IT8249727A1 - PEPTIDE COMPOUND WITH HYPOTENSIVE ACTIVITY AND PROCEDURE FOR ITS SYNTHESIS. - Google Patents

Description

SIB-83 675 Description of the industrial invention entitled: '<?>'Peptide compound with hypotensive activity and procedure for its synthesis'

of the Italian company POLIFARMA S.p.A.

based in ROME and

of the Italian National Research Council based in Rome

SUMMARY

A new peptide has been described, isolated from the venom of the snake species "Crotalus Atrox", which has the following sequence of left-handed amino acids;

PYR-LEU-THP-PRO-ARG-PRO-GLN-IL?-PRO-PRO

as well as a procedure for its synthesis and its use for the treatment of hypertension*

DESCRIPTION

The present invention relates to a new peptide whose formula is represented by the following amino acid sequence:

PYR-LEU-TRP-PRQ-ARG-PRO-GLN--ILE-PRO-PF.Q

<'>where FYR indicates pyroglutamic acid,

LEU leucine,

TRP tryptophan,

PRO proline,

ARG arginine,

GLN glutamine,

ILE isoleucine,

The invention also refers to a process for the production of this compound and to its use in the pharmaceutical field for the treatment of hypertension,

State of the art

It has been known for several years that the venoms of several snake species contain peptide fractions capable of lowering blood pressure in mammals.

For example, in 1970 Ferreira and colleagues (Biochemistry 9? 2583-93) isolated from the venom of "Bothrops Jararaea" a peptide with the structure: FYR-LYS-TRP-ALA-PRO capable of potentiating the effect of hradikinin.

In 1971 Rato and Suzuki (Biochemistry 10, 972-980) reported the presence in the venom of "Agkistrodon Haly3 Blomhoffii" of two peptides that potentiate the effect of bradykinin, called potentiator B and potentiator C and with the formula, respectively, PYH-GLY-LEU-PRO-PRO-ARG-PRO-LYS-ILE-PRO-PRO and PYR-C-LY--LEU-PRO-FRQ-GLY-PRO-PRO-ILE-PRO-PRO.

Also in 1971, Ondetti and collaborators (Biochemistry 10, 4033-39) reported having identified in the venom of "Bothrops-Jararaca<11> six peptides having the property of inhibiting the enzyme that transforms angiotensin I into angiotensin II. Of these peptides, one, called in the work V-6--I and in subsequent publications "teprotide<,i> shows a great affinity with the peptide which is the object of the present invention since it lacks only the leucine in position 2 (forum la: PYR-TRP-PRO-ARG-PRO-GLN-ILE-PHO-PRO). The others have the following formulas: V2: PYR-TRP-PRO-ARG-PRO-THR-PRO-GLN-ILE-FRO-PR?; V*6-II: PYR-?SN-TRP?PRO-ARG-PRO-GLN-ILE-E?O-FRO; V-?: PYR-ASN-TRP-PR0-1IIS~PR0-PR0-PRO; V-9: PYR-GLY-GLY-TRP-PRO-ARG-PRO-GLY-PRO-GLU-ILE-PRO-PRO.

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In 1973 Ondetti's group (Experientia 29, 1052-35) proceeded to the synthesis of over 50 peptides, modifying the original structures of the \teprotide and the pentapeptide identified by Perreira.

More recently, B?nilla (FEBS Le?t. 68, 297-302, 1976) described a peptide of 2G residues, isolated from the "venom of Orotalus Atrox", capable of lowering the blood pressure of experimental animals, but did not describe its sequence.

Summary of the <8>invention

It has now been found that the venom of "Cr? talus Atrox" contains a peptide of the following structure:

PYE-LETJ-TRP-PRO -ARG-PRO -GLN-ILE-PR?- PR?

peptide that has been isolated by chromatography from the lyophilized venom and whose structure is determined by amino acid analysis, and, after Rf = 0.9, negative for the ninhydrogen ion reaction (for the free aramidic groups), but positive for the Ehrlich reaction (for the tryptophan) and the peptide bond reaction (for the peptide bond).

Analysis of amino acids/after hydrolysis in 6 N HCl at 110?C, for 24 hours gives the following results:

- glutamic acid (2.0);

- proline (3*5);

- isoleucine (1.0);

- leucine (0.9);

- arginine (1.0);

(moles per mole of peptide, assuming the presence of an arginine residue in the peptide). The presence of tryptophan is confirmed by the characteristic UV spectrum of the fraction

An attempt to determine the presence of a free terminal amino group by reaction with dansyl chloride, acid hydrolysis, and subsequent identification of any dansyl residue on a thin layer of polyamide yields a negative result.

An aliquot of P^ is treated with methanol/hydrochloric acid for 24 hours at room temperature (Anal. Biochem, 48, 5^6? 1972) and shows, after chromatography on a cellulose thin layer, a single ninhydrin-positive component, as well as positive for the Ehr?ich reaction and for chlorination and, after reaction with dansyl chloride, the presence of li-terminal guta?niic acid*.

