JPH0687887A - Enamine derivative of peptide and peptide preparation containing same as prodrug - Google Patents

Abstract

PURPOSE: To provide the prodrug of pharmaceutical peptide of which absorption at mucous membranes of bowel and nose is improved.

CONSTITUTION: The objective prodrug is obtained by subjecting N-terminal amino group of a peptide to the reaction to form an enamine. As the suitable pharmaceutical peptides, angiotensin II, bradykinin, caerulein, carnosine, cholecystokinin-octapeptide, enkephalin, Met-enkephalin, Leu-enkephalin, kallidin, octocin, vasopressin, phyllocerulein, U-72102E (merchandise No.; Upjohn Co.), insulin, calcitonin or the like are exemplified. The enamine derivative of the peptide is obtained by reacting the peptide with a diketone such as ethyl acetoacetate, propyl acetoacetate, butyl acetoacetate and hexyl acetoacetate.

COPYRIGHT: (C)1994,JPO

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an enamine derivative of a drug peptide and a peptide preparation containing the same as a prodrug.

[0002]

2. Description of the Related Art In order for a drug to exert its action, absorption from the administration site is required as the first step of delivery to the target site. However, drug peptides are known to be difficult to absorb through the mucous membrane of the small intestine (including the rectum and colon),
The reason is believed to be that the oil / water partition coefficient is small and it cannot diffuse and permeate through the mucosal lipid layer. Many peptide drugs have poor solubility in water.

Therefore, various proposals have been made in order to improve the permeation of peptides from the intestinal mucosa and nasal mucosa, and one of them is the development of absorption enhancers. This absorption enhancer changes the transmucosal permeation defense mechanism when used in combination with the main peptide, thereby increasing the permeability of the peptide. Typical absorption enhancers include surfactants [E. Touitou et al., Journal of Pharmacy and Pharmacology (J.
Pharm.Pharmacol.), 30, p.662 (1978);
Shichimoto Akira, Journal of Pharmacy and Pharmacology (J.Pharm.Pharmacol.), 30
Vol., 806 (1978)] and salicylic acid and its derivatives as non-surfactants [Toshiaki Nishihata et al., Journal of Pharmacy and Pharmacol. (J.Pharm.Pharmacol.), 33, 334 (198).
1); Diabetes, Volume 30, 10
65 (1981); Journal of Pharmacy and Pharmacology (J.Pharm.Pharmacol.),
35, p. 148 (1983)] is well known, and some results have been obtained. However, its clinical trials are limited [see Toshiaki Nishihata et al., Journal of Pharmacy and Pharmacol., Vol. 41, p. 799 (1989)]. Therefore, absorption enhancers are not yet practical.

Another method for improving the transmucosal permeability of peptides is chemical modification aimed at increasing the oil / water partition coefficient. As a study on the prodrug formation by this chemical modification, a prodrug by esterification of the C-terminal carboxylic acid group has only recently been published [A. Kahn et al., International Journal of. Pharmachutics (Int.J.Ph
arm.), 76, 99 (1991); Ji J.
GJRasmussen et al., International Journal of Pharmaceuticals (In
tJPharm.), 76, 113 (1991)].

[0005]

DISCLOSURE OF THE INVENTION An object of the present invention is to provide a prodrug of a drug peptide having enhanced absorption from the mucous membrane of the nose and intestines. Another object of the present invention is to
It is intended to provide a prodrug of a drug peptide as an injection that can be easily adjusted.

[0006]

As a result of intensive studies, the present inventors have found that the subject can be achieved by performing specific chemical modification on the N-terminal amino group or amino group residue of a peptide, The present invention has been completed.

That is, the present invention provides an enamine derivative of a drug peptide, characterized in that the N-terminal amino group or amino group residue of the drug peptide is enamined, or a pharmaceutically acceptable salt thereof, and a prodrug thereof. To provide a peptide preparation containing Since the enamine derivative of the present invention has a significantly increased solubility and dissolution rate in water as compared with the parent peptide, peptide injections that conventionally required a solubilizing agent can be easily converted into physiological saline by using the enamine derivative. Can be dissolved.

First, the target drug peptide in the enamine derivative of the present invention refers to a peptide having physiological activity, and specific examples thereof include angiotensin II, bradykinin, cerulein, carnosine, cholecystokinin octapeptide, enkephalin, Met-enkephalin, Leu-enkephalin, kallidin, octocin, vasopressin, phylloserlein, U-72102.
E (Product number of Upjohn Co.), insulin, calcitonin and the like can be mentioned.

