JP2000095794A - Enkephalin degrading enzyme inhibitors - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a synthetic inhibitor having a higher inhibitory activity on enkephalin-degrading enzymes, particularly dipeptidyl aminopeptidase than conventional ones. A formula (I) in _{X 1 -Val-Val-Tyr-} Pro-X 2 (I) ( the following formula, X ₁ is indicates no a is either or amino acid residue Leu, X ₂ Indicates Trp or no amino acid residue.) However, Leu-Va
Except for l-Val-Tyr-Pro, a peptide having enkephalin degrading enzyme inhibitory activity or a salt thereof, and a pharmaceutical composition containing such a peptide or a salt thereof in a therapeutically effective amount.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a peptide that inhibits enkephalin-degrading enzymes, particularly dipeptidyl aminopeptidase. The invention also relates to a pharmaceutical composition comprising such a peptide.

[0002]

BACKGROUND OF THE INVENTION Enkephalin has the formula Tyr-Gly-Gly-Phe-
A type of opioid peptide represented by X (X = Met or Leu), which is an endogenous peptide that binds to an enkephalin receptor and exhibits a morphine-like analgesic action. Although the details of its physiological actions are not clear, pharmacological actions such as control of pain and body temperature and regulation of neuroendocrine function are known. On the other hand, some enkephalin-degrading enzymes that are presumed to be involved in regulation of enkephalin metabolism or activity in vivo are also known. In each case, the specific site of enkephalin is specifically cleaved, leading to its inactivation. Especially Tyr-Gl
Enzymes that cleave between y, Gly-Gly, and Gly-Phe are known, and aminopeptidase, dipeptidyl aminopeptidase, enkephalinase, and angiotensin converting enzyme have been found, respectively.

[0003] When enkephalin is inactivated in vivo by these degrading enzymes, the above-mentioned pharmacological action,
In particular, inhibition or suppression of the analgesic effect occurs. In contrast, when inhibiting enkephalin degrading enzymes,
Enkephalin's analgesic effect is enhanced. For example, kelatorphan inhibits enkephalinase and dipeptidyl aminopeptidase (MCFo
urnie-Zaluski et al., Eur. J. Pharmacol. 102: 525, 198.
4) Captopril inhibits angiotensin converting enzyme (Tanaka and Kato eds., "NEW Pharmacology (Revised 2nd ed.)", 164-165, 1994, Nankodo, spinorphin) Is aminopeptidase,
Inhibiting enkephalinase, angiotensin converting enzyme and dipeptidyl aminopeptidase (Japanese Patent Application Laid-Open No. 3-167199; Anesthesia Vol. 42, 1497-1503 and 1663-16)
70, 1993). In particular, spinorphin has the formula Leu-Val-Val-Tyr-Pro-Tr-Tr isolated from bovine spinal cord.
A heptapeptide represented by p-Thr, which is an endogenous pain regulator that specifically inhibits enkephalin degrading enzymes present in the central nervous system. Thus, it has been suggested that an inhibitor of an enkephalin degrading enzyme may enhance endogenous enkephalin activity and participate in a control mechanism of enkephalin action, and its application to medicine is expected.

[0004]

Under such circumstances, the present inventors have conducted a search for a peptide having enkephalin degrading enzyme inhibitory activity. That is, an object of the present invention is to provide a synthetic inhibitor having a higher inhibitory activity on enkephalin-degrading enzymes, particularly dipeptidyl aminopeptidase than conventional ones. Another object of the present invention is to provide a pharmaceutical composition comprising such a synthetic inhibitor.

[0005]

According to the present invention, there is provided a compound represented by the following formula (I) X ₁ -Val-Val-Tyr-Pro-X ₂ (I) wherein X ₁ is Leu or an amino acid residue. indicates no group, X ₂ is represented by.) indicating no or, or amino acid residue is Trp, however Leu-Va
Provided is a peptide having enkephalin degrading enzyme inhibitory activity, except l-Val-Tyr-Pro, or a salt thereof.

In an embodiment of the present invention, the present invention relates to the following formulas (II), (III) and (IV): Val-Val-Tyr-Pro-Trp (SEQ ID NO: 1) (II); Leu-Val -Val-Tyr-Pro-Trp (SEQ ID NO: 2) (III); Val-Val-Tyr-Pro (SEQ ID NO: 3) (IV); or a salt thereof.