50 ml of E? are methanolized, and the amino acid sequence is determined according to the Edman-dansi1e manual degradation technique (Biochem. J* 119, 805, 1970).

The structure of the peptide is: PYB-LEU -TSP-FRO-ARG-PRO -GLN-ILE-FilQ- PRO The presence of tryptophan in position 3 is assigned only on the basis of indirect evidence obtained from the Edman-dansilejin degradation and is confirmed by the results of the cleavage at the level of tryptophan of an aliquot of F- after incubation in a mixture of dimethyl sulfoxide/HCl/HBr (Meth. Ensymol. 47, 459-463, 1977), and subsequent purification and analysis of the fragments* The amidation state of glutamine in position 7 is assigned following the determination of the electrophoretic mobility of at pH 6.5 according to Offord (Nature 211, 591, 1966).

The peptide which is the object of the invention has also been synthesized and the synthesis process and a production example are described below.

Peptide synthesis

The basic premise of this method is that amino acids can be linked together into peptides of the desired length by anchoring one of them (the -COOH terminal) to an insoluble support.

After the desired sequence of amino acids has been ligated together, a reagent is used that releases the chain from the support, leaving the peptide in solution.

The solid support is a synthetic polymer containing reactive groups. These groups are chosen to react with the carboxyl groups of the amino acids, so that the amino acid is covalently bonded to the polymer.

During this reaction, the amino group of the amino acid must be protected by suitable substituent groups, so that the amine does not react with the polymer.

The protecting groups must also be such that they can be removed selectively, without damaging the bond between the amino acid and the solid support.

After removing the protecting group, the first amino acid bound to the solid support is made to react with a second amino acid protected on the amino group, by adding suitable coupling agents.

We then proceed to the unlocking of the second amino group and so on, in an alternation of coupling between amino acids and unlocking of the amino groups until the desired sequence of amino acids is reached.

At the end of the synthesis, a different reagent is used to specifically break the bond between the first amino acid and the polymer, and the peptide is released into solution.

The object of the invention is therefore a process for the production of the peptide according to the present invention comprising the steps of:

react proline, protected on the amine by a protecting group, with a reagent capable of covalently binding the carboxyl group of the proline.

and supported on an activated insoluble resin, in anhydrous solution, and in the presence of a catalyst;

recover the resin carrying the proline protein bound to it;

remove the said protective group;

reacting further proline protected on the amine by a protecting group, with the amino group of the proline bound to said resin, in anhydrous solution and in the presence of a coupling agent, so as to form the bond of the first two amino acids of the amino acid chain of the peptide;

recover the resin carrying the chain of two proline groups, linked to it;

repeat the coupling operations between amino acids in the sequence required to form the entire desired peptide chain;

detach said peptide from said resin;

and recover said peptide.

A detailed example of the synthesis of the new peptide is now described.

Summary example

2.5 g of activated resin (chloromethy? Bio-Beade S-X 1 from Bio-Rad) are reacted in a flask with 215 mg of 1-tert.butoxycarbonylproline, 20 ml of absolute ethanol and 0.15 ml of triethylamine.

A water condenser connected to a drying flask containing CaCl^ is placed on the flask. The flask is placed in an oil bath at 90°C and left to reflux for 24 hours. The resin is then filtered through a Buchner filter, washed with ethanol, water, methanol, and dichloromethane*, and brought to dryness under vacuum.

The proline resin is placed in a cylindrical glass container, equipped with an opening at the top for the introduction of reagents, and equipped, in the lower part, with a filter coupled to a tap, with Teflon inserts. 20 ml of dioxane are added, and the mixture is shaken for 5 minutes with a special oscillating apparatus in 105°C.

The dioxane is filtered off and a solution of 4 M HCl in dioxane (20 ml) is added. After 30 minutes, the acid is filtered off and the reaction mixture is washed with dioxane, then with chloroform. Triethylamine in chloroform (1:9, 20 ml) is added, then washed with chloroform and then with dichloromethane. 430 mg of N-tert.-butoxycarbonylproline in dichloromethane and 1.2 ml of dicyclohexylcarbodimide (DCC) are added. The reaction mixture is allowed to react for 2 hours, then washed with dichloromethane. The cycle is then repeated, starting with the dioxane wash.

To prevent the proline-proline sequence from giving rise to diketopiperazine, the addition of DCC vij3 was performed before N-tert.butoxycarbonylsoleucine (460 mg).

In the next cycle, 490 mg of N-tert.butoxycarbonylglutamine is added, and in the further cycle, another 430 mg of N-tert.butoxycarbonylproline is added.

1/<1>arginine is introduced as Ha-tert.butos sicarbonyl without groups protecting the guanidinium group, but in large excess (820 mg).

After another cycle of tert.butoxycarbonylproline (430 mg), N-tert.butoxycarbonyltryptophan (620 mg) is added in dichloromethane containing indole (1 mg/ml) and traces of trifluoroacetic acid, then:* in the next cycle 460 mg of N-tert.butoxycarbonyllucine, finally in the last cycle, 260 mg of pyroglutaric acid.

