. Get High lyophilization gave 270 mg of the desired peptide ESI-MS: [M] 4186 (Theoretical: 4186.6) Leu Standard Amino Acid Composition after Hydrolysis with 6N Hydrochloric Acid: Asx: 1.99 (2), Thr: 1.97 ( 2), Ser: 4.77 (5), Glx: 6.06 (6), Gly: 4.97 (5), Ala: 2.02 (2), Val: 0.94 (1), Met: 0.68 (1), Ile: 0.98 (1 ), Leu: 3 , Phe: 1.90 (2), Lys: 2.17 (2), His: 0.98 (1), Arg: 0.92 (1), Pro: 3.96 (4).
[241] Preparation Example 28 Preparation of Nasal Absorption Pharmaceutical Composition of GLP-1 (7-37) Containing Corn Starch (5%) and Benzalkonium Chloride (0.01%)
[242] About 269 mg of calcium carbonate (average particle size: 53.6 μm) was mixed with about 15 mg of corn starch (average particle size: 13.3 μm) and the mixture was thoroughly mixed. Purified water was then added to the mixture and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-37) powder (approximately 10.8 mg) corresponding to 9.03 mg of GLP-1 (7-37) was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 2.2 mg) corresponding to 1.0% of the total weight was mixed together to give about 252 mg of a powder sample. The content of GLP-1 (7-37) in the powder sample was measured by reversed phase HPLC, and 3 mg of the powder sample containing 107 µg of GLP-1 (7-37) was prepared as a nasal absorption pharmaceutical composition.
[243] Preparation Example 29 Preparation of Nasal Absorption Pharmaceutical Composition of GLP-1 (7-37) Containing Benzalkonium Chloride (0.01%)
[244] As the peptide component, the amount of GLP-1 (7-37) powder (approximately 10.5 mg) corresponding to 8.75 mg of GLP-1 (7-37) was gradually mixed with about 284 mg of calcium carbonate (average particle size: 53.6 µm). Mixed. After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 2.9 mg) corresponding to 1.0% of the total weight was mixed together to give about 258 mg of a powder sample. The content of GLP-1 (7-37) in the powder sample was measured by reverse phase HPLC, and 3 mg of the powder sample containing 102 µg of GLP-1 (7-37) was prepared as a nasal absorption pharmaceutical composition.
[245] Preparation Example 30 [Val] Containing Corn Starch (5.0%) and Benzalkonium Chloride (0.01%) ⁸] -GLP-1 (7-37) Preparation of Nasal Absorption Pharmaceutical Composition
[246] About 269 mg of calcium carbonate (average particle size: 53.6 μm) was mixed with about 15 mg of corn starch (average particle size: 13.3 μm) and the mixture was thoroughly mixed. Purified water was then added to the mixture and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. A peptide component, and ^{[Val 8] -GLP-1 [} Val 8] corresponding to (7-37) -GLP-1 (7-37 ) the amount of the powder (about 13mg) of the obtained dry product of 9.38mg Mix slowly. After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 3.1 mg) corresponding to 1.0% of the total weight was mixed together to give about 261 mg of a powder sample. The amount of the powder samples of ^{[Val 8] -GLP-1 (} 7-37) is that the powder sample containing 3mg was measured by reverse phase ^{HPLC, [Val 8] -GLP-} 1 (7-37) 108㎍ It was prepared as a nasal absorption pharmaceutical composition.
[247] Preparation Example 31 [Val containing benzalkonium chloride (0.01%) ⁸] -Expense of GLP-1 (7-37) Preparation of absorption pharmaceutical composition
[248] As a peptide component, the amount of [Val ⁸ ] -GLP-1 (7-37) powder (about 13 mg) corresponding to 9.31 mg of [Val ⁸ ] -GLP-1 (7-37) was calculated as about 284 mg of calcium carbonate ( Average particle size: 53.6 μm). After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 2.6 mg) corresponding to 1.0% of the total weight was mixed together to give about 252 mg of a powder sample. The content of [Val ⁸ ] GLP-1 (7-37) in the powder sample was measured by reversed phase HPLC, and 3 mg of the powder sample containing 111 µg of [Val ⁸ ] GLP-1 (7-37) was nasal absorbed. It was prepared as a pharmaceutical composition.
[249] Preparation Example 32 Preparation of Non-absorbing Pharmaceutical Composition of Exendin-4 Containing Corn Starch (5.0%) and Benzalkonium Chloride (0.01%)
[250] About 270 mg of calcium carbonate (average particle size: 53.6 µm) was mixed with about 15 mg of corn starch (average particle size: 13.3 µm) and the mixture was thoroughly mixed. Purified water was then added to the mixture and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of exendin-4 powder (about 12 mg) corresponding to 9.08 mg exendin-4 powder was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 3.0 mg) corresponding to 1.0% of the total weight was mixed together to give about 255 mg of a powder sample. The content of exendin-4 in the powder sample was measured by reverse phase HPLC, and 3 mg of the powder sample containing 107 μg of exendin-4 was prepared as a nasal absorption pharmaceutical composition.
