JPH0349920B2 - - Google Patents

Description

[Detailed description of the invention]

[Industrial Application Field] The present invention relates to a polypeptide and a pharmaceutical composition for pressurization containing the polypeptide as an active ingredient. [Prior Art] It has long been recognized that naturally occurring vasopressin has biological effects such as antidiuretic activity. However, its use is limited by its relatively short half-life in the bloodstream and by its well-known pressurizing effect. Accordingly, various vasopressin analogs have been synthesized in an effort to obtain products that modify the properties of vasopressin and increase its pharmaceutical uses. In an attempt to extend the half-life of vasopressin, attempts have been made to modify it to inhibit enzymatic cleavage and the resulting inactivation of the vasopressin molecule. For example, [8-D-arginine]vasopressin can be deaminated at the 1-position to produce "desmopressin" that is immune to aminopeptidase cleavage of the 1-2 carbon-nitrogen bond, resulting in desmopressin. It was found to have high antidiuretic activity. US Patent No.
See No. 3497491. Furthermore, a disulfide bridge is linked with a thioether bond (-CH ₂ S- or -SCH ₂ -)
Further analogs with higher potency and half-life are generated by substitution with . US Patent No.
See No. 3980631. Yet another approach to new and potentially more effective vasopressin analogs involves the preparation of hormone source forms, ie, vasopressin analogs, that have a splittable or cleavable peptide residue attached to the active molecule. These analogs are themselves inactive, but serve as reservoirs for the active molecule, gradually releasing it by enzymatic cleavage of the additional residue. This approach involves a series of analogs obtained by attaching one or more peptide residues to the N-terminus of the molecule, as shown in US Pat. No. 3,558,590. Of these analogs, N-glycyl-glycyl-glycyl-8-lysine-vasopressin, or tGLVP, is the most interesting. Although this modification is effective in providing long-lasting drugs, it is still not as effective as desired. For example, only about 1-5% of the administered tGLVP is present because partitioning of the hormone source also occurs at various positions in the active nonapeptide portion of the molecule.
is actually released in active form. As a result, large doses of tGLVP are required. [Problems to be Solved by the Invention] As mentioned above, vasopressin and its analogues can be protected from enzymatic degradation and released in sufficient yield of the active hormone so that relatively small doses can be used. It would be desirable to modify the compounds and their analogs. It is an object of the present invention to provide polypeptides that are novel analogues of vasopressins with long-term activity. Another object of the present invention is to provide a pharmaceutical composition for increasing blood pressure, which contains the above-mentioned polypeptide as an active ingredient and is effective for treating gastrointestinal bleeding, uterine bleeding, burns or bleeding shock, bleeding during surgical operations, etc. It is in. Still other objects of the present invention will be apparent from the description and claims that enable the invention to be carried out. [Means for Solving the Problem] According to the present invention, the formula (): (In the formula, the configuration of the 8-position lysine moiety is D or L) and a pharmaceutical composition containing the same as an active ingredient are provided. The polypeptides of the invention are characterized by an active capacity that confers high activity and prolongs the useful life of the active hormone. These are derivatives of vasopressin and its analogues that have vasopressin-like activity [hereinafter collectively referred to as vasopressins].
