JP2009539853A - Antiplasmin cleaving enzyme substrates and inhibitors and uses thereof - Google Patents

Abstract

The present invention relates to inhibitors of antiplasmin cleaving enzymes for use in various therapies related to fibrin and α ₂ -antiplasmin, and to substrates of APCE that can be used, for example, in screening methods to identify such inhibitors. . The present invention relates to methods for treating or inhibiting atherosclerosis and thrombotic disorders by altering the ratio of the plasma α ₂ -antiplasmin form. In one embodiment, the present invention is plasma Met-alpha ₂ - changing the antiplasmin Ratio - pre-treatment levels of anti-plasmin and plasma Asn-alpha ₂ - plasma in subjects with pre-treatment levels of anti-plasmin alpha ₂ Provide a method.

Description

The present invention relates to, but is not limited to, substrates and inhibitors of α ₂ -antiplasmin cleaving enzyme, screening methods for identifying such inhibitors, and plaque and clot formation and atherosclerosis. Including methods for treating conditions involving fibrin and α ₂ -antiplasmin.

α ₂ -antiplasmin (α ₂ AP) is an enzyme that digests the blood clot, whether it initially appears as an intravascular platelet-fibrin deposit or as a partially or fully occlusive thrombus Is a plasma protein that inhibits rapidly and specifically. Similarly, the activities of plasmin and α ₂ AP are important for inflammation, wound healing, and the development and survival of fibrin that occurs in virtually all forms of cancer and its metastases.
alpha ₂ - human alpha _2, also known as plasmin inhibitor - antiplasmin (α ₂ AP) is the main inhibitor of plasmin. Plasmin plays a critical role in fibrin proteolysis and tissue remodeling. The physiological relevance of plasmin inhibition by α ₂ AP and homeostasis of blood clotting and fibrinolysis is supported by the following findings: (1) The rate of free plasmin inactivation by circulating α ₂ AP is Is much faster than fibrin (fibrinogen) digestion by, thus eliminating the systemic dissolution state and the possibility of resulting bleeding; (2) α ₂ AP is being formed by activated blood coagulation factor XIII (FXIIIa) Cross-linked to fibrin and inhibits plasmin-mediated lysis in direct proportion to the amount taken up; (3) Patients with homozygous α ₂ AP deficiency show severe bleeding tendency, but heterozygotes It tends to bleed only after major trauma or major surgery. Human α ₂ AP is first synthesized in the liver and secreted precursor Met-α ₂ -antiplasmin (Met), a 464 residue protein with methionine as the N-terminus while circulating in plasma. -Α ₂ AP) is proteolytically cleaved between Pro12 and Asn13 (P ₁ -P ₁ ′ scissile bond), and the Asn-α ₂ − in the form of 452 residues having asparagine as the N-terminus. Produces antiplasmin (Asn-α ₂ AP). Met-α ₂ AP accounts for approximately 30% of circulating α ₂ AP and Asn-α ₂ AP accounts for approximately 70%.

When Met-type α ₂ AP was found in plasma and the gene was sequenced, it initially seemed that there was a mismatch in one of the nucleotides encoding the sixth amino acid. Two groups found that cytidine (C) became Arg as the sixth amino acid, and one group found that thymidine (T) became Trp at that position. This difference was shown to be due to one group using liver cancer cells as a DNA source and the other two groups using normal cells. It has now been determined that both Arg6 and Trp6 Met-α ₂ AP are present in normal human plasma samples. Studies of mutant α ₂ APs in families with a tendency to bleed include mutations in this α ₂ AP gene that cause invalid α ₂ APs, and also this C / T single nucleotide polymorphism (SNP) 3 Identified two polymorphisms. This study examined 30 healthy blood donors and reported an allelic frequency of 0.81 / 0.19 for C / T SNP. In a study of a large healthy population, it was examined whether the frequency of homozygotes and heterozygotes, or whether the genotype could affect the ratio of Met-α ₂ AP to Asn-α ₂ AP in plasma. Not. Arg6Trp SNP is probably a silent polymorphism, but biochemical examination of two polymorphic forms of Met-α ₂ AP during production of its derivative form Asn-α ₂ AP, its incorporation into fibrin and The effect on fibrinolysis has not been evaluated prior to this study.

SUMMARY OF THE INVENTION The present invention is directed to inhibitors of antiplasmin cleaving enzymes for use in various therapies related to fibrin and α ₂ -antiplasmin, and is used, for example, in screening methods to identify such inhibitors. It is intended to be an APCE substrate. The present invention is further directed to methods of treating or suppressing atherosclerosis and thrombotic disorders by modifying, but not limited to, plasma alpha ₂ -antiplasmin form ratios and inhibiting APCE. .

Met-α ₂ AP by genotype as a percentage of total α ₂ AP. Α ₂ AP was purified from the plasma of each of the persons with RR (n = 15), RW (n = 15), and WW (n = 5) genotypes, and the amino terminal sequence was determined by Edman degradation. The ratio of Met-α ₂ AP was calculated from the picomole recovery of Met and Asn in the first cycle. Plasma APCE values sorted by genotype in healthy population. APCE values in plasma samples were determined by ELISA. Panel A is a histogram of APCE concentration in a healthy population (n = 109). Panel B shows APCE values according to Met-α ₂ AP genotype. APCE cleavage of the polymorphic form of Met-α ₂ AP. Equal amounts (40 μg) of purified Met-α ₂ AP (Arg6) and Met-α ₂ AP (Trp6) were digested with APCE. After stopping the reaction at the selected time, the samples were subjected to electrospray mass spectrometry to evaluate the amount of amino terminal 12 amino acid peptide produced from each Met-α ₂ AP form. Effect of Met-α ₂ AP genotype over time on plasma Asn-α ₂ AP production. Plasma samples from individuals with RR, RW, or WW genotypes were incubated at 29 ° C. and α ₂ AP was purified from each sample at selected times for amino-terminal sequence analysis. The ratio of Asn-α ₂ AP / Met-α ₂ AP was calculated from the picomolar recovery of Met and Asn in the first cycle. Plasma clot lysis time (PCLT) by Met-α ₂ AP genotype. PCLT was determined in plasma samples from RR, RW, and WW individuals. In panel A, the PCLT values are divided by genotype, and the mean ± S.D. E. M.M. As plotted. Panel B is the proportion of undissolved plasma (n = 14) compared to the proportion in the total population (n = 200) within each genotype. Effect of APCE removal over time on the conversion of Met-α ₂ -AP to Asn-α ₂ -AP. Plasma was collected from individuals with Met-α ₂ AP genotype RR and divided into 3 aliquots. One aliquot was mixed with APCE F19 mAb (F19) bound to chromatography beads and incubated at 4 ° C. to remove APCE. A second aliquot was incubated at 4 ° C. with non-specific rabbit anti-goat Ab (RAG) conjugated to beads. The third aliquot (RR) was not treated. After removal of the beads, each sample was incubated at 29 ° C. and the Asn-α ₂ -AP / Met-α ₂ -AP ratio was determined at selected times. In addition, F19-bound beads and RAG-bound beads were boiled with SDS to remove antibody-bound protein. Samples were electrophoresed on a 10% Bis-Tris SDS-PAGE gel and blotted onto nitrocellulose. APCE (97 kDa) was identified and visualized with a chemiluminescent substrate by Western blotting with goat Ab against its amino terminal region. FIG. 7 shows the inhibitory effect of Val-boroPro on the conversion of Met-α ₂ AP to Asn-α ₂ AP by APCE. FIG. 8 shows the sequence of the FRET peptide, which is a non-limiting example (SEQ ID NO: 5). _9, a variety of substitutions which may be made to the position 'P1 groups' P _2- P 1 -P1, residue and P1' residues downstream (C-terminal direction of _{P 2} residues upstream (N-terminal direction) ) And the various substitutions that can be made to the residues (SEQ ID NO: 6). P ₇ position to determine the substrate preference in _(P 1 pro 6 amino acid upstream), LC / MS analysis of antiplasmin peptides (SEQ ID NO: 7). Those with His (H), Lys (K), and Arg (R) with positive charge improved the cleavage rate of the Pro ₁₂ -N ₁₃ bond. Importance of Arg position for the scissile bond of Pro-Asn. Four similar peptides based on the antiplasmin amino terminal sequence (4-17) were digested by APCE followed by LC / MS analysis. Each reaction solution contains one experimental peptide and a control peptide, and any given peptide has an Arg at the P ₈ , P ₇ , P ₆ , P ₅ , or P ₄ (not shown) position and the other 3 It had glycine at two variable positions. Substitution at P ₄ led to results similar to P ₈ . LC / MS analysis of anti-plasmin peptides to determine substrate selectivity at P ₄ , P ₃ , P ₂ , P ₁ positions. A peptide library representing positions 6-17 of the human antiplasmin sequence was treated with APCE (also known as soluble FAP). Red phenylalanine was added to improve binding to the reverse phase HPLC column. A smaller amount of cysteine, which is an amino acid common to proteins, was substituted one by one at each of positions P _{1 to} P ₄ (SEQ ID NO: 8). The relative rate of Pro-Asn bond cleavage was determined by LC / MS analysis. FIG. 13 shows an alternative embodiment of the amino acid sequence (SEQ ID NO: 9) comprising a FRET peptide or inhibitor portion of the invention. This sequence can be extended in the C-terminal direction by, for example, the sequence shown in FIG. 9 extending from N ₁₃ to the N-terminal direction or from N ₁₃ to the C-terminal direction. FIG. 14 shows the APCE inhibitory effect of various inhibitory peptidomimetics (SEQ ID NOs: 11 to 13) with proline substituted with pipecolic acid at an inhibitor concentration of 20 μM.

DETAILED DESCRIPTION OF THE INVENTION Human Met-α ₂ AP is proteolytic cleavage between Pro12-Asn13 produces the more active derivative Asn-α ₂ AP, which is significantly faster than Met-α ₂ AP by FXIIIa. It is a physiologically important APCE substrate because it crosslinks to fibrin and consequently improves fibrin's resistance to digestion by plasmin. Human APCE potentiates the inhibition of fibrinolysis by increasing the availability of a more rapidly cross-linked form (ie Asn-α ₂ AP) and thus the potent and potent APCE found as described herein. Selective inhibitors allow titration of Asn-α ₂ AP production to lower in vivo values, thereby enhancing endogenous and exogenous fibrinolysis (fibrin removal). Based on the finding that the risk of bleeding in a heterozygous person with respect to α ₂ AP deficiency is negligible, it is unlikely to use the treatment described herein for hemorrhagic complications. Therefore, there is a safe dosage to reduce α ₂ AP function in healthy people. In particular, in clinical situations where fibrin formation is likely, APCE inhibitors will result in a decrease in the amount of Asn-α ₂ AP that can be used for cross-linking to fibrin as thrombus develops or inflammation progresses. Endogenous levels of plasmin produced, or plasmin produced by the administration of small amounts of plasminogen activator, can remove fibrin so that vascular patency and organ function are maintained and the risk of bleeding is minimized. It is considered sufficient to bring. As described above, APCE cleaves Metα ₂ AP into the derivative Asnα ₂ AP, which is more efficiently incorporated into fibrin, resulting in significant resistance to plasmin digestion. Thus, APCE is a pharmacological agent because less production of Asn-α ₂ AP, ie less uptake, leads to more rapid fibrin removal by plasmin during atherogenesis, thrombosis, and inflammation conditions. New targets for regulation and inhibition of the fibrinolytic system.

