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MX2008015649A - Substrates and inhibitors of antiplasmin cleaving enzyme and methods of use. - Google Patents

Substrates and inhibitors of antiplasmin cleaving enzyme and methods of use.

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Publication number
MX2008015649A
MX2008015649A MX2008015649A MX2008015649A MX2008015649A MX 2008015649 A MX2008015649 A MX 2008015649A MX 2008015649 A MX2008015649 A MX 2008015649A MX 2008015649 A MX2008015649 A MX 2008015649A MX 2008015649 A MX2008015649 A MX 2008015649A
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antiplasmin
plasma
asn
met
level
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MX2008015649A
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Patrick A Mckee
Kyung N Lee
Kenneth W Jackson
Victoria J Christiansen
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Patrick A Mckee
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Abstract

The present invention is directed toward inhibitors of antiplasmin cleaving enzyme for use in various therapies related to fibrin and alpha2-antiplasmin, and to substrates of APCE, which may be used, for example, in screening methods for identifying such inhibitors. The present invention is further directed to methods of treating or inhibiting atherosclerosis and thrombus disorders by altering the rations of types of plasma alpha2-antiplasmin.

Description

SUBSTITUTION ENZYME SUBSTRATES AND INHIBITORS OF ANTIPLASMINE AND METHODS OF USE Field of the Invention The present invention relates to, but is not limited to, substrates and inhibitors of the a2-antiplasmin cleavage enzyme and selection methods for identifying these inhibitors. , and methods for treating conditions comprising fibrin and a2-antiplasmin, including plaque and clot formation and atherosclerosis. BACKGROUND OF THE INVENTION The a.2-antiplasmin (a2AP) is a blood plasma protein that rapidly and specifically inhibits the enzyme, plasmin, which digests blood clots, if they occur early as intravascular platelet-fibrin deposits or as thrombi. partial or completely occlusive. Similarly, the activities of plasmin and a2AP are important in the development and survival of fibrin as it occurs in inflammation, wound healing and virtually all forms of cancer and their metastases. Human a2-antiplasmin (a2AP), also known as a2-plasmin inhibitor, is the main inhibitor of plasmin. Plasmin plays a critical role in the proteolysis of fibrin and tissue remodeling. The physiological relevance of plasmin inhibition by a2AP Ref .: 198910 to blood coagulation and fibrinolytic hemostasis is supported by the following observations: (1) the rate of inactivation of free plasmin by a2AP in circulation is much faster than digestion with fibrin (ogen) by plasmin, thus eliminating the possibility of a systemic lithic state and consequent bleeding; (2) a2AP is cross-linked to form fibrin by activated blood coagulation factor XIII (FXIIIa) and inhibits plasmin-mediated lysis in direct proportion to the amount incorporated; and (3) patients with homozygous a2AP deficiency show serious hemorrhagic tendencies, while heterozygotes tend to bleed only after major trauma or surgery. Human a2AP is synthesized mainly in the liver, and during circulation in plasma, the secreted precursor form, et-a2-antiplasmin (Met-a2AP), a protein of 464 residues that have methionine as the N-terminus, is subjected to Proteolytic cleavage between Prol2 and Asnl3 (the cleavable union Pi_Pi ') to produce Asn-a2-antiplasmin (Asn-a2AP), a 452-residue version with asparagine as the N-terminus. The Met-a2AP accounts for approximately 30% of the a2AP in circulation, and the Asn-a2AP accounts for approximately 70%. When the Met form of a2AP was found in plasma and its gene was sequenced, it initially appeared to be a discrepancy in one of the nucleotides coding for the sixth amino acid. The two groups found a cytidine (C) that results in Arg as the sixth amino acid, and one group found thymidine (T), which results in Trp in that position. It was suggested that the difference was due to a group that used hepatic carcinoma cells as a source of DNA, while the other two groups used normal cells. Now, it has been determined that both the Arg6 and Trp6 forms of et-a2AP exist in normal samples of human plasma. An investigation of mutant a2AP in a family with bleeding tendencies identified the mutation responsible for the ineffective a2AP together with three polymorphisms in the a2AP gene including this individual C / T nucleotide polymorphism (SNP); this study examined 30 normal blood donors and reported an allelic frequency of 0. 81 / 0.19 for the SNP C / T. No studies of a normal population have been conducted any longer to examine the frequency of homozygotes and heterozygotes, or whether the genotype can affect the Met- a Asn-a2AP plasma relationships. The SNg of Arg6Trp was apparently assumed to be an inactive polymorphism, but the biochemical examination of the two polymorphic forms of et-a2AP in the production of the derivative form, Asn-a2AP, its incorporation into fibrin and the impact on fibrinolysis they have been evaluated before this work.
Brief Description of the Invention The present invention relates to inhibitors of the antiplasmin cleavage enzyme for use in various therapies related to fibrin and a2-antiplasmin, and to the APCE substrate, which may be used, for example, in methods of selection to identify these inhibitors. The present invention further relates to, but is not limited to, methods of treating or inhibiting atherosclerosis and thrombotic disorders by altering the ratios of the a2-antiplasmin types in plasma and inhibiting PACE. Brief Description of the Figures Figure 1. Met-a2AP as percent of total a2AP and by genotype. A2AP was purified from each plasma of people with the RR (n = 15), RW (n = 15) and WW (n = genotypes) genotypes. 5) 'and the amino-terminal sequences determined by Edman degradation. The percent of Met-a2AP was calculated from picomol recoveries of Met and Asn in the first cycle. Figures 2a-2b. Plasma levels of PACE in a normal population, and divided by genotype. The levels of APCE in plasma samples were determined by ELISA. Figure 2a. Histogram of APCE concentrations in a normal population (n = 109). Figure 2b. Levels of APCE by Met-a2AP genotype.