The enamine derivative of the present invention is obtained by enamine-forming the N-terminal amino group or amino group residue of the drug peptide as described above, and generally has the formula:

[Chemical 1] It is represented by. The C-terminal carboxylic acid group of the peptide or the functional group capable of forming a salt on the side chain may be in the form of a salt, and the enamine derivative in the form of a salt is also within the scope of the present invention.

The enamine derivative of the peptide of the present invention is generally obtained by reacting the peptide with a β-diketone. Examples of β-diketones that can be used include ethylacetoacetate, propylacetoacetate, butylaceteacetate, isobutylacetoacetate, hexylacetoacetate and the like.

In the enamine reaction, generally, 1 molar equivalent of peptide and 1 molar equivalent of alkali such as caustic soda and caustic potash are mixed in a solvent such as methanol or ethanol, and 1 to 1.2 mol of β-diketone is added thereto. Add the equivalent amount. Generally, the reaction temperature is 30 to 60 ° C. and the reaction time is 3 to 24 hours.

The reaction is usually carried out under shaking until the reaction solution becomes transparent. The obtained solution is filtered, and the filtrate is dried under reduced pressure heat retention (about 40 ° C.). Then, toluene and / or dioxane is added, and excess β-diketone is removed by azeotropy under reduced pressure heating to obtain a powdery enamine derivative.

In the presence of caustic soda, a salt form of the carboxylic acid at the C-terminal of the peptide or at the side chain is obtained. The salt form is fat-soluble and water-soluble. This salt form can be appropriately converted into a carboxylic acid by a method such as dissolving in dehydrated ethanol, adding an acidic ion exchange resin (bead form), shaking, filtering, and drying the filtrate. Further, it may be in the form of a pharmaceutically acceptable salt of another type.

The thus obtained enamine derivative of the peptide of the present invention or a pharmaceutically acceptable salt thereof has the same toxicity as that of the drug peptide and can be used as a prodrug. , Enteric coated tablets, enteric coated granules, enteric coated capsules, rectal suppositories, vaginal suppositories, transpulmonary aerosols, nasal drops, eye drops,
It can be formulated into injections and the like, and such peptide formulations can be administered nasally, rectally, vaginally, or orally. For injection administration, put the peptide enamine derivative powder in a vial, heat sterilize it (or make a vial under the sterilization process), and put the peptide enamine derivative powder in a vial or saline before administration. Dissolve in glucose solution to make an injection.

For nasal administration, in addition to the enamine derivative of the peptide, ingredients such as glycerin, chlorobutanol, phosphate buffer (pH = 7.5), etc. are blended and adjusted according to the general rules of formulation of the 12th Pharmacopoeia of Japan. As a result, a nasal preparation is obtained.

For rectal administration and vaginal administration, in addition to an enamine derivative of a peptide, an oleaginous base such as cacao butter, laurin butter, nikkei butter, hydrogenated oil or a semi-synthetic fat or oil base,
If desired, a surfactant such as lecithin, or an ingredient such as an aqueous base of polyethylene glycol is mixed, and a suppository formulation can be obtained by a conventional method such as a cold pressure method and a melting method.

For oral administration, the enamine of peptide returns to the peptide of the parent compound in an acidic solution, so that it is used as an enteric preparation (enteric tablet or enteric granule). Such enteric preparations include, for example, enamine derivatives, starch, lactose, mannitol, hydroxypropyl cellulose and the like, and the components such as molded tablets or granules are mixed with cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, and lumethacrylic acid. It can be obtained by applying a coating based on ethyl acrylate copolymer, methacrylic acid / methyl methacrylate copolymer, and the like. Further, in oral administration, particularly when the peptide is decomposed in the lumen of the small intestine by a protease, the effect can be enhanced by the combined use of a protease inhibitor. Examples of such protease inhibitors include phenylmethyl-sulfonyl-fluoride and the like.

The dose of the prodrug in the peptide preparation of the present invention for the purpose of the pharmacological action of the parent peptide is, for example, when the peptide is angiotensin II, 10 times as much as the injection by rectal administration and 2 times as much as the injection by oral administration. In the case of double dose, enkephalin, it is in the range of 5 times the injection dose for rectal administration and the same dose for oral administration.