The present invention is directed to inhibitors of antiplasmin cleaving enzymes for use in various therapies associated with fibrin and α ₂ -antiplasmin and APCE substrates, for example screening methods for identifying such inhibitors Can be used. The present invention is further directed to methods of treating or inhibiting atherosclerosis and thrombotic disorders by altering the ratio of the plasma α ₂ -antiplasmin form, but is not limited thereto. In a preferred embodiment, inhibition of APCE is defined herein as an inhibition of at least 50% of APCE activity, for example at an inhibitor concentration of 20 μM.

Asn-α ₂ AP crosslinks to fibrin at a rate about 13 times faster than Met-α ₂ AP. The faster cross-linking rate allows a greater number of antiplasmin molecules to bind to newly formed fibrin, resulting in improved resistance to fibrinolysis. Inhibition of plasma APCE can therefore reduce the number of cross-linked antiplasmin molecules, thereby resulting in a clot that is more easily removed during fibrinolysis. Thus, fibrinolysis can be adjusted using inhibitors specific for APCE. As noted above, human Met-α ₂ AP exists in two polymorphic forms at position 6 of the mature sequence, with arginine predominating and tryptophan accounting for a smaller proportion. As used herein, inhibition of APCE results in an α ₂ AP ratio in plasma of approximately 30% Met-α ₂ AP: 70% Asn-α ₂ P, approximately 60% to 70% Met-α ₂ AP: 40%. It has been shown that it can be changed to ˜30% Asn-α ₂ AP.

Modulation of α ₂ -antiplasmin ratio In this study, we determined the number of polymorphic patients in a larger healthy population and then evaluated whether it was related to the inhibitory function of α ₂ AP. In the present specification, results relating to the following are shown: (1) Genotype frequency of Arg6Trp SNP in Met-α ₂ Ap; (2) How each form affects the sensitivity to cleavage by APCE; (3) Proportion of Met-α ₂ AP in plasma for each of the two polymorphisms; (4) Plasma clot lysis time associated with genotype; (5) Removal of circulating APCE is the Asn of Met-α ₂ AP evidence of preventing the conversion of-.alpha. ₂ AP.

Materials and Methods Materials Fresh frozen human plasma for protein purification was purchased from Sylvan Goldman Blood Institute (Oklahoma City, Oklahoma, USA). Hybridoma cells secreting F19 antibody were purchased from American Type Culture Collection (ATCC) (Manassas, VA, USA), grown in serum-free medium, and cultured using MEP-Hypercel chromatography (Pall, East Hills, NY, USA). F19 antibody was purified from the solution. These studies were approved by the Institutional Review Board (IRB) from the University of Oklahoma Health Sciences Center (IRB # 10142 and 12189).

using positive kringles le 1-3 attached to Sepharose 4B as isolation of affinity matrix alpha ₂ AP, the modification of purification steps is published, Met-α ₂ AP and Asn-α ₂ AP mixture single of Released. Met-α ₂ AP and Asn-α ₂ AP were separated by immunoaffinity chromatography as described above. The ratio of Met-α ₂ AP to Asn-α ₂ AP in plasma samples was determined by picomole recovery of Met vs. Asn at cycle 1 in automated protein sequencing by Edman degradation (Applied Biosystems Protype 492, Foster City, Calif., USA). Determined by rate comparison.

APCE Isolation APCE was purified from human plasma as previously described (US patent application Ser. No. 10 / 774,242). Briefly, a combination of ammonium sulfate precipitation, hydrophobic interaction, and immunoaffinity chromatography was used for purification. Before storage at −80 ° C., glycerol was added to pure APCE to a final concentration of 20%.

α ₂ AP genotype determination Self-reported to be healthy and free of acute disease to determine genotype frequency and plasma clot lysis time (PCLT; see next section) in a healthy population, optionally 200 healthy volunteers were recruited for blood donation. DNA was isolated from each donor's whole blood using the AquaPure Genomic DNA Blood Kit (Bio-Rad, Hercules, CA). Oligonucleotide primers (5'-GACCTCTCTCTCCATCCCCTTTT (SEQ ID NO: 1) and 5'-CTGGTTCGGCCCCCGTAGTAG (SEQ ID NO: 2)), dNTP (Takara Miras Bio, Madison, Wis., USA), and Platinum Taq Carlen, California, USA A portion containing DNA containing Arg6Trp SNP was amplified by polymerase chain reaction using. After amplification, PCR products were purified using MinElute PCR Purification Kit (Qiagen Operan, Alameda, Calif., USA) and sequenced using an ABI 3730 automated DNA sequencer.

Measurement of plasma clot lysis time To determine plasma clot lysis time (PCLT), each volunteer applied a mixture of 1 unit / ml thrombin, 16 mM CaCl ₂ , and 45 IU / ml urokinase (uPA) (Abbott, Chicago, IL, USA). Added to the person's plasma to essentially catalyze instant fibrin clot formation and initiate fibrinolysis. Plasma clot dissolution rate was determined by microtiter plate turbidimetry.

Determination of cleavage rate A reaction mixture containing the same amount (40 ug) of pure Met-α ₂ AP (Arg6) and pure Met-α ₂ AP (Trp6) was digested by APCE. At selected times, digestion was stopped by reducing the pH from 7.5 to 4.0 with trifluoroacetic acid. Protein was removed from the mixture by Microcon (Millipore, Bedford, Mass.) Centrifugal ultrafiltration using a 30 kDa cut-off membrane. Isolation of peptides from the ultrafiltered digestion mixture by binding to POROS-50 reversed phase media (Applied Biosystems, Foster City, Calif., USA) loaded into a glass purification capillary (Proxeon, Odense, Denmark) did. The peptide was then eluted from POROS-50 directly into a metal-coated glass nanospray capillary containing 2.0 μl of 0.5% acetic acid in methanol / water 1: 1. Nanospray capillary is included in the nanospray ionization source of the QSTAR ESI-Quad-TOF mass spectrometer operated at 1400 volts under Analyst QS software version 1.0 (Applied Biosystems, Foster City, CA) did. Data was collected over a mass range of 300-1500 Da. The amino terminal 12 amino acid peptides of the two α ₂ APs produced, MEPLGRQLTSGP (SEQ ID NO: 3), Met-α ₂ AP (Arg6) Mr = 1284.65, MEPLGWQLTSGP (SEQ ID NO: 4), Met-α The relative amount of ₂ AP (Trp6) with Mr = 1314.63 was determined by summing the areas of the four most abundant isotope peaks of the observed charge form of each peptide. Data for each time point was normalized by similar quantification of an inactive internal standard peptide that did not contain added proline.

Determination of APCE antigen level Enzyme-linked immunosorbent assay (ELISA) was developed to determine the antigen level in human plasma. A multi-antigenic peptide (MAP) constructed in our laboratory to contain eight copies of the amino terminal peptide linked to the seven lysine core peptides via the carboxyl terminus was used as an immunogen and the amino acid of APCE A goat antibody against the terminal 15 amino acid sequence was prepared. The goat MAP (amino terminal 15 residue APCE peptide) antibody was bound to a white high binding polystyrene assay plate (Corning, Corning, NY, USA) and used as a capture antibody. After incubation with plasma dilutions, a monoclonal antibody purified from a commercially available F19 hybridoma (ATCC, Manassas, VA, USA) was applied, followed by a peroxidase-conjugated anti-mouse antibody (Sigma, St. Louis, MO, USA). A chemiluminescent substrate, SuperSignal ELISA Pico (Pierce, Rockford, Ill., USA) was added and luminescence was monitored using a BIO-TEK FL600 plate reader (Winooski, Vermont, USA). Antigen values were quantified using purified human APCE as a standard substance.

APCE removal from plasma F19 mAb was linked to POROS EP 20 poly (styrenedivinylbenzene) perfusion chromatography beads (Applied Biosystems, Foster City, Calif., USA), and a non-specific antibody rabbit anti-goat was linked to the same medium. It was. Plasma from a single donor of RR genotype was divided into 3 aliquots and diluted 1: 1 with PBS and separately at 4 ° C. with each of the two non-specific Ab or F19 mAb two bead-linked antibodies. Incubated overnight. The third aliquot was not treated. The beads were removed from the plasma by filtration and then the plasma was incubated at 29 ° C. Aliquots were removed at zero, 24, and 48 hours. Α ₂ AP was purified from each aliquot and the Asn-α ₂ AP / Met-α ₂ AP ratio was determined as described above. After removal from plasma, F19 mAb beads were washed with 25 mM Na PO ₄ /0.5 M NaCl and then boiled with SDS running buffer. The SDS buffer extract was then separated by electrophoresis on a 10% Bis-Tris gel (Invitrogen, Carlsbad, CA) and blotted onto nitrocellulose. As previously described in this method section, APCE was identified by Western blotting using goat amino-terminal MAP antibody, and SuperSignal West Femto Maximum Sensitivity Chemiluminescent Substrate (Pierce Biotechnology, USA) was used. And visualized.

Results α ₂ AP genotyping A group of 201 healthy volunteers, recruited in two groups of approximately 100 each about 2 years apart, provide blood samples to determine C / T SNP frequency of α ₂ AP did. The entire population consists of 61 males and 139 females aged 21-69 years old, and ethnicity is exactly the same as the Oklahoma population demographics described for 2000 on the US Census website (factfinder.census.gov) Fits. Genotypes were determined for 200 subjects. Only one DNA sample was not amplified by the polymerase chain reaction, which appears to be due to a mutant that prevented binding of one of the primers, but this was not further investigated. The genotype frequencies of the two healthy populations were essentially similar, with differences of less than 1% for both genotypes. After mixing the two sets, the frequency of the entire population was RR 62.5%, RW 34.0%, WW 3.5%. The frequency of the R allele was 79.5% and the frequency of the W allele was 20.5%. There was no difference in genotype frequency between men and women. Since 72% of the population was Caucasian and the remaining 28% was divided into 6 ethnic categories, it was impossible to determine if the genotypes differed between ethnic groups.

Plasma Met-α ₂ AP and Asn-α ₂ AP levels α ₂ AP was purified from plasma of 15 RR genotypes, 15 RW genotypes, and 5 WW genotypes. α ₂ AP was sequenced by Edman degradation and picomolar recovery of Met and Asn was determined for the first cycle to calculate the ratio of Met-α ₂ AP to Asn-α ₂ AP. FIG. 1 shows genotypes divided by WW with the highest proportion of Met-α ₂ AP (56.4%), lowest RR (23.6%), and intermediate RW (40.6%). Shows significantly different (p <0.001) results.

In order to understand the mechanism of variation in the proportion of Met-α ₂ AP in human plasma, we determined that the variation in the value of the enzyme that converts Met-α ₂ AP into Asn-α ₂ AP, ie APCE, was genotyped. It was examined whether the difference between them could be explained. To study this possibility, we developed an ELISA method to quantify APCE antigen levels in plasma, and then determined APCE concentrations in plasma from 109 subjects in the healthy population of this study. As shown in FIG. 2a, the distribution of APCE values in normal human plasma ranges from 38 to 159 ng / ml, which is not correlated with age or gender. Moreover, there is a possibility that the APCE value is related to the genotype (RR: 70.4 ± 26; RW: 66.6 ± 24.5; WW: 64.7 ± 10.1). The difference is not statistically significant (Figure 2b). Thus, APCE values do not appear to account for variations in Met-α ₂ AP values between genotypes.

Another explanation for the proportion of Met-α ₂ AP that differs in the three genotypes examined is that Met-α ₂ AP (Arg6) is a better substrate for APCE than Met-α ₂ AP (Trp6). there were. To test this hypothesis, Met-α ₂ AP (Arg6) was used to purify α ₂ AP from RR and WW plasma and to use mass spectrometry to monitor the production of 12-residue amino-terminal peptides over time. And the cleavage rate of each polymorphic form of Met-α ₂ AP (Trp6) was determined. As shown in FIG. 3, the reaction rate comparison was based on linear regression analysis at an early time point, and APCE cleaved Met-α ₂ AP (Arg6) approximately 8 times faster than Met-α ₂ AP (Trp6). Showed that to do.