[0007] The peptide of the present invention is characterized in that it has an enkephalin-degrading enzyme inhibitory activity and is also easily soluble in water. For example, it is easily dissolved in water even at a concentration of 10 mg / ml. Many drug candidates having the inhibitory activity against enkephalin-degrading enzyme have been hindered from being poorly soluble or insoluble in water, but the peptide of the present invention solves the problem of solubility in water. The present invention also provides a pharmaceutical composition comprising a peptide as defined above or a salt thereof in a therapeutically effective amount. The pharmaceutical composition of the present invention can be used for the purpose of exhibiting an analgesic effect such as, for example, pain reduction. As used herein, therapeutically effective means that a pharmacological effect such as an analgesic effect can be exhibited.

[0008]

BEST MODE FOR CARRYING OUT THE INVENTION The peptide represented by the formula (I) of the present invention can be synthesized by using either a solid phase synthesis method or a liquid phase synthesis method. Usually, it is convenient to use the solid phase synthesis method of Merrifield. In a general method, α-
A series of operations such as selective removal of the amino protecting group and condensation of the protected amino acid are repeated to construct a protected peptide chain, followed by deprotection of the protecting group of the side chain functional group to obtain the desired peptide.

In peptide synthesis, the α-amino group of the constituent amino acids and the phenolic OH of tyrosine (Tyr)
The groups must be protected beforehand with any protecting groups. There are mainly two methods for protecting the α-amino group and the side chain functional group, namely, protecting the α-amino group with a t-butoxycarbonyl (Boc) group and protecting the side chain functional group with a benzyl alcohol-based protecting group. The Boc method and the Fmoc method in which the α-amino group is protected with a 9-fluorenylmethoxycarbonyl (Fmoc) group and the side chain functional group is protected with a t-butyl alcohol-based protecting group can be used. In the Boc method, trifluoroacetic acid (TFA)
While removing the c group, the Boc amino acids are sequentially condensed, and finally the entire peptide can be removed with a strong acid such as hydrogen fluoride to obtain the target peptide.In the Fmoc method, the Fmoc group is used as the second peptide. While being deprotected by the secondary amine piperidine,
The desired peptide can be obtained by condensing Fmoc amino acids and finally deprotecting the side chain protecting group with a weak acid such as TFA.

The formation of an acid amide bond requires activation of either the amino group or the carboxyl group,
Due to the problem of side reactions such as racemisation, activation methods for carboxyl groups are generally used. For example, a protected amino acid is treated with a dehydrating agent such as dicyclohexylcarbodiimide (DCC) to form a protected amino acid anhydride, which is used for a condensation reaction (a symmetric acid anhydride method). A method of producing a mixed acid anhydride by reacting such an alkyl chloroformate in the presence of a tertiary amine and using it in a condensation reaction (mixed acid anhydride method), a protected amino acid and a phenol having an electron withdrawing group, The condensation reaction can be carried out by a method such as a method of preparing an active ester by condensing N-hydroxyamines with DCC and using the active ester for the condensation reaction (active ester method). In constructing the peptide chain, the protected amino acids may be condensed one by one, or protected amino acids may be separately synthesized and condensed.

The synthetic intermediates and final products are purified by chromatography such as reverse phase HPLC (high performance liquid chromatography), gel filtration, ion exchange chromatography, adsorption chromatography and the like.
The structure can be confirmed by elemental analysis, mass spectrometry, spectroscopic means such as NMR, and the like. For details on chemical synthesis of peptides, see, for example, The Society of Biochemistry, Japan, Lectures on Experimental Chemistry 1, Protein VI Synthesis and Expression, Chapter 1 Current Status of Chemical Synthesis (Nobutaka Fujii, Akira Otaka), Tokyo Kagaku Dojin, 1992 And the peptide of the present invention can be synthesized in the same manner.

