Radiolabelled peptide compounds

A diagnostic composition suitable for administration to a warm-blooded animal comprising hirudin or a molecule capable of interacting with the hirudin receptor labeled with a radionuclide by means of a chelate ligand capable of administration to an animal to produce reliable visual imaging of thrombus.

FIELD OF THE INVENTION 
This invention relates generally to novel compounds for use in diagnostic 
tissue imaging and more particularly, to site specific radiolabelled 
peptides, to methods of preparing such site specific radiolabelled 
peptides, and to pharmaceutical compositions comprising these site 
specific radiolabelled peptides for diagnostic imaging. 
BACKGROUND OF THE INVENTION 
Scintigraphic imaging and similar radiographic techniques for visualizing 
tissues in vivo are finding ever-increasing application in biological and 
medical research and in diagnostic and therapeutic procedures. Generally, 
scintigraphic procedures involve the preparation of radioactive agents 
which, upon introduction to a biological subject, become localized in the 
specific organ, tissue or skeletal structure of choice. When so localized, 
traces, plots or scintiphotos depicting the in vivo distribution of 
radiographic material can be made by various radiation detectors, e.g., 
traversing scanners and scintillation cameras. The distribution and 
corresponding relative intensity of the detected radioactive material not 
only indicates the space occupied by the targeted tissue, but also 
indicates a presence of receptors, antigens, aberrations, pathological 
conditions, and the like. 
In general, depending on the type of radionuclide and the target organ or 
tissue of interest, the compositions comprise a radionuclide, a carrier 
agent designed to target the specific organ or tissue site, various 
auxiliary agents which affix the radionuclide to the carrier, water or 
other delivery vehicles suitable for injection into, or aspiration by, the 
patient, such as physiological buffers, salts, and the like. The carrier 
agent attaches or complexes the radionuclide to the peptide carrier agent, 
which results in localizing the radionuclide being deposited in the 
location where the carrier agent concentrates in the biological subject. 
Technetium-99m(.sup.99m Tc) is a radionuclide which is widely known for its 
uses in tissue imaging agents. Due to its safety and ideal imaging 
properties, this radionuclide is conveniently available commercially in 
the oxidized pertechnetate form (.sup.99m TcO.sub.4.sup.-) hereinafter 
"pertechnetate-Tc99m". However, pertechnetate will not complex with the 
most commonly used biological carriers for radionuclide tissue imaging. 
Thus, technetium-labelled imaging agents are generally prepared by 
admixing a pertechnetate-Tc99m isotonic saline solution, a technetium 
reductant (reducing agent) such as stannous chloride or sodium dithionite, 
and a chelate conjugated to the desired peptide carrier agent for 
targeting the organ of interest. Alternatively, an intermediate transfer 
liquid-technetium 99m complex may be prepared prior to addition to the 
chelate-biological molecule to maintain the oxidation state within a 
desired level. Examples of such include 99m Tc-tartrate or 99m 
Tc-gluconate. 
Another problem is that technetium-containing scintigraphic imaging agents 
are known to be unstable in the presence of oxygen, primarily since 
oxidation of the reductant and/or the technetium-99m destroys the reduced 
technetium-99m/targeting carrier complex. Accordingly, such imaging agents 
are generally made oxygen-free by saturating the compositions with 
oxygen-free nitrogen gas or by preparing the agents in an oxygen-free 
atmosphere. Stabilization of imaging agents can also be achieved through 
chemical means. U.S. Pat. No. 4,232,000, Fawzi, issued Nov. 4, 1980, 
discloses the use of gentisyl alcohol as a stabilizer for technetium 
imaging agents. Similarly, U.S. Pat. No. 4,233,284, Fawzi, issued Nov. 11, 
1980 discloses the use of gentisic acid as a stabilizer. 
SUMMARY OF THE INVENTION 
The present invention discloses novel radiolabelled peptide compounds, 
methods of preparing these compounds, pharmaceutical compositions 
comprising these compounds and the use of these compounds in kits for the 
diagnostic imaging of thrombotic diseases. Thrombus hirudin contain large 
numbers of receptors having a high affinity for hirudin and derivatives 
thereof. 
