Anticoagulant hirudin variants and methods for their production

The present invention relates to hirudin variants having high anti-thrombin and anti-platelet activity, methods for producing them, and anti-coagulants having said variants as active ingredients. Hirudin variants shown in formula (I) having tyrosine residues or having their hydroxyl group sulfated. Methods for producing hirudin variants by sulfating hydroxyl group of said tyrosine residues, and anti-coagulants having hirudin variants shown in formula (I) as active ingredients. EQU Phe-Glu-A-Ile-Pro-B-Tyr(R)-Tyr(R) (I) PA1 In the formula, A represents Glu or Pro, B represents Glu, Tyr (R), Glu-Asp or Glu-Tyr(R), and (R) represents the hydroxy group or its sulfated ester (--O--SO.sub.3 H) of tyrosine residue.!

TECHNICAL FIELD 
The present invention relates to hirudin variants or their salts, methods 
for their production and anti-coagulants having said compounds as active 
ingredients. The hirudin variants or their salts in the present invention 
are useful as drugs for pharmacological therapy of acute deep venous 
thrombosis, pulmonary thromboembolism, acute arterial embolism of limbs, 
myocardial infarction and intravascular coagulation on infection. 
BACKGROUND ART 
Natural hirudin is a mixture of peptides composed of 65 or 66 amino acids 
and is secreted from salivary glands of medicinal leeches in very small 
amounts. A variant called HV-1 is the first hirudin isolated from leeches. 
A variant called HV-2 differs from the aforesaid HV-1 in 9 amino acids, 
and HV-3 is identical with HV-2 up to the 32nd serine and differs in 10 
amino acids including an additional 63rd alanine on the C-terminal end. 
Further, the existence of tyrosine in which the phenolic hydroxide residue 
is sulfated as shown in the following formula 
##STR1## 
or --Tyr(SO.sub.3 H)!-- has been confirmed. 
It has been reported that the anti-thrombin activity increases about 10 
times by existence of the sulfate on the tyrosine residue. 
So far, it is very difficult to manufacture polypeptides having sulfated 
tyrosine in the molecule. When chemical introduction of a sulfate group on 
the tyrosine residue in polypeptide obtained by methods such as 
recombinant DNA technology is considered, in which case the amino acid 
sequence is long, it is very difficult to sulfate targeted tyrosine 
selectively without influencing other amino acids. It also requires 
drastic reaction conditions which often cause disruption of peptide bonds 
and it is difficult to obtain the sulfated compound in satisfactory yield. 
For this reason, hirudins under development at present as anti-coagulants 
are non-sulfated ones and have low activities. 
Although the therapeutic application of hirudin as an anti-coagulant is 
thought to be effective, being a foreign compound, allergic symptom such 
as shock and eczema are possible. However, it is thought to be possible to 
reduce allergic responses by decreasing the administration dose or 
shortening the amino acid sequence of the polypeptide. The aim of the 
present invention is to provide hirudin variants with higher anti-thrombin 
activity by sulfating the hydroxyl group of tyrosine residue in the 
molecule. 
DISCLOSURE OF INVENTION 
The present invention relates to hirudin variants or their salts having the 
following amino acid sequence in formula (I) (SEQ. ID. NO:1) as a part of 
their sequence or as all of their sequence. 
EQU Phe-Glu-A-Ile-Pro-B-Tyr(R)-Tyr(R) (I) 
In the formula, A represents Glu or Pro, B represents Glu, Tyr(R), Glu-Asp 
or Glu-Tyr(R), and (R) represents the hydroxy group or its sulfated ester 
(--O--SO.sub.3 H) of the tyrosine residue. 
Further, the present invention relates to the method of manufacturing of 
the hirudin variants or their salts characterized by sulfating the 
hydroxyl group of the tyrosine residue in the amino acid sequence of 
hirudin variants or their salts having the following formula (II) SEQ. ID. 
NO:1 as a part or as all of the amino acid sequence. 