Figure 3. Cleavage of APCE from polymorphic forms of Met-a2AP. Equal amounts (40 μg) of Met-a2AP (Arg6) and Met-a2AP (Trp6) purified by APCE were digested. After stopping at selected times, the reaction, the samples were assessed by mass spectrometry by electro-spraying for the amount of the amino-terminal 12 amino acid peptide produced from each form of Met-a2AP. Figure 4. Effect of the Met-a2AP genotype on the generation of Asn-a2AP in plasma over time. A plasma sample from a person of the RR, RW or WW genotype was incubated at 29 ° C; at selected times, a2AP was purified from each sample and subjected to amino-terminal sequence analysis. The ratio of Asn-a2AP / Met-a2AP was calculated from picomol recoveries of Met and Asn in the first cycle. Figures 5a-5b. Plasma clot lysis time (PCLT) by the Met-a2AP genotype. The PCLT of plasma samples from RR, RW and WW persons were determined. Figure 5a. The PCLT values were divided by genotype and were plotted as mean ± S.E.M., for the total population, men only and women only. Figure 5b. Percent of plasmas that did not lyse (n = 14) in comparison to the percentage of total population (n = 200) within each genotype. Figure 6. Effect of removal of PACE in the Conversion of Met-a2-AP to Asn-a2-AP over time. Plasma was extracted from a person of the Met-a2AP RR genotype and divided into three aliquots. An aliquot was mixed with APCE F19 mAb (F19) bound to chromatography beads and incubated at 4 ° C to remove APCE. The second aliquot was incubated at 4 ° C with non-specific rabbit a-goat Ab (RAG) bound to beads. The third aliquot (RR) did not receive treatment. After the removal of the beads, each sample was incubated at 29 ° C and the Asn-a2AP / Met-a2AP ratios were determined at selected times. In addition, the beads bound to F19 and the beads bound to RAG were boiled with SDS to remove the antibody bound protein. The samples were subjected to electrophoresis in 10% Bis-Tris SDS-PAGE gels and transferred to nitrocellulose. PACE (97 kDa) was identified by western blotting using a goat Ab to its amino-terminal region, and visualized with a chemiluminescent substrate. Figure 7 shows the inhibitory effect of Val-boroPro on the conversion of Met-a2AP to Asn-a2AP by APCE. Figure 8 shows a non-limiting example sequence of the peptide FRET (SEQ ID NO: 5). Figure 9 shows several substitutions that can be made at position Pi 'of the group P2-Pi-i', and several substitutions that can be made to residues in the direction 5 '(N-terminal address) of the residue P2, and to the residues in the 3' direction (C-terminal direction) of the residue ?? ' (SEQ ID NO: 6). Figure 10. LC / MS analysis of antiplasmin peptides (SEQ ID NO: 7) for determination of different substrate in position P7 (sixth amino acid in the 5 'direction of Pi pro). Those with His (H), Lys (K) and Arg (R) positively charged improved the cleavage rate of the Proi2-Ni3 junction. Figure 11. Importance of the position of Arg in relation to the cleavable union Pro-Asn. Four similar peptides were digested based on the amino-terminal antiplasmin sequence (4-17") by APCE and then the LC / MS analysis was performed.Each reaction solution contained an experimental peptide and the control peptide, with Arg in position P, 7,? ß? 5 or P4 (not shown) and glycines in the other three variable positions for any given peptide The substitution in P4 gave a result that was approximately the same as Ps Figure 12. LC analysis / MS of antiplasmin peptides for determination of substrate preference at positions P4, P3, P2, Pi Peptide libraries representing position six to 17 of the human antiplasmin sequence were treated with PACE (also known as soluble FAP). Phenylalanine in red was added to improve the binding to the inverted phase HPLC column. The amino acids common to the proteins, minus cysteine were substituted one at a time in each of the positions Pi up to P4 (SEQ ID NO: 8). The relative cleavage rates of the Pro-Asn junction were determined by LC / MS analysis. Figure 13 shows an alternative embodiment of an amino acid sequence (SEQ ID NO: 9) comprising a portion of a FRET peptide or inhibitor of the present invention. The sequence can be extended in the C-terminal direction, for example, by the sequences shown in Figure 9 which extend from Ni3, in the N-terminal direction or extending from Ni3 in the C-terminal direction. Figure 14 shows the inhibitory effect of PACE of several inhibitory peptidomimetics (SEQ ID NO: 11-13) at inhibitor concentrations of 20 μ ?, where proline has been replaced with pipecolic acid. Detailed Description of the Invention Human Met-a2AP is a physiologically important substrate of PACE, since proteolytic cleavage between Prol2-Asnl3 produces the most active derivative, Asn-a2AP, which is significantly more rapidly crosslinked to fibrin by FXIIIa than Met-a2AP, and as a consequence, improves the fibrin resistance to plasmin digestion. Since human APCE increases the inhibition of fibrinolysis by increasing the availability of the faster cross-linking form (ie, Asn-a2AP), the potent and selective inhibitors of APCE that have been discovered as described herein allow titration. of the production of Asn-a2AP at lower levels in vivo, and thus improve both endogenous and exogenous fibrinolysis (fibrin removal). Bleeding complications using the treatments described here are unlikely, based on the observation that people heterozygous for a2AP deficiency have minimal bleeding risk. Therefore, there is a safety advantage to decrease the function of a2AP in healthy people. Particularly in clinical situations where fibrin formation is likely, the APCE inhibitors will result in a decreased amount of Asn-2AP available for cross-linking to fibrin as the development of thrombi or inflammation progresses. Then, the endogenous levels of plasmin generated, or plasmin produced by administering small amounts of a plasminogen activator, may be sufficient to effect the removal of fibrin so that the opening of the vessels and the function of the organs is maintained and reduced. to the minimum the risk of bleeding. How I know noted above, APCE cleaves Met-a2AP to the Asn-a2AP derivative, which is incorporated more efficiently