[251] Preparation Example 33 Preparation of Nasal Absorption Pharmaceutical Composition of Exendin-4 Containing Benzalkonium Chloride (0.01%)
[252] As the peptide component, the amount of Amylin: Exendin-4 powder (about 12 mg) corresponding to 9.0 mg of Exendin-4 (about 12 mg) was slowly mixed with about 285 mg of calcium carbonate (average particle size: 53.6 μm). After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 2.6 mg) corresponding to 1.0% of the total weight was mixed together to give about 260 mg of a powder sample. The content of exendin-4 in the powder sample was measured by reverse phase HPLC, and 3 mg of the powder sample containing 104 µg of exendin-4 was prepared as a nasal absorption pharmaceutical composition.
[253] Preparation Example 34 [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7- ) containing corn starch (5.0%) and benzalkonium chloride (0.01%) 37) of nasal absorption pharmaceutical composition
[254] Produce
[255] About 270 mg of calcium carbonate (average particle size: 53.6 µm) was mixed with about 15 mg of corn starch (average particle size: 13.3 µm) and the mixture was thoroughly mixed. Purified water was then added to the mixture and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. A peptide component, of ^{9.23mg [Lys 26, ε-NH} {γ-Glu (N-α- palmitoyl)}] - GLP-1 for the ^{(7-37) [Lys 26, ε} -NH {γ- The amount of Glu (N-α-palmitoyl)}]-GLP-1 (7-37) powder (about 12 mg) was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 2.7 mg) corresponding to 1.0% of the total weight was mixed together to give about 245 mg of a powder sample. The content of [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37) in the powder sample was measured by reversed phase HPLC, [Lys ²⁶ , ε- 3 mg of the powder sample containing 113 μg of NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37) was prepared as a nasal absorption pharmaceutical composition.
[256] Preparation Example 35 [Lys] containing benzalkonium chloride (0.01%) ²⁶ , ε-NH {γ-Glu (N-α-
[257] Palmitoyl)}]-GLP-1 (7-37) Preparation of Nasal Absorption Pharmaceutical Composition
[258] A peptide component, of ^{9.08mg [Lys 26, ε-NH} {γ-Glu (N-α- palmitoyl)}] - GLP-1 for the ^{(7-37) [Lys 26, ε} -NH {γ- The amount of Glu (N-α-palmitoyl)}]-GLP-1 (7-37) powder (about 12 mg) was slowly mixed with about 285 mg of calcium carbonate (average particle size: 53.6 μm). After the mixture was thoroughly mixed, a solution containing 0.03 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a desiccator under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (ca. 2.4 mg) corresponding to 1.0% of the total weight was mixed together to give about 265 mg of a powder sample. The content of [Lys ²⁶ , ε-NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37) in the powder sample was measured by reversed phase HPLC, [Lys ²⁶ , ε- 3 mg of the powder sample containing 103 µg NH {γ-Glu (N-α-palmitoyl)}]-GLP-1 (7-37) was prepared as a nasal absorption pharmaceutical composition.
[259] Example 5: Non-absorption promoting effect of starch acid polypeptide as an additive
[260] This example shows the effect of improving the absorption of starch additives for nasal absorption of human insulin using Chinese monkeys. Human insulin was used as one of the examples of acidic polypeptides other than the incretin hormones.
[261] Fine powder of aluminum sulphate (sucralate) of calcium carbonate or sucrose with an average particle size of 50 μm was used as a carrier. According to the procedure disclosed in Preparation Examples 10 to 18, a nasal absorption pharmaceutical composition containing Domyo-ji powder (1.0%) was prepared. The dose of human insulin was adjusted to allow about 25 IU (international units) / monkey / (40 mg of the composition) to be administered. Two formulations without additives, one containing calcium carbonate and human insulin, and one containing sucralate and human insulin were synthesized as controls.
[262] Using a nebulizer (UNISIA JECS Co., Ltd.), about 40 mg of the compositions of Preparation Examples 36-38 were individually administered to Chinese monkeys (each body weight about 3 kg) into their nasal cavity. Concentrations of insulin and glucose in plasma were determined by insulin-RIA beads II (Dinabott Co., Ltd.) and blood glucose level measuring devices (FREESTYLE-KISSEI; KISSEI Pharmaceutical Co., Ltd.). It was measured at 15, 20, 30, 45, 60, 90 and 120 minutes.