EQU Phe-Glu-A-Ile-Pro-B-Tyr-Tyr (II) 
In the formula, A represents Glu or Pro, and B represents Glu, Tyr, Glu-Asp 
or Glu-Tyr, respectively. 
The sulfation in the present invention maybe carried out by reacting 
hirudin variants or their salts with aryl sulfotransferase in the presence 
of sulfate group donors, with sulfur trioxide complex, or with sulfuric 
acid and dicyclohexylcarbodiimide 
One of the important points of the present invention is that the 
recognition activity against tyrosine by aryl sulfotransferase is improved 
by substituting 1 or 2 amino acids adjacent to the tyrosine residue in the 
C terminal of natural hirudin to tyrosine. With the polypeptides used in 
the present invention, the present inventors have confirmed that the 
sulfation reaction by aryl sulfotransferase will hardly take effect 
without the aforesaid treatment. 
Another important point of the present invention is, if a peptide contains 
a sequence of 8 amino acids starting from the 56th amino acid from the 
N-terminal to the C-terminal of natural hirudin, or if it is composed 
solely aforesaid sequence of 8 amino acids, or if both ends of it are 
substituted by a protecting group such as a succinyl group or an amino 
group, it should show a high anti-thrombin activity as long as it is 
sulfated. Particularly, a compound composed of only 8 amino acids 
according to formula (I), having the N-terminal protected by a succinyl 
group and two tyrosine residues sulfated in both hydroxyl group thereof, 
is confirmed to have an extremely high anti-thrombin activity. 
The hirudin variants in the present invention are the peptides having 
aforesaid amino acid sequence and having the high anti-thrombin activity. 
The C-terminal of the compounds in the present invention are the 
abovementioned -Tyr(R), -Tyr(R)-Leu or -Tyr(R)-Asp. Further, the aforesaid 
-Tyr(R), -Tyr(R)-Len or -Tyr(R)-Asp mayhave the following substituents. 
Amide, lower alkyl(C.sub.1 -C.sub.5) amide, e.g. --NHCH.sub.3, --NHC.sub.2 
H.sub.5 !, amino acids e.g. natural amino acids, D-Glu, .alpha.-amino 
adipic acid, .alpha.-amino suberic acid(Asu)!, lower alkyl(C.sub.1 
-C.sub.5) ester of amino acids e.g. Glu--OC.sub.2 H.sub.5, Glu(OC.sub.2 
H.sub.5)OC.sub.2 H.sub.5, Asu (OMe)--OMe!, amino acid amnide e.g. 
Glu--NH.sub.2, --Gln--NH.sub.2, Asu(NH.sub.2)NH.sub.2 !, lower 
alkyl(C.sub.1 -C.sub.5) amide of amino acids e.g. Glu--NHC.sub.2 H.sub.5, 
--Gln--NHC.sub.2 H.sub.5 !, amino sulfonic acids e.g. --NH--CH.sub.2 
--SO.sub.3 H, taulin (Tau), --NH--(CH.sub.2).sub.3 --SO.sub.3 H!, 
aminosulfone amide e.g. --NH--CH.sub.2 --SO.sub.2 NH.sub.2, Tau--NH.sub.2 
!, amino alcohol e.g. --NH(CH.sub.2).sub.2 --OH, --NH(CH.sub.2).sub.3 
--OH, Leu--o1!, amino phosphoric acid e.g. --NHPO(OH).sub.3 !, amino 
phosphoric acid ester e.g. --NHPO(OC.sub.2 H.sub.5).sub.3, 
--NHPO(OPh).sub.2 ! or amino phosphone amide e.g. 
--NHPO--(NH.sub.2).sub.2 !. 