In diagnostic thrombus imaging, a radiolabelled compound must be easily 
detectable and highly selective and have low blood binding. High 
selectivity, which is essential in these compounds means that the 
diagnostic compound, after having been introduced into the body, 
accumulates to a greater degree in the target tissue or tissues, i.e. a 
thrombi, than in surrounding tissues. In using hirudin or derivatives 
thereof as carrier agents in radiolabelled compounds, the specific high 
selectivity of the particular peptide used provides for the strong 
accumulation of the diagnostic compound in the target tissue or tissues, 
such as in thrombus in the case of hirudin, compared with the 
concentration thereof in non-target tissues. 
The radiolabelled peptide compounds of the present invention employ the 
hirudin peptide having sequence identification number 1 NH.sub.2 
-Ile-Thr-Thr-Thr-Asp-Cys-Thr-Glu-Ser-Gly-Gln-Asn-Leu-Cys-Leu-Cys-Glu-Gly-S 
er-Asn-Val-Cys-Gly-Lys-Gly-Asn-Lys-Cys-Ile-Leu-Gly-Ser-Asn-Gly-Lys-Gly-Asn- 
Gln-Cys-Val-Thr-Gly-Gly-Gly-Thr-Pro-Lys-Pro-Glu-Ser-His-Asn-Asn-Gly-Asp-Phe 
-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln-COOH; 
hirulog-1 peptide having sequence identification number 2 NH.sub.2 
-D-Phe-Pro-Arg-Pro-(Gly).sub.4 
-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-COOH; 
hirulog-64 peptide having sequence identification number 3 NH.sub.2 
-D-Phe-Pro-Arg-Pro-(Gly).sub.4 
-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gly-Gly-Lys-COOH; 
hirulog-133 peptide having sequence identification number 4 NH.sub.2 
-D-Phe-Pro-Arg-Pro-(Gly).sub.4 
-Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gly-Gly-Cys-COOH; and 
like derivatives. 
In targeting particular receptors with radiolabelled hirudin or its 
derivatives, it is not necessary that the complete sixty-five (65) residue 
sequence of hirudin (Seq. I.D. No. 1) be present. Binding is thought to 
reside primarily in the anion binding exosite. Through substitution in the 
hirudin sequence, including some limited substitutions in the anion 
binding exosite and perhaps incorporating D-amino acid enantiomorphs, 
additional useful peptides are developed without affecting the binding 
specificity and affinity desired. Likewise peptidomimetic molecules may be 
prepared to duplicate this specific binding function. 
In the present invention, the hirudin peptide itself, or a molecule having 
hirudin receptor specificity, such as hirulog-1(Seq. I.D. No. 2), 
hirulog-64 (Seq. I.D. No. 3) and hirulog-133 (Seq. I.D. No. 4) may be 
radiolabelled using more than one method. The reaction generally takes 
place between the amino groups in the peptide and the carbonyl group in 
the active ester to form an amide bond. In particular, the peptides can be 
radiolabelled using either a conventional method referred to as 
"post-formed chelate approach" or by a recent method referred to as 
"pre-formed chelate approach" developed by Fritzberg et al., U.S. Pat. 
Nos. 4,965,392 and 5,037,630 incorporated herein by reference. In the 
"preformed approach," the desired ligand is complexed with the 
radionuclide and then conjugated to hirudin or a molecule having hirudin 
receptor specificity. In the "post-formed approach," the desired ligand is 
first conjugated to the peptide and the resulting conjugate is incubated 
with 99mTc sodium pertechnetate solution obtained from .sup.99 Mo/.sup.99m 
Tc generator along with a reducing agent. In the present invention, the 
latter approach has the additional advantage of allowing preparation of 
the complex in kit form. Users merely add Na.sup.99m TcO.sub.4 to the 
ligand-hirudin conjugate or a derivative thereof for labelling to occur. 
It is important to note the unique mechanism of the present invention 
whereby the conjugation reaction will be successful only when the 
alpha-amino group is not affected. If the alpha-amino group is affected, 
the specificity and affinity of the peptide is altered. Therefore, in the 
molecules of the present invention it is important to perform the 
conjugation while protecting the alpha-amino group possibly by using 
blocking agents. For example, in the conjugation of hirulog-133, 
D-phenylalanine must be protected to ensure specificity. Therefore, in the 
case of labelling hirulog-1, hirulog-64 or hirulog-133 the epsilon-amino 
group or the sulfahydryl groups are the groups preferably targeted for 
labelling. Avoiding the deprotonation of the alpha-amino group involved in 
binding with the receptor prevents the formation of a chelate complex 
which interferes with the binding site of the peptide and thus protects 
the peptide's ability to bind to the receptor. In short, in the present 
invention, binding preferably occurs on the epsilon-amino group in order 
to avoid interference with the binding specificity of the hirudin peptide 
or derivatives thereof. 