[263] The results are shown in Tables 9 and 10 below. As can be seen from the results, the uptake of insulin and a decrease in blood glucose levels were observed by administration of the composition without additives. However, for administration of a composition containing 1.0% of Domyo-ji powder (partially starch), an increase in human insulin uptake was observed and significantly lower blood glucose levels were observed at an early stage.
[264]
[265]
[266] Preparation Example 36 Preparation of a Pharmaceutical Composition of Insulin / Sucralate
[267] To 120 mg of sucralate, 1 mL of human insulin preparation (Humulin R: Shionogi Pharmaceutical Co., Ltd.) was added and the mixture was thoroughly mixed. The mixture was then dried overnight under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 1.2 mg) corresponding to 1.0% of the total weight was mixed together to provide a powder sample. 40 mg of the powder sample containing about 25 IU of human insulin was filled in a # 2 capsule to prepare a nasal absorption pharmaceutical composition.
[268] Preparation Example 37 Preparation of Pharmaceutical Composition of Insulin / Calcium Carbonate
[269] To 120 mg of calcium carbonate (average particle size: 53.6 mu m), 1 mL of human insulin preparation (Humulin R: Shionogi Pharmaceutical Co., Ltd.) was added and the mixture was thoroughly mixed. The mixture was then dried overnight under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 1.2 mg) corresponding to 1.0% of the total weight was mixed together to provide a powder sample. 40 mg of the powder sample containing about 25 IU of human insulin was filled in a # 2 capsule to prepare a nasal absorption pharmaceutical composition.
[270] Preparation Example 38 Preparation of a Pharmaceutical Composition of Insulin / Calcium Carbonate Containing Domyo-ji Powder (1.0%)
[271] To 120 mg of calcium carbonate (average particle size: 53.6 mu m), a suspension of 1 mL of human insulin preparation (Humulin R: Shionogi Pharmaceutical Co., Ltd.) and 1.2 mg of Domyo-ji powder was added and the mixture was kneaded. After kneading, the mixture was dried overnight under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 1.2 mg) corresponding to 1.0% of the total weight was mixed together to provide a powder sample. 40 mg of the powder sample containing about 25 IU of human insulin was filled in a # 2 capsule to prepare a nasal absorption pharmaceutical composition.
[272] Example 6 Influence of Starch Particle Size on Nasal Absorption as Additive of Pharmaceutical Composition
[273] In this Example, the effect of reducing starch particle size on the nasal absorption of the pharmaceutical composition containing GLP-1 (7-36) NH ₂ was investigated using the activity of reducing blood glucose levels in rats as an indicator. .
[274] Two different fine powder starches having the following average particle size were used as additives in the pharmaceutical composition of this example.
[275] Corn Starch:
[276] Average (d50) of integrated particle size: 13.3 μm
[277] Center distribution: 4㎛
[278] Distribution range: 5.5 μm to 30 μm
[279] Potato Starch:
[280] Average (d50) of integrated particle size: 37.7 μm
[281] Center distribution: 45㎛
[282] Distribution range: 5.5 μm to 105 μm
[283] CD (SD) rats weighing about 300 g were anesthetized using pentbarbital. The front edge of the pipette (GPS-250, RAININ) tip was filled with 3 mg of the pharmaceutical composition (containing 90 μg polypeptide) prepared by the preparation example described below, and the tip was mounted in a syringe. The composition was sprayed into the nasal cavity of the rat with 1 mL of air. Then, 5 minutes after nasal administration of the composition, 0.5 g / kg glucose was administered intravenously into the tail of the rat. Blood samples were taken from the aorta 15 minutes after nasal administration of the composition (10 minutes after glucose administration). Blood glucose levels were measured using a blood glucose level measuring instrument (FREESTYLE-KISSEI; Kissei Pharmaceutical Co., Ltd.).
[284] The results are shown in Table 11 below. As can be seen from the results, the blood glucose level of the control group not administered GLP-1 (7-36) NH ₂ was 196 mg / dL (mean value of 3 rats). The blood glucose level in the groups containing GLP-1 (7-36) NH ₂ without additives, containing 5% corn starch and containing 5% potato starch was 170 mg / dL, respectively. , 157 mg / dL and 182 mg / dL. The blood glucose level is lower than that of the control group not administered GLP-1 (7-36) NH ₂ .