Based on the presence of R or W in position 6, genotype determined to be RR, RW, and WW to investigate whether the cleavage rate of Met-α ₂ AP in whole plasma shows similar results Fresh plasma samples were obtained from the individuals who were incubated and incubated at 29 ° C., and the respective Asn-α ₂ AP / Met-α ₂ AP ratios were analyzed after 0, 24, and 48 hours. α ₂ AP was purified from each sample and the ratio of the two polymorphic forms was determined by picomolar recovery of the amino terminal residue in the first cycle of Edman degradation. As shown in FIG. 4, Asn-α ₂ AP increased in plasma from healthy volunteers with RR genotype at a much faster rate than either RW or WW genotype patients. These results show that APCE cleaves Met-α ₂ AP (Arg6) approximately 8 times faster than Met-α ₂ AP (Trp6), the results from a reaction mixture of pure α ₂ AP and APCE Clearly matches.

Plasma clot lysis Plasma clot lysis time (PCLT) was determined in blood samples from 200 women and men, including a healthy population. We intend to control the variability of clot lysis known to occur by activator release, activator-inhibitor interactions, fibrinogen levels, or by lipid effects on enzyme-substrate mechanisms. There wasn't. Therefore, gender, age, adrenergic status, smoking, obesity, alcohol intake, serum lipid and fibrinogen measurements, medication, daytime activity, etc. were ignored as qualifications for volunteer participation in this study. Instead, we believe that the lysis time of the study participants should effectively reflect the “average” of all physiological / pharmacological effects within each genotype at any point in time, and in fact the polymorphism We enrolled “all applicants” as long as they self-reported their health as we thought their potential importance would be emphasized if they would detectably affect plasma clot lysis under our conditions. It should be emphasized that the average PCLT for men was significantly longer than the value for women (p = 0.0002). As shown in FIG. 5, the mean PCLT for the RR, RW, and WW genotype groups showed a marked linear decrease, but the mean PCLT difference between the three genotypes was not statistically significant . As shown in FIG. 5 panel A, after genotyping by gender, the difference in mean PCLT between genotypes is a clear tendency for WW genotypes to have shorter dissolution times in both men and women. It has been shown. Due to the asymmetric distribution of PCLT, data was analyzed using non-parametric statistical methods. The median values of RR and RW are similar, the distribution is overlapping, indicating that the R allele is dominant. After revealing gender variation, when the RR and RW groups were combined and compared to WW, the difference reached almost significant (p = 0.061). As shown in panel B of FIG. 5, 12 of the RR genotypes (10%) and 2 of the RW genotypes (3%) remained completely intact over the entire 1 hour assay time. None of the WW genotypes had a plasma clot with a lysis time> 2100 seconds.

APCE Removal from Plasma In an attempt to clearly demonstrate that APCE is an enzyme involved in the conversion of Met-α ₂ AP to Asn-α ₂ AP, we have developed FAP covalently bound to POROS chromatography beads. APCE was removed from plasma by incubating the plasma with F19, a specific monoclonal antibody. After removal of the beads, incubation of plasma at 29 ° C. was continued for a selected time to convert Met-α ₂ AP to Asn-α ₂ AP. The graph in FIG. 6 shows the Asn-α ₂ AP / Met-α ₂ AP ratio in control plasma not incubated with POROS-conjugated antibody, which is 6.12 after incubation from 2.62 to 48 hours. Rose to. In the second control, ie plasma incubated with non-specific antibodies conjugated to POROS chromatography beads, there was a significant increase in the Asn-α ₂ AP / Met-α ₂ AP ratio from 2.54 to 5.2, Occurs after 48 hours. However, plasma incubated with F19 antibody did not show an increase in the ratio from 2.4 to 2.17 over the same incubation period, thereby indicating removal of APCE by F19 antibody. These results clearly demonstrate that removal of APCE from plasma prevents Met-α ₂ AP from converting to Asn-α ₂ AP, a shorter and faster fibrin cross-linked form. In further support of this conclusion, we also demonstrated by Western blot analysis that it was indeed APCE that was bound to and removed from the bead-linked F19 mAb. The Western blot analysis shown in FIG. 6 shows a 97 kDa band of APCE monomer in the sample eluted from the F19 linked beads, but such a band is on the rabbit anti-goat (RAG) Ab linked beads. Did not exist. The other visible band was the result of non-specific binding of secondary Ab to the immunoglobulin in the sample leaching from the beads during extraction by boiling in SDS.

In this study, the two populations of healthy volunteers analyzed at different time periods were essentially the same, with genotype frequencies collected of RR 62.5%, RW 34.0%, and WW 3.5%. It was. The allele frequency values of 0.795 / 0.205 are consistent with the only other study we are aware of, but this is the study of Lind and Thorsen, in a single group of 30 healthy blood donors. Nucleotide mutation values of 0.81 / 0.19 are reported. Since this polymorphism occurs in a 12-residue amino-terminal peptide that is removed from the longer precursor form of α ₂ AP, and positively charged hydrophilic arginine (R) is a hydrophobic amino acid tryptophan (W). Given the substitution, we questioned whether such differences could affect the rate of peptide cleavage by APCE and subsequent incorporation into fibrin during formation. We have found that pure APCE cleaves WW genotype pure Met-α ₂ AP about 8 times slower when compared to pure Met-α ₂ AP from plasma of individuals with RR. Figure 4 is a naturally occurring precursor Met-α ₂ AP / derivative Asn-alpha ₂ AP ratio in plasma samples containing each of the two polymorphic forms of Met-alpha ₂ AP is at 29 ° C. The freshly collected plasma It clearly shows that it changes with time spontaneously when incubated. This cleavage, as shown in FIG. 6, resulted in natural APCE plasma values, since removal of APCE with a specific monoclonal antibody completely interrupted the production of the derivative Asn-α ₂ AP during the same incubation period. Must be attributed. Since this cleavage occurs spontaneously in the circulating blood, the ratio of precursor Met-α ₂ AP / derivative Asn-α ₂ AP should be different in circulating plasma from the three genotypes, In fact, we demonstrated by quantitative amino-terminal analysis of the 35 precursor / derivative α ₂ AP forms analyzed. As expected, individuals with RR genotype had the least amount of circulating Met-α ₂ AP (23.6%), RW was intermediate (40.6%), and WW was the highest (56.4%). The results of this study cannot be explained by the variation in APCE values because the antigen values are not significantly different among the three genotypes as shown in FIG. 2b.

The relationship between RR, RW, and WW genotypes, and the corresponding Met-α ₂ AP / Asn-α ₂ AP ratio, affects the individual's fibrinolytic activity, thereby producing in life vascular Increased the vulnerability of endogenous fibrin to endogenous fibrinolysis and hence the possibility that its survival was differentially affected by the Met-α ₂ AP genotype. As a result, individuals with the WW genotype will have Met-α ₂ AP that is difficult to convert to Asn-α ₂ AP by cleavage by APCE, and thus are less efficiently incorporated into the forming fibrin and are thereby generated Makes any fibrin easier to digest with plasmin. To demonstrate this, experiments were performed with whole plasma to approximate natural conditions as much as possible. As stated in the previous section, if the fibrinolysis time of the WW genotype group is actually short, in many cases, this situation is likely to be established, and all samples are collected from healthy volunteers. Only minimal effort was made to standardize the conditions. Most perturbation factors known to affect fibrinolysis time either acutely or chronically act throughout life, but regardless of such effects, we, on average, show the state of fibrinolysis. Was assumed to be separated by Met-α ₂ AP genotype. It should be noted that in all our analyses, the RR genotype person had the longest average PCLT, the RW group was intermediate, and the WW genotype was the shortest, which is the WW person Indicates that it has a fibrinolytic system that is chronically more active than the RW group and is certainly more active than individuals with the RR genotype. The RR genotype contains a higher percentage of people who do not dissolve fibrin than expected and is given a PCLT value that is 1 second above the maximum measured value for any person in this study, for men the average lysis time Association with RR and RW became significant at the p <0.05 level. The results of this study show that the W allele is a “protective factor” (well-known) by increasing its sensitivity to plasmin removal of growing intravascular thrombi throughout life, thereby reducing the risk of atherosclerosis. It has been shown that it can function as a term (in contrast to the term “risk factor”).

Furthermore, these results demonstrate the utility of increasing the Met-α ₂ AP / Asn-α ₂ AP ratio closer to that that accelerates fibrinolysis. As illustrated by the heterozygous deficiency of α ₂ AP action and sustained chronic levels of endogenous lytic activity, the action of α ₂ AP, essentially the only plasmin in vivo inhibitor, is By reducing to a level with little risk, the survival and involvement of intravascular fibrin / platelet thrombus in the atherosclerosis process can be reduced. In another study, plasma samples were collected from patients with metastatic colon cancer who were experimentally treated with oral Val-boroPro (Talabost), which we demonstrated to be a non-specific inhibitor of APCE. FIG. 7 shows that the APCE inhibitor Val-boroPro increased the proportion of Met-α ₂ AP in plasma and decreased the conversion of Met-α ₂ AP to Asn-α ₂ AP by APCE. ing.

Fibrin is the key to platelet stabilization when platelets adhere and aggregate at the site of arterial wall injury in the earliest stages of plaque development. Fibrin continues to be deposited as oxidized lipids and macrophages infiltrate the site of injury, and plaques grow and gradually invade the lumen diameter, with the risk of rupture and acute occlusive thrombus formation. In all of these steps, if the alpha ₂ plasmin content of fibrin, the higher or maximum vulnerability of fibrin to removal by endogenous fibrinolytic system, fibrin contains lesser amounts of alpha ₂ AP inhibitor It is lower than the case. A higher circulating precursor α ₂ AP value (Met-α ₂ AP) reduces the total α ₂ AP that crosslinks to fibrin, thereby more easily digesting fibrin by the fibrinolytic enzyme plasmin, Removed from plaque site. However, fibrin is more resistant to removal by fibrinolysis when its derivative Asn-α ₂ AP is dominant. By inhibiting APCE, the blood concentration of Met-α ₂ AP is increased and any fibrin that forms is more easily removed by its own fibrinolytic system.

In a fluorescence resonance energy transfer peptide substrate present invention one embodiment, P-N - has the scissile bond (proline asparagine) _(P 1 -P1 '), G to _{P 2} position _{P 1} proline upstream (glycine) A fluorescent resonance energy transfer peptide (FRET peptide) is contemplated. The FRET peptide contains a quenching group such as DABCYL on one side of the P ₁ P ₁ ′ bond and a fluorophore group such as EDANS on the opposite side of this fragile bond. Preferably, the FRET peptide has a maximum of 13 amino acid residues upstream of P ₁ proline and a maximum of 13 residues downstream of the P ₁ 'asparagine residue (ie, the entire peptide has a maximum of 28 Including amino acids). The amino acid having a quenching group (eg, lysine) may be _one of up to 13 amino acids upstream or downstream of the P ₁ -P ₁ ′ group, and the amino acid having a fluorophore group (eg, glutamic acid) is P ₁ -P _{1 '1} Tsude good of upstream or downstream of up to 13 amino acid groups. Preferably, the quenching group and the fluorophore group are at remote ends of the FRET peptide, and preferably at least one end of the peptide terminates with an arginine residue (or other positively charged amino acid) or at one end It may have an amino acid having a positive charge and an amino acid having a negative charge at the other end.

  The term “heterocycle” or “heterocyclic” refers to a cyclic structure, but is preferably a 4, 5, 6, or 7-membered cyclic structure, more preferably the cyclic structure is nitrogen, sulfur, or oxygen A 5- to 6-membered ring containing 1 to 4 heteroatoms. Heterocyclic groups include, but are not limited to, thiophene, furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, and pyridazine.