The following acyl group may be used as protecting groups of the N-terminal 
amino group: 
Alkanoyl e.g.acetyl(CH.sub.3 CO--), butyryl(CH.sub.3 CH.sub.2 CH.sub.2 
CO--), isobutyryl ((CH.sub.3).sub.2 CHCO--)!, substitutedalkanoyl 
e.g.lactinyl(CH.sub.3 CH(OH)CO--)!, carboxy alkanoyl 
e.g.succinyl(HOOCCH.sub.2 CH.sub.2 CO--), glutaryl (HOOC(CH.sub.2).sub.3 
CO--)!, substituted carboxy alkanoyl e.g. malicyl (HOOCCH(OH)CH.sub.2 
CO--)!, alkoxycarbonyl alkanoyl e.g. ethoxy carbonyl propionyl (EtOOCCH 
.sub.2 CH.sub.2 CO--)!, carbamoyl alkanoyl e.g.carbamoyl propionyl 
(H.sub.2 NOCCH.sub.2 CH.sub.2 CO--)!, alkenoyl e.g.acryl (CH.sub.2 
.dbd.CHCO--), oleonyl (CH.sub.3 (CH.sub.2).sub.7 CH.dbd.CH(CH.sub.2).sub.7 
CO--)!, carboxy alkenoyl e.g. 3-carboxy-cis-propenoyl, 
3-carboxy-trans-propenoyl (HOOCCH.dbd.CHCO--)!, alkoxycarbonyl alkenoyl 
e.g.ethoxy carbonyl acryloyl EtOOCCH.dbd.CHCO--)!, or carbamoylalkenoyl 
e.g. carbamoylacryloyl (H.sub.2 NOCCH.dbd.CHCO--)! 
The hirudin variants of the present invention may form salts with acids or 
bases etc. 
Salts in the present invention may be the following: 
hydrochloride,sulfate, p-toluene sulfonate, phosphate, formic acid salt, 
malonic acid salt, succinic acid salt, lactic acid salt, oxalic acid salt, 
tartaric acid salt, acetic acid salt, trifluoroacetic acid salt, sodium 
salt, potassium salt, magnesium salt, barium salt, calcium salt, ammonium 
salt, piperidine salt, morpholine salt, dimethyl amine salt, diethyl amine 
salt, etc. 
A preferred genus of peptides in accordance with the present invention is 
represented by the following formula: 
EQU D-R1-Phe-Glu-A-Ile-Pro-B-Tyr(R.sup.a)-Tyr(R.sup.b)-R2-C (III) 
wherein the peptide has an amino acid sequence consisting of 12 amino acids 
or less; 
A is Glu or Pro; 
B is Glu, Tyr or Tyr (SO.sub.3 H); 
Tyr(R.sup.a) and Tyr(R.sup.b) are independently selected from Tyr and Tyr 
(SO.sub.3 H); 
Tyr (SO.sub.3 H) is a sulfated ester of tyrosine; 
R1 is -Gly-Asp-, -Asp-, or a bond; 
preferably at least one of B, Tyr(R.sup.a) and Tyr(R.sup.b) is Tyr 
(SO.sub.3 H); 
D is bonded to the N-terminal amino group of R1 and is selected from the 
group consisting of a hydrogen atom, an alkanoyl group, an alkanoyl group 
bearing an OH group, a carboxyalkanoyl group, a carboxyalkanoyl group 
bearing an OH group, an alkoxycarbonyl alkanoyl group, an alkenoyl group, 
a carboxyalkenoyl group, an alkoxycarbonyl alkenoyl group, and a 
carbamoylalkenoyl group; 
R2 is optionally present and is Leu or Asp; and 
C optionally replaces the C-terminal hydroxyl of the peptide and is 
selected from the group consisting of an --NH.sub.2 group, a (C.sub.1 
-C.sub.5 -alkyl)amino group, an amino acid, a C.sub.1 -C.sub.5 alkyl ester 
of an amino acid, an amino acid amide group, a (C.sub.1 -C.sub.5 
-alkyl)amide of an amino acid, an amino sulfonic acid group, an 
aminosulfonamide group, an amino alcohol group, an amino phosphoric acid 
group, an amino phosphoric acid ester, and an aminophosphonamide group. 