[285] The absorption enhancement effect on GLP-1 (7-36) NH ₂ absorption was observed in the case of the group using the small particle size corn starch compared with the group using the large particle size potato starch.
[286] As is clear from the results in Table 11 below, although preferred nasal absorption of GLP-1 (7-36) NH ₂ is observed when using smaller particle size carriers, administration of starch of larger particle size is clearly absorbed. No improvement effect.
[287]
[288] Preparation Example 39 Preparation of Pharmaceutical Composition Without Participant
[289] As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 108 mg) corresponding to 90.2 mg of GLP-1 (7-36) NH ₂ was mixed with about 2.86 g of calcium carbonate. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, and then purified water was added by adding air, and the mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 30 mg) corresponding to 1.0% of the total weight was mixed together to give 2.95 g of a powder sample. GLP-1 (7-36) NH ₂ content in the powder sample was measured by reverse phase HPLC, and 3 mg of the powder sample containing 92 μg of GLP-1 (7-36) NH ₂ was prepared as a nasal absorption pharmaceutical composition. It became.
[290] Preparation Example 40 Preparation of a Pharmaceutical Composition Containing Corn Starch (5.0%)
[291] To about 2.71 g of calcium carbonate (average particle size: 53.6 µm), about 150 mg of corn starch (average particle size: 13.3 µm) is added, the mixture is thoroughly mixed, and purified water is then added to the mixture and obtained. The resulting mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As the peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 107 mg) corresponding to 89.2 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 29 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.90 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reverse phase HPLC, and 3 mg of the powder sample containing 92 µg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[292] Preparation Example 41 Preparation of a Pharmaceutical Composition Containing Potato Starch (5.0%)
[293] To about 2.71 g of calcium carbonate (average particle size: 53.6 µm), about 150 mg of potato starch (average particle size: 37.7 µm) is added, the mixture is thoroughly mixed, and purified water is then added to the mixture and obtained. The resulting mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed through a 180 μm sieve to give a dry product. As peptide component, the amount of GLP-1 (7-36) NH ₂ powder (about 107 mg) corresponding to 89.5 mg of GLP-1 (7-36) NH ₂ was slowly mixed with the dry product obtained above. After the mixture was thoroughly mixed, a solution containing 0.3 mg of benzalkonium chloride was added, followed by further addition of purified water, and the obtained mixture was kneaded. After kneading, the mixture was dried overnight in a freeze-dryer under reduced pressure and passed the dried product through a 180 μm sieve. To the sieved mixture, an amount of calcium stearate (about 29 mg) corresponding to 1.0% of the total weight was mixed together to give about 2.88 g of a powder sample. The content of GLP-1 (7-36) NH ₂ in the powder sample was measured by reverse phase HPLC, and 3 mg of the powder sample containing 93 µg of GLP-1 (7-36) NH ₂ was used as a nasal absorption pharmaceutical composition. Was prepared.
[294] As described above, the present invention provides a novel pharmaceutical composition for non-absorption, which includes a bioactive polypeptide, a water insoluble or poorly water soluble carrier, and an additive capable of uniformly dispersing and embedding the polypeptide on the surface of the carrier. It contains. The new pharmaceutical composition has an absorption rate of a bioactive polypeptide, particularly an acidic bioactive polypeptide having low solution stability since it has an isoelectric point of 7 or less (in other cases, difficult to administer via other non-injection routes including oral administration). To improve.
[295] Therefore, the nasal absorption pharmaceutical composition of the present invention enables the application of nasal mucosa or nasal mucosa of insulin secretion-promoting polypeptide (in the form of a powder composition, a practical non-injection route of administration has not yet been established). . In this manner, the composition can be used as a clinically effective medicament by enhancing the bioavailability of the polypeptide. For this reason, the pharmaceutical composition of the present invention has a remarkable medical effect.