The pharmacological tests for the representative prodrugs of the present invention will be described below. The intravenous and rectal administration of angiotensin II ethyl acetoacetate enamine was compared with angiotensin II itself in rats. Rats were anesthetized with pentobarbital, and changes in blood pressure were measured by a conventional method. Angiotensin II 0.3μ
When g / kg (dissolved in physiological saline) was administered and when the enamine derivative (corresponding to angiotensin II 0.3 μg / kg) was intravenously administered, as shown in Table 1, the blood pressure-increasing effect was similar, and It was found that the derivative quickly returned to angiotensin II. Angiotensin
II rectal administration of 7.5 μg / kg did not show any increase in blood pressure, but the enamine derivative 7.5 μg / kg
The blood pressure increase rate when administered kg was intravenous (0.3 μg /
kg), and the blood pressure increase time was prolonged 3 times as compared with intravenous administration (0.3 μg / kg). This result shows that the enamine derivative became a prodrug capable of rectal absorption.

[0020]

[Table 1] Raising blood pressure of rat after administration of angiotensin II enamine derivative Dose and dosage form of enamine Blood pressure (μg / kg) Systolic blood pressure, mgHg Pressurization period Intravenous administration Saline 0.3 30 10 sec Rectal administration Phosphorus Acid buffer 7.5 25 30 seconds

As described above, the enamine derivative of the drug peptide according to the present invention exhibits a superior pharmacological effect as compared with the case where it is administered in the unmodified form, and therefore exhibits the function as a prodrug sufficiently. It is considered that this is based on the fact that the enamine derivative is fat-soluble and has improved absorbability.

[0022]

EXAMPLES The present invention will be described in more detail with reference to the following examples. Example 1 Enamine synthesis from tripeptide (phe-gly-gly) and ethyl acetoacetate Tripeptide (10 g) and sodium hydroxide (1.35)
g) is dissolved in 500 ml of ethanol, ethyl acetoacetate (5 g) is added, and the mixture is reacted at 40 ° C. until it becomes clear. The ethanol solution is filtered and the filtrate is put under reduced pressure 4
After removing ethanol at 0 ° C., 50 ml of toluene was added, and the operation of removing excess ethyl acetoacetate again under reduced pressure was repeated 5 times to obtain the enamine represented by the following structural formula as a powder.

[Chemical 2] UV spectrum shown in attached FIG. 1 and NMR shown in FIG.
From the spectrum, it is clear that the enamine is formed.

Example 2 Synthesis of enamine from tripeptide (phe-gly-gly) and propylacetoacetate Tripeptide (10 g) and sodium hydroxide (1.35)
g) is dissolved in 500 ml of ethanol, propyl acetoacetate (5.8 g) is added, and the mixture is reacted at 40 ° C. until it becomes clear. This ethanol solution was filtered, ethanol was removed from the filtrate at 40 ° C. under reduced pressure, 50 ml of toluene was added, and excess propylacetoacetate was removed again under reduced pressure. This operation was repeated 5 times to obtain the following structure. The title enamine with the formula is obtained as a powder.

[Chemical 3] The formation of enamine is clear from the UV spectrum of the attached FIG.

Example 3 Synthesis of enamine from tripeptide (phe-gly-gly) and butyl acetoacetate Tripeptide (10 g) and sodium hydroxide (1.35)
g) and 500 ml of ethanol are dissolved, butyl acetoacetate (6.4 g) is added, and the mixture is dissolved at 40 ° C. until it becomes clear. This ethanol solution was filtered, ethanol was removed from the filtrate at 40 ° C. under reduced pressure, 50 ml of a mixed solution of toluene and dioxane was added, and the operation of removing excess butyl acetoacetate again under reduced pressure was repeated 5 times. , The enamine having the following structural formula is obtained as a powder.

[Chemical 4] The formation of enamine is clear from the UV spectrum of the attached FIG.

Example 4 Synthesis of enamine from tetrapeptide (phe-phe-phe-phe) and ethyl acetoacetate Tetrapeptide (1 g) and sodium hydroxide (0.06)
g) is dissolved in 100 ml of ethanol, ethyl acetoacetate (0.23 g) is added, and the mixture is reacted at 40 ° C. until it becomes clear. The ethanol solution was filtered, ethanol was removed from the filtrate at 40 ° C. under reduced pressure, 50 ml of toluene was added, and excess ethyl acetoacetate was removed again under reduced pressure. This operation was repeated 5 times to obtain the following structural formula. The indicated enamine is obtained as a powder.

[Chemical 5] The formation of enamine is clear from the UV spectrum of the attached FIG.