Heterocyclic rings are in one or more positions, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amide, phosphonic acid, phosphinic acid, carbonyl, carboxyl , Silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclyl moiety, aromatic moiety, or heterocyclic aromatic moiety, CF ₃ , CN and the like.

  As used herein, the term “carbocycle” or “carbocyclic” refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon, preferably a 4, 5, 6, or 7 carbocycle. And more preferably 5 and 6 carbocycles.

  Proline analogs and derivatives of the peptidomimetic compounds of the invention may exist in certain geometric or stereoisomeric forms. The present invention includes all cis and trans isomers, R and S enantiomers, diastereomers, (D) -isomers, (L) -isomers, their racemic mixtures, and other mixtures thereof Such compounds are intended to be included within the scope of the present invention. Additional asymmetric carbon atoms may be present in a substituent such as an alkyl group. All such isomers, and mixtures thereof, are intended to be included in the present invention.

FIG. 8 shows an example of a FRET peptide (SEQ ID NO: 5) having a natural amino acid sequence around the P ₁ -P ₁ ′ bond of Arg6 form of Met-α ₂ -AP. In this example, the overall preferred maximum sequence of the FRET peptide used to mimic the substrate properties of the N-terminal peptide of Met-α ₂ -AP is shown. The rationale for including up to 13 residues on either side of the cleavage bond is that this level of symmetry is due to the steric nature when the enzyme and substrate bind to cleave the P ₁₂ -N ₁₃ (P ₁ -P ₁ ′) bond. This is because it contains a similar number of amino acid residues on either side of the P ₁ -P ₁ ′ bond to minimize potential problems with configuration. In this embodiment, any arginine group at each end improves the solubility of the peptide derivative upon cleavage of the -P-N- bond. G ₁₁ and P ₁₂ (P ₂ -P ₁ ) are preferred amino acids that cannot be substituted by other natural amino acids when the peptide is intended to be a cleavable substrate. Proline can be substituted with another proline derivative or analog or a proline-like amino acid in a peptide intended for use as an inhibitor. P ₁₂ -N ₁₃ -is a bond that is easily broken. As will be described, it is possible to replace the P _12, to form the inhibitor. The fluorescent group and the quenching group of the FRET peptide are each bonded to an amino acid, which in this embodiment is E or K near the end. M ₁ is the N-terminal methionine residue of the native sequence of the precursor α ₂ AP.

  FIG. 9 shows an alternative FRET peptide sequence (SEQ ID NO: 6) with possible permutations of substitution amino acids allowed at specific positions of the peptide comprising amino acids 8-18 of the peptide of FIG.

In a preferred embodiment of the FRET peptide, the quenching group and the fluorophore group each bind to an amino acid residue on either side of the scissile bond, preferably within 1.0-5.0 nm of the scissile bond. ing. In another preferred embodiment, the quenching group and the fluorophore group are each attached to a residue either upstream of the P ₂ glycine residue or downstream of the P ₁ ′ (asparagine or appropriately substituted amino acid) residue. And the quencher group is on one side of the P ₂ or P ₁ ′ residue and the fluorophore group is on the opposite side of the P ₂ or P ₁ ′ residue.

  In order to investigate how the structure of the antiplasmin cleavage site and its vicinity (amino acid substitution) affects the cleavage of antiplasmin by APCE, experiments were performed using peptide substrates.

Using the amino acid sequence of Met-α ₂ AP around the P ₁ -P ₁ ′ bond cleaved by anti-plasmin cleaving enzyme (Pro-Asn) as a guide, the same synthetic peptides of various lengths were prepared and cleaved Assuming that the rate is proportional to binding, critical residues were determined to enhance peptide cleavage. Proline at position ₁ (or carbocyclic or heterocyclic proline analog or substitution) is a residue necessary for binding to APCE, and P adjacent to the upstream of the proline or proline substitution or analog residue. _{The same} applies to glycine at the _2nd position. Considering a series of substitutions at various residues upstream from P ₁ proline, we have P ₇ (ie, the 6th residue upstream of proline) or an amino acid with a positive charge at position P ₅ or P ₆ (eg, , Arginine, lysine, or histidine) was found to be particularly advantageous in maximizing binding to APCE, ie, the rate of cleavage (FIGS. 10 and 11). The designations presented herein for P ₅ , P ₆ , P ₇ refer to residues that increase in number from the pro-residue of a scissile bond in the N-terminal direction. For example, Pro is _{P 1,} the subscript number increases in the upstream direction.

FIG. 10 shows that upstream arginine, or any positively charged amino acid, is important for maximizing the rate of cleavage of the P ₁ -P ₁ ′ bond by APCE in the peptide having SEQ ID NO: 7. ing. FIG. 11 shows that by replacing the arg of P ₄ , P ₅ , P ₆ , and P ₈ , P ₅ and P ₆ are at least as effective as the natural P ₇ arg, with P ₆ being the most effective It is shown that it was determined. P ₄ and P ₈ is significantly inferior, it did not promote cleavage of scissile bonds. In addition to the presence of positive charges at P ₅ , P ₆ , or P ₇ that have an accelerating effect on the breakage of fragile bonds, there is also a requirement for spacing to maximize the effect of positive charges.

In P ₇ position, arg was optimal amino acid with lys other relative cutting speed of about 5-10 times faster than amino acid except (arg about 70% of the rate of) (Figure 10). Arginine _{P 6} or _{P 5-position (i.e.,} P 1 -P ₁ '4 or 5 residues upstream binding) enhancement of (6-th residues upstream from _{P 1} proline) _{P 7} arginine at position when in Although an effect was also observed (FIG. 11), the cleavage rate decreased when arg was in the P ₈ , P ₄ , P ₃ , or P ₂ position of the P ₁ -P ₁ ′ bond (FIGS. 11 and 12). FIG. 12 shows, for example, the effect of substituting each of 19 amino acids in each of amino acid sequences P _{1 to} P ₄ in the peptide (SEQ ID NO: 8), and the P ₁ -P ₁ ′ bond cleavage of the peptide by APCE. Shows the effect on the reaction rate. The positively charged residues lys and his can replace arg at positions P ₇ , P ₆ , and P ₅ (see FIG. 10 for P ₇ ), the cleavage rate of P ₁ -P ₁ ′ Shows that the effect on phenotype is substantially dependent on positive charge (however, pro, phe, tyr, trp, and some other amino acids have partial levels of activity (FIG. 10)) .

In an alternative embodiment, the FRET peptide of the invention comprises the peptide sequence shown in FIG. 13 (SEQ ID NO: 9). Position of the X ₁ to X ₅ and _{X 8 may} comprise an amino acid selected from the group of amino acids shown below each X 1 to X ₅ and _{X 8,} provided that at least one of _X 1 to X ₃ One is selected from R, H, and K (a positively charged amino acid). Furthermore, at least one of X ₁ , X ₂ and X ₃ may be absent. As mentioned above, the FRET peptide (or any peptide or peptidomimetic) comprising SEQ ID NO: 9 preferably comprises no more than 28 amino acids.

  The FRET peptide comprising SEQ ID NO: 9 preferably comprises a quenching group and a fluorophore on opposite sides of the gly-pro bond. SEQ ID NO: 9 may further comprise a 1-lOmer peptide in the N-terminal direction upstream of the N-terminal amino acid, which may include any of the 20 natural amino acids and downstream of the C-terminal amino acid It may further comprise a 1-lOmer peptide in the C-terminal direction, which may include any of the 20 natural amino acids.

As described above, fluorescence resonance energy transfer peptide substrate, P 1 _-P 1 _'upstream to the quenching group of the coupling, P 1 _{-P 1'} may include a fluorophore downstream of the coupling, or fluorescence resonance energy transfer peptide May comprise a quencher group downstream of the P ₁ -P ₁ ′ bond and a fluorophore upstream of the P ₁ -P ₁ ′ bond. P ₁ is preferably proline or an effective proline analog or substitution, as described herein, P ₁ ′ is preferably asparagine, and the P ₂ group upstream of P ₁ is preferably glycine.

Screening for APCE Inhibitors In certain embodiments, the present invention includes methods for screening for inhibitors of anti-plasmin cleaving enzymes. The _method 'includes a _{bond, P 1 -P 1' P 1} -P 1 comprises fluorophore group are separated by bonds (e.g., EDANS) and quenching groups (e.g., DABCYL), whereby alpha ₂ - antiplasmin Providing a fluorescent resonance energy transfer peptide contemplated herein, capable of cleaving the peptide with a cleaving enzyme, providing an amount of α ₂ -antiplasmin cleaving enzyme, and α ₂ -antiplasmin Exposing the cleaving enzyme to an α ₂ -antiplasmin cleaving enzyme inhibitor candidate to form a test mixture, combining the test mixture with a fluorescence resonance energy transfer peptide, and the α ₂ -antiplasmin cleaving enzyme inhibitor candidate α _2- measuring fluorescence emission from the test mixture to determine when to inhibit the activity of the antiplasmin cleaving enzyme; including.

APCE Inhibitors As described herein, various sequence variations of peptide sequences around the FRET peptide and the scissile bond of α ₂ AP have been used herein to generate inhibitors. Specifically, substitution of proteolytically bound proline with proline analogs led to the generation of specific APCE inhibitors described herein. Accordingly, the present invention relates to proline analogs for use as P ₁ proline substitutes, including proline analogs and derivatives having a maximum of 28 or more amino acids, including but not limited to those shown in Table I. APCE-inhibitory peptidomimetics, including derivatives, are contemplated. The peptidomimetics are preferably 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 amino acids, combined or conjugated to extend the serum life of the peptide, such as spacer compounds to separate some amino acids of the compound, or PEG or other polymeric materials or carriers Contains no compounds.

Preferably, P ₁ -P ₁ ′ remaining positively charged at a distance (3.5 to 24 mm, ie 0.35 to 2.4 nm) corresponding to the length of 4 to 7 residues upstream of the cleavable bond APCE inhibitors with groups can be used as effective APCE inhibitors that bind rapidly and strongly. Furthermore, such inhibitors are useful for the inhibition of fibroblast activating protein (FAP), a similar enzyme, ie for the treatment of disorders related to FAP, as described for example in US Pat. No. 6,949,514. Can be.

The distance from the scissile bond of P ₁ -P ₁ ′ to the positive charge at any position of P ₅ , P ₆ , or P ₇ is also a related determinant and is therefore an approximate length (0.35 nm-2 Any spacer built up with any amino acid or inert or neutral material that fills this interval to reach .4 nm) would be effective in building APCE / FAP inhibitors. In a particularly advantageous embodiment of the FRET peptide or APCE inhibitors, arginine (or amino acids with other positive charge) from the scissile bond of the proline (or _{the P 1} residue substitutions) 6~21Å (0.6nm~ 2.1 nm).

A preferred variant of the inhibitor comprises a sequence having 2 to 6 amino acids in the N-terminal direction from the P ₁ -P ₁ ′ bond, including a positively charged amino acid (eg, arginine, lysine, or histidine), and Preferably, but not necessarily, P ₁ -P ₁ ′ contains a pro-asn linkage, preferably P ₂ contains glycine and has a negative charge downstream (C-terminal direction) from the P ₁ -P ₁ ′ bond. Includes amino acids or aromatic amino acids (eg, asp, glu, trp, tyr, and phe).