权利要求:
Claims (22)
[1" claim-type="Currently amended] A nasal absorption pharmaceutical composition containing a bioactive acidic polypeptide having an isoelectric point of 7 or less, a water-insoluble or poorly water-soluble carrier, and an additive for dispersing and embedding the polypeptide on the surface of the carrier.
[2" claim-type="Currently amended] 2. A bioactive acidic polypeptide having an isoelectric point of 7 or less, a water insoluble or poorly water soluble carrier, and an additive having an average particle size of 1 µm to 20 µm for dispersing and embedding the polypeptide on the surface of the carrier. Pharmaceutical composition for nasal absorption containing.
[3" claim-type="Currently amended] 3. A bioactive acidic polypeptide having an isoelectric point of 7 or less, a water-insoluble or poorly water-soluble carrier, and an average particle size of 1 µm to 4 for dispersing and embedding the polypeptide on the surface of the carrier. A non-absorbing pharmaceutical composition containing a water insoluble or poorly water-soluble additive of 20㎛.
[4" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to any one of claims 1 to 3, wherein the water-insoluble or poorly water-soluble carrier is a polyvalent metal compound.
[5" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to claim 4, wherein the polyvalent metal compound is an aluminum compound, calcium compound, magnesium compound, silicon compound, iron compound or zinc compound.
[6" claim-type="Currently amended] The method of claim 5, wherein the aluminum compound is a dry aluminum hydroxide gel, aluminum chloride hydroxide, synthetic aluminum silicate, hard aluminum oxide, colloidal hydrous aluminum silicate, aluminum magnesium hydroxide, aluminum hydroxide, aluminum hydroxide gel, aluminum sulfate, A non-absorbing pharmaceutical composition which is selected from the group consisting of dihydroxy aluminum acetate, aluminum stearate, natural aluminum silicate, aluminum monostearate, and potassium aluminum sulfate.
[7" claim-type="Currently amended] 6. The method of claim 5, wherein the calcium compound is apatite, hydroxyapatite, calcium carbonate, calcium disodium edetate, calcium chloride, calcium citrate, calcium glycophosphate, calcium gluconate, calcium silicate, calcium oxide, calcium hydroxide, stearic acid Calcium acid, calcium triphosphate, calcium lactate, calcium pantothenate, calcium oleate, calcium palmitate, calcium D-pantothenate, calcium alginate, anhydrous calcium phosphate, calcium hydrogen phosphate, calcium dihydrogen phosphate, calcium acetate, calcium saccharide A pharmaceutical composition for absorption of nasal absorption, which is selected from the group consisting of calcium sulfate, calcium dihydrogen phosphate, para-aminosalicylate, and biologically calcined (calcified) compounds.
[8" claim-type="Currently amended] 6. The magnesium compound according to claim 5, wherein the magnesium compound is magnesium L-aspartate, magnesium chloride, magnesium gluconate, magnesium aluminosilicate, magnesium silicate, magnesium oxide, magnesium hydroxide, magnesium stearate, magnesium carbonate, magnesium aluminometasilicate , Magnesium sulfate, magnesium sodium silicate, and synthetic magnesium sodium silicate is selected from the group consisting of nasal absorption pharmaceutical composition.
[9" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to claim 5, wherein the silicon compound is selected from the group consisting of hydrated silicon dioxide, hard silicic anhydride, synthetic hydrotalcite, diatomaceous earth, and silicon dioxide.
[10" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to claim 5, wherein the iron compound is iron sulfate.
[11" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to claim 5, wherein the zinc compound is one selected from the group consisting of zinc chloride, zinc stearate, zinc oxide, and zinc sulfate.
[12" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to any one of claims 4 to 11, wherein the polyvalent metal compound has an average particle size of 100 µm or less.
[13" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to claim 12, wherein the polyvalent metal compound has an average particle diameter of 20 to 60 µm.