It is. The above objects of the present invention are achieved by introducing a "side peptide chain" as defined below into vasopressins containing alpha-amino acids with basic side chains. These amino acid residues can be represented by the following general formula. (In the formula, R is a divalent saturated aliphatic or aromatic group, preferably a saturated aliphatic group having up to 6 carbon atoms, and one hydrogen atom of the amino side group can be used as an amide bond.) Representative examples of amino acid residues include lysine, homolysine, ornithine, and the like. Vasopressins having the above-mentioned basic amino acid residue or a guanidine residue such as arginine that can be substituted with the above-mentioned basic amino acid residue in the vasopressin chain are susceptible to enzymatic action by trypsin-like enzymes. These enzymes cleave polypeptide bonds at one of the adjacent carbon-nitrogen bonds. In accordance with the present invention, it has been discovered that when the ω-terminal amine proton (hydrogen) of one of the basic residues is replaced by a "side peptide chain", the resulting polypeptide becomes immune to said cleavage. . However, we have also discovered that this side peptide chain can be removed by amino-peptidases, thereby releasing the active vasopressin or analogue molecule.
In this way, the polypeptides of the invention can release the active hormone over an extended period of time, increasing the useful half-life of the active molecule. As used herein, the term "side peptide chain" refers to a chain of 1 to about 3 amino acid residues. Preferred amino acid residues are alpha-amino acids consisting exclusively of saturated or aromatic moieties, such as glycine (Gly), alanine (Ala), valine (Val), leucine (Leu), isoleucine (Ile) and phenylalanine (Phe). It is derived from. Furthermore, residues other than the Gly residue must be L in order to obtain the derivatives of the present invention with useful levels of activity.
- Must be in configuration. When D-alanine was side-substituted, the resulting compound was relatively inactive and did not act as a hormone source. Glycine residues are particularly preferred as side peptide chains, and tripeptide Gly-Gly-Gly chains are most preferred. Polypeptides of the invention of particular interest are 1-
8- of deamino-vasopressin and its analogues
N-peptide hormone sources, which are more potent in their action and have a longer duration of activity than the N-peptide hormone sources of U.S. Pat. No. 3,558,590 or their analogues. The novel polypeptides of the invention can be readily prepared by common methods for polypeptide synthesis. As a preferred procedure, Stewart and
Young’s “Solid Phase Peptide Synthesis”
WHFreeman & Co., San Francisco (1969)
Examples include stepwise solid-phase synthesis methods, such as those described in . The principles and procedures of this synthetic method are well known and will not be described further here. [Example] Synthesis examples of N-substituted analogs according to the present invention are listed below, but it goes without saying that these are merely illustrative and do not limit the scope of the present invention. Example 1-Desamino-[2-Phe,8-N〓-Lys
(Gly)]-Vasopressin The vasopressin analog according to the present invention was produced as follows. Benzhydrylamine-HCl resin (Beckman) with 0.51 meq of available amine per gram of resin.
3.0 g of the sample was charged to a 135 ml reaction vessel of a Vega Model 96 automatic programmable synthesizer. Gly, N〓-(carbobenzoxy)Lys, Pro, S
-(p-methoxybenzyl)Cys, Asn, Gln,
Phe and N-(t-butyloxy-carbonyl) of S-(p-methylbenzyl)-β-propionic acid
A peptide chain was prepared by sequentially reacting the [N〓-(t-Boc)] derivatives, and 4.52 g of the resin-bound peptide S-p-MeBzl-β-mercaptopropionic acid-Phe-Phe-Gln- Asn-S-p-
MeOBzlCys-Pro-Lys(CBZ)-Gly- _NH2 -resin was produced. The overall yield was 87%. t
- All Boc amino acids are from Vega Laboratories,
Inc. and dissolved in an appropriate solvent. That is, p
-t-Boc derivatives of MeOBzlCys, Gly, Pro and Phe are dissolved in dichloromethane, while Asn and
The derivative of Gln is dimethylformamide (DMF)
and 2-fold excess of hydrobenzotriazole monohydrate (HOBzt). Synthesis is the first
The procedure in Tables and Table 2 was followed using a 3-fold molar excess of double coupling for each of the protected residues. In all cases, Anal.
Biochem., 34 , 595 (1970),
The resin was negative for free amino groups after the second coupling, as determined by the method of Kaiser et al.