In the peptide of the present invention, even if the amino terminus of the peptide forms an acid addition salt with an inorganic acid such as hydrochloric acid, sulfuric acid or nitric acid, or an organic acid such as acetic acid, oxalic acid, citric acid, succinic acid or tartaric acid. Alternatively, the carboxy terminal may form a salt with a metal such as sodium or potassium, or a salt with a pharmaceutically acceptable amine. Thus, in addition to the peptides of formulas (II), (III) and (IV), Pro-Trp-Thr, Val-Tyr-Pro-Trp, Tyr-Pro-Tr
p, Val-Val-Tyr-Pro and Val-Tyr-Pro were synthesized, and their inhibitory activities against monkey brain membrane-derived enkephalin degrading enzymes (aminopeptidase, dipeptidyl aminopeptidase, enkephalinase, angiotensin converting enzyme) were investigated. Norphine, Val-Val-Tyr-Pro-Trp-Thr, Val
-Tyr-Pro-Trp-Thr, Tyr-Pro-Trp-Thr, Leu-Val-Val-Tyr
Comparison with known Enkephalin degrading enzyme inhibitors of -Pro (Kinya Nishimura et al., Anesthesia, 42, 1663-1670, 1993) (see Example 4 below).

As a result, in particular, the formulas (II) and (II)
That the peptide represented by I) inhibits aminopeptidase, dipeptidyl aminopeptidase and angiotensin converting enzyme almost as much as spinorphin, especially higher for dipeptidyl aminopeptidase than any known inhibitor Having inhibitory activity,
It was also found that the peptide represented by the formula (IV) inhibited dipeptidyl aminopeptidase, enkephalinase, and angiotensin converting enzyme at almost moderate levels. It has been found that the peptide of the present invention structurally has a part of a spinorphin skeleton, but has an activity of inhibiting dipeptidyl aminopeptidase at a lower concentration than conventionally known peptides. In particular, the IC ₅₀ (the amount of inhibitor required for 50% inhibition) of the peptides of formula (II) and formula (III) of the present invention for dipeptidyl aminopeptidase is 23.5
ng / ml and 620 ng / ml, which correspond to about 60-fold and 2.3-fold stronger inhibitory activity than spinorphin (IC ₅₀ : 1420 ng / ml), respectively. Also, the Ki value of the peptide of the formula (II) of the present invention determined by the Lineweaver-Burk plot is
57 nM. Due to the above-described inhibitory properties on enkephalin-degrading enzymes, the peptide of the present invention can be expected to have the same pain-reducing effect as known enkephalin-degrading enzymes such as spinorphin and keratorphan (Tadahiko Hari, Biochemistry, 66, No. 1, pp. 57-60).

Thus, a pharmaceutical composition or medicament for reducing pain, for example, by combining a therapeutically effective amount of the peptide of the present invention with a suitable pharmaceutically acceptable carrier such as a diluent or excipient. It can be a formulation. The dose may be any amount as long as it reduces or eliminates pain. Formulated by conventional methods in the form of liquids, suspensions, emulsions, tablets, powders, granules, capsules, sprays, enteric agents, liposome encapsulating agents, suppositories, sustained-release preparations, and It can be administered orally (intravenously, intraarterially, intramuscularly, intraperitoneally, subcutaneously, etc.), or rectally, nasally, vaginally, etc. to animals including humans. Administration can be performed once or several times a day, and once to several times a day. As the carrier, purified water, physiological saline, buffer, ethanol, castor oil, sodium carboxymethylcellulose, talc, lactose, starch, mannitol, polylactic acid, polyglycolic acid, and the like,
A suitable carrier should be selected according to the dosage form or administration form. The pharmaceutical composition also includes a solubilizing agent (such as Tween 80 and Polysorbate 80), a disintegrant, a lubricant, a preservative (such as thimerosal and benzyl alcohol), an antioxidant (such as ascorbic acid), a fragrance, Additives such as sweeteners can also be appropriately compounded. In addition, enkephalin and
It can also be used as an analgesic for pain relief or alleviation in combination with known enkephalin degrading enzyme inhibitors.