To separate the peptide from the resin, the latter is suspended in 20 ml of trifluoroacetic acid. It is bubbled with anhydrous HBr for 90 minutes, then the solution is filtered into a round-bottom flask. It is brought to dryness under vacuum without heating, then washed with methanol:water (1:1). The residue is brought to dryness and weighed: 915 mg of peptide.

The synthesized peptide and the one obtained by extraction from the venom of "Crotalus ?trox" are now compared, with particular attention to their chemical properties.

1) Both peptides, loaded onto a Bondapack phenyl resin column (Waters) measuring 0.44 x 50 cm and eluted with a hydroalcoholic solution, gave a retention time of 9.1 minutes.

2) Both peptides show, upon amino acid analysis, to possess 4 prolines, 2 glutamic acids, 1 arginine, 1 tryptophan, 1 leucine, 1 isoleucine.

5) Both peptides, by thin layer chromatography on silica gel, and using as eluent a-butanol/ethyl acetate/acetic acid/water, show to have Rf = 0*54.

4) Both peptides, by electrophoresis at pH 1.9 on Whatman 5 ?1? paper, after 1 hour at 1000 volts, show a single spot with Rf=0.30.

5) After opening of the pyroglutamine by methane lysis, both peptides are shown to have the sequence; FYR-LEU?TSP-PRO-ARG-PRO-- GLN-ILE~PR0~RR0 .

Study of pharmacological activity

Tests were conducted to test the enzymatic and pharmacological activity of the peptide according to the invention.

Proye^enziiaatic^ ''in vitro?

Both the extractive and synthetic peptides were tested in vitro on the enzyme that converts angiotensin I to angiotensin II.

The method used is that of Cushman and Cheung (Biochem. Pharmacol.. 20, 1637-48, 1971)?

The IC^Q for the synthetic peptide was found to be 3*5x10~6 M, while equimolar concentrations of the synthetic and extractive peptide (calculated based on amino acid analysis) showed enzyme inhibition of 52% and 44%, respectively. Tested under the same conditions, the teprotide also had an IC^Q = 5?XxlQ~^ H.

"In vivo" pharmacological tests

1) Effect on blood pressure.

Male Vistar rats, anesthetized with ethyl urethane, weighing 200–500 g were used. After tracheal cannulation, the right carotid artery was isolated and connected via a cannula to a HP pressure transducer. Flow from the isolated left carotid artery was recorded using a Biotronest electromagnetic flowmeter. Other parameters recorded: dp/dtj ECO, ????.

All values were recorded on a Hewlett-Packard eight-channel polygraph. Particular attention was paid to carotid arterial pressure. The peptide, synthetic or extractive, dissolved in physiological solution and injected into the femoral vein, causes a long-lasting dose-dependent hypotensive effect, evident even at a dose of 0.1 mg/kg.

2) Bradykinin enhancement.

Male Patti Vistar patients, treated as above, were tested with bradykinin. This physiological peptide, injected into the femoral vein, causes rapid and transient hypotension, as it is rapidly degraded in the pulmonary circulation by ACE (angiotensin-converting enzyme).

The peptide in question, injected into the vena cava immediately before bradykinin, enhances and prolongs its effect on blood pressure. This phenomenon is evident starting from a dose of 0.5 mg/kg.

the potentiating effect on bradykinin au

Isolated ileum of guinea pigs has also been observed, immersed in an isolated organ bath containing thyroxine solution at 30°C. The contractions induced by the addition of bradykinin to the bath were potentiated by the _Cs

peptide, right? at a dose of 5 x 10 'M.

5. Inhibition of the effect of angiotensin I. Angiotensin I was injected into the right femoral vein of male Wistar rats treated as above. The peptide in question was then injected and after 90 seconds, the same dose of angiotensin I was injected again. Already at a dose of 0.5 mgAg, the peptide was able to substantially attenuate the pressor effect caused by the second injection of angiotensin I.

The effect of <s>angiotensin I remains inhibited for more than 2 hours.

4) DL50.

The toxicity of the peptide was tested on albino Swiss mice by intrapertussive administration of the substance dissolved in physiological solution. The BLQ appears to be 5 mg/kg.

Claims (1)

Therefore, the peptide according to the invention, in particular the one obtained synthetically, proves useful for the clinical treatment of hypertension. The invention therefore also includes pharmaceutical compositions containing the peptide according to the invention. The peptide according to the invention can be used in clinical use for protected oral administration in the form of tablets, pills, granules, capsules, and the like, for rectal administration, in the form of suppositories and for repeated administration in the form of injectable solutes together with compatible pharmaceutical vehicles. The daily dosage of the active ingredient, which can be administered in clinical use in the ways mentioned above, is preferably t 1) 2D0 up to 1000 mg/day orally, 2) 50 up to 500 mg/day intramuscularly, 3) 20 up to 250 mg/day intravenously, 4) 500 up to 2000 mg/day rectal. Preferred amounts of active ingredient per single oral dose are 200 mg and 50 mg^ together with usual pharmaceutical excipients*