into fibrin and consequently makes it surprisingly resistant to plasmin digestion. In this way, PACE represents a new objective for pharmacological modulation and inhibition of the fibrinolytic system, since less generation and therefore less incorporation of Asn-a2AP results in a faster removal of fibrin by plasmin during atherogenesis, thrombosis, and inflammatory states. The present invention relates to inhibitors of the antiplasmin cleavage enzyme for use in various therapies related to fibrin and a2-antiplasmin, and to substrates of APCE (antiplasmin cleavage enzyme, for its acronym in English), which can be used , for example in detection methods to identify these inhibitors. The present invention further relates to, but is not limited to, methods of treating or inhibiting atherosclerosis and thrombus disorders by altering the ratios of a2-antiplasmin types in plasma. In a preferred embodiment, inhibition of PACE is defined herein as at least 50% inhibition of PACE activity, for example, at a 20 μ inhibitor concentration. Asn-a2AP is cross-linked to fibrin at a rate of approximately 13 times faster than Met-a2AP. A faster rate of crosslinking results in a greater number of antiplasmin molecules bound to the newly formed fibrin and an improved resulting resistance to fibrinolysis. In this way, the inhibition of APCE in plasma can decrease the number of crosslinked antiplasmin molecules, thus resulting in clots that are more easily removed during fibrinolysis. Therefore, a specific inhibitor for PACE can be used to regulate fibrinolysis. As noted above, human Met-a2AP exists in two polymorphic forms at position six in the mature sequence, with predominant arginine and tryptophan accounting for a smaller percentage. It has been discovered herein that the inhibition of PACE can alter the a2AP ratio from about 30% et-a2AP: 70% Asn-a2AP to about 60% -70% Met-a2AP: 40% -30% Asn-a2AP in the plasma. Modulation of a2-Antiplasmin Ratio In the present work, the prevalence of the polymorphism in a much larger normal population was determined and then it was evaluated if it is related to the inhibitory function of a2AP. Results are provided herein with respect to (1) genotype frequencies of the Arg6Trp SNP in Met-a2AP; (2) how each form affects susceptibility to excision by PACE; (3) the percent of Met-a2AP in plasma for each of the two polymorphisms; (4) times of clot lysis in plasma relative to the genotype; and (5) evidence that the removal of PACE in circulation prevents the conversion of Met- to Asn-a2AP. Materials and Methods Materials Frozen, fresh human plasma was purchased for protein purification from Sylvan Goldman Blood Institute (Oklahoma City, OK). Hybridoma cells secreting the F19 antibody of the American Type Culture Collection (ATCC) (Manassas, VA) were purchased and cultured in serum-free medium; the F19 antibody was purified from the culture medium using MEP-Hypercel chromatography (Pall, East Hills, NY). Approval was obtained from the Institutional Review Board (IRB) of the University of Oklahoma Health Sciences Center for these studies (IRB # 10142 and 12189). Isolation of a2AP Met-a2AP and Asn-a2AP mixtures were isolated by a modification of a published purification procedure using the peptide domains 1-3 of plasminogen bound to Sepharose 4B as an affinity matrix. Met-a2AP and Asn-a2AP were separated by immunoaffinity chromatography as described above. The ratio of Met-a2AP to Asn-a2AP in Plasma samples were determined by comparing the picomol recovery of Met versus Asn in cycle one during the automated protein sequencing by Edman degradation (Applied Biosystems Procise model 492, Foster City, CA). APCE Isolation PACE was purified from human plasma as described above (U.S. Serial No. 10 / 774,242). Briefly, a combination of ammonium sulfate precipitation, hydrophobic interaction, and immunoaffinity chromatography were used for the purification. Before storage at -80 ° C, glycerol was added to the pure APCE to give a final concentration of 20%. Determination of a2AP genotype Two hundred normal volunteers who responded randomly, who self-reported as healthy and free of acute disease, were recruited to donate blood for the determination of genotype frequency and plasma clot lysis time (PCLT see next section) in a normal population. DNA was isolated from the whole blood of each donor using an AquaPure genomic DNA blood kit (Bio-Rad, Hercules, CA). The DNA portion spanning the Arg6Trp SNP was amplified by polymerase chain reaction using the oligonucleotide primers (5'-GACCTCCTATCCTCATCCCTTT (SEQ ID NO: 1) and 5'- CTGGTTCGGCCCGCTAGTTAG (SEQ ID NO: 2)), dNTP (Takara Mirus Bio, adison, WI), and Platinum Taq-DNA polymerase (Invitrogen, Carlsbad, CA). After purification, the PCR product was purified, using the MinElute PCR purification kit (Qiagen Operon, Alameda, CA) and sequenced, using an automated ABI3730 DNA sequencer. Measurement of Plasma Clot Lysis Time To measure plasma clot lysis time (PCLT), a mixture of 1 unit / mL of thrombin, 16 mM CaCl 2 and 45 IU / mL of urokinase (μ) was added (Abbott, Chicago, IL) to each plasma of the volunteers to catalyze the essentially instantaneous formation of fibrin clot and to initiate fibrinolysis; The speed of plasma clot lysis was determined by a turbidimetric method of microtiter plate. Determination of Cleavage Speeds Reaction mixtures containing equal amounts (40 μg) of pure Met-a2AP (rg6) and pure et-a2AP (Trp6) were digested by APCE. At selected times, digestion was stopped by lowering the pH from 7.5 to 4.0 with trifluoroacetic acid. Proteins were removed from the mixture by Microcon centrifugal ultrafiltration (Millipore, Bedford, MA) using a 30 kDa cut-off membrane. The peptides were isolated from the ultrafiltered digestion mixture by binding to the inverted phase POROS-50 medium (Applied Biosystems, Foster City, CA) packed in a glass purification capillary (Proxeon, Odense, Denmark). The peptides were then eluted from the POROS-50 directly into a nano-sprayed capillary of glass coated with 2.0 μ metal. of 0.5% acetic acid in methanol / water 1: 1. The nano-spray capillary was mounted on the nano-spray ionization source of a QSTAR ESI-Quad-TOF mass