Isohirudins

DESCRIPTION
The invention relates to novel thrombin inhibitors from the hirudin family which differ from the previously known hirudins on account of mutation in the protein chain.
Hirudins are known, for example, from EP-A 142,860; EP-A 158,564; EP-A 158,986; EP-A 168,342; EP-A 171,024; EP-A 193,175; EP-A 200,655; EP-A 209,061; EP-A 227,938; Chang, FEBS, Vol. 164 (1983) 307; J. Dodt et al., Biol. Chem. Hoppe-Seyler 367 (1986) 803-811 and D. Tripier, Folia Haematol. Leipzig 115 (1988) 1-2, 30-35. However, these hirudins may be partially bonded via active centers to carriers or via amino acids which participate in the bonding to thrombin. In addition, the known hirudins have limited transdermal properties and are very short-lived for reasons of high elimination rates so that stationary or ambulant thrombosis prophylaxis is made difficult.
The aim is therefore to find hirudins having high specific activity, high stability and good pharmacokinetics. In addition, these hirudins should be distinguished by better chemical manageability on immobilization on carriers.
This aim is achieved according to the invention by the isohirudins of the formula (A) ##STR1## in which A denotes Val or Ile,
B denotes Val or Thr,
C denotes Gln or Glu,
E denotes Asn or Asp,
F denotes Glu or Gln,
G denotes Asp or Gly,
I denotes Asp or Asn,
J denotes Gln, Glu, Asn or Lys,
K denotes Ile or Lys,
L denotes Asp or Asn,
M denotes Lys or Glu,
N denotes Gln or Glu and
R denotes hydrogen or SO.sub.3 H,
with the proviso that if J stands for Gln, L is not Asp, and their mutants and their physiologically tolerable salts, if such can be formed.
The following isohirudins may be mentioned in particular, where R denotes hydrogen or SO.sub.3 H: ##STR2##
The invention in addition relates to a process for the preparation of isohirudins which comprises isolating and purifying isohirudins from leeches with the aid of a combination of extraction methods, precipitation methods and chromatographic methods, if desired hydrolytically splitting off an optionally present phenol ester group R with the formation of the phenolic hydroxyl group and optionally converting the resulting polypeptides into their physiologically tolerable salts.
The isohirudins are preferably first extracted as described by F. Markwardt in Biomed. Biochem. Acta 44 (1985) 1007-1013 or in EP-A 158,986 and EP-A 209,061 and the crude isohirudins are subsequently precipitated. The further isolation and purification is carried out by means of a combination of chromatographic methods as described, for example, by P. Walsmann in Pharmazie 36 (1981) 860-861. Preferably, however, the following combination of chromatography methods is used:
ion exchange chromatography.fwdarw.gel permeation chromatography.fwdarw.affinity chromatography.fwdarw.gel permeation chromatography.fwdarw.ion exchange chromatography.fwdarw.microbore RP-HPLC.
The individual chromatographic methods are generally known per se.
The process according to the invention is distinguished by the combination of the different chromatography methods including the final microbore RP-HPLC, in which columns of 1 mm internal diameter are used. Due to a smaller plate height and the lower diffusion in the column, a higher resolution results in this case than in conventional 4.6 mm columns (R. P. W. Scott, J. Chromatographic Science 23 (1986) 233-237; R. Gill, J. Chromatography 16 (1986) 281).
The microbore RP-HPLC unit used here consists of two Beckman 114M solvent delivery modules which are controlled by a 421 a controller. The buffer line to each pump--made of passivated 1/8 inch V8 steel tube--is connected via a T-piece with a dynamic microgradient mixer which homogenizes the buffer solutions, the resulting pulsations being absorbed by a pulsation damper inserted on-line (Waters part No. 98060) and the buffer solution being introduced into the microbore column via the Reodyne valve of a sample applicator (Gilson Abimed model 231, dilutor model 401). The microbore column packed with Aquapore RP-300 (300 .ANG. pore size) 7 .mu.m spherical silica (Applied Biosystems, Cat. No. 400422 Rp) is in a furnace (Sykam S 4110, temperature 40.degree. C.). Detection is at 205 nm in a UV detector (Sykam S 3300 which is equipped and adjusted for 0.08 AUFS). Recording is carried out by means of a compensating recorder (10 mv FS), and integration using the LAS system (Hewlett Packard) after A/D conversion (HP 18625 A A/D convertor). Linear gradient operation is optimized.
The following mixtures A and B are used as buffers:
A: 90% by weight of water, 10% by weight of acetonitrile (ACCN)+0.1% by volume of trifluoroacetic acid (TFA)
B: 90% by weight of ACCN, 10% by weight of water+0.1% by volume of TFA.
In this connection, 3 different gradients are used for the different separation and purification steps:
a) for the separation of the isohirudins from one another in analytical and preparative operation: 0% B to 40% B (gradient 0.06% B/min)
b) for rechromatography and final purification: 0% B to 35% B (gradient 0.4% B/min)
c) for peptide separation: 0% B to 30% B (gradient 1.7% B/min)
Chromatography is carried out using a flow rate of 80 .mu.l/min and manual fractionation.