【table】

[Table] The amino acid was a 3.0M excess solution of t-Boc derivative. DCC was equimolar with the protected amino acid.

【table】

[Table] Amino acids are t-Boc in a 3.0-fold molar excess in DMF.
It was a derivative. HOBzt was used in 2-fold molar excess relative to the amino acid concentration. DCC was equimolar. 400mg sample of the peptide resin obtained in this way
is supplied by Peninsula Laboratories. A 50 ml Teflon-Kel-F container of the HF apparatus was charged and a small Teflon coated magnetic stir bar and 1 ml of anisole were added. A frit was secured near the top of the vessel and then connected to the HF apparatus. The HF apparatus was evacuated with a vacuum pump and the sample container was immersed in a dry ice/acetone bath. After 20 minutes, the sample container was disconnected from the vacuum system and connected to the HF reservoir. HF was distilled into the vessel until the total liquid volume was approximately 10 ml (approximately 5 minutes). The dry ice/acetone bath was replaced with a water/ice bath, a magnetic stirrer was placed under the vessel, and the vessel was sealed from the rest of the system. After 75 minutes, the vessel was carefully pumped to allow the HF to evaporate over approximately 60 minutes. The remaining sample container was immersed at 0° C. during this time. The system was then flushed with nitrogen back to atmospheric pressure and the cleavage vessel was quickly removed and sealed with parafilm. Coarse fritted from the container
The contents were washed out into the funnel several times (total volume: about 100 ml) with degassed ethyl acetate. The funnel and container were then placed in a vacuum desiccator and vacuumed for 30 minutes to remove residual ethyl acetate. The resin in the cleavage vessel and funnel was then washed several times with degassed IM acetic acid (100 ml total) and then in several portions with 300 ml of degassed water.
The solution was adjusted to pH 6.9 with 3M _NH4OH and 25ml of 0.01M potassium ferricyanide solution was added. Stir this yellow solution for about 30 min and PH with 50% acetic acid.
was adjusted to 5, and AG-3 anion exchange resin (TFA
) was added and the mixture was stirred for an additional 20 minutes. The slurry was then filtered to produce a clear colorless solution, the resin was washed with a small amount of water, and the solution was lyophilized. The resulting powder was taken up in approximately 15 ml of 50% acetic acid, filtered to obtain a clear pale yellow solution, and this was transferred to a Sephadex G-15 column (2.6
cm). Elution rate is 1 ml/mm, fraction 3 ml.
Collected. Two overlapping peaks were eluted by detection at OD ₂₆₅ . Peak 1 is a slightly yellow fraction
It appeared in fractions 45-53, and peak 2 appeared in fractions 54-79. The material in peak 2 was rechromatographed under the same conditions, and a second peak was found to produce the desired product, (1-desamino[2-Phe,8-
[Lys] vasopressin) and the yield was 62.5 mg. Amic acid analysis after 24 hours of hydrolysis with 6MHCl showed Lys l.1, Pro 1.0, Glu 1.2, Phe 1.8,
Asp 1.0, hemy Cys (unreliable after HCl hydrolysis) 0.6.

[Table]
1-Desamino-[2-Phe,
Triethylamine (0.007 ml) was added to a solution of 50 mg of 8-Lys]-vasopressin, then N-t-
Boc-glycine p-nitrophenyl ester (69
mg) and 1-HOBzt (10 mg) were added. at room temperature
After stirring for 1.5 h, TLC on silica gel ( _CHCl3−
Visualization using ninhydrin (MeOH- _H2O , 200:75:13) revealed that all peptides had reacted. Next, mix this reaction mixture with 2-propanol (10 ml).
and ether (50ml), cooled to 4°C and filtered. The product was treated with hot acetone (15ml), diluted with cold ether (50ml) and collected by centrifugation. The yield was 38 mg. The protected decapeptide was protected in trifluoroacetic acid (1 ml) for 30 minutes and isolated by ether precipitation followed by centrifugation. 1-desamino-[2
-Phe,8-N〓-Lys(Gly)]-The final yield of vasopressin was 29.14 mg (54.5%). Amino acid analysis: (hydrolyzed in 6M HCl for 24 hours)
Lys1.0, Asp 1.0, Glu1.0, Pro0.9, Gly2.1,
Phe1.7, Hemi-Cys 0.6.