spectrometer operated under version 1.0 of the Analyst QS software (Applied Biosystems, Foster City, CA) with an ion spray voltage of 1400 volts. Data were collected on a mass range of 300 to 1500 Da. The relative amounts of the two peptides of 12 amino-terminal amino acids of a2AP, produced, (MEPLGRQLTSGP (SEQ ID NO: 3), Mr = 1284.65 for Met-a2AP (Arg6) and MEPLGWQLTSGP (SEQ ID NO: 4), Mr = 1314.63 for Met-a2AP (Trp6), were determined by adding the areas of the four most abundant isotope peaks for the charge forms observed for each peptide.The data for each time point was normalized by a similar quantification of a normal peptide inert internal supplement that did not contain proline Antibody Level Determination of APCE An enzyme-linked immunosorbent assay (ELISA) was developed to determine plasma antigen levels human. A goat antibody was prepared to the amino-terminal 15 amino acid sequence of APCE, using as the immunogen, a multiple antigenic peptide (MAP) constructed in our laboratory to contain eight copies of the amino-terminal peptide linked by its carboxyl-terminus to a core peptide of seven Usinas. This goat MAP antibody (amino-terminal 15-residue APCE peptide) was bound to white high-binding polystyrene assay plates (Corning, Corning, NY) and used as the capture antibody. After incubation with plasma dilutions, a purified monoclonal antibody from commercially available F19 hybridomas was applied (ATCC, Manassas, VA), and followed by anti-mouse antibody conjugated with peroxidase (Sigma, St. Louis, MO). A chemiluminescent substrate, SuperSignal ELISA Pico (Pierce, Rockford, IL) was added, and the luminescence was monitored using a BIO-TEK FL600 plate reader (Winooski, VT). The level of antigen was quantified using purified human PACE as the norm. Plasma APCE Removal F19 mAb was linked to poly (styrenedivinylbenzene) POROS EP 20 perfusion chromatography (Applied Biosystems, Foster City, CA) beads and a non-specific, rabbit anti-goat antibody was bound to the same type of medium. The plasma of an individual donor of the RR genotype was divided into 3 aliquots, diluted 1: 1 with PBS and incubated in a manner separated with each of the two antibodies bound to beads, non-specific Ab or mAb F19, overnight at 4 ° C. The third aliquot did not receive treatment. The beads were removed from the plasma by filtration and the plasma was then incubated at 29 ° C. The aliquots were removed at time zero, 24 hours and 48 hours. A2AP was purified from each aliquot and the Asn-a2AP / Met-a2AP ratio was determined as described above. After plasma removal, mAb F19 beads were washed with 25 mM NaP04 / 0.5 M NaCl and then boiled with SDS charge buffer. The SDS buffer extract was then separated by electrophoresis in a 10% Bis-Tris gel (Invitrogen, Carlsbad, CA) and transferred to nitrocellulose. PACE was identified by Western blot using the goat amino-terminal MAP antibody as described above in this Methods section and visualized using the West Femto SuperSignal Maximum Sensitivity Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL). Results Determination of a2AP Genotype A group of 201 normal volunteers, who were recruited into two sets of approximately 100 people each, separated by approximately two years, provided blood samples to determine the frequency of SNP C / T from a2 ?? The total population consisted of 61 men and 139 women from 21 to 69 years of age with an ethnicity that corresponded closely to the demographic for an Oklahoma population as it was listed for the year 2000 on the US Census website (factfinder.census. gov). The genotype was determined for 200 of the subjects. Only one DNA sample was not amplified by the polymerase chain reaction, possibly due to a mutation that prevented the binding of one of the primers, although this has not been further explored. The frequencies of the genotypes for the two normal populations were essentially the same with less than 1% difference for any of the genotypes. After combining the two sets, the frequencies for the entire population were RR 62.5%; RW 34.0%; WW 3.5%. The R allele has a frequency of 79.5% and the W allele has a frequency of 20.5%. There was no difference in genotype frequency between men and women. Because the population was 72% Caucasian, with the other 28% divided among 6 ethnic categories, it was not possible to determine whether the genotype varied between ethnic groups. Levels of Met-a2AP and Asn-a2AP in Plasma A2AP was purified from plasmas of 15 people of the RR genotype; 15 people with RW; and 5 with the WW genotype. The a2AP was sequenced by Edman degradation and determined the picomol recoveries of Met and Asn for the first cycle to calculate the percent of Met-a2AP and Asn-a2AP. Figure 1 shows significantly different results (p <0.001) that were divided by the genotype with WW that has the highest percentage of Met-ot2AP (56.4%); RR, the lowest (23.6%), and RW that falls between, in (40.6%). To understand the mechanism for the variable percentages of Met-a2AP in human plasma, we examined whether a variation in the level of the enzyme that converts Met-a2AP to Asn-a2AP, that is, PACE, to explain the differences between the genotypes. To investigate this possibility, an ELISA method was developed to quantify the antigen level of PACE in plasma and then the plasma PACE concentrations of 109 subjects in our normal population were determined. As seen in Figure 2a, there is a distribution of levels of APCE in normal human plasma, ranging from 38 to 159 ng / ml that does not correlate with age or gender, and despite the suggestion of a possible association of levels of PACE with the genotype (RR: 70.4 ± 26, RW: 66.6 ± 24.5, WW: 64.7 ± 10.1), the differences were not statistically significant (Figure 2b). Therefore, it seems that PACE levels do not account for the variation of Met-a2AP levels between genotypes. Another explanation for the different percentages of Met-a2AP in the three genotypes that was explored was that Met-a2AP (Arg6) is a better substrate for PACE than Met-a2AP (Trp6). To test this hypothesis, a2AP was purified from RR plasma and y the cleavage rate of each polymorphic form, Met-a2AP (Arg6) and Met-a2AP (Trp6), was determined using