It is only possible to carry out further novel peptide separations owing to the use, according to the invention, of microbore RP-HPLC--additionally to the known isolation and purification methods. Unfortunately, however, cases still occur in which for reasons of too large an affinity of the individual components, separation remains incomplete even here. This is, for example, the case with the isohirudins II and II'; IIIa, IIIa'; IIIb and IIIb'. The addition ' thus means that these mutations were found, i.e. were additionally sequenced, during the sequence analysis of the related proteins. Thus, for example, isohirudin II' was found in the analysis of isohirudin II in degradation step 20.
The novel isohirudins described in this application are initially purified by methods known from the literature, including affinity chromatography on thrombin-sepharose columns. The resulting isohirudin mixture is then separated into its components with the aid of microbore RP-HPLC. In the case of serious separation problems, the isolated fractions are rechromatographed under slightly changed conditions. Each novel isohirudin is purified to homogeneity or virtual homogeneity and its sequence is completely elucidated, in some cases not only the amino acid sequence being determined by sequence analysis, but additionally protein chemistry methods, such as specific chemical cleavages, being used in order to prove the novel sequences.
The amino acid analysis is carried out using a Beckmann 6300 amino acid analyzer according to the instructions of the manufacturer. To do this, the proteins (30-50 pmol) were previously dried in a 4.times.40 mm quartz test tube and subsequently hydrolyzed at 110.degree. C. in the vapor phase using azeotropic HCl/0.8% phenol under a nitrogen atmosphere. For each batch, 500 pmol of insulin standard are additionally analyzed. The results are integrated using the LAS system (Hewlett Packard).
The N-terminal amino acid sequence is determined by automated sequence analysis on the native protein. This is carried out using a 477 A Pulsed Liquid-Phase (TM) protein sequencer from Applied Biosystems, which is linked on-line with a 120 A PTH analyzer with a data analysis system, according to the instructions of the manufacturer.
In some cases, i.e. for the isohirudins I, II, IIb, III and IIIa, a protease-specific peptide cleavage is additionally carried out. To do this, the "disulfide bridges" of the particular proteins are first oxidized using performic acid, by means of which cystine is converted into cysteic acid. This oxidation causes the proteins to lose their spatial configuration and to form a polymer (a knot) of amino acids without a rigidly definable configuration. After the oxidation, the reaction mixture is freeze-dried, the resulting protein is cleaved using trypsin in a suitable buffer, the peptide mixture is separated into the individual components by means of microbore RP-HPLC and isolated in a preparative procedure.
The primary sequence is subsequently determined by amino acid analysis of these peptides. A serine protease, preferably trypsin, which cleaves at a basic amino acid, i.e. at Arg or Lys, is employed for the specific cleavage.
The new isohirudins of the formula A may additionally be prepared analogously to the process described in EP-A 171,024.
The isohirudins according to the invention are specific thrombin inhibitors. The quantitative inhibition of thrombin by the inhibitors according to the invention showed that the thrombin inhibitor/thrombin complex remains virtually undissociated.
The activity and thus the degree of purity of the isohirudins according to the invention can be determined during working up and purification with the aid of the thrombin inhibition test described below. The isohirudins of the general formula (A) thus purified in this case have a thrombin inhibition of 12-16 AT-U/.mu.g (antithrombin units/.mu.g).
Thrombin inhibition test for the determination of activity
The test is carried out at room temperature in a total volume of 200 .mu.l in a 96-well microtiter plate. After preincubating the thrombin (bovine thrombin 50 NIH-U/mg (Merck, Item No. 12374); stock solution: 40 NIH-U/ml in 20 mM MES pH 6, 0.154 mM NaCl, 0.2% PEG 6000, 1% BSA) in a final volume of 100 .mu.l with 1 to 50 .mu.l of isohirudin, the reaction is started by addition of substrate. Since the activities tie between 0.03 and 0.15 AT-U in the measuring region of the test, the samples to be determined must be correspondingly diluted using a test buffer.
The required amount of test buffer (50 mM tris/HCl, pH 8; 154 mM NaCl; 0.2% polyethylene glycol 6000) per microtiter plate well is initially introduced. 50 .mu.l each of 4 NIH-U/ml thrombin solution (see above) and the sample are subsequently added. After an incubation time of 10 minutes, the reaction is then started by the addition of 100 ul of 1 mM Chromozym TH (Boehringer, Item No. 206849; 10 .mu.m in water/HCl, pH 6; working solution: 1 mM diluted with test buffer). After incubation of the residual thrombin with the chromogen substrate for 5-10 minutes, the nitroaniline reteased is then determined photometrically at 405 nm. A reaction terminator is not required, but an .RTM.ELISA reader (Easy Reader EAR 400 (SLT Labinstruments, Austria) is available for rapidly reading the microtiter plate wells. A hirudin standard is in principle also unnecessary, since an absolute determination of the antithrombin units is possible by means of the use of a defined thrombin activity. Nevertheless, the gradual inactivation of the working solution leads to a systematic error which can be eliminated if the measured values are related to a defined hirudin standard. The test is then independent of the absolute thrombin concentration and permits reproducible determinations.