[Table] Visualization with ninhydrin. O-toluidine showed no additional spots. Example 1-Desamino-[8-N〓-Lys(Gly-Gly-
Gly)]-Vasopressin 1-Desamino-[8-Lys]-vasopressin was synthesized in the same manner as in the example except that tyrosine (Tyr) was used in place of phenylalanine (Phe) at position 2. Amino acid analysis: Lys1.0, Asp1.0,
Glu1.0, Pro0.9, Gly0.9, Phe0.9, Tyr1.1, Hemi-Cys0.7. Gly−Gly−Gly (Vega Laboratories, Inc.)
Suspend 2.98g in 100ml of 50% dioxane and adjust the pH to approx.
The suspension was adjusted to 10.0 to dissolve the suspended matter. G-t
2.2 g of -butyl-dicarbonate was added and the reaction was analyzed by TLC (butanol-pyridine-acetic acid-water, 15:
10:3:6). After 3 hours, an additional 1 g of di-tert-butyl-dicarbonate was added, and after a further 3 hours the reaction to protect the N-terminus of the tripeptide was complete. This solution was then extracted three times with 30 ml portions of hexane. The pH of the aqueous phase was adjusted to 2.5, extracted with three 50 ml portions of ethyl acetate, dried over MgSO ₄ and concentrated to an oil. The residue was dissolved in ethyl acetate.
Crystallized from ether. The yield was 750 mg.
This substance was converted into N-t-Boc-Gly-
Conversion to Gly-Gly p-nitrophenyl ester yielded 750 mg of ester. N-t-Boc using a method similar to the example procedure
-Gly-Gly-Gly p-nitrophenyl ester is coupled to the epsilon amino group of 8-Lys, and then the N-t-Boc group is removed to form 1-desamino-[8-N〓-Lys(Gly-Gly -Gly)]-vasopressin was produced. Amino acid analysis: Lys1.0,
Asp1.0, Glu1.0, Pro0.9, Gly3.8, Phe1.0,
Tyr1.1, Hemi-Lys0.6. Example 1-Desamino-[8-D-N〓-Lys(Gly-
Gly−Gly)]-vasopressin 1-Desamino-[8-D-N〓-Lys(Gly- Gly-Gly)]-vasopressin was prepared. The polypeptide according to the present invention, which is represented by the formula () and is preferably administered to mammals, is NΩ-
It is activated by splitting (or cleavage) of the peptide chain to release the active original polypeptide, which of course still has its original activity. Compounds such as and are expected to have prolonged pressor activity and are useful in the treatment of shock therapy, gastrointestinal bleeding, uterine bleeding, burns, disorders in the pregnancy process, hematuria, and pancreatic disease. For example, compounds such as Examples and Examples are expected to have prolonged pressor activity and may be useful in treating conditions such as shock therapy, gastrointestinal bleeding, uterine bleeding, burns, disorders of the pregnancy process, hematuria, and pancreatic disease. be. The evaluation analysis of the pressor and antidiuretic effects of the compounds in Examples ~ is based on Schaechtelin et al.'s “Enhancement of the
Activity of Angiotensin by Certain
"Cations", Molec. Pharmacol., 10 , 57-67
(1974) and “Role of the Disulfide” by Cort et al.
Bridge and the C-Terminal Tripeptide in
the Anti-diuretic Action of Vasopressin in
Man and the Rat”, Kidney Internat., 8 ,
292-302 (1975) using the method described in
male Sprague-Dawley (Sprague-
Dawley) rats. Highly purified arginine-vasopressin with known activity was used to assay the response to a three-point curve. For comparison, similar tests were conducted on known vasopressin analogs. Furthermore, the factor release properties of the compounds in Examples ~ were evaluated using the clotling assay of Prowse et al., described in Brit. J. Haematol., 41 , 437 (1979).
was tested in citrated plasma of male dogs, and the results were assayed for factor-releasing activity of desmopressin. In all cases the half-life of the response is at equi-peak-potent levels.
compared to the standard. These test results are summarized in Tables 3 to 5 below.

[Table] From the results in Table 3, it is clear that all the compounds in Example and No. 3 have more potent pressor activity than tGLVP and last for a longer period of time.