Using either method of labelling the hirudin peptide or its derivatives, 
any suitable ligand can be used to incorporate the preferred radionuclide 
metal ion such as technetium, iodine, rhenium, indium, gallium, samarium, 
holmium, yttrium, copper, or cobalt. The choice of the ligand entirely 
depends on the type of metal ion desired for diagnostic purposes. For 
example, if the radionuclide is a transition element such as technetium or 
rhenium, then ligands containing amine, amide, and thiols are preferred to 
form a stable complex whereas if the radionuclide is a lanthanide element, 
then polyaminocarboxyates or phenolate type ligands are preferable. 
The above-described unique characteristics of the present invention make 
radiolabelled hirudin and its derivatives very attractive for diagnostic 
purposes. The compounds of the present invention may be labelled with any 
radionuclide favorable for these purposes. For diagnostic purposes the 
most suitable radionuclides include but are not limited to the transition 
metals as exemplified by technetium-99m, and copper-62 and. 
Due to the unique mechanism employed in the present invention to label by 
means of a chelate ligand the epsilon amino group of hirudin and avoid the 
alpha amino group(s) (which would inhibit the ability of hirudin or 
derivative peptides to bind to its receptor) a significantly advantageous 
radiolabelled peptide compound for diagnostic imaging of thrombus and 
thrombotic diseases is achieved. 
It is therefore an object of the present invention to provide a selective 
agent, both for the diagnostic imaging and for the therapeutic treatment 
of thrombotic diseases containing high-affinity hirudin receptors having a 
significantly high target to background ratio. 
DETAILED DESCRIPTION OF THE INVENTION 
The preferred peptide employed in the present invention is a hirudin 
peptide as described in German Pat. Nos. 136,103 (1902) and 150,805 (1903) 
incorporated herein by reference or derivatives thereof. The hirudin 
peptide is radiolabelled using a pre-formed or post-formed methodology. In 
a preferred embodiment according to the present invention, the hirudin or 
a molecule having hirudin receptor specificity is first bonded to the 
N.sub.3 S aminothiol ligand which is illustrated in FIG. 1 
##STR1## 
wherein m is a whole number less than eleven and preferably 3; p is either 
0 or 1; PG.sub.1 is a suitable sulfur protecting group selected from the 
group consisting of C.sub.1-20 S-acyl such as alkanoyl, benzoyl and 
substituted benzoyl--whereby alkanoyl is preferable, C.sub.1-20 S-acyl 
groups such as benzyl, t-butyl, trityl, 4-methoxybenzyl and 
2,4-dimethoxybenzyl--whereby 2,4-dimethoxybenzyl is preferable, C.sub.1-10 
alkoxyalkyl such as methoxymethyl, ethoxyethyl and tetrahydropyranyl 
--whereby tetrahydropyranyl is preferable, carbamoyl, and C.sub.1-10 
alkoxy carbonyl such as t-butoxycarbonyl and methoxycarbonyl--whereby 
t-butoxycarbonyl is preferable; and X is a coupling moiety selected from 
the group consisting of carboxyl, amino, isocyanate, isothiocyanate, 
imidate, maleimide, chlorocarbonyl, chlorosulfonyl, 
succinimidyloxycarbonyl, haloacetyl and C.sub.1-10 
N-alkoxycarbamoyl--whereby N-methylcarbamoyl is preferable. 
In another preferred embodiment according to the present invention, hirudin 
or a molecule having hirudin receptor specificity is bonded to the N.sub.2 
S.sub.2 aminothiol ligand which is illustrated in FIG. 2; 
##STR2## 
wherein n is a whole number less than eleven and preferably 3; PG.sub.2 
and PG.sub.3 may be the same or different sulfur protecting groups 
selected from the group consisting of C.sub.1-20 S-acyl such as alkanoyl, 
benzoyl and substituted benzoyl--whereby alkanoyl is preferable, 
C.sub.1-20 alkyl groups such as benzyl, t-butyl, 4-methoxybenzyl, trityl 
and 2,4dimethoxybenzyl--whereby 2,4-dimethoxybenzyl is preferable, 
C.sub.1-10 alkoxyalkyl such as for example methoxymethyl, ethoxyethyl, and 
tetrahydropyranyl--whereby tetrahydropyranyl is preferable, carbamoyl and 
C.sub.1-10 alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl and 
t-butoxycarbonyl--whereby t-butoxycarbonyl is preferable; and Y is a 
coupling moiety selected from the group consisting of carboxyl, amino, 
isocyanate, isothiocyanate, imidate, maleimide, chlorocarbonyl, 
chlorosulfonyl, succinimidyloxycarbonyl, haloacetyl, and C.sub.1-10 
N-alkoxycarbamoyl--whereby N-methylcarbamoyl is preferable. 