The following controls are used:
a) Blank
The thrombin solution is replaced by a buffer (photometer zero equatization)
b) Thrombin value
A microtiter plate well is left without inhibitor.
c) Hirudin standard
0.07-0.1 AT-U of a known hirudin standard (Pentapharm hirudin "purified by affinity chromatography", specific activity: 10 AT-U/.mu.g, concentration: 0.1 AT-U/50 .mu.l) is added to one microtiter plate well.
In order to determine the concentrations of the samples, a calibration curve for 1 to 20 .mu.g of isohirudin was determined for the microbore HPLC.
Table 1 which follows compares the concentration values determined on the microbore HPLC with those obtained by the thrombin inhibition test:
TABLE 1______________________________________ Concentration [.mu.g/ml] ThrombinIsohirudin Microbore HPLC inhibition test AT-U/.mu.g______________________________________Ia 56 59 14I 56 62 15Ib 34 38 16IIa 68 72 15II 120 140 16IIb 137 145 16IIIa 84 87 12III 397 533 16IIIb 97 109 14______________________________________
The invention therefore also relates to the use of isohirudins of the formula (A) as blood coagulation inhibitors for use in the prophylaxis and therapy of thromboembolic processes and also to their use as diagnostics and reagents. In addition, the isohirudins according to the invention may also be coupled to a carrier, by means of which derivatives having sustained thrombin-inhibitory action are formed, such as are proposed in German Patent Application P 38 19 079.6.
The invention furthermore relates to pharmaceutical preparations which contain an isohirudin of the formula (A) in a pharmaceutically acceptable excipient.
These preparations may be used, in particular, in the abovementioned indications, when they are administered, for example, parenterally (such as intravenously, intracutaneously, intramuscularly or subcutaneously), orally or topically. The dosage depends primarily on the specific administration form and on the purpose of the therapy or prophylaxis. The size of the individual doses and the administration scheme can best be determined by an individual evaluation of the particular disease case; the methods required for this for the determination of relevant blood factors are familiar to the expert. In the normal case, with one injection the therapeutically effective amount of the isohirudins according to the invention is in the dose range from about 0.005 to about 0.1 mg/kg of body weight. The range from about 0.01 to about 0.05 mg/kg of body weight is preferred. Administration is carried out by intravenous, intramuscular or subcutaneous injection. Accordingly, pharmaceutical preparations for parenteral administration in single-dose form contain about 0.4 to about 7.5 mg of the isohirudin according to the invention depending on the type of administration. In addition to the active compound, these pharmaceutical preparations customarily contain a buffer, for example a phosphate buffer, which should keep the pH between about 3.5 and 7, and in addition sodium chloride, mannitol or sorbitol for adjusting the isotonicity. They may be present in freeze-dried or dissolved form, it being possible for solutions to advantageously contain an antibacterially active preservative, for example 0.2 to 0.3% of methyl or ethyl 4-hydroxybenzoate.
A preparation for topical application may be present as an aqueous solution, lotion or get, an oily solution or suspension, or a fat-containing or, in particular, emulsion ointment. A preparation in the form of an aqueous solution is obtained, for example, by dissolving the active compounds according to the invention or a therapeutically utilizable salt thereof in an aqueous buffer solution of pH 4 to 6.5 and, if desired, adding a further active compound, for example an antiinflammatory, and/or a polymeric adhesive, for example polyvinylpyrrolidone, and/or a preservative. The concentration of the active compound is about 0.08 to about 1.5 mg, preferably 0.25 to 1.0 mg, in about 10 ml of a solution or 10 g of a gel.
An oily administration form for topical application is obtained, for example, by suspending the active compounds according to the invention or a therapeutically utilizable salt thereof in an oil, if desired using swelling agents, such as aluminum stearate, and/or surface-active agents (surfactants), the HLB value ("hydrophilic-lipophilic balance") of which is under 10, such as fatty acid monoesters of polyhydric alcohols, for example glycerol monostearate, sorbitan monolaurate, sorbitan monostearate or sorbitan monooleate. A fat-containing ointment is obtained, for example, by suspending the active compounds according to the invention or their salts in a spreadable fatty foundation, if desired using a surfactant of HLB value under 10. An emulsion ointment is obtained by triturating an aqueous solution of the active compounds according to the invention or their salts in a soft, spreadable fatty foundation with the addition of a surfactant, the HLB value of which is under 10. All these topical application forms may also contain preservatives. The concentration of the active compound is about 0.08 to about 1.5 mg, preferably 0.25 to 1.0 mg, in about 10 g of the foundation.
In addition to the pharmaceutical compositions described above and their analogues, which are intended for direct medicinal use on the human or mammalian body, the present invention also relates to pharmaceutical compositions and preparations for medicinal use outside the living human or mammalian body. Such compositions and preparations are used primarily as coagulation-inhibiting additives for blood which is subjected outside the body to a circulation or treatment (for example dialysis in artificial kidneys), preservation or modification (for example hemoseparation). In their composition, preparations of this type, such as stock solutions or else preparations in single-dose form, are similar to the injection preparations described above; expediently, however, the amount or concentration of active compound is related to the volume of the blood to be treated or, more exactly, to its thrombin content. In this connection, it is to be observed that the active compounds according to the invention (in free form)
(a) deactivate an approximately 5-fold amount by weight of thrombin completely;
(b) are physiologically harmless even in relatively large amounts; and
(c) are excreted very rapidly from the circulating blood even in high concentrations, so that no risk of overdosage exists, even, for example, with transfusions. Depending on the specific purpose, the suitable dose is about 0.01 to about 1.0 mg of active compound/l of blood, it still being possible for the upper limit to be far exceeded without risk.