In a preferred embodiment of the inhibitor or FRET peptide of the present invention, the compound may contain a spacer (linker or filler) group between the scissile bond and the N-terminal positively charged amino acid, eg arg, his Or a positively charged amino acid such as lys is in a position corresponding to P ₇ , P ₆ , or P ₅ (see, eg, FIG. 14). The spacer (linker or filler) between the scissile linking group and the positively charged amino acid at the P ₅ , P ₆ , or P ₇ position is, for example, a neutral amino acid that does not have multiple charges (eg, glycine , Alanine, leucine, isoleucine, valine, proline, methionine, tryptophan, tyrosine, threonine, serine, and most preferably serine, glycine, alanine, β-alanine, γ-aminobutyric acid, epsilon aminocaproic acid; or 8-, 11 -, Or amino-PEG derivatives such as 14-amino-3,6,9,12-tetraoxatetradecanoic acid, and PEG oligomers (eg n = 2-6)). These spacers may be homologous (eg, all glycines, alanines, etc., or other single amino acids) or heterogeneous (eg, one or more amino acids or hybrid amino acids / PEG molecules). Preferably, the length is 3.5 to 21 mm (0.35 nm to 2.1 nm) (or 1 to 7 amino acids). The spacer may be composed of a neutral monomer such as ethylene glycol, or other similar monomer unit (for example, propylene glycol), and has a length of 3.5 to 21 mm (0.35 nm to 2.1 nm). The arginine (or other positively charged amino acid) is placed within about 5 to 25 mm (0.5 nm to 2.5 nm) of the proline, or proline substitution product or analog with a spacer that can be easily broken.

Thus, the presence of positively charged amino acids within the distance defined herein is an aspect of the invention that contributes significantly to the specificity of binding to the APCE sequence. Furthermore, when amino acids in this space are substituted with polyethylene glycol of length 3.5-21 mm (0.35 nm-2.1 nm), the cleavage rate of the P12-N13 bond is still maximal for the following peptides: X—R ₆ — (PEG ₃ ) —G ₁₁ —P ₁₂ —N ₁₃ —Q ₁₄ —E ₁₅ (SEQ ID NO: 10) ((PEG ₃ ) represents polyethylene glycol containing three ethylene glycol units; X is any amino acid). Considering the specificity of the positive charge effect that must be within a certain distance from P12 (P ₁ ), this pegylated peptide selectively selects proline (P12) to inhibit the proteinase activity of APCE. This is the basis for the generation of inhibitors by modification or substitution. FIG. 14 shows the APCE inhibitory action of several peptides (SEQ ID NOs: 11-13) having the indicated sequence when Prol2 is replaced with the proline analog pipecolic acid (each peptide is 20 μM).

In preferred embodiments, the present invention includes compounds having the formula: Xaa ₁ -Sp ₁ -Gly-Cyc-Xaa ₂ -Sp ₂ -Xaa ₃ (Formula I). In this embodiment, Xaa ₁ is a positively charged amino acid including but not limited to arginine, histidine, or lysine. Sp ₁ is preferably serine, glycine, alanine, or β-alanine, or gamma aminobutyric acid, epsilon aminocaproic acid, 8-amino-3,6,9,12-tetraoxatetradecanoic acid, 11-amino-3,6 , 9,12-tetraoxatetradecanoic acid, 14-amino-3,6,9,12-tetraoxatetradecanoic acid, or neutral containing PEG _n or PPG _n (n = 1-6 ethylene glycol or propylene glycol units) It is a spacer molecule containing 1 to 6 amino acids, which is an amino acid, and Sp ₁ has a length of 3 to 21 (0.3 to 2.1 nm). Gly is glycine. Cyc is a carbocyclic or heterocyclic ring. A carbocyclic ring may contain 4, 5, 6, or 7 carbon atoms, and a heterocyclic ring is an atom 4, 5, for example where at least one atom is a heteroatom such as nitrogen. Six or seven may be included. Xaa ₂ is preferably any natural amino acid that can serve as a scissile bond for glutamine, asparagine, serine, histidine, tyrosine, alanine, or phenylalanine, or P ₁ -P ₁ ′ of the compounds of the present invention. . Sp ₂ is a spacer molecule and may be absent, preferably serine, glycine, alanine, β-alanine, aspartic acid, glutamic acid, or gamma aminobutyric acid, epsilon aminocaproic acid, 8-amino-3,6,9 , 12-tetraoxatetradecanoic acid, ll-amino-3,6,9,12-tetraoxatetradecanoic acid, 14-amino-3,6,9,12-tetraoxatetradecanoic acid, or PEG _n or PPG _n (n = 1-6 ethylene glycol or propylene glycol units), which may contain 1 to 6 amino acids, Sp ₁ having a length of 3 to 21 Å (0.3 nm to 2.1 nm). Xaa ₃ is glutamic acid, aspartic acid, tryptophan, tyrosine, or phenylalanine, or any negatively charged amino acid or aromatic amino acid. The compound may further comprise an N-terminal oligopeptide having 1 to 10 amino acids extending from Xaa _{1 in the} N-terminal direction and a C-terminal oligopeptide having 1 to 10 amino acids extending from Xaa _{3 in the} C-terminal direction; The N-terminal oligopeptide and C-terminal oligopeptide may comprise one or more of the 20 natural amino acids. The compound is preferably placed in a pharmaceutically acceptable carrier as described elsewhere herein.

In one embodiment, the present invention is plasma Met-alpha ₂ - changing the antiplasmin ratio - plasma alpha ₂ in subjects with pre-treatment levels of anti-plasmin - pre-treatment levels of anti-plasmin and plasma Asn-alpha ₂ Is the method. Specifically, the method includes treating a subject with an inhibitor of an antiplasmin cleaving enzyme, wherein the inhibitor cleaves the Pro-Asn cleavage site of Met-α ₂ -antiplasmin by an antiplasmin cleaving enzyme. Specifically inhibit and after treatment the subject has a post-treatment level of plasma Met-α ₂ -antiplasmin that is at least 5% higher than the pre-treatment level of plasma Met-α ₂ -antiplasmin, and plasma Asn -.alpha. ₂ - at least 5% than the level before treatment antiplasmin low plasma Asn-alpha ₂ - has a level after the treatment of the anti-plasmin, whereby plasma alpha ₂ in the subject - to change the antiplasmin ratio. In the present method, changing the α ₂ -antiplasmin ratio in the subject preferably improves fibrinolysis in the subject. Specifically, the method is a treatment for inhibiting or treating atherosclerosis, arterial thrombosis, venous thrombosis, stroke, or pulmonary embolism.

In one embodiment of the present method, plasma Met-alpha ₂ - levels after treatment of anti-plasmin, plasma Met-alpha ₂ - at least 10% higher than pre-treatment levels of anti-plasmin, plasma Asn-α ₂ - Anti The post-treatment level of plasmin is at least 10% lower than the pre-treatment level of plasma Asn-α ₂ -antiplasmin. In another embodiment of the method, plasma Met-alpha ₂ - levels after treatment of anti-plasmin, plasma Met-alpha ₂ - at least 15% higher than pre-treatment levels of anti-plasmin, plasma Asn-α ₂ - The post-treatment level of antiplasmin is at least 15% lower than the pretreatment level of plasma Asn-α ₂ -antiplasmin. In another embodiment of the method, plasma Met-alpha ₂ - levels after treatment of anti-plasmin, plasma Met-alpha ₂ - at least 20% higher than the level before treatment antiplasmin, plasma Asn-α ₂ - The post-treatment level of antiplasmin is at least 20% lower than the pretreatment level of plasma Asn-α ₂ -antiplasmin. In another embodiment of the method, plasma Met-alpha ₂ - levels after treatment of anti-plasmin, plasma Met-alpha ₂ - at least 25% higher than pre-treatment levels of anti-plasmin (Also, for example, any ratio higher), plasma Asn-α ₂ - levels after treatment of anti-plasmin, plasma Asn-α ₂ - at least 25% lower than pre-treatment levels of anti-plasmin (also, for example, lower any proportion).

In one embodiment, the present invention is preferably a plasma Met-alpha ₂ - levels and plasma pretreatment antiplasmin Asn-alpha ₂ - in subjects with pre-treatment levels of anti-plasmin, alpha in the subject ₂ - Anti A method of treating or suppressing atherosclerosis, arterial thrombosis, venous thrombosis, stroke, or pulmonary embolism by altering plasmin levels. The method includes treating a subject with an inhibitor of an antiplasmin cleaving enzyme, wherein the inhibitor specifically inhibits the cleavage of the Pro-Asn cleavage site of Met-α ₂ -antiplasmin by an antiplasmin cleaving enzyme. After treatment, the subject is at least 5%, 10%, 15%, 20%, or 25% higher (or any percentage higher) plasma Met-α ₂ than the pre-treatment level of plasma Met-α ₂ -antiplasmin. Have post-treatment levels of antiplasmin and at least 5%, 10%, 15%, 20%, or 25% lower (or any percentage) than the pre-treatment levels of Asn-α ₂ -antiplasmin, respectively (Lower) Asn-α ₂ -antiplasmin level after treatment, thereby altering the plasma α ₂ -antiplasmin ratio in the subject. The change in the α ₂ -antiplasmin ratio in the subject preferably occurs by improving fibrinolysis in the subject.

  The present invention further contemplates therapeutic compounds for treating cancer and disorders related to FAP, such as those described in US Pat. No. 6,949,514, incorporated herein by reference. The therapeutic compound includes an APCE inhibitor of the present invention described herein conjugated to a carrier compound that can cross the cell membrane, including but not limited to a protein transduction domain (PTD). A PTD is a positively charged peptide or peptide-like molecule that passes through the cell membrane lipid bilayer. Typically, a PTD contains several arginine residues and delivers another substance, such as a peptide, protein, oligonucleotide, or small molecule, through the cell membrane into the cytosol. Some PTDs are very efficient molecular transporters that replace εACA formed by synthesizing arginine oligomers. Examples of PTDs that can be used in the present invention include US Pat. Nos. 7,166,692; 7,217,539; 7,053,200, the entire disclosure of which is incorporated herein by reference. No. 6,835,810; 6,645,501; and US Patent Application Publication Nos. 2002/0009491; 2003/0032593; 2003/0162719; 2006/0159719; 2006/0293234. And 2007/0105775.

Utility The utility of the present invention includes, but is not limited to:
(1) Prevention and reduction of the development and progression of atherosclerotic plaques, especially in high-risk patients.
(2) Prevention and reduction of the occurrence of arterial or venous blood clot formation (atherothrombosis and venous thrombosis disorders), especially in conditions where the risk of such clots is perceived to be high (ie, heart attack or stroke).
(3) Improvement of vascular patency maintenance by continued administration of an inhibitor of APCE, optionally associated with co-administration of a low dose of plasminogen activator.
(4) Prevention and reduction of fibrin formation that can develop inflammatory conditions such as all forms of arthritis, organ fibrosis, unwanted scars, and persistent acute or chronic symptoms associated with cancer and its metastases.
(5) The risk of bleeding when an excess fibrinolytic state is induced on the premise that α ₂ AP _Pro inhibits plasmin in the solution state as well as α ₂ AP _act when it is not crosslinked in fibrin. Reduction.
(6) interfere with atherothrombotic diseases such as coronary artery thrombosis, stroke, pulmonary embolism, all other forms of arterial and venous thrombosis, inflammatory conditions, and organ function that may be seen in cancer and its metastases Assist in the prevention and treatment of fibrin deposition.
(7) Determination of whether the subject has a Trp6 or Arg6 polymorphism in α ₂ -antiplasmin.
(8) Screening for inhibitors of antiplasmin cleaving enzyme.