Example 5 Synthesis of enamine from pentapeptide (phe-phe-phe-phe-phe) and ethyl acetoacetate Pentapeptide (1 g) and sodium hydroxide (0.05)
3 g) and 100 ml of ethanol are dissolved, ethyl acetoacetate (0.2 g) is added, and the mixture is dissolved at 40 ° C. until it becomes clear. This ethanol solution is filtered, ethanol is removed from the filtrate at 40 ° C. under reduced pressure, 50 ml toluene is added, and excess ethylacetoacetate is removed again under reduced pressure. This operation is repeated 5 times, and has the following structural formula. The indicated enamine is obtained as a powder.

[Chemical 6] The formation of enamine is clear from the UV spectrum of the attached FIG.

Example 6 Synthesis of Enamine from Angiotensin II and Ethylacetoacetate Angiotensin II (100 mg) and sodium hydroxide (3.8 g) were dissolved in 100 ml of ethanol, ethylacetoacetate (15 mg) was added, and the mixture was added at 40 ° C. React until clear. After filtering this ethanol solution and removing ethanol from the filtrate under reduced pressure at 40 ° C., 50 m
1 Toluene was added, and the operation of removing excess ethyl acetoacetate again under reduced pressure was repeated 5 times to obtain the enamine represented by the following structural formula as a powder.

[Chemical 7] The formation of enamine is clear from the UV spectrum of the attached FIG.

Example 7 Synthesis of enamine from insulin and ethyl acetoacetate Insulin (50 mg) and sodium hydroxide (2 mg)
And are dissolved in 100 ml of ethanol, ethyl acetoacetate (20 mg) is added, and the mixture is dissolved at 40 ° C. until it becomes clear. The ethanol solution was filtered, and the filtrate was put under reduced pressure for 4
After removing ethanol at 0 ° C., 50 ml of toluene was added, and the operation of removing excess ethyl acetoacetate again under reduced pressure was repeated 5 times to obtain a powder. Attached Figure 8 U
The formation of enamine is clear from the V spectrum.

[0029]

INDUSTRIAL APPLICABILITY The present invention provides an enamine derivative of a drug peptide having improved absorption from intestinal / nasal mucosa and a peptide preparation containing the same as a prodrug.

[Brief description of drawings]

FIG. 1 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 1.

FIG. 2 is an NMR spectrum diagram of an enamine derivative of the peptide obtained in Example 1.

FIG. 3 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 2.

FIG. 4 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 3.

FIG. 5 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 4.

FIG. 6 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 5.

FIG. 7 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 6.

FIG. 8 is a UV spectrum diagram of an enamine derivative of the peptide obtained in Example 7.

─────────────────────────────────────────────────── ─── Continuation of the front page (51) Int.Cl. ⁵ Identification code Office reference number FI Technical display area A61K 37/34 8314-4C C07K 5/10 ZNA 8318-4H 7/06 Z 8318-4H 7/12 7306-4H 7/14 7306-4H 7/16 7306-4H 7/18 7306-4H 7/36 7306-4H 7/40 7306-4H // C07K 99:00

Claims (5)

[Claims] 1. An enamine derivative of a drug peptide or a pharmaceutically acceptable salt thereof, wherein the N-terminal amino group of the drug peptide or the amino group residue of the peptide is enamined. 2. The drug peptide is angiotensin I.
I, bradykinin, cerulein, carnosine, cholecystokinin octapeptide, enkephalin, Met-enkephalin, Leu-enkephalin, kallidin, octocin, vasopressin, phylloserrein, U-72
The compound according to claim 1, which is selected from the group consisting of 102E (product number of Upjohn Company), insulin and calcitonin. 3. A peptide preparation containing the enamine derivative according to claim 1 or a pharmaceutically acceptable salt thereof as a prodrug. 4. The drug peptide is angiotensin I.
I, bradykinin, cerulein, carnosine, cholecystokinin octapeptide, enkephalin, Met-enkephalin, Leu-enkephalin, kallidin, octocin, vasopressin, phylloserrein, U-72
The peptide preparation according to claim 3, which is selected from the group consisting of 102E (product number of Upjohn Co.), insulin and calcitonin. 5. A dosage form selected from the group consisting of enteric coated tablets, enteric coated granules, enteric coated capsules, rectal suppositories, vaginal suppositories, pulmonary aerosols, nasal drops, eye drops and injections. The peptide preparation according to claim 3 or claim 4.