mass spectrometry to monitor the generation of the peptide Amino-terminal of 12 residues with time. As seen in Figure 3, comparisons of reaction rates were based on linear regression analysis of early time points and showed that PACE cleaves Met-a2AP (Arg6) approximately 8 times faster than Met-a2AP (Trp6 ). To examine whether the rate of cleavage of Met-a2AP in whole plasma gives similar results based on the presence of R or in position 6, fresh plasma samples were obtained from individuals whose genotype was determined to be RR, RW and WW, they were incubated at 29 ° C, with each one analyzed at 0, 24 and 48 hours for the Asn-a2AP / Met-a2AP ratios. A2AP was purified from each sample and the ratio of the two polymorphic forms was determined by picomole recovery of the amino-terminal residue in the first cycle of Edman degradation. As seen in Figure 4, Asn-a2AP was increased at a much higher plasma velocity than a normal volunteer RR genotype in either patients with R or WW genotype. These results obviously concur with those of reaction mixtures of pure a2AP and APCE that showed that APCE cleaves Met-a2AP (Arg6) approximately 8 times faster than Met-a2AP (Trp6). Plasma Clot Lysis Plasma clot lysis times (PCLT) were determined in blood samples of 200 women and men comprising the normal population. No attempt was made to control the variations in clot lysis that are known to occur due to activator release, activator-inhibitor interactions, fibrinogen level or lipid effects, and enzyme-substrate mechanisms; therefore, gender, age, adrenergic status, smoking, obesity, alcohol consumption, measurements of fibrinogen and serum lipids, medication, daytime activities, etc., were ruled out as qualifiers for the participation of volunteers in the study. In contrast, the "participants" were introduced as long as they reported themselves to be in good health, believing that the lysis times of the study participants should reflect more or less the "average" of all the physiological / pharmacological effects within each genotype at any point of time and that if in reality the polymorphism detectably affected the lysis of plasma clot under the present conditions, then will accentuate its potential importance. Important to emphasize is that the average PCLT for men was significantly longer compared to values for women (p = 0.0002). As shown in Figure 5, the average PCLT for the RR, RW and WW genotype groups exhibited an impressive linear decrease, but the differences between the average PCLT among the three genotypes were not statistically significant. As shown in panel A of Figure 5, after separating the genotypes by gender, the differences for the average PCLT between the genotypes showed an obvious tendency towards shorter lysis times for the WW genotype in both men and women. . Since the distribution of the PCLT was biased, nonparametric statistical methods were used to analyze the data. The means for RR and RW were similar and the distributions overlapped, suggesting that the R allele is dominant. When the RR and RW groups were merged and compared to WW, after counting the variation due to gender, the differences approached the meaning (p = 0.061). As shown in Figure 5, panel B, 12 people (10%) of the RR genotype and 2 (3%) of the RW genotype have plasma clots that remained totally intact during the entire one-hour test period; no person of the genotype WW has a time of lysis > 2100 seconds Removal of APCE from Plasma In an effort to establish definitively that APCE is the enzyme responsible for the conversion of Met-a2AP to Asn-a2AP, APCE was removed from the plasma by incubating the plasma with the FAP-specific monoclonal antibody, F19 , covalently bound to POROS chromatography beads. After the removal of the beads, incubation of the plasma was continued for selected times at 29 ° C to allow the conversion of Met-a2AP to Asn-a2AP. The graph of Figure 6 shows that the Asn-a2AP / Met-a2AP ratio in the control plasma, not incubated with the antibody bound to POROS, was increased from 2.62 to 6.12 after 48 hours of incubation. In a second control, specifically, the plasma incubated with a non-specific antibody bound to POROS chromatography accounts, after 48 hours a significant increase in the Asn-a2AP / et-a2AP ratio from 2.54 to 5.2 was presented. However, the plasma incubated with the F19 antibody showed no increase in the Asn-a2AP / et-a2AP ratio, 2.4 to 2.17, during the same incubation period, thus indicating the removal of APCE by the F19 antibody. These results clearly demonstrate that the removal of plasma APCE abolishes the conversion of Met-a2AP to the shorter, faster fibrin-crosslinking form, Asn-a2AP. As additional support for this conclusion, it was also demonstrated by Western transfer analysis that actually APCE that was joined and removed by mAb F19 attached to accounts. The Western blot analysis shown in Figure 6 shows a 97 kDa monomeric band of APCE in the sample eluted from the beads linked to F19; this band was not present in the accounts linked to rabbit anti-goat Ab (RAG). Other visible bands were the result of the specific binding of the secondary Ab to immunoglobulin in the samples, which leaches the beads during the boiling extraction in SDS. In the present work, the two populations of normal volunteers that were analyzed in separate tables of time were essentially identical, with the frequency of the mixed genotype that is RR, 62.5%; RW, 34.0%; and WW, 3.5%. The allele frequency values of 0.795 / 0.0205 are in agreement with the other single study of which we are aware, specifically that of Lind and Thorsen, who reported values of 0.81 / 0.19 for the individual nucleotide transition in 30 normal blood donors. Because this polymorphism occurs in the 12-residue amino-terminal peptide that is removed from the longer precursor form of a2AP, and given that a positively charged hydrophilic (R) arginine is replaced with a hydrophobic amino acid, tryptophan (W) , it was questioned whether this difference can affect the rate of cleavage of the peptide by PACE and the subsequent incorporation in fibrin formation. HE found that pure APCE cleaved et-a2AP pure genotype approximately 8X slower compared to