Accordingly, various embodiments of the invention include, but are not limited to, the following.
(1) A therapeutic method for promoting fibrin digestion in vivo, comprising a step of administering an effective amount of an antiplasmin cleaving enzyme inhibitor to a subject.
(2) A fluorescence resonance energy transfer (FRET) peptide that functions as a substrate for APCE.
(3) An inhibitor of antiplasmin cleaving enzyme, wherein the inhibitor may comprise or be linked to a polymer spacer, linker, or carrier.
(4) anti-plasmin cleavage enzyme alpha ₂ - antiplasmin binding site or alpha _2, - the antiplasmin pro-asn cleavage site inhibitor effective antiplasmin-cleaving enzyme to cut off to, or their binding.
(5) including a step of simultaneously or sequentially providing a certain amount of a plasminogen activator and an anti-plasmin cleaving enzyme inhibitor to a subject in need of clot digestion or clot prevention. A method of improving fibrin digestion in vivo that is less than that provided by standard treatment protocols without cleaving enzyme inhibitors.
(6) DNA encoding the FRET peptide or inhibitor peptide contemplated herein, and a plasmid vector or host containing the DNA.

  Another embodiment of the present invention comprises one or more of the compounds described herein in a therapeutically effective amount, and one or more pharmaceutically acceptable carriers (additives) and / or diluents. Provided is a pharmaceutically acceptable composition formulated together. As detailed below, the pharmaceutical compositions of the present invention can be specially formulated for administration in the solid or liquid state, including those adapted to: (1) For example, Oral administration of aqueous or non-aqueous solutions or suspensions, tablets for oral, sublingual, systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) For example, parenteral administration, eg, as a sterile solution or suspension, or as a sustained release formulation, eg, subcutaneous, intramuscular, intravenous, or epidural injection; (3) a cream applied to the skin, Topical administration as an ointment, or a controlled release patch or spray; (4) vaginal or rectal administration, for example as a cream or foam; (5) sublingual administration; (6) intraocular administration; (7) transdermal Administration; or (8 Nasal administration.

  As used herein, the phrase “therapeutically effective amount” refers to an animal in which the amount of the compound, material, or composition comprising a compound of the invention is a reasonable effect / risk ratio that can be applied to any medical procedure. Means effective in producing a desired therapeutic effect in at least a subpopulation of cells.

  As used herein, the phrase “pharmaceutically acceptable” is within the scope of normal medical judgment and is reasonable without undue toxicity, irritation, allergic reactions, or other problems or complications. Refers to compounds, materials, compositions, and / or dosage forms suitable for use in contact with human and animal tissues, to meet an effective effect / risk ratio.

  As used herein, the phrase “pharmaceutically acceptable carrier” refers to a liquid or solid involved in transporting or transporting a subject compound from one organ or body part to another organ or body part. Means a pharmaceutically acceptable substance, composition or vehicle, such as a filler, diluent, excipient, or solvent encapsulating material. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of substances that can serve as pharmaceutically acceptable carriers include (1) sugars such as lactose, glucose, and sucrose; (2) starches such as corn starch and potato starch; (3) Cellulose and its derivatives, sodium carboxymethylcellulose, ethylcellulose, and cellulose acetate, etc .; (4) tragacanth powder; (5) malt; (6) gelatin; (7) talc; (8) cocoa butter and suppository wax, etc. (9) Oils such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols such as propylene glycol; (11) glycerin, sorbitol, mannitol, and Polyols such as polyethylene glycol; ( 2) Esters such as ethyl oleate and ethyl laurate (13) Agar; (14) Buffering agents such as magnesium hydroxide and aluminum hydroxide; (15) Alginic acid; (16) Pyrogen-free water; (17) etc. (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) others used in pharmaceutical formulations Non-toxic compatible substances.

  As described above, some embodiments of the compounds include a basic functional group such as amino or alkylamino, thus forming a pharmaceutically acceptable salt with a pharmaceutically acceptable acid. can do. In this regard, the term “pharmaceutically acceptable salts” refers to the relatively non-toxic, inorganic and organic acid addition salts of the compounds of the present invention. These salts react separately with a suitable organic or inorganic acid in its free base form, either in situ in the administration vehicle or in the dosage form manufacturing process, or during subsequent purification, thus in its free base form. It can be prepared by isolating the salt formed. Typical salts include hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, lauric acid Salt, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate, mesylate, glucoheptonate, lactobionic acid Salt, and lauryl sulfonate.

  Pharmaceutically acceptable salts of the subject compounds include the conventional non-toxic salts or quaternary ammonium salts of the compounds, eg, from non-toxic organic or inorganic acids. For example, such conventional non-toxic salts include those derived from inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, sulfamic acid, phosphoric acid, nitric acid, and acetic acid, propionic acid, succinic acid, glycolic acid , Stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, palmitic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluene There are salts prepared from organic acids such as sulfonic acid, methanesulfonic acid, ethanedisulfonic acid, oxalic acid, and isothionic acid.

  In other cases, the compounds of the present invention may contain one or more acidic functional groups and thus can form pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term “pharmaceutically acceptable salts” in these examples refers to the relatively non-toxic inorganic or organic base addition salts of the compounds of the invention. These salts may likewise be used in situ in the administration vehicle or dosage form manufacturing process or in purified form of the purified compound in its free acid form, as a pharmaceutically acceptable metal cation hydroxide salt, carbonate or bicarbonate. It can be prepared by reacting separately with a suitable base such as a salt and ammonia or a pharmaceutically acceptable organic primary, secondary or tertiary amine. Exemplary alkali or alkaline earth salts include lithium, sodium, potassium, calcium, magnesium, and aluminum salts. Representative organic amines useful for the formation of base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.

  Wetting agents such as sodium lauryl sulfate and magnesium stearate, emulsifiers and lubricants, as well as colorants, mold release agents, coating agents, sweeteners, flavors and fragrances, preservatives, and antioxidants are also present in the composition. May be.

  Examples of pharmaceutically acceptable antioxidants include (1) water-soluble antioxidants such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite; (2) ascorbyl palmitate, butyl Oil-soluble antioxidants such as hydroxanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol; and (3) citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, There are metal chelating agents such as phosphoric acid.

  As mentioned above, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal, and / or parenteral administration. These formulations may be conveniently provided in a unit dosage form and may be prepared by any method well known in the pharmaceutical art. The amount of active ingredient that can be combined with the carrier material to produce a single dosage form will vary depending upon the host treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound that produces a therapeutic effect. Generally, in 100 percent, this amount is the active ingredient ranging from about 1% to about 99%, preferably from about 5% to about 70%, and most preferably from about 10% to about 30%. .

  In some embodiments, the formulations of the present invention are excipients selected from the group consisting of cyclodextrins, liposomes, micelle forming agents such as bile acids, and polymeric carriers such as polyesters and polyanhydrides. including.

  The methods of preparing these formulations or compositions may include the step of bringing the compound of the invention into association with a carrier and optionally with one or more accessory ingredients. In general, formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

  Formulations of the present invention suitable for oral administration are capsules, cachets, pills, tablets, lozenges (flavored bases, usually containing each of the compounds of the present invention as an active ingredient) Sucrose and acacia or tragacanth), in the form of powders, granules, or as a solution or suspension in an aqueous or non-aqueous liquid, or as a water-in-oil or oil-in-water liquid emulsion, or an elixir or It may be as a syrup or as a pasty tablet (using gelatin and glycerin or inert bases such as sucrose and acacia) and / or as a mouthwash. The compounds of the present invention can also be administered as boluses or pastes.

  In solid dosage forms for oral administration of the present invention (capsules, tablets, pills, powders, granules, etc.), the active ingredient is one or more pharmaceutically acceptable, such as sodium citrate or dicalcium phosphate. And / or mixed with any of the following: (1) fillers or extenders such as starch, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) for example, carboxymethylcellulose, Binding agents such as alginate, gelatin, polyvinylpyrrolidone, sucrose, and / or acacia; (3) humectants such as glycerol; (4) agar, calcium carbonate, potato or tapioca starch, alginic acid, some silicates, And disintegrants such as sodium carbonate; (5) dissolution retardants such as paraffin; (6 Absorption promoters such as quaternary ammonium compounds; (7) wetting agents such as, for example, cetyl alcohol, glycerol monostearate, and nonionic surfactants; (8) absorbents such as kaolin and bentonite clay; (9) Lubricants such as talc, calcium stearate, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, and mixtures thereof; and (10) colorants. In the case of capsules, tablets and pills, the pharmaceutical composition may also comprise a buffer. Similar types of solid compositions can also be used as fillers in soft and hard shell gelatin capsules with excipients such as lactose or lactose and high molecular weight polyethylene glycols.

  A tablet may be made by compression or molding, optionally with one or more accessory ingredients. Compressed tablets use binders (eg, gelatin or hydroxypropylmethylcellulose), lubricants, inert diluents, preservatives, disintegrants (eg, sodium starch glycolate or cross-linked sodium carboxymethylcellulose), surfactants or dispersants. Can be prepared. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.

  Dragees, capsules, pills, granules and the like, which are tablets and other solid dosage forms of the pharmaceutical composition of the present invention, are optionally coated with enteric coatings and other coatings well known in the pharmaceutical formulation field. It can also be divided or prepared by the shell. They also provide sustained or controlled release of the active ingredient they contain, for example using various proportions of hydroxypropylmethylcellulose, other polymer matrices, liposomes, and / or microspheres to provide the desired release characteristics. It can also be formulated to provide. They may be formulated for rapid release, for example lyophilized. They can be sterilized, for example, by filtration through a bacteria-retaining filter, or by incorporating a sterilant in the form of a sterile solid composition that can be dissolved in sterile water or some other sterile injectable medium immediately before use. . Also, these compositions may optionally contain opacifiers and are compositions that release the active ingredient only in certain parts of the gastrointestinal tract or preferentially there in some cases. It may be. Examples of embedding compositions that can be used are polymeric substances and waxes. The active ingredient can also be in microencapsulated form, if appropriate, with one or more of the above-described excipients.

  Liquid dosage forms for oral administration of the compounds of the invention include pharmaceutically acceptable emulsions, microemulsions, solutions, suspensions, syrups, and elixirs. In addition to the active ingredient, the liquid dosage form may be an inert diluent commonly used in the art, such as water or other solvents, ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate , Propylene glycol, 1,3-butylene glycol, oils (specifically, cottonseed oil, peanut oil, corn oil, germ oil, olive oil, castor oil and sesame oil), glycerol, tetrahydrofuryl alcohol, polyethylene glycol, and sorbitan Solubilizing and emulsifying agents such as fatty acid esters, and mixtures thereof may be included.

  In addition to inert diluents, oral compositions may contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preservatives.

  Suspensions, in addition to active compounds, are suspensions of, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar and tragacanth, and mixtures thereof An agent may be contained.

  Formulations of the pharmaceutical composition of the present invention for rectal or vaginal administration may be suppositories, which are one or more suitable non-residues including, for example, cocoa butter, polyethylene glycol, suppository waxes or salicylic acid. Can be prepared by mixing one or more compounds of the invention in an irritating excipient or carrier, and melts in the rectum or vaginal cavity because it is solid at room temperature but liquid at body temperature; The active compound will be released.

  Formulations of the present invention suitable for vaginal administration also include tampons, creams, gels, pastes, foams or spray formulations containing carriers known to be suitable in the art.

  Dosage forms for topical or transdermal administration of the compounds of this invention include powders, sprays, ointments, pastes, creams, lotions, gels, solutions, patches, and inhalants. The active compound can be mixed under sterile conditions with a pharmaceutically acceptable carrier, and with any preservatives, buffers, or propellants that may be required.

  Ointments, pastes, creams and gels include animal and vegetable fats, oils, waxes, paraffins, starches, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites in addition to the active compounds of the present invention. And excipients such as silicic acid, talc, and zinc oxide, or mixtures thereof.