The present invention also relates to the bioanalytical application of the compounds according to the invention and their salts for the determination of thrombin, and also preparations serving this purpose and containing the active compounds according to the invention, for example solid mixtures and above all solutions, in particular aqueous solutions; these may also expediently contain inert auxiliaries, for example those mentioned above in connection with injection preparations which, for example, have a stabilizing and/or preserving function, in addition to an exact amount or concentration of the active compounds according to the invention, also in the form of a salt. These preparations are used in bioanalyses in analogous, known ways like the hirudin preparations, for example for the determination of thrombin.
The compounds according to the invention can moreover be used for blood preservation. For this purpose, the compounds according to the invention are advantageously added to blood preserves in an amount of 0.1-2% by weight.

BRIEF DESCRIPTION OF THE DRAWING
The Drawing depicts HPLC resolution of an active hirudin fraction.

The examples which follow serve to illustrate the invention. The one-letter code is used as an abbreviation for amino acids here in contrast to the foregoing description and the claims.
______________________________________Three- and one-letter code for amino acidsAmino acid Abbreviation Amino acid Abbreviation______________________________________Alanine Ala (A) Proline Pro (P)Arginine Arg (R) Serine Ser (S)Cysteine Cys (C) Threonine Thr (T)Glycine Gly (G) Tryptophan Trp (W)Histidine His (H) Tyrosine Tyr (Y)Isoleucine Ile (I) Valine Val (V)Leucine Leu (L) Asparaginic acid Asp (D)Lysine Lys (K) Asparagine Asn (N)Methionine Met (M) Glutamic acid Glu (E)Phenylalanine Phe (F) Glutamine Gln (Q)______________________________________
EXAMPLE 1
Isolation of isohirudins Ia, I, Ib, IIa, II, IIb, IIIa, III, IIIb, oxidation, tryptic cleavage, sequence analysis and amino acid analysis
a) Isolation
The isolation of the new isohirudins initially followed the methods known from the Literature (P. Walsmann, Thrombosis Research 40 (1985) 563-569). The most important steps should only be mentioned here. After the extraction of the front part of the leeches with 40% acetone and precipitation of ballast materials with glacial acetic acid, the enriched hirudin extract was precipitated using acetone. The precipitate was purified via ion exchange (for example .RTM.DEAE Sephacel) in tris/HCl buffer (pH 7.4) with the aid of a saline gradient. The fractions active in the antithrombin test were combined and freeze-dried. The buffer components were then separated in portions via .RTM.Sephadex G 25 in water, the active fractions were applied to a thrombin-sepharose column in 0.1M tris/HCl buffer (PH 8.0) and, after washing with the same buffer, eluted with 0.1M sodium acetate 0.5M sodium chloride (pH 5.0). Part of the hirudin was also eluted in the course of this. The pure hirudin fractions were then eluted using 0.1M tris/HCl/1.5M benzamidine (pH 8.0). By coupling the thrombinsepharose to a .RTM.Sephadex G 25 column, the elution of the hirudin was subsequently performed together with separation of the thrombin inhibitor benzamidine at reduced flow. After freeze-drying, the active fractions were purified by ion exchange chromatography over SE-.RTM.Sephadex in 0.01M ammonium acetate buffer (pH 3.8) and elution with a gradient of sodium chloride. Three active fractions were isolated. Fraction II was desalted over .RTM.Sephadex and freeze-dried. The specific activity was 12 AT-U/ug.
The non-homogeneous fraction II was employed for microbore RP-HPLC. Using very shallow gradients (0.06% of B per minute), the mixture was resolved into three principal peaks having very different retention times (RT) (peak I: RT about 89 minutes; peak II: RT about 113 minutes; peak III: RT about 134 minutes; see Drawing). These principal peaks are accompanied by smaller satellite peaks (a and b). Peak IIIa designates, for example, the satellite peak having an RT of about 130 minutes which is eluted immediately before the principal peak III. Correspondingly, the satellite peak IIIb (RT about 139 minutes) is used which is eluted after the principal peak III.
By carefully increasing the amount injected and collecting the fractions (peaks), the microbore RP-HPLC unit was used for preparative isolation of the different isohirudin variants. Up to 50 .mu.g were injected and the various fractions (peaks) were collected without noting losses in the resolution of the peaks. In order to isolate a sufficient amount, this procedure was repeated several times and the identical fractions were combined and freeze-dried. The different rechromatographed peaks were then finally purified by "narrower" fractionations. In this way, the isohirudins Ia, I, Ib, IIa, II, IIb, IIIa, III and IIIb were separated.