[14" claim-type="Currently amended] The method according to any one of claims 1 to 13, wherein the physiologically active acid polypeptide is calcitonin, catacalcin, cholecystokinin-12, cholecystokinin-8, corticotropin-lipotropin precursor, corticotropin-like intermediate peptide. , Reportropin-β, Reportropin-γ, Melanotropin-β, Corticoberine, Endothelin-1, Endothelin-2, Endothelin-3, Galinine message-associated polypeptide, Gastrin-71, Gastrin-34, gastrin-17, gastric inhibitory polypeptide, glycentin-related polypeptide, glucagon, glucagon-like peptide-1, glucagon-like peptide-1 amide, glucagon-like peptide-1 (7-36) amide, glucagon- Similar peptide-1 (7-37), [Val ⁸ ] -glucagon-like peptide-1 (7-36) amide, [Val ⁸ ] -glucagon-like peptide-1 (7-37), [Lys ²⁶ , ε -NH {γ-Glu (N-α-palmitoyl)}]-GLP-1- (7-37), glucagon-like peptide-1 (9-36) amide, glucagon-like peptide -1 (9-37), glucagon-like peptide-2, exendin-3, exendin-4, insulin β-chain, insulin α-chain, insulin, progonadoliberin-I, gonadoriberine -II, gonadoruririne-related peptide-I, neuromedin C, insulin-like protein (INSL) A-chain, motiline-related peptide E, leucine-enkephalin, methionine-enkephalin, leumorphine, oxytocin, neuropisin- 1, neuropisin-2, copeptin, neuromedin B, neuromedin N, neuropeptide Y, neuropeptide AF, PACAP-related peptide, pancreatic hormone, pancreatic 20-peptide (icosapeptide), peptide YY, tyroriberine, neuro A quinine A, urocortin, urotensin II, enteric peptide (PHM-27), and enteric peptide-42 is selected from the group consisting of nasal absorption pharmaceutical composition.
[15" claim-type="Currently amended] The method according to claim 14, wherein the physiologically active acid polypeptide is glucagon-like peptide-1, glucagon-like peptide-1 amide, glucagon-like peptide-1 (7-36) amide, glucagon-like peptide-1 (7- 37), [Val ⁸ ] -glucagon-like peptide-1 (7-36) amide, [Val ⁸ ] -glucagon-like peptide-1 (7-37), [Lys ²⁶ , ε-NH {γ-Glu ( N-α-palmitoyl)}]-GLP-1- (7-37), glucagon-like peptide-1 (9-36) amide, glucagon-like peptide-1 (9-37), glucagon-like peptide- 2, exendin-3, exendin-4, glucagon, gastric inhibitory polypeptide, insulin and derivatives thereof.
[16" claim-type="Currently amended] 14. A non-absorbable medicament according to any one of claims 1 to 13, comprising a peptide incretin, a water insoluble or poorly water soluble carrier, and an additive for dispersing and embedding the peptide incretin on the surface of the carrier. Composition.
[17" claim-type="Currently amended] The method of claim 16, wherein the peptide incretin is Glucagon-like peptide-1, Glucagon-like peptide-1 amide, Glucagon-like peptide-1 (7-36) amide, Glucagon-like peptide-1 (7-37) ), [Val ⁸ ] -glucagon-like peptide-1 (7-36) amide, [Val ⁸ ] -glucagon-like peptide-1 (7-37), [Lys ²⁶ , ε-NH {γ-Glu (N -α-palmitoyl)}]-GLP-1- (7-37), glucagon-like peptide-1 (9-36) amide, glucagon-like peptide-1 (9-37), glucagon-like peptide-2 , Exendin-3, exendin-4, glucagon, gastric inhibitory polypeptide, insulin and derivatives thereof.
[18" claim-type="Currently amended] The pharmaceutical composition for nasal absorption according to any one of claims 1 to 17, wherein the additive is starch selected from the group consisting of amylopectin, amylose, or a mixture containing amylopectin and amylose in any ratio.
[19" claim-type="Currently amended] 19. The method according to any one of claims 1 to 18, wherein the additive is selected from rice flour, rice starch, rice beta-starch (non-rice type), rice beta-starch (glucorice type), pregelatinized rice starch (non-rice type). ), Luxury Rice Starch (Glutinous Rice Type), Corn Starch, Corn Beta-Starch (Glutinous Rice Type), Corn Beta-Starch (Glutinous Rice Type), Luxury Corn Starch (Glutinous Rice Type), Luxury Corn Starch (Glutinous Rice Type), Potato Starch, A pharmaceutical composition for nasal absorption, which is potato beta-starch (non-rice type), luxury potato starch (non-rice type), luxury wheat starch (non-rice type), or the above partial luxury starch.
[20" claim-type="Currently amended] The pharmaceutical composition for non-absorption according to any one of claims 1 to 19, wherein the additive is oligosaccharide, carboxyvinyl polymer, povidone, hydroxypropyl cellulose, xanthan gum, pectin, sodium alginate, powdered gum arabic or gelatin.
[21" claim-type="Currently amended] A pharmaceutical composition comprising a nasal absorption pharmaceutical composition according to any one of claims 1 to 20 together with a DPP-IV inhibitor.
[22" claim-type="Currently amended] The pharmaceutical composition according to claim 21, wherein the DPP-IV inhibitor is diprotein A, baccitracin, or isoleucine thiazolidide.