In another preferred embodiment of the present invention, hirudin or a 
molecule having hirudin receptor specificity is conjugated with the ligand 
illustrated in FIG. 3, 
##STR3## 
wherein n varies from 1 to 10, and Y is a coupling moiety selected from 
the group consisting of carboxyl, amino, isocyanate, isothiocyanate, 
imidate, maleimide, chlorocarbonyl, chlorosulfonyl, 
succinimidyloxycarbonyl, haloacetyl, and C.sub.1-10 N-alkoxycarbamoyl such 
as N-methoxycarbamoyl and t-butoxycarbamonyl--whereby t-butoxycarbamonyl 
is preferable; and R is selected from the group consisting of hydrogen and 
C.sub.1-10 alkyl such as methyl and t-butyl--whereby t-butyl is 
preferable. 
In another preferred embodiment, the hirudin or a molecule having hirudin 
receptor specificity can be conjugated with the metal complex illustrated 
in FIG. 4 
##STR4## 
wherein m is a whole number less than eleven and more preferably 3; p is 
either 0 or 1; X' is a coupling moiety selected from the group consisting 
of carboxyl, amino, isocyanate, isothiocyanate, imidate, maleimide, 
chlorocarbonyl, chlorosulfonyl, succininimidyloxycarbonyl, haloacetyl and 
C.sub.1-10 N-alkoxycarbamoyl such as N-methoxycarbamoyl and 
t-butoxycarbamoyl--whereby t-butoxycarbamoyl is preferable and M is a 
radionuclide suitable for diagnostic imaging or therapeutic use such as 
technetium, rhenium, copper, cobalt, indium, gallium, samarium, yttrium 
and holmium. 
In another preferred embodiment, the hirudin or a molecule having hirudin 
receptor specificity can be conjugated with a metal complex as illustrated 
in FIG. 5 wherein Y' and n are defined the same respectively as Y and n in 
FIG. 3 and M is defined the same as M in FIG. 4. 
##STR5## 
In another preferred embodiment, the hirudin or a molecule having hirudin 
receptor specificity can be conjugated with a metal complex as shown in 
FIG. 6. 
##STR6## 
wherein Z', q and R are defined the same respectively as Y, n and R of 
FIG. 3 and M is defined the same as M in FIG. 4. 
In another preferred embodiment, the hirudin or a molecule having hirudin 
receptor specificity can be conjugated with a metal complex as shown in 
FIG. 7. 
##STR7## 
wherein M is defined the same as M in FIG. 4. 
Common esters which have been found useful in this labelling technique are 
o- and p- nitrophenyl, 2-chloro- 4-nitrophenyl, cyanomethyl, 
2-mercaptopyridyl, hydroxybenztriazole, N-hydroxysuccinimide, 
trichlorophenyl, tetrafluorophenyl, thiophenyl, tetrafluorothiophenyl, 
o-nitro-p-sulfophenyl, N-hydroxyphthalimide and the like. For the most 
part, the esters will be formed from the reaction of the carboxylate with 
an activated phenol, particularly, nitro-activated phenols, or a cyclic 
compound based on hydroxylamine. 
The advantages of using sulfur protecting or blocking groups include the 
fact that a separate step for removal of the sulfur-protective group is 
not necessary. The protecting groups are displaced from the compound 
during the labelling in what is believed to be a metal-assisted acid 
cleavage: i.e., the protective groups are displaced in the presence of a 
radionuclide at an acid pH and the radionuclide is bound by the chelating 
compound. The radiolabeling procedure thus is simplified, which is a 
significant advantage when the chelating compounds are to be radiolabelled 
in a hospital laboratory shortly before use. Additionally, another 
advantage of the present invention is that the basic pH conditions and 
harsh conditions associated with certain known radiolabeling procedures or 
procedures for removal of other sulfur protected groups are avoided. Thus, 
base-sensitive groups on the chelating compounds survive the 
radio-labelling step intact. Suitable sulfur-protecting groups, when taken 
together with the sulfur atom to be protected, include hemithioacetal 
groups such as ethoxyethyl, tetrahydrofuranyl, methoxymethyl, and 
tetrahydropyranyl. Other suitable sulfur protecting groups are C.sub.1-20 
acyl groups, preferably alkanoyl or benzoyl. Other possible formulas for 
the chelating compounds are described in the European Patent Application 
assigned publication number 0 284 071 incorporated herein by reference. 