The pure substances were oxidized according to need and cleaved with trypsin.
b) Oxidative cleavage of the disulfides
100 .mu.l of performic acid solution (prepared from 50 .mu.l of hydrogen peroxide and 950 .mu.l of formic acid; left to stand at room temperature for 2.5 hours) were added to the isohirudins. After 30 minutes, the mixture was diluted with 200 .mu.l of water and then freeze-dried.
c) Trypsin cleavage
The oxidized isohirudins were taken up in 50 .mu.l of NMM buffer (0.2M N-methyloorpholine adjusted to pH 8 with glacial acetic acid). A solution of trypsin in NMM buffer in the ratio substrate:enzyme=1:100 was added to this. After 30 minutes at room temperature, the reaction was stopped by addition of 100 .mu.l of glacial acetic acid. This resulting mixture was then used further for microbore RP-HPLC.
d) Sequence analysis
The isolated and purified isohirudins were sequenced using a 477A Pulsed liquid Phase (TM) protein sequencer (Applied Biosystems) which is coupled on-line with a 120A PTH analyzer having a data analysis system. The sequencing was carried out according to the instructions of the manufacturer. The results are shown in Tables 3 to 15.
e) Amino acid analysis
The amino acid composition of the individual isohirudins was determined using a Beckman 6300 amino acid analyzer. In this connection, the analysis was carried out according to the instructions of the manufacturer. The reagents and the separating column were likewise bought from the manufacturer. Subsequent integration was carried out using the LAS system (Hewlett Packard). The proteins (about 30-50 pmol) were dried in a 4.times.40 mm quartz test tube and hydrolyzed at 110.degree. C. in the vapor phase by azeotropic HCl/0.8% phenol under a nitrogen atmosphere. 500 pmol of insulin standard were additionally analyzed for each batch.
Table 2 which follows shows the results obtained.
TABLE 2__________________________________________________________________________Amino acid analysisIsohirudinsIa I IIa II IIb IIIa III IIIbIsohirudine a b a b a b a b a b a b a b a b__________________________________________________________________________Asx 9.6 9 9.8 10 9.9 10 9.1 9 9.2 9 10 10 10 10 9.5 10Thr 3.8 4 3.7 4 4.7 5 4.5 5 4.6 5 3.9 4 3.8 4 3.7 4Ser 3.4 4 3.4 4 3.6 4 5.6 4 3.7 4 3.7 4 4 4 3.2 4Glx 12.2 12 13.3 12 13.2 13 13.9 13 13.2 13 13.6 13 13.7 13 13.1 13Pro 2.3 3 2 3 2.2 3 2.1 3 2.1 3 2.1 3 2.2 3 2.4 3Gly 9.3 9 9 9 9.6 9 10.1 9 9.1 9 9.4 9 9.4 9 9.1 9Cys/2 4 6 4.5 6 3.9 6 3.6 6 4.5 6 4.8 6 3.9 6 4.1 6Val 2.9 4 3.0 4 1.9 2 1.9 2 1.8 2 2.9 4 2.5 4 2.8 4Ile 1.8 2 1.8 2 1.7 2 2.3 2 1.8 2 1.9 2 1.7 2 1.9 2Leu 3.7 4 4.0 4 3.8 4 3.4 4 4 4 4.2 4 3.9 4 4 4Tyr 1.9 2 1.9 2 1.8 2 0.6 2 1.9 2 1.7 2 1.2 2 1.7 2Phe 0.9 1 0.9 1 1 1 0.9 1 0.9 1 1 1 1 1 1 1His 1 1 0.9 1 0.9 1 1.3 1 0.1 1 0.9 1 1 1 0.9 1Lys 2.8 4 2.9 3 3.1 3 2.5 3 2.8 3 3 3 2.9 3 2.7 3__________________________________________________________________________ a: found b: calculated
In Examples 2 to 9 which follow the analysis was carried out analogously to the methods indicated in Example 1. The corresponding data concerning the specific activity and the amino acid analyses are indicated in Tables 1 and 2.
EXAMPLE 2
Isohirudin I
Isohirudin I was isolated from the peak I (RT about 89 minutes) and purified by rechromatography. N-terminal sequence analysis made it possible to determine the structure up to 30 amino acids (Table 3). After oxidation and tryptic cleavage, the fragments T2 and T3 (C-terminal peptide) were isolated and the sequences elucidated. The results are shown in Table 4.