  Powders and sprays may contain excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicate, and polyamide powder, or mixtures of these substances, in addition to the compounds of the present invention. The spray may further contain conventional propellants such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons such as butane and propane.

  Transdermal patches have the additional advantage of allowing controlled delivery of the compounds of the invention to the body. Such dosage forms can be made by dissolving or dispersing the compound in the proper medium. Absorption enhancers can be used to increase the flux of the compound across the skin. Such flow rate can be controlled by providing a rate limiting membrane or by dispersing the compound in a polymer matrix or gel.

  Eye drops, ophthalmic ointments, powders, solutions, and the like are also within the scope of the present invention.

  Pharmaceutical compositions of the present invention suitable for parenteral administration comprise one or more compounds of the present invention as one or more pharmaceutically acceptable sterile isotonic aqueous or non-aqueous solutions, dispersions, suspensions. Or emulsion, or a sterile powder that can be reconstituted into a sterile injectable solution or dispersion just prior to use, which is intended for sugars, alcohols, antioxidants, buffers, bacteriostats, formulations Solutes that are isotonic with the recipient's blood, or suspending or thickening agents may be included.

  Suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical compositions of the present invention include water, ethanol, polyols (glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils such as olive oil, and Injectable organic esters such as ethyl oleate. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.

  These compositions can also contain adjuvants such as preservatives, wetting agents, emulsifying agents, and dispersing agents. The action of microorganisms on the target compound can be reliably prevented by including various antibacterial and antifungal agents such as parabens, chlorobutanol, and phenol sorbic acid. It may also be desirable to include isotonic agents such as sugars, sodium chloride in the composition. In addition, sustained absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin.

  In some instances, it may be desirable to delay the absorption of the drug from subcutaneous or intramuscular injections in order to sustain the effect of the drug. This can be achieved by using a liquid suspension of crystalline or amorphous material with low water solubility. The drug absorption rate depends on its dissolution rate, which can depend on crystal size and crystal shape. Alternatively, delayed absorption of a parenterally administered dosage form is also achieved by dissolving or suspending the drug in an oil medium.

  Injectable sustained release forms are made by forming microencapsule matrices of the subject compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of drug to polymer, and the nature of the particular polymer used, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Long-acting injectable formulations can also be prepared by entrapping the drug in liposomes or microemulsions that are compatible with body tissues.

  When the compound of the present invention is administered to humans and animals as a pharmaceutical, it is pharmaceutically acceptable by itself or, for example, 0.1 to 99.5% (more preferably 0.5 to 90%) of the active ingredient. It can be administered as a pharmaceutical composition comprising a carrier.

  The formulations of the present invention can be administered orally, parenterally, topically, or rectally. Needless to say, they are administered in a form suitable for each route of administration. For example, they are administered in tablet or capsule form and by injection, inhalation, eye drops, ointment, suppository, etc. (injection, infusion, administration by inhalation; topical administration by lotion or ointment; and rectal administration by suppository). Oral administration is preferred.

  As used herein, the phrases "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, including Intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, epidermal, intraarticular, subcapsular, subarachnoid, intraspinal, and This includes, but is not limited to, intrasternal injection and infusion.

  As used herein, the phrases “systemic administration”, “administered systemically”, “peripheral administration”, and “administered peripherally” refer to the patient's condition without entering the central nervous system directly. By administration of a compound, drug, or other substance that enters the system and thereby undergoes metabolism or other similar processes, such as subcutaneous administration.

  These compounds can be administered therapeutically to humans and other animals by any suitable route of administration, including oral, eg, nasal, rectal, intravaginal, parenteral, large, by spray. For example, in the bath, and topical administration including, for example, in the mouth and sublingually with powders, ointments, or drops.

  Regardless of the route of administration selected, the compounds of the present invention and / or the pharmaceutical compositions of the present invention that can be used in a suitable hydrate form are pharmaceutically acceptable by conventional methods known to those skilled in the art. Blended into the dosage form.

  The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention is such that for each individual patient, composition, and mode of administration, an amount of the active ingredient effective to achieve the desired therapeutic response is obtained. Furthermore, it can be changed so as not to be toxic to the patient.

  The selected dosage level depends on the activity of the particular compound of the invention used, or its ester, salt, or amide, route of administration, administration time, excretion or metabolic rate of the individual compound used, duration of treatment Other drugs, compounds and / or substances used in combination with the individual compounds used, the age, sex, weight, condition, overall health, and previous medical history of the patient being treated, as well known in the medical field Will depend on a variety of factors, including

  A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, a physician or veterinarian can start a dose of a compound of the invention used in a pharmaceutical composition at a level lower than that required to achieve the desired therapeutic effect, and achieve the desired effect. The dosage may be gradually increased until done.

  In general, a suitable daily dose of a compound of the invention will be the lowest dose of compound that is effective to produce a therapeutic effect. Such an effective dose will generally depend on the factors discussed above. In general, intravenous, intraventricular, and subcutaneous doses of a compound of the invention to a patient will be about 0.0001 to about 100 mg per kilogram body weight per day when used for the required analgesic effect. It becomes the range.

  If desired, an effective daily dose of the active compound may be administered in unit dosage forms, optionally in 2, 3, 4, 5, 6 or more sub-doses that are administered separately at appropriate intervals throughout the day. May be administered.

  While it is possible for a compound of the present invention to be administered alone, it is preferable to administer the compound as a pharmaceutical formulation (composition).

  In another aspect, the invention includes a therapeutically effective amount of one or more subject compounds formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents as described above. A pharmaceutically acceptable composition is provided. As detailed below, the pharmaceutical compositions of the present invention can be specially formulated for administration in the solid or liquid state, including those adapted to: (1) For example, oral administration of aqueous or non-aqueous solutions or suspensions, tablets, boluses, powders, granules, pastes for application on the tongue; (2) eg, subcutaneous, intramuscular, or intravenous injection (3) topical administration, for example as a cream, ointment, or spray applied to the skin, lungs, or mouth; (4), for example, pessaries, creams, Or vaginal or rectal administration as foam; (5) sublingual administration; (6) intraocular administration; (7) transdermal administration; or (8) nasal administration.

  The compounds according to the invention can be formulated for administration in any convenient manner for use in human or veterinary medicine, by analogy with other pharmaceutical products.

  The term “treatment” is also intended to encompass prophylaxis, therapy, and cure.

  Patients receiving this treatment are any animal in need, including primates (especially humans) and other mammals such as horses, cows, pigs, and sheep, and general poultry and pets. .

  The compounds of the present invention can be administered by themselves or as a mixture with a pharmaceutically acceptable carrier, and in combination with antibacterial agents such as penicillins, cephalosporins, aminoglycosides, and glycopeptides. You can also Combination treatment includes sequential administration, simultaneous administration and separate administration of the active compounds in such a way that the therapeutic effect of the first dose is not completely abolished when the next dose is administered.

  As used herein, the term “subject” or “patient” preferably refers to a warm-blooded animal, such as a mammal, having a particular inflammatory condition. It will be understood that guinea pigs, dogs, cats, rats, mice, horses, cows, sheep, and humans are examples of animals within the meaning of the term.

  The therapeutically effective amount of a compound used in the treatments described herein can be determined by the attending diagnostician as one of ordinary skill in the art using conventional techniques and observing results obtained under similar circumstances. Can be easily determined. A number of factors are considered by the attending diagnostician in determining the therapeutically effective dose. These factors include: mammalian species; its size, age, and overall health; the specific disease or condition involved; the extent or involvement or severity of the disease or condition; the response of the individual subject; Individual compounds; modes of administration; characteristics of bioavailability of the administered formulation; selected usage; use of concomitant drugs; and other related situations.

  A therapeutically effective amount of a composition of the invention is generally sufficient to deliver about 0.1 μg / kg to about 100 mg / kg (weight of active ingredient / patient body weight) (ie APCE or an inhibitor thereof). ) Is almost contained. The composition will preferably deliver at least 0.5 μg / kg to 50 mg / kg, more preferably at least 1 μg / kg to 10 mg / kg.

  The practice of the method of the invention includes administering to the subject a therapeutically effective amount of the active ingredient in any suitable systemic or topical formulation in an amount effective to deliver the above dosage. It is. This dosage can be administered once, or (for example) 1 to 5 times a day, or once or twice a week, or continuously by intravenous infusion or other means, depending on the desired therapeutic effect. .

  As noted, the preferred amount and mode of administration can be determined by one skilled in the art. Those skilled in the art of formulation preparation will be able to determine the selected compounds using formulation techniques known in the art, for example, as described in the latest issue of “Remington's Pharmaceutical Sciences” (Mack Publishing Co.). Appropriate modes and modes of administration can be readily selected depending on the individual characteristics, the condition being treated, the stage of the disease, and other relevant circumstances.

  The pharmaceutical composition can be manufactured using techniques known in the art. Typically, a therapeutically effective amount of the compound will be admixed with a pharmaceutically acceptable carrier.

  The half-life of the molecules described herein is extended by joining them to a molecule, such as another polymer, to form a drug-polymer complex using methods known in the art. be able to. For example, the molecule can be conjugated to an inert polymer molecule known in the art, such as a polyethylene glycol (PEG) molecule in a method known as “pegylation”. PEGylation can consequently extend the in vivo lifetime of the molecule, ie the therapeutic effectiveness.

  PEG molecules are described, for example, in Harris et al. , “Pegylation, A Novel Process for Modifying Pharmacakonetics”, Clin Pharmacokitet, 2001: 40 (7); 539-551, which can be modified by a functional group, or present at the amino terminus of the molecule. If present, cysteine residues or other linking amino acids contained therein can be linked, and the PEG molecule can have a plurality of one or more types of molecules.

  “Polyethylene glycol” or “PEG” involves derivatization of a coupling agent, or coupling or activation moiety (eg, with a thiol, triflate, tresylate, aziridine, oxirane, or preferably with a maleimide moiety). Or a polyalkylene glycol compound or a derivative thereof, without or with it. Compounds such as maleimide monomethoxy PEG are exemplary or activated PEG compounds of the invention. Other polyalkylene glycol compounds such as polypropylene glycol may be used in the present invention. Other suitable polymer conjugates include non-polypeptide polymers, the following types of charged or neutral polymers: dextran, colominic acid, or other carbohydrate-based polymers, biotin derivatives, dendrimers, etc. It is not limited to. The term PEG is intended to include other polymers of polyalkylene oxides.

  PEG may be linked to any N-terminal amino acid of the molecules described herein and / or are amino acid residues downstream of the N-terminal amino acid, such as lysine, histidine, tryptophan, aspartic acid, glutamic acid, And cysteine, or other such amino acids known to those skilled in the art.

  The PEG carrier moiety attached to the peptide may range in molecular weight from about 200 to 20,000 MW. The PEG moiety will preferably be about 1,000 to 8,000 MW, more preferably about 3,250 to 5,000 MW, and most preferably about 5,000 MW. The actual number of PEG molecules covalently attached to each molecule of the invention may vary greatly depending on the desired stability (serum half-life). Molecules contemplated herein are, for example, U.S. Pat. Nos. 4,179,337; 5,382,657; 5,566, each of which is incorporated herein by reference. Nos. 972,885; 6,177,087; 6,165,509; 5,766,897; and 6,217,869, but not limited thereto. Can be used to link to PEG molecules.

  Alternatively, the molecules can be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (eg, hydroxymethylcellulose or gelatin-microcapsules and poly (methyl methacrylate) microcapsules, respectively), colloids It can be encapsulated in a drug delivery system (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or encapsulated in a macroemulsion. Such techniques are disclosed in the latest issue of “Remington's Pharmaceutical Sciences”.