[0015]

The present invention will be described below with reference to examples, but these examples do not limit the present invention. Example 1 Production of Val-Val-Tyr-Pro-Trp Boc-Trp-M bound to the fifth protected amino acid Boc-Trp-OH
One equivalent of errifield resin is placed in a reaction vessel, washed and swelled repeatedly with DCM (dichloromethane), and then subjected to 50% (v / v) TFA.
The Boc group was removed by contact with a deblocking solution containing (trifluoroacetic acid) / DCM for 30 minutes. T remaining in isopropanol
After removing the FA, it was washed with DCM. The resin was neutralized with DCM containing 5% (v / v) diisopropylethyleneamine and washed with DCM. Then 4 equivalents of the fourth protected amino acid Boc-
The reaction was carried out at room temperature for 2 hours in a small amount of DCM-DMF mixture containing Pro-OH and 4 equivalents of DCC (dicyclohexylcarbodiimide). After washing with DMF and DCM sequentially, Boc-Pr
o-Trp-Merrifield resin was obtained. Hereinafter, the third to first amino acids were sequentially coupled using the following protected amino acids.

[0016] Here, BrZ represents a benzyloxycarbonyl bromide protecting group.

The protected peptide resin coupled as described above is treated for 1 hour in anhydrous hydrogen fluoride containing 10% anisole and 5% 1,2-ethanedithiol (only when Trp is contained).
After the reaction at ℃, hydrogen fluoride was distilled off and washed with ether. The peptide was extracted from the resulting mixture of the peptide and the resin with 10% acetic acid, and lyophilized to obtain a crude peptide. After dissolving the crude peptide in a 0.1% TFA solution, a high-performance liquid chromatograph connected to an octadecyl silica (ODS) column yields a linear gradient of acetonitrile containing 0.1% TFA (10 to 50% / 20 minutes). And elute the fraction corresponding to the target peptide from the column,
The peptide was obtained by lyophilization.

The results of amino acid analysis of the obtained peptide were as follows. Pro (1) 1.05, Trp (1) 0.00 ^{* 1} , Tyr (1) 1.00, Val
(2) 1.95 ( ^{* 1} : Trp is not detected by amino acid analysis by hydrochloric acid hydrolysis.) The mass spectrum signal of the obtained peptide is 66.
It was 3.36.

1.0 mg / ml solution of the obtained peptide, 20 μl
High-performance liquid chromatography connected to an octadecyl silica (ODS) column (Vydac C18) was applied to a linear concentration gradient of triacetylamine / phosphate buffer to acetonitrile (10-40% / 20 min, 1.5 ml / min). When expanded
At a detection wavelength of 215 nm, a peak was detected at a retention time of 11.865 minutes. Further, under the same conditions, a linear concentration gradient of acetonitrile containing 0.1% TFA (10 to 40% / 20 minutes, 1.5 ml /
Min), a peak was detected at a retention time of 13.410 minutes.

Example 2 Preparation of Leu-Val-Val-Tyr-Pro-Trp A coupling reaction similar to that in Example 1 was carried out, but Boc-Leu-OH was added to further attach Leu to the N-terminal. And the same procedure was repeated. By this, Boc-Leu-Val-
Example 1 Val-Tyr (BrZ) -Pro-Trp-Merrifield resin was obtained.
The peptide was purified from the resin to obtain the title peptide.

The results of the amino acid analysis were as follows. Leu (1) 1.00, Pro (1) 1.10, Trp (1) 0.00 ^{* 1} , Tyr
(1) 1.05, Val (2) 1.85 ( ^{* 1} : Trp is not detected by amino acid analysis by hydrochloric acid hydrolysis.) The mass spectrum signal of the obtained peptide is 77.
5.83.

A 1.0 mg / ml solution of the obtained peptide, 20 μl
High-performance liquid chromatography connected to an octadecyl silica (ODS) column (Vydac C18) was applied to a linear concentration gradient of triacetylamine / phosphate buffer to acetonitrile (10-40% / 20 min, 1.5 ml / min). When expanded
A peak was detected at a retention time of 15.740 minutes at a detection wavelength of 215 nm. Further, under the same conditions, a linear concentration gradient of acetonitrile containing 0.1% TFA (10 to 40% / 20 minutes, 1.5 ml /
Min), a peak was detected at a retention time of 14.170 minutes.