TABLE 3______________________________________Quantitative sequence analysis of isohirudin I______________________________________ 1 5 10 Amino acid V V Y T D C T E S G Q N pmol 107 90 76 48 30 nd 21 29 13 30 19 20 15 20 Amino acid L C L C E G S N V C G N* pmol 18 nd 6 nd 11 16 12 17 15 nd 12 13 25 30 35 Amino acid G N K C I L G S D G E K pmol 11 8 6 nd 5 5 40 45 Amino acid N Q C V T G E G T P K P pmol 50 55 60 Amino acid Q S H N D G D F E E I P pmol 65 Amino acid E E Y L Q pmolV V Y T D C T E S G Q N L C L C E G S N (1)V C G Q G N K C I L G S D G E K N Q C VT G E G T P K P Q S H N D G D F E E I PE E Y* L Q______________________________________ *amino acid exchange in comparison to (1) nd: not determinable Y* Sulfatotyrosine
TABLE 4__________________________________________________________________________Quantitative sequence analysis of the tryptic cleavageproducts of isohirudin I__________________________________________________________________________Peptide T2 28 30 35Amino acid C I L G S D G E KT2pmol nd 204 156 128 40 63 68 23 8Peptide T3 37 40 45Amino acid N Q C V T G E G T P K PT3pmol 159 178 nd 105 89 122 89 86 63 57 58 25 50 55 60Amino acid Q S H N D G D F E E I PT3pmol 36 12 6 12 9 10 11 7 7 3 4 5 65Amino acid E E Y L QT3pmol 4 4 1 1 3__________________________________________________________________________ nd: not determinable
EXAMPLE 3
Isohirudin Ia
Isohirudin Ia was isolated from the protein mixture which was obtained before the peak I (RT about 85 minutes) analogously to Example I. The product was purified by rechromatography. Subsequent N-terminal sequence analysis made it possible to determine the total structure (Table 5).
TABLE 5__________________________________________________________________________Quantitative sequence analysis of isohirudin Ia__________________________________________________________________________ 1 5 10Amino acid V V Y T D C T E S G Q Npmol 336 334 297 614 161 nd 339 114 241 185 196 181 15 20Amino acid L C L C E G S N V C G K*pmol 188 nd 173 nd 123 156 130 149 146 nd 132 118 25 30 35Amino acid G N K C I L G S D G E Kpmol 122 118 78 nd 71 84 71 53 69 76 60 69 40 45Amino acid N Q C V T G E G T P K Ppmol 60 61 nd 46 39 43 37 39 24 16 28 19 50 55 60Amino acid Q S H N D G D F E E I Ppmol 17 11 6 16 12 9 11 13 6 5 5 3 65Amino acid E E Y L Qpmol 4 4 3 3 2__________________________________________________________________________ *amino acid exchange in comparison to (1) nd: not determinable
EXAMPLE 4
Isohirudin IIa
Isohirudin IIa was isolated from the peak IIa (RT about 107 minutes) and purified by rechtomatography. The structure was elucidated by N-terminal sequence analysis (Table 6).
TABLE 6__________________________________________________________________________Quantitative sequence analysis of isohirudin IIa__________________________________________________________________________ 1 5 10Amino acid I* T* Y T D C T E S G Q Npmol 423 372 377 310 368 nd 392 274 295 261 287 271 15 20Amino acid L C L C E G S N V C G N*pmol 258 nd 241 nd 230 228 227 226 218 nd 223 210 25 30 35Amino acid G N K C K* L G S D G E E*pmol 191 149 200 nd 171 182 169 176 162 145 135 125 40 45Amino acid N Q C V T G E G T P K Ppmol 115 126 nd 107 105 96 93 92 79 72 71 62 50 55 60Amino acid Q S H N D G D F E E I Ppmol 60 49 55 52 50 41 43 47 30 31 30 16 65Amino acid E E Y L Qpmol 15 15 17 12 12__________________________________________________________________________ *amino acid exchange in comparison to (1) nd: not determinable
EXAMPLE 5
Isohirudins II and II'
Isohirudins II and II' were isolated as a mixture from fraction II (peak II; RT about 113 minutes) and purified by rechromatography analogously to Example 1. The sequence of the isohirudins up to amino acid 35 was elucidated by N-terminal sequence analysis. The C-terminal peptide (28-65) was isolated by oxidation and tryptic cleavage and the sequence was determined (Table 8). The total sequence analysis of the isohirudins II and II' is indicated in Table 7. Isohirudin II' was in this case characterized during the sequence analysis of the isohirudin mixture II and II', as already explained previously. Isohirudin II' differs from isohirudin II by the exchange of asparagine for asparaginic acid in position 20.
TABLE 7__________________________________________________________________________Quantitative sequence analysis of isohirudins II and II'__________________________________________________________________________ 1 5 10Amino acid I* T* Y T D C T E S G Q D*IIpmol 311 268 278 267 242 nd 169 175 165 144 165 163 15 20Amino acid L C L C E G S N V C G K*IIpmol 150 nd 132 nd 124 109 116 124 110 nd 111 110Amino acid D**II'pmol 4 25 30 35Amino acid G N K C I L G S N* G E E*IIpmol 98 89 74 nd 66 55 43 70 49 51 45 28 40 45Amino acid N C V T G E G T P K PIIpmol 20 22 nd 17 12 13 10 3 15 50 55 60Amino acid Q S H N D G D F E E I PIIpmol 65Amino acid E E Y L QIIpmol__________________________________________________________________________ *amino acid exchange in comparison to (1) **amino acid exchange in comparison to II nd: not determinable
TABLE 8__________________________________________________________________________Quantitative sequence analysis of the tryptic cleavageproducts of isohirudins II and II'__________________________________________________________________________ 28 30 35Amino acid C I L G S N* G E E* N Q Cpmol nd 266 242 250 223 217 184 178 178 169 145 nd 40 45 50Amino acid V T G E G T P K P Q S Hpmol 144 125 138 126 125 137 118 112 102 125 91 113 55 60Amino acid N D G D F E E I P E E Ypmol 117 100 106 97 99 83 79 80 68 59 63 41 65Amino acid L Qpmol 38 16__________________________________________________________________________ *amino acid exchange in comparison to (1) nd: not determinable
EXAMPLE 6
Isohirudin IIb
Isohirudin IIb was isolated from the protein mixture after peak II (RT about 115 minutes) and purified by rechromatography analogously to Example 1. The total sequence shown in Table 9 was determined by N-terminal sequence analysis. A trypsin cleavage was additionally carried out which led to the isolation of the peptide 28-65 (Table 10). The corresponding sequence was determined.