Synthesis of the Tc-99m bifunctional chelate and subsequent conjugation to 
a hirudin peptide, or a derivative thereof, can be performed as described 
in the European Patent Application assigned publication number 0 284 071 
and U.S. Pat. No. 4,965,392 incorporated herein by reference and related 
technologies as covered by U.S. Pat. Nos. 4,837,003, 4,732,974 and 
4,659,839, each incorporated herein by reference. 
After purification, technetium-99m labelled hirudin peptide, or derivatives 
thereof, may be injected into a patient for diagnostic imaging. The 
technetium-99m hirudin compound is capable of reliably visualizing 
thrombus within minutes of post-injection. The hirudin peptide when 
radiolabelled with the technetium-99m triamide thiolate bifunctional 
chelate is efficacious as an in vivo diagnostic agent for the imaging of 
thrombus of the type described above. The radiolabelled hirudin compound 
of the present invention are described in still greater detail in the 
illustrative examples which follow.

EXAMPLE 1 
A solution of hirudin, or derivatives thereof, (0.01 mmol) in 2 mL of 
carbonate/bicarbonate buffer at pH 8.5 .+-.0.5 is treated with a solution 
of 0.1 mmol of the ligand in FIG. 1 (wherein m=2, p=1, PG.sub.1 is 
benzoyl, and X is succinimidyloxycarbonyl) in dimethylformamide (0.5 mL) 
and the entire mixture is kept at room temperature for 2 hours. The 
mixture is then diluted with water (2.5 mL) and dialyzed extensively 
against water. After dialysis, the solution is lyophilized to give the 
desired hirudin conjugate. 
EXAMPLE 2 
A solution of hirudin, or derivatives thereof, (0.01 mmol) in 2 mL of 
carbonate/bicarbonate buffer at pH 8.5.+-.0.5 is treated with a solution 
of 0.1 mmol of the ligand in FIG. 2 (wherein n=2, PG.sub.2 and PG.sub.3 
are benzoyl, and Y is succinimidyloxycarbonyl) in dimethylformamide (0.5 
mL) and the entire mixture is kept at room temperature for 2 hours. The 
mixture is then diluted with water (2.5 mL) and dialyzed extensively 
against water. After dialysis, the solution is lyophilized to give the 
desired hirudin conjugate. 
EXAMPLE 3 
A solution of hirudin, or derivatives thereof, (0.01 mmol) in 2 mL of 
carbonate/bicarbonate buffer at pH 8.5 .+-.0.5 is treated with a solution 
of 0.1 mmol of the ligand in FIG. 3 (wherein q=4, and Z is 
succinimidyloxycarbonyl) in dimethylformamide (0.5 mL) and the entire 
mixture is kept at room temperature for 2 hours. The mixture is then 
diluted with water (2.5 mL) and dialyzed extensively against water. After 
dialysis, the solution is lyophilized to give the desired hirudin 
conjugate. 
EXAMPLE 4 
To 100 uL of a solution containing 5 mg of sodium gluconate and 0.1 mg of 
stannous chloride in water, 500 ul of 99m-Tc04 (pertechnetate) is added. 
After incubation at room temperature for about 10 minutes at room 
temperature, a solution of 500 uL of the hirudin, or derivatives thereof, 
conjugates (1 mg/mL in 0.1M carbonate/bicarbonate buffer, pH 9.5) in 
Examples 1 or 2 is then added and the entire mixture is incubated at 
37.degree. C. for about 1 hour. The desired labelled peptide is separated 
from unreacted 99mTc-gluconate and other small molecular weight impurities 
by gel filtration chromatography (Sephadex G-50) using phosphine buffered 
physiological saline, (hereinafter PBS), 0.15M NaCl, pH 7.4 as eluent. 