Example 3 Production of Val-Val-Tyr-Pro Instead of the resin bonded with Boc-Trp-OH, a resin bonded with Boc-Pro-OH was used, and the resin was directed toward the N-terminal side on the peptide chain.
Protected amino acids corresponding to Tyr, Val, Val in the order: B
Using oc-Tyr (BrZ) and Boc-Val-OH, a coupling reaction was carried out in the same manner as in Example 1, and the protected peptide Boc-Val-Val-Tyr was obtained.
(BrZ) -Pro-Merrifield resin was obtained. The peptide was cleaved from the resin in the same manner as in Example 1, and the peptide was purified to obtain the title peptide. The results of the amino acid analysis were as follows.

Pro (1) 1.08, Tyr (1) 1.00, Val (2)
1.92 The mass spectrum signal of the obtained peptide was 47.
7.72. 1.0 mg / ml solution of the obtained peptide, 20 μ
l Using a high performance liquid chromatograph connected to an octadecyl silica (ODS) column (Vydac C18), linear concentration gradient of triacetylamine / phosphate buffer to acetonitrile (10-40% / 20 min, 1.5 ml / min) As a result, a peak was detected at a retention wavelength of 12.640 minutes at a detection wavelength of 215 nm. Further, under the same conditions, a linear concentration gradient of acetonitrile containing 0.1% TFA (5-35% / 20 minutes, 1.5 ml
/ Min), a peak was detected at a retention time of 11.327 minutes.

Example 4 Measurement of Enkephalin Degrading Enzyme Inhibitory Activity The enkephalin degrading enzyme inhibitory activity of the peptide of the present invention was determined by Kinya Nishimura and Tadahiko Hari, Anesthesia Vol. 42, pp. 1497-1503.
P., 1993. The method described here is based on the following document: That is, the activity of aminopeptidase (AP) was measured by M. Shimamur
a et al., Biochem Biophys Acta 756: 223-229 (1983),
For measuring the activity of dipeptidyl aminopeptidase (DPP)
T. Hazato et al., Biochem Biophys Res Commun 105: 470-47.
5 (1982), Enkephalinase (ENA) activity measurement
C. Gorenstein et al., Life Sci 25: 2065-2070 (1979)
In addition, the activity measurement of angiotensin converting enzyme (ACE)
ayakari et al., Anal Biochem 84: 361-369 (1978), respectively.

According to the methods described in these documents, each enkephalin-degrading enzyme was extracted from the monkey brain membrane-bound fraction with the surfactant Triton X-100 and partially purified using a DE52-cellulose column. The peptide sample was added in the presence of a buffer (neutral region), enkephalin-degrading enzyme, and substrate, and the enzyme activity was measured as described below to determine% inhibition.

(A) Measurement of aminopeptidase activity Aminopeptidase enzyme (5 units), each peptide sample (5 μg) and 1 M Tris-HCl buffer (pH 7.0) 5
Water was added to 0 μl to make the total volume 1 ml. After 3 minutes of pre-incubation (37 ° C.), 200 μl of substrate 2 mM Tyr-β-naphthylamide (Sigma) was added and reacted for 30 minutes. Thereafter, β-naphthylamine released from the substrate is
It was diazotized with 1 ml of 0.1% fast garnet GBC solution, left at room temperature, and measured at an absorbance of 525 nm.

(B) Measurement of dipeptidyl aminopeptidase activity Dipeptidyl aminopeptidase enzyme (5 units), each peptide sample (5 μg), bestatin (30 μg) and 1
Add water to 50 μl of M Tris-HCl buffer (pH 7.0),
The total volume was 1 ml. After 3 minutes of pre-incubation (37 ° C.), 200 μl of 2 mM substrate Arg-Arg-β-naphthylamide (Sigma) was added and reacted for 30 minutes. Thereafter, β-naphthylamine released from the substrate was diazotized with 1 ml of 0.1% fast garnet GBC solution, left at room temperature, and measured at an absorbance of 525 nm.

(C) Measurement of angiotensin converting enzyme activity Water is added to angiotensin converting enzyme (25 units), each peptide sample (1.25 μg), bestatin (30 μg) and phosphoramidone (10 μg) to make 250 μl, and preincubation for 3 minutes ( 37 ° C), then 5mg / ml Hip substrate
50 μl of 0.5 M Tris-HCl buffer (pH 7.4) containing puryl-His-Leu (Sigma) was added. After 8 minutes reaction, 1N
The reaction was stopped with 20 μl of NaOH and left at room temperature for 30 minutes. 1 ml of 60 mM phosphate buffer (pH 7.2)
1 ml of 1% cyanuric chloride (dissolved in ethylene glycol monomethyl ether) was added, centrifuged, and the amount of hippuric acid in the released supernatant was measured at an absorbance of 382 nm.