Met-a2AP pure plasma from individuals with RR. Figure 4 clearly shows that the ratios of Met-a2AP native precursor / Asn-a2AP derived in plasma samples containing each of the two polymorphic forms of Met-a2AP changed spontaneously with time when the newly extracted plasma was allowed to incubate at 29 ° C. This cleavage may be due to plasma levels that occur naturally in the PACE, since as shown in Figure 6, the removal of PACE with a specific monoclonal antibody completely aborted the generation of Asn-a2AP derived during the same incubation time. Since this cleavage occurs spontaneously within circulating blood, the relationships of et-a2AP precursor / Asn-a2AP derivative must vary within the circulating plasma of the three genotypes, which was actually demonstrated by the quantitative amino-terminal analysis of the precursor / derivative a2AP forms for each of the 35 people analyzed. As predicted, people of the RR genotype have the lowest amount of Met-a2AP in circulation (23.6%); with intermediate RW, (40.6%); and WW the highest, (56.4%). The present results can not be explained by the variation in PACE levels, since as shown in Figure 2b, antigen levels are not were significantly different among the three genotypes. The relationship between the RR, RW and WW genotypes and the corresponding et-a2AP / Asn-a2AP relationships increased the possibility that the latter would impact the fibrinolytic activities of the individuals so that during the course of someone's life, the vulnerability of the Fibrin intravascularly generated to endogenous fibrinolysis, and consequently their survival, are affected differently by the Met-a2AP genotype. As a consequence, people of the WW genotype would have Met-a2AP that is less susceptible to conversion to Asn-a2AP due to excision by PACE and therefore incorporated less effectively in fibrin formation, thus making any fibrin generated more susceptible to plasmin digestion. Experiments were carried out to demonstrate this using whole plasma to approximate the native conditions as much as possible. As indicated in the previous section, only minimal effort was made to standardize the conditions under which all samples of healthy volunteers were extracted, on the basis that if in fact the WW genotype group has shortened fibrinolysis times, then the probability to favor this which is the case for most of the time. Most known disturbances that affect fibrinolysis times, either from Exactly or chronically, they are at stake during anyone's lifetime, and despite these influences, it is postulated that on average, the fibrinolytic state would segregate according to the Met-a2AP genotype. Notable is that in all of the present analyzes, people of the RR genotype have the longest average PCLT, with the intermediate RW group, and those in the WW genotype the shortest, suggesting that people with WW have chronic a fibrinolytic system more active than the RW group, and certainly more active than those with the RR genotype. The RR genotype contained a higher than expected percentage of people whose fibrin was never smooth, and if these were assigned PCLT values, one second above the maximum value measured for any person in the present study, then, for men, the association of Average times of lysis with RR and RW achieved significance at the p < 0.05. The present results indicate that during the lifetime of someone, the W allele can serve as a "protection factor" (in contrast to the term well understood, "risk factor") by increasing the susceptibility to develop intravascular thrombi for removal by plasmin, thus decreasing the risk of atherosclerosis. Additionally, these results demonstrate the use in the increase of Met- a2AP / Asn-a2AP to approximate those that accelerate fibrinolysis. By decreasing the role of a2AP, essentially the only in vivo inhibitor of plasmin, at levels that carry little risk of major bleeding, as exemplified in the heterozygous deficiencies of a2AP function, and a chronic level of endogenous sustained lithic activity, then the survival and participation of intravascular fibrin-platelet thrombi in the atherosclerotic process can be reduced. In another work, plasma samples were taken from patients with metastatic colon cancer who were treated experimentally with oral Val-boroPro (talabostat) which has been shown to be a non-specific inhibitor of PACE. Figure 7 shows that the APCE inhibitor Val-boroPro increases the percentage of Met-a2AP in plasma indicating less conversion of et-a2AP to Asn-a2AP by APCE. Fibrin is key to the stabilization of platelets as they adhere and aggregate at a site of injury to an arterial wall during the early stage of plaque development. Fibrin continues to be left as oxidized lipid and macrophages infiltrate the site of injury, and the plaque grows to gradually enter the diameter of the lumen with risk of rupture and acute occlusive thrombus formation. During all these stages, if the fibrin contains high or maximum a2plasmin, then the vulnerability of the fibrin upon removal by the endogenous fibrinolytic system, which if fibrin contains less amounts of a2AP inhibitor. With higher circulating blood levels of precursor a2AP (Met-a2AP) minus total a2AP, it becomes cross-linked to fibrin, causing fibrin to be more easily digested and cleared from the plaque site by the fibrinolytic enzyme, plasmin. However, if its derivative, Asn-a2AP, dominates, then fibrin is more resistant to removal by fibrinolysis. By inhibiting APCE, then et-a2AP increases in blood concentration and any fibrin that forms is more easily removed by the fibrinolytic system of anybody. Fluorescence Resonance Energy Transfer Peptide Substrates The present invention in one embodiment contemplates a fluorescence resonance energy transfer peptide (FRET-peptide) having a cleavable PN (proline-asparagine) (? -? ? ') and having a G (glycine) at position P2 in the 5' direction of the Pi proline. He FRET-peptide comprises a cleavage group, eg, DABCYL, on one side of the junction ???? ' and a fluorophore group, for example, EDANS, on the other side of the cleavable linkage. Preferably, the FRET-peptide has up to 13 amino acid residues in the 5 'direction of the proline? and up to 13 