Table: As is evident from the data in Table 4, the example compounds had considerably higher antidiuretic activity, and all compounds had greater antidiuretic activity than tGLVP. Considering also the results in Table 3, the compounds of Example and No. 3 have low antidiuretic activity and are coupled with
It has higher peak potency and longer duration of pressor activity than tGLVP.

[Table] Based on the data summarized in Tables 3 to 5, the compounds in Examples and above are useful for treating conditions such as shock, gastrointestinal bleeding, uterine bleeding, burns, disorders during pregnancy, hematuria, and pancreatic disease. The compounds in the examples have particularly high activity as antidiuretics and can therefore be used as antidiuretics. Polypeptides of the invention are administered to the host to be treated according to generally known methods. For example, it can be administered by intravenous injection or infusion, or intranasally in the form of nasal drops. In either case, the polypeptide is administered in solution in a suitable solvent, preferably water.
This solution can contain various additives commonly known in the industry. A preferred medium is saline solution. This solution is preferably
The polypeptide is stabilized by an acidic pH of about 3 to about 5, particularly preferably about 4. Also, a small amount of antibacterial agent,
It may also be desirable to include, for example, chlorobutanol to minimize bacterial contamination of intranasal formulations. The concentration of the polypeptide in the solution is not particularly narrowly limited and can vary over a wide range from about 1 μg/ml to 1000 μg/ml or more, depending on the intended dosage form and dosage. Generally, solutions for intranasal administration are more concentrated than solutions for injectable administration. Thus, solutions for intranasal administration contain about 100 to about 400 μg of polypeptide per ml, whereas injectable solutions contain about 10 to about 400 μg per ml.
Contains about 40 μg of polypeptide. The dosage of polypeptide administered depends to a large extent on the specific action of the polypeptide, the level of its action, the intended effect and the mode of administration. For example, when administered in the form of nasal drops, the dose administered should be about 10 times the dose administered by the intravenous route. When used to increase factor levels, the compounds of the invention can be used in methods of preparing high potency factor formulations. In such methods, compounds of the invention can be administered to a volunteer blood donor to increase factor levels in the donor's bloodstream. The increase in factor factor levels begins within about 15 minutes after administration of the polypeptide, and the increase in levels persists for at least 4 hours. When circulating levels of factor reach the desired level, generally 30 minutes or so after administration, the donor's blood can be collected by any conventional method.
The blood thus obtained is processed by conventional methods, such as cryo-precipitating, to produce a factor-containing preparation (AHG).
Because the concentration of factor in the blood increases, the amount of factor in the preparation recovered from a given volume of blood is commensurate with that obtained from volunteers not treated with the polypeptides of the invention. It is increasing. Furthermore, since the amounts of blood constituent factors other than factor (and plasminogen activator) are not affected, the proportion of impurities in factor is reduced. Furthermore, these same factor-releasing polypeptides are associated with von Willebrond's disease.
It can be used to treat menorrhagia associated with low factor factor levels in women with cancer.

Claims (1)

[Claims] 1 Formula (): (In the formula, the configuration of the lysine moiety at position 8 is D or L.) 2. The polypeptide according to claim 1, which is nodesamino [N-N-ε-triglycyl)-8-L-lysine] vasopressin. 3 Formula (): (In the formula, the steric configuration of the lysine moiety at position 8 is D or L.) A pharmaceutical composition for pressurization comprising an effective amount of a polypeptide represented by the formula: and a pharmaceutical carrier. 4. The pharmaceutical composition for increasing blood pressure according to claim 3, which is used to treat hypotension associated with gastrointestinal bleeding. 5. The pharmaceutical composition for increasing blood pressure according to claim 3, which is used to treat hypotension associated with uterine bleeding. 6. The pharmaceutical composition for increasing blood pressure according to claim 3, which is used to treat hypotension associated with burns or hemorrhagic shock. 7. The pharmaceutical composition for increasing blood pressure according to claim 3, which is used for the treatment of bleeding during surgical operations.