(D) Measurement of enkephalinase activity Enkephalinase (2 units) was added to a reaction system containing 50 μl of 250 mM Tris-HCl buffer (pH 7.4).
Each peptide sample (25 μg), water, captopril 10
0 μg and the substrate Glutaryl-Ala-Ala-Phe-β-
Add 50 µl of naphthylamide (NOVA), 250 µl in total volume
And After 2 hours incubation at 37 ° C, 90 ° C
The enzyme was inactivated by leaving it inside for 10 minutes to stop the reaction. After cooling, preincubation was performed at 37 ° C. for 3 minutes, and then 10 μl of aminopeptidase M (Sigma) was added, followed by incubation at 37 ° C. for 30 minutes. Further, 750 μl of a 0.1% fast garnet GBC solution was added, the released β-naphthylamine was diazotized, left at room temperature, and measured at an absorbance of 525 nm.

(E) Results In addition to the peptides of the formulas (II), (III) and (IV), Pro-Trp-Thr, Val-Tyr-Pro-Trp, Tyr-Pro-Trp, V
al-Val-Tyr-Pro and Val-Tyr-Pro were synthesized in the same manner as in Example 1, and their inhibitory activities on enkephalin-degrading enzymes were determined by spinorphin, Val-Val-Tyr-Pro-Trp-Thr, V
al-Tyr-Pro-Trp-Thr, Tyr-Pro-Trp-Thr, Leu-Val-Val-T
This was compared with a known enkephalin-degrading enzyme inhibitor of yr-Pro (Kinya Nishimura et al., Anesthesia, 42, 1663-1670, 1993). Table 1 shows the results.

[0032]

[Table 1]

From Table 1, it can be seen that the peptides represented by the formulas (II) and (III) are aminopeptidases derived from monkey brain membrane,
Inhibiting dipeptidyl aminopeptidase and angiotensin converting enzyme almost as much as spinorphin, in particular having a higher inhibitory activity on dipeptidyl aminopeptidase than any known inhibitor, and having the formula (IV ) Was found to inhibit dipeptidyl aminopeptidase, enkephalinase, and angiotensin converting enzyme at almost moderate levels.

[0034]

The peptide of the present invention or a salt thereof has an activity of strongly inhibiting enkephalin-degrading enzymes, particularly dipeptidyl aminopeptidase, and thus can be used alone or as an enkephalin- and / or enkephalin-degrading enzyme inhibitor. It can be used for pain relief or alleviation in combination with known enkephalin degrading enzyme inhibitors.

[0035]

[Sequence list]

 ────────────────────────────────────────────────── ─── Continuing on the front page (72) Inventor Yukio Yamamoto F-term (reference) 4C084 AA01 AA02 AA07 BA01 BA16 BA17 DC33 MA13 MA17 MA22 MA23 MA24 MA31 MA35 MA37 MA41 MA43 MA52 MA56 MA60 MA66 NA05 ZA082 ZC202 4H045 AA10 AA30 BA13 BA14 DA55 EA21 FA33

Claims (5)

[Claims] 1. A compound represented by the following formula (I) X ₁ -Val-Val-Tyr-Pro-X ₂ (I) wherein X ₁ is Leu or has no amino acid residue; ₂ indicates Trp or no amino acid residue), provided that Leu-Va
Peptides having enkephalin degrading enzyme inhibitory activity or salts thereof, excluding l-Val-Tyr-Pro. 2. The peptide according to claim 1, which is represented by the following formula (II): Val-Val-Tyr-Pro-Trp (II) or a salt thereof. 3. The peptide according to claim 1, which is represented by the following formula (III): Leu-Val-Val-Tyr-Pro-Trp (III) or a salt thereof. 4. A pharmaceutical composition comprising the peptide according to claim 1 or a salt thereof in a therapeutically effective amount. 5. The pharmaceutical composition according to claim 4, which has an analgesic effect.