The polypeptides including the amino acid sequence shown in aforementioned 
formula (II) can easily be produced by various known methods. Such methods 
include chemical synthesis methods like the solid phase method and the 
liquid phase method, recombinant DNA method and the combination of these 
methods. 
The method of sulfation using aryl sulfotransferase in the present 
invention has the advantage that it can sulfate tyrosine residues 
specifically in polypeptides of long amino acid sequence under mild 
condition so that it does not affect other amino acids. There is no 
particular limitation to the enzyme used for the sulfation as long as it 
has aryl sulfotransferase activity, for instance, the one derived from 
human enterobacteria (Eubacterium A-44). 
Preferable examples of sulfate group donors are aryl sulfates or their 
salts, for example, phenyl sulfate, p- or m-nitro phenyl sulfate, p- or m- 
acetyl phenyl sulfate, tyramine sulfate, p-nitro catechol sulfate, p-nitro 
catechol disulfate, pico sulfate, phenolphthalein disulfate, 4-methyl 
unberiferril sulfate, 1- or 2-naphthyl sulfate, 4-nitro-l-naphthyl 
sulfate, 4-fenantoryl sulfate, or their alkali metal salts. 
Reaction temperature and pH should be optimized depending on the nature of 
the polypeptide and ayl sulfotransferase; however, the present inventors 
have found preferable conditions at the temperature of 25-37 .degree. C. 
and at the pH of 8-9. 
When the reaction is over, the sulfated compound may easily be separated 
and collected from unreacted materials by high performance liquid 
chromatography under appropriate conditions. Recovered unreacted materials 
may be re-used for sulfation of the polypeptide by feeding them back to 
the reaction system. 
On the other hand, those polypeptide chains of relatively short amino acids 
including the amino acid sequence shown in aforementioned formula (I) may 
be generally chemically synthesized by solid phase or liquid phase 
methods. The hydroxyl group of the tyrosine residue of these peptides may 
be sulfated by chemical methods using sulfating reagents. The sulfation 
may be carried out by treating the aforementioned polypeptide chain at 
around room temperature, in the presence of solvents, such as pyridine or 
dimethyl formamide, with a 10-500 equivalent amount excess of sulfur 
trioxide complex, such as pyridine-sulfur trioxide, dioxane-sulfur 
trioxide, trimethylamine-sulfur trioxide, triethylamine-sulfur trioxide, 
dimethylaniline-sulfur trioxide, thioxane-sulfur trioxide, bis 
(2-chloroethyl)ether-sulfur trioxide and, 2-methylpiridine-sulfur 
trioxide, quinoline-sulfur trioxide, dimethylformamide-sulfur trioxide. As 
an alternative method, sulfation may be done by condensation by treating 
an excess of sulfuric acid and dicyclohexylcarbodiimide with the 
aforementioned polypeptide chain at around room temperature. 
When compounds obtained in the present invention are going to be used as 
pharmaceuticals, for example, they may be administered orally, or 
subcutaneously, intravenously, intramuscularly, or intraarterially by 
injection, or non-orally through mucous membranes. The dose of 0.1-1000 mg 
for an adult per a day is suitable. The total amount may be administered 
one to several times. However, naturally, the amount may be properly 
increased or decreased upon necessity. For oral administration, they may 
be used in form of a tablet, capsule or granule, which are prepared by 
using pharmaceutically acceptable additives, diluents, carrier, 
excipients, etc. For non-oral administration, the compounds may be 
prepared in injectable formulations of solutions or suspensions, using 
sterilized solutions of water or oils, detergents, other pharmaceutically 
acceptable additives, pharmacologically acceptable diluents or carriers, 
etc. Similarly, using pharmaceutically acceptable additives, diluents, 
carriers or excipients etc., the compounds maybe prepared in suppository 
form or a into formulation that enables absorption through the skin or 
mucous membranes.