residues in the 3 'direction of the asparagin residue Pi' (ie, the complete peptide including up to 28 amino acids). The amino acid having the cleavage group (for example, lysine) can be one of up to 13 amino acids in the 5 'direction or in the 3' direction of the group ?? - ?? ' and the amino acid having the fluorophore group (eg, glutamic acid) can be one of up to 13 amino acids in the 5 'direction or 3' direction of the group ?? - ?? ' . Preferably, the cleavage and fluorophore groups are at the distant ends of the FRET-peptide, and preferably at least one end of the peptide is terminated with an arginine residue (or other positively charged amino acid), or alternatively, one end may have a positively charged amino acid and the other end may have a negatively charged amino acid. The terms "heterocycle" or "heterocyclic" refer to ring structures, preferably ring structures of 4, 5, 6, or 7 members, and more preferably rings of 5 to 6 members, whose ring structures include one to four heteroatoms such as nitrogen, sulfur or oxygen. Heterocyclic groups include, but are not limited to, thiophene, furan, pyran, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine and pyridazine. The heterocyclic ring may be substituted one or more positions with substituents such as, for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, heterocyclic, an aromatic or heteroaromatic portion, CF3, CN, or the like. The term "carbocycle" or "carbocyclic", as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon, preferably to rings of 4, 5, 6 or 7 carbons, and more preferably to rings of 5 and 6 carbons. The proline analogs, and derivatives, of the peptidomimetic compounds of the present invention may exist in particular geometric or stereoisomeric forms. The present invention contemplates all of these compounds, including cis and trans isomers, R and S enantiomers, diastereomers, (D) -isomers, (L) -isomers, racemic mixtures thereof, and other mixtures thereof, as they fall within of the scope of the invention. Additional, asymmetric, carbon atoms may be present in a substituent such as an alkyl group. All these isomers, as well as the mixture thereof, are proposed to be included in this invention. Figure 8 shows an example of a FRET-peptide (SEQ ID NO: 5) that has the native amino acid sequence that surrounds the junction ?? - ?? ' of the Arg6 form of Met-a2-AP. In this case, the maximum, preferred, complete sequence of a FRET-peptide to be used to mimic the substrate characteristics of the N-terminal peptide of Met-a2-AP is shown. The rationale for including up to 13 residues on either side of the cleavage junction is to include an equal number of amino acid residues on either side of the junction ?? - ?? ', since this level of symmetry minimizes potential steric problems as enzyme and substrate bind to cleave the Pi2_ i3 junction (ie, ?? - ?? ') · In this embodiment, the optional arginine groups in each term improve the solubility of the peptide derivatives when the -PN bond is cleaved -. Gn and P12 (ie, P2-Pi) are preferred amino acids that can not be substituted by other amino acids that occur naturally if the peptide is proposed to be a cleavable substrate. The proline may be substituted with other proline derivatives or analogs or proline-like amino acids in peptides proposed for use as inhibitors. Pi2-Ni3- is the cleavable bond. Pi2, as noted, can be substituted to form an inhibitor. The fluorescent and cleavage groups of the FRET-peptide are each coupled to amino acids, such as E and K near the terms in this embodiment. Mi is the methionine residue N- terminal of the native sequence of the a2AP precursor. Figure 9 shows an alternative sequence of FRET-peptide (SEQ ID NO: 6) having potential permutations of acceptable substitute amino acids at specific positions of a peptide comprising amino acids 8-18 of the peptide of Figure 8. In a preferred embodiment of the FRET-peptide, the cleavage group and the fluorophore group are each coupled to an amino acid residue which are on either side of the cleavable linkage and which are preferably within 1.0 to 5.0 nm of the cleavable linkage. In another preferred embodiment, each cleavage and fluorophore group is attached to any of the residues in the 5 'direction of the glycine residue of P2, or in the 3' direction of the residue of ?? ' (Asparagine or an appropriately substituted amino acid), wherein the cleavage group is on one side of the residue P2 or ?? ', and the fluorophore group is on the residue side of P2 or Pi 1, opposite the cleavage group. Experiments were performed using peptide substrates to investigate how structural variations (amino acid substitutions) in and near the antiplasmin cleavage site affect the cleavage of antiplasmin by APCE. The amino acid sequence of Met-a2AP surrounding the P1-P1 'junction cleaved by the cleavage enzyme of antiplasmin (i.e., Pro-Asn) was used as a guide to produce identical synthetic peptides of variable length to determine important residues to improve cleavage of the peptide, assuming that cleavage rates are proportional to binding. Proline (or a carbocyclic or heterocyclic proline analogue or substitute) in the Pi position is a residue necessary for binding to PACE, as is glycine in the P2 position in the 5 'direction and adjacent to the proline or proline substitute or analogous residue. Working through a set of substitutions at various residues in the 5 'direction from the Pi proline, a positively charged amino acid (eg, arginine, lysine or histidine) was found in P7 (ie, the sixth residue in the direction 5 'of the proline) or in positions P5 or P6 were particularly favorable to maximize the binding to PACE and thus the rate of cleavage (Figures 10 and 11). The labeling shown therein with reference to P5, P6, P7 refers to the residues as they are numbered increasingly from the Pro residue in the cleavable linkage in the N-terminal direction. Pro for example is Pi, with the subscript number that increases in the 5 'direction. Figure 10 shows that an arginine in the 5 'direction, or any positively charged amino acid, is critical to maximizing the cleavage rate of the junction ?? - ??' by APCE in a peptide having SEQ ID NO: 7.