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同族专利:

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公开号 | 公开日

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MXPA04005025A|2005-04-08|

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AU2002349513A1|2003-06-10|

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US20050014681A1|2005-01-20|

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IL161446D0|2004-09-27|

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CA2468250A1|2003-06-05|

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JPWO2003045418A1|2005-04-07|

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BR0214438A|2004-11-03|

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CN1596120A|2005-03-16|

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AU2002349513B2|2007-10-18|

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EP1466610A1|2004-10-13|

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EP1466610A4|2007-03-21|

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WO2003045418A1|2003-06-05|

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RU2004119427A|2005-03-27|

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RU2327484C2|2008-06-27|

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JP4124734B2|2008-07-23|

引用文献:

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公开号 | 申请日 | 公开日 | 申请人 | 专利标题

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法律状态:
2001-11-26|Priority to JP2001359559

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2001-11-26|Priority to JPJP-P-2001-00359559

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2002-11-26|Application filed by 다이이찌 산토리 파마 가부시키가이샤

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2002-11-26|Priority to PCT/JP2002/012337

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2004-07-03|Publication of KR20040058324A

优先权:

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申请号 | 申请日 | 专利标题

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JP2001359559|2001-11-26|

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JPJP-P-2001-00359559|2001-11-26|

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PCT/JP2002/012337|WO2003045418A1|2001-11-26|2002-11-26|Medicinal compositions for nasal absorption|