EXAMPLE 5 
A mixture of gentisic acid (25 mg), inositol (10 mg), and the hirudin, or 
derivatives thereof, conjugate (500 uL, 1 mg/mL in water) was treated with 
In-111 indium chloride in 0.05M HCl. The solution was allowed to incubate 
at room temperature for about 30 minutes. The desired labelled peptide is 
separated from unreacted In-111 indium salts and other small molecular 
weight impurities by gel filtration chromatography (Sephadex G-50) using 
phosphine buffered physiological saline, (PBS), 0.15M NaCl as eluent. 
After the hirudin or a derivative thereof is prepared and labelled 
according to the procedure described above, the compound is used with a 
pharmaceutically acceptable carrier in a method of performing a diagnostic 
imaging procedure using a gamma camera or like device. This procedure 
involves injecting or administering, for example in the form of an 
injectable liquid, to a warm-blooded animal an effective amount of the 
present invention and then exposing the warm-blooded animal to an imaging 
procedure using a suitable detector, e.g. a gamma camera. Images are 
obtained by recording emitted radiation of tissue or the pathological 
process in which the radioactive peptide has been incorporated, which in 
the present case are thrombus, thereby imaging thrombus in the body of the 
warm-blooded animal. Pharmaceutically acceptable carriers for diagnostic 
use include those that are suitable for injection or administration such 
as aqueous buffer solutions, e.g. tris (hydroxymethyl)aminomethane (and 
its salts), phosphate, citrate, bicarbonate, etc., sterile water for 
injection, physiological saline, and balanced ionic solutions containing 
chloride and/or bicarbonate salts of normal blood plasma cations such as 
Ca.sup.2 +, Na.sup.+, K.sup.+ and Mg.sup.2 +. Other buffer solutions are 
described in Remington's Practice of Pharmacy, 11th edition, for example 
on page 170. The carriers may contain a chelating agent, e.g. a small 
amount of ethylenediaminetetraacidic acid, calcium disodium salt, or other 
pharmaceutically acceptable chelating agents. 
The concentration of labeled peptide and the pharmaceutically acceptable 
carrier, for example in an aqueous medium, varies with the particular 
field of use. A sufficient amount is present in the pharmaceutically 
acceptable carrier in the present invention when satisfactory 
visualization of the thrombi is achievable. 
The composition is administered to the warm-blooded animals so that the 
composition remains in the living animal for about six to seven hours, 
although shorter and longer residence periods are normally acceptable. 
The radiolabelled hirudin compounds of the present invention or derivatives 
thereof, prepared as described herein, provide means of in vivo diagnostic 
imaging of thrombus which provides advantages over prior known procedures 
for diagnosis of thrombotic disease. 
After consideration of the above specification, it will be appreciated that 
many improvements and modifications in the details may be made without 
departing from the spirit and scope of the invention. It is to be 
understood, therefore, that the invention is in no way limited, except as 
defined by the appended claims. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 4 
(2) INFORMATION FOR SEQ ID NO: 1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 65 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hirudin 
(B) STRAIN: Human 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
IleThrTyrThrAspCysThrGluSerGlyGlnAsnLeuCysLeuCys 
151015 
GluGlySerAsnValCys GlyLysGlyAsnLysCysIleLeuGlySer 
202530 
AsnGlyLysGlyAsnGlnCysValThrGlyGlyGlyThrProLysPro 
35 4045 
GluSerHisAsnAsnGlyAspPheGluGluIleProGluGluTyrLeu 
505560 
Gln 
65 
(2) INFORMATION FOR SEQ ID NO: 2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hirudin 
(B) STRAIN: Human 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: 
PheProArgProGlyGlyGlyGlyAsnGlyAspPhe GluGluIlePro 
151015 
GluGluTyrLeu 
20 
(2) INFORMATION FOR SEQ ID NO: 3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
( D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hirudin 
(B) STRAIN: Human 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: 
PheProArgProGlyGlyGlyGlyAsnGlyAspPheGluGluIlePro 
15 1015 
GluGluTyrLeuGlyGlyLys 
20 
(2) INFORMATION FOR SEQ ID NO: 4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 amino acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
(ii) MOLECULE TYPE: protein 
(iii) HYPOTHETICAL: NO 
(vi) ORIGINAL SOURCE: 
(A) ORGANISM: Hirudin 
(B) STRAIN: Human 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: 
PheProArgProGlyGlyGlyGlyAsnGlyAspPheGluGluIlePro 
151015 
GluGluTyrLeuGlyGlyCys 
20