TABLE 9__________________________________________________________________________Quantitative sequence analysis of isohirudin IIb__________________________________________________________________________ 1 5 10Amino acid I* T* Y T D C T E S G Q Npmol 378 502 244 465 445 nd 377 400 396 303 291 281 15 20Amino acid L C L C E G S N V C G K*pmol 279 nd 269 nd 240 248 248 235 235 nd 259 231 25 30 35Amino acid G N K C I L G S N* G E E*pmol 215 230 219 nd 221 222 217 210 206 200 195 195 40 45Amino acid N Q C V T G E G T P K Ppmol 185 174 nd 163 156 145 132 140 129 112 105 91 50 55 60Amino acid Q S H N D G D F E E I Ppmol 86 80 72 71 65 54 51 42 46 33 24 26 65Amino acid E E Y L Qpmol 18 18 15 12 10__________________________________________________________________________ *amino acid exchange in comparison to (1)
TABLE 10__________________________________________________________________________Quantitative sequence analysis of the tryptic peptide ofisohirudin IIb__________________________________________________________________________ 28 30 35Amino acid C I L G S N* G E E* N Q Cpmol 300 379 381 368 347 337 332 316 305 293 271 nd 40 45 50Amino acid V T G E G T P K P Q S Hpmol 255 249 234 228 212 205 197 167 154 143 133 127 55 60Amino acid N D G D F E E I P E E Ypmol 112 102 115 100 91 81 78 64 54 32 22 18 65Amino acid L Qpmol 14 12__________________________________________________________________________ *amino acid exchange in comparison to (1) nd: not determinable
EXAMPLE 7
Isohirudins IIIa and IIIa'
Isohirudins IIIa and IIIa' were isolated from the peak having an RT of about 126 minutes and purified by rechromatography. N-terminal sequence analysis made it possible to determine the structure up to the carboxyl terminus (Table 11). Isohirudin IIIa' was characterized during the sequence analysis as already explained previously. Isohirudin IIIa' differs from isohirudin IIIa by exchange of glutamic acid for glutamine in position 11, asparaginic acid for asparagine in position 12, glycine for asparaginic acid in position 18 and asparaginic acid for asparagine in position 33.
TABLE 11__________________________________________________________________________Quantitative sequence analysis of the isohirudins IIIaand IIIa'__________________________________________________________________________ 1 5 10Amino acid V V Y T D C T E S G Q NIIIapmol 660 675 666 477 407 nd 321 458 238 390 269 282Amino acid E** D**IIIa'pmol 46 33 15 20Amino acid L C L C E D* S N V C G E*IIIapmol 318 nd 284 nd 269 247 239 227 218 nd 204 185Amino acid G** E*IIIa'pmol 11 25 30 35Amino acid G N K C I L G S N* G E KIIIapmol 178 169 161 nb 150 157 154 137 131 128 125 120Amino acid D**IIIa'pmol 4 40 45Amino acid N E* C V T G E G T P K PIIIapmol 88 84 nd 73 62 69 63 59 51 45 53 38 50 55 60Amino acid Q S H N D G D F E E I PIIIapmol 32 33 21 22 18 15 13 10 10 11 8 6 65Amino acid E E Y L QIIIapmol 6 5 5 4 3__________________________________________________________________________ *amino acid exchange in comparison to (1) **amino acid exchange in comparison to IIIa nd: not determinable
EXAMPLE 8
Isohirudin III
Isohirudin III was isolated from the peak having an RT of about 139 minutes and purified by rechromatography.
N-terminal sequence analysis in combination with sequence analysis of the tryptic cleavage products (peptide 28-65) made possible the structure determination of the complete protein (Tables 12 and 13).
Compared to all isohirudins described here, isohirudin III was isolated in a relatively large amount so that the protein-chemical structure proof of the mutation asparaginic acid to asparagine was additionally carried out with this substance.
Bornstein (Methods in Enzymol. 47 (1977) 132-145) described a selective cleavage of Asn-Gly-peptide bonds using the nucleophilic agent hydroxylamine. Accordingly, a buffer was initially prepared which contained 6M guanidinium hydrochloride and 2M hydroxylamine. To this end, 23 g of guanidinium hydrochloride and 5.5 g of hydroxylamine were combined in an icebath with some water and dissolved with vigorous stirring using 4.5M LiOH, the pH not exceeding 6.5. The pH was then adjusted to 9 and the solution was made up to 50 ml with water.