  US Pat. No. 4,789,734 describes a method for encapsulating biochemicals in liposomes, the entire disclosure of which is hereby incorporated by reference. In principle, the substance is dissolved in an aqueous solution, the appropriate phospholipids and lipids are added with a surfactant if necessary, and the substance is dialyzed or sonicated if necessary. A review of known methods can be found in G. Chapter 14 by Gregoriadis. “Liposomes”, Drug Carriers in Biology and Medicine, pp. 287-341 (Academic Press, 1979). Microspheres formed from polymers or proteins are well known to those skilled in the art and can be tailored to pass directly from the gastrointestinal tract into the bloodstream. Alternatively, the drug can be incorporated and microspheres or microsphere complexes can be infused and released over a period ranging from days to months. See, for example, US Pat. Nos. 4,906,474; 4,925,673; and 3,625,214, incorporated herein by reference.

  Where the composition is to be used as an injectable material, it is formulated into a conventional injectable carrier. Suitable carriers include biocompatible and pharmaceutically acceptable phosphate buffered saline, which is preferably isotonic.

  For the reconstitution of the lyophilized product according to the invention, sterile diluents can be used, but this is determined by substances generally accepted to approximate physiological conditions and / or government regulations. May be included. In this regard, the sterile diluent is sodium chloride, saline, phosphate buffered saline, and / or other physiologically acceptable and / or to obtain a physiologically acceptable pH value. Or you may contain buffer agents, such as a substance which can be used safely. In general, substances for intravenous injection in humans should comply with regulations stipulated by the Food and Drug Administration that can be consulted by those skilled in the art.

  The pharmaceutical composition may be in the form of an aqueous solution containing many of the same materials described above for reconstitution of the lyophilized product.

  Compounds include inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid By reaction with organic acids such as succinic acid, maleic acid, and fumaric acid, or inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and monoalkyl, dialkyl, trialkyl, and arylamines and substitutions It may be administered as a pharmaceutically acceptable acid or base addition salt formed by reaction with an organic base such as ethanolamine.

  As mentioned above, the compounds of the invention can be incorporated into pharmaceutical formulations that can be used for therapeutic purposes. However, the term “pharmaceutical formulation” is to be interpreted herein in a broader sense and used not only for therapeutic purposes, but also for reagents or diagnostic purposes known in the art, or for tissue culture. And a preparation containing the glycosulfopeptide composition according to the present invention. A pharmaceutical formulation intended for therapeutic use should contain a “pharmaceutically acceptable” or “therapeutically effective amount” of a molecule as defined herein.

  All assay methods described herein are well within the scope of one of ordinary skill in the art based on the teachings provided herein.

  All references, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. In particular, US patent application Ser. Nos. 10 / 774,242, 60 / 445,774, 60 / 811,568, and 60 / 836,365 are hereby incorporated by reference in their entirety. The

  Although the invention and its advantages have been described in detail, it will be understood that various changes, substitutions and modifications can be made without departing from the spirit or scope of the invention as defined by the appended claims. Should be understood. Furthermore, the scope of the present application is not limited to the specific embodiments of the processes, manufacture, compositions, means, methods, and steps described herein. As one of ordinary skill in the art will readily appreciate from the disclosure of the present invention, an existing, performing substantially similar function or achieving substantially similar results to the corresponding embodiments described herein, Alternatively, processes, manufactures, compositions, means, methods, or steps that will be developed later can be used by the present invention. Accordingly, the appended claims are intended to include within their scope such processes, manufacture, compositions of matter, means, methods, or steps.

  References

表Ｉ．環状アミンおよびプロリンアナログ

Table I. Cyclic amine and proline analogs

Claims (26)

A method of altering a plasma α ₂ -antiplasmin ratio in a subject having a pre-treatment level of plasma Met-α ₂ -antiplasmin and a pre-treatment level of plasma Asn-α ₂ -antiplasmin, the method comprising: Treating the subject with an inhibitor of an antiplasmin cleaving enzyme,
The inhibitor specifically inhibits cleavage of the Pro-Asn cleavage site of Met-α ₂ -antiplasmin by an antiplasmin cleaving enzyme, and after treatment, the subject is treated before treatment with the plasma Met-α ₂ -antiplasmin. Plasma Asn-α having a post-treatment level of plasma Met-α ₂ -antiplasmin that is at least 5% higher than the level of and at least 5% lower than the pre-treatment level of the plasma Asn-α ₂ -antiplasmin _{A method} having a post-treatment level of ₂ -antiplasmin, thereby altering the plasma α2-antiplasmin ratio in the subject. The method of claim 1, wherein altering the α ₂ -antiplasmin ratio in the subject enhances fibrinolysis in the subject.   The treatment of a subject with an inhibitor of the antiplasmin cleaving enzyme is a treatment for suppressing or treating atherosclerosis, arterial thrombosis, venous thrombosis, stroke, or pulmonary embolism. The method described. The plasma Met-alpha ₂ - levels after treatment of antiplasmin, the plasma Met-alpha ₂ - at least 10% higher than pre-treatment levels of anti-plasmin, the plasma Asn-alpha ₂ - antiplasmin after treatment _2. The method of claim 1, wherein the level is at least 10% lower than the pre-treatment level of the plasma Asn- [alpha] ₂ -antiplasmin. The plasma Met-alpha ₂ - levels after treatment of antiplasmin, the plasma Met-alpha ₂ - at least 15% higher than pre-treatment levels of anti-plasmin, the plasma Asn-alpha ₂ - antiplasmin after treatment The method of claim 1, wherein the level is at least 15% lower than the pre-treatment level of the plasma Asn-α ₂ -antiplasmin. The plasma Met-alpha ₂ - levels after treatment of antiplasmin, the plasma Met-alpha ₂ - at least 20% higher than the level before treatment antiplasmin, the plasma Asn-alpha ₂ - antiplasmin after treatment _2. The method of claim 1, wherein the level is at least 20% lower than the pre-treatment level of the plasma Asn- [alpha] ₂ -antiplasmin. The plasma Met-alpha ₂ - levels after treatment of antiplasmin, the plasma Met-alpha ₂ - at least 25% higher than pre-treatment levels of anti-plasmin, the plasma Asn-alpha ₂ - antiplasmin after treatment _2. The method of claim 1, wherein the level is at least 25% lower than the pre-treatment level of the plasma Asn- [alpha] ₂ -antiplasmin. A compound having the formula _{Xaa 1 -Sp 1 -Gly-Cyc-} Xaa 2 -Sp 2 -Xaa 3,
Where
Xaa ₁ is arginine, histidine, or lysine;
Sp ₁ is 8-, 11-, or 14-amino-3,6,9,12-tetraoxatetradecanoic acid, PEG _n or PPG _n (n = 1-6), gamma amino butyric acid, epsilon amino caproic acid, serine , Glycine, alanine, or β-alanine, or a spacer comprising at least one of these combinations, wherein Sp ₁ has a length of 0.3 nm to 2.1 nm,
Gly is glycine;
Cyc is a carbocyclic or heterocyclic compound,
Xaa ₂ is glutamine, asparagine, serine, histidine, tyrosine, alanine, or phenylalanine;
Sp ₂ is PEG _n or PPG _n (n = 1-5), 8-, 11-, or 14-amino-3,6,9,12-tetraoxatetradecanoic acid, gamma aminobutyric acid, epsilon aminocaproic acid, serine , A spacer comprising at least one of glycine, alanine, β-alanine, aspartic acid, glutamic acid, or combinations thereof, or absent,
A compound wherein Xaa ₃ is glutamic acid, aspartic acid, tryptophan, tyrosine, or phenylalanine.   9. The compound of claim 8, wherein Cyc is a 4, 5, 6, or 7 membered carbon carbocycle.   9. The compound of claim 8, wherein Cyc is a 4, 5, 6, or 7 membered carbon heterocycle.   11. The compound of claim 10, wherein the carbon heterocycle contains a nitrogen heteroatom. 9. The compound of claim 8, wherein Sp ₁ is PEG _n and n = 2-5. 9. The compound of claim 8, wherein Sp ₁ has a length of 0.6 nm to 17.5 nm. 9. The compound of claim 8, comprising a 1-8mer oligopeptide extending from Xaa _{1 in the} N-terminal direction. 9. The compound of claim 8, comprising a 2-10mer oligopeptide extending from Xaa _{3 in the} C-terminal direction.   9. A compound according to claim 8 disposed in a pharmaceutically acceptable carrier.   9. A compound according to claim 8, which is linked to a polyethylene glycol carrier molecule via an amino acid residue. A method of screening for an inhibitor of an antiplasmin cleaving enzyme, the method comprising:
alpha ₂ - cleavable through antiplasmin-cleaving enzyme, _'include binding, the _{P 1 -P 1' P 1 -P} 1 providing a fluorescence resonance energy transfer peptide comprising a fluorophore and a quenching group are separated by binding Wherein P ₁ comprises proline and P ₁ ′ comprises asn, phe, gln, ser, tyr, his, or ala, and has a P ₂ residue comprising gly;
Providing an amount of α ₂ -antiplasmin cleaving enzyme;
Forming a test mixture by exposure to antiplasmin-cleaving enzyme inhibitor candidate, - the alpha ₂ - antiplasmin cleaving enzyme alpha ₂
Combining the test mixture with the fluorescence resonance energy transfer peptide;
The alpha ₂ - ₂ antiplasmin-cleaving enzyme inhibitor candidates alpha - to identify when to inhibit the activity of antiplasmin cleaving enzyme, and measuring the fluorescence emission from the test mixture,
Including a method. The method of claim 18, wherein the fluorescence resonance energy transfer peptide comprises the quenching group upstream of the proline-asparagine bond and the fluorophore downstream of the P ₁ -P ₁ ′ bond. Wherein fluorescence resonance energy transfer peptide _'includes the quenching group downstream of the coupling, the P ₁ -P _1' wherein P ₁ -P 1 containing the fluorophore upstream of binding, The method of claim 18.   19. The method of claim 18, wherein the fluorescence resonance energy transfer peptide comprises SEQ ID NO: 9. An inhibitor of α ₂ -antiplasmin cleaving enzyme identified by the screening method according to claim 18. _{X 1 -X 2 -X 3 -X 4} -X 5 -Gly-Pro-X 8 ( SEQ ID NO: 9)
An oligopeptide substrate of α ₂ -antiplasmin cleaving enzyme comprising:
Where
X ₁ is selected from arg, his, lys, thr, ser, trp, gly, ala, gln, asn, ile, leu, met, phe, val, pro, and tyr;
X ₂ is selected from arg, his, lys, thr, ser, trp, gly, ala, gln, asn, ile, leu, met, phe, val, pro, and tyr;
X ₃ is selected from arg, his, lys, thr, ser, trp, gly, ala, gln, asn, ile, leu, met, phe, val, pro, and tyr;
X ₄ is selected from thr, ser, trp, gly, ala, gln, ile, leu, met, phe, val, pro, tyr, asn, and his,
X ₅ is selected from thr, ser, trp, gly, ala, gln, ile, leu, met, phe, val, pro, tyr, asn, and his,
X ₈ is selected from asn, ser, his, tyr, ala, phe, gln,
At least one of X ₁ , X ₂ , and X ₃ is selected from arg, his, and lys;
One or two of X ₁ , X ₂ , and X ₃ may be absent, and the oligopeptide consists of up to 28 amino acids;
Oligopeptide substrate of α ₂ -antiplasmin cleaving enzyme. Further comprising the oligopeptide 1~8mer extending from X ₁ to N-terminal direction, an oligopeptide as claimed in claim 23. From X ₈ further comprising a 2~10mer oligopeptide extending C-terminal direction, an oligopeptide as claimed in claim 23.   24. The oligopeptide of claim 23, further comprising a quenching group and a fluorophore on opposite sides of the Gly-Pro bond.

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