Isohirudin III (about 2 nmol) was lyophilized, taken up using 200 .mu.l of the buffer prepared and then incubated for 4 hours at 45.degree. C. in a waterbath. 200 .mu.l of glacial acetic acid (pH 3) was added to complete the reaction and the sample was frozen. The sample was then concentrated to 200 .mu.l and desalted on a reversed-phase column (4.6 mm.times.250 mm), packed with C-18 modified silica gel. The gradient in this case ran from 0% to 100% of B in 30 minutes with a flow of 1 ml/min (buffer A: 100% of water, 0.1% of TFA; buffer 8: 90% of ACCN, 10% of water, 0.1% of TFA). The resulting main fraction was then rechromatographed with the aid of microbore HPLC. A fraction F1 was isolated which contained two N terminals after characterization by sequence analysis (Table 14), namely Val Val Tyr and Gly Glu Lys.
Isohirudin III was held together even after hydroxyl cleavage by the cysteine linkage (Cys6-Cys14, Cys16-Cys28, Cys22-Cys39).
TABLE 12__________________________________________________________________________Quantitative sequence analysis of isohirudin III__________________________________________________________________________ 1 5 10Amino acid V V Y T D C T E S G Q NIIIpmol 615 569 468 377 265 nd 178 250 108 214 147 195 15 20Amino acid L C L C E D* S N V C G QIIIpmol 158 nd 83 nd 102 88 72 64 54 nd 50 48 25 30 35Amino acid G N K C I L G S N* G E KIIIpmol 32 37 29 nd 28 30 23 19 14 17 16 12 40 45Amino acid N Q C V T G E G T P K PIIIpmol 10 9 nd 5 3 5 6 4 2 1 1 3 50 55 60Amino acid Q S H N D G D F E E I PIIIpmol 1 0.5 1 1 5 1.5 0.3 0.8 2 2 1.5 1 65Amino acid E E Y L QIIIpmol 1 0.4 0.6 0.8 0.6__________________________________________________________________________ *amino acid exchange in comparison to (1) nd: not determinable
TABLE 13__________________________________________________________________________Quantitative sequence analysis of the tryptic peptidesof isohirudin III__________________________________________________________________________ 28 30 35Amino acid C I L G S N* G E K N Q Cpmol nd 301 295 286 234 273 246 242 219 194 139 nd 40 45 50Amino acid V T G E G T P K P Q S Hpmol 151 136 141 149 117 99 82 87 71 68 64 58 55 60Amino acid N D G D F E E I P E E Ypmol 51 41 36 30 25 24 27 21 12 16 15 12 65Amino acid L Qpmol 0.7 2__________________________________________________________________________ *amino acid exchange in comparison to (1) nd: not determinable
TABLE 14______________________________________Quantitative sequence analysis of the fraction F1 fromthe hydroxylamine cleavageDegradation step 1 2 3 4______________________________________Sequence position 1 2 3 4Amino acid: N-terminal V V Y Tpmol 17 17 24 22Sequence position 34 35 36 37Amino acid: cleavage site G E K Npmol 41 50 37 34______________________________________
The finding of two N terminals for F1 is clear proof for hydroxylamine cleavage in the sequence of isohirudin III on the peptide bond N-G
EXAMPLE 9
Isohirudins IIIb and IIIb'
Isohirudins IIIb and IIIb' were isolated analogously to Example 1 from the peak having an RT of about 134 minutes and purified by rechromatography. The structure of isohirudin IIIb to the carboxyl end was characterized by N-terminal sequence analysis (Table 15). The structure of isohirudin IIIb', which was likewise determined in the sequence analysis, is also shown in Table 15.
TABLE 15__________________________________________________________________________Quantitative sequence analysis of isohirudins IIIb and IIIb'__________________________________________________________________________ 1 5 10Amino acid V V Y T D C T E S G Q NIIIbpmol 530 511 499 433 460 nd 395 332 339 326 321 278 15 20Amino acid L C L C Q D* S N V C G QIIIbpmol 264 nd 258 nd 246 240 221 224 195 nd 185 175Amino acid G**IIIb'pmol 56 25 30 35Amino acid G N K C I L G S N* G E KIIIbpmol 168 154 154 nd 131 134 128 126 118 106 102 106 40 45Amino acid N Q C V T G E G T P K PIIIbpmol 106 95 nd 87 82 80 74 75 71 61 55 52 50 55 60Amino acid Q S H N D G D F E E I PIIIbpmol 48 12 36 31 30 24 21 23 18 16 18 12 65Amino acid E E Y L QIIIbpmol 11 11 8 5 3__________________________________________________________________________ *amino acid exchange in comparison to (1) **amino acid exchange in comparison to IIIb nd: not determinable

Number	Name	Date	Kind
4668662	Tripier	May 1987
4791100	Kramer et al.	Dec 1988

Number	Date	Country
4136485	Oct 1985	AUX
4365585	Dec 1985	AUX
4597785	Feb 1986	AUX
6023386	Jan 1987	AUX
6569386	Jun 1987	AUX
1828488	Aug 1989	AUX
7368091	Oct 1991	AUX
1239606	Jul 1988	CAX
0142860B1	May 1985	EPX
0142860	May 1985	EPX

Isohirudins

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