Figure 11 indicates that by substituting arg in P4, P5, P6 and Pe r it was determined that P5 and ß are at least as effective as arg in the native P7 position, with P6 being better. P4 and P8 are significantly lower and did not show improvement to excision of the cleavable linkage. In addition to the presence of a positive charge at P5, P6, or P7 which has an accelerating effect on cleavage of the scissile bond, there is also a requirement for spacing so that the effect of the positive charge is maximum. At position P7, Arg was the most optimal amino acid with a relative cleavage rate of approximately 5-10 times faster than other amino acids except lys, which was approximately 70% of the arg rate (Figure 10). The enhancing effect of arginine at the P7 position (the sixth residue in the 5 'direction of the Pi proline) was also observed (Figure 11) when arginine was in the P6 or P5 position (ie, 4 or 5 residues in the direction 5 'of the junction Pi_Pi') / but the cleavage rates decreased when arg was in the positions P8, Pi, P3 or P2 of the junction P1-P1 '(Figures 11 and 12). Figure 12, for example, shows the effect of substituting each of the 19 amino acids in each of the positions P3.-P4 of amino acid sequence in a peptide (SEQ ID NO: 8) and the effect on the reaction rate of cleavage of the P1-P1 'junction of the peptide by APCE. Positively charged waste, lys and his, can be replace by arg at positions P7, P6 and P5 (see Figure 10 for P7), indicating that the effect on the cleavage rate of P1-P1 'is substantially dependent on the positive charge (although pro, phe, tyr, trp, and some other amino acids have partial levels of activity (Figure 10)). In an alternative embodiment, the FRET-peptide of the present invention comprises a peptide sequence as shown in Figure 13 (SEQ ID NO: 9) wherein the X1-X5 and ¾ positions may comprise an amino acid selected from the group of amino acids indicated below, each of X1-X5 and Xa, with the proviso that at least one of X1-X3 is selected from R, H and K (ie, it is a positively charged amino acid). Additionally, at least one of Xi, X2 and X3 may be absent. As noted above, the FRET-peptide comprising SEQ ID NO: 9 (or any peptide or peptidomimetic) preferably comprises up to and including 28 amino acids. The FRET-peptide comprising SEQ ID NO: 9 preferably comprises a cleavage group and a fluorophore on opposite sides of the gly-pro linkage. SEQ ID NO: 9 may further comprise a 1-1Orner peptide in the 5 'direction of the N-terminal amino acid in the N-terminal direction and which may comprise any of the 20 natural amino acids, and may further comprise an l-10mer peptide in the 3 'direction of the C-terminal amino acid in the C-terminal direction and which can understand any of the 20 natural amino acids. As noted above, the substrate of the fluorescent resonance energy transfer peptide can comprise the cleavage group in the 5 'direction of the Pi-Pi' junction and the fluorophore in the 3 'direction of the? -pi bond. , or the fluorescent resonance energy transfer peptide may comprise the cleavage group in the 3 'direction and the fluorophore in the 5' direction of the? - ?? 'junction . The Pi is preferably proline or an effective analogue or substitute for proline, as described herein, Pi 1 is preferably asparagine, and a P2 group in the 5 'direction of ?? it is preferably glycine. Detection for APCE inhibitors In a particular embodiment, the invention comprises a method for selecting inhibitors of the antiplasmin cleavage enzyme, comprising: providing a fluorescence resonance energy transfer peptide as contemplated herein, comprising a binding Px-Pi 'and comprising a fluorophore (e.g., EDANS) and a cleavage group (e.g., DABCYL) separated by the coupling ?? - ??' such that the peptide can be cleaved by the a2-antiplasmin cleavage enzyme, provide an amount of a2-antiplasmin cleavage enzyme, expose the a2-antiplasmin cleavage enzyme to an inhibitor candidate of a2-antiplasmin. cleavage enzyme of a2-antiplasmin to form a test mixture, combine the test mixture with the fluorescent resonance energy transfer peptide, and measure the fluorescence emission of the test mixture to identify when the activity of the enzyme is inhibited. a2-antiplasmin cleavage enzyme by the a2-antiplasmin cleavage enzyme inhibitor candidate. APCE Inhibitors As described herein, several sequence variations of the FRET-peptide and the peptide sequence surrounding the cleavable a2AP junction have been used herein for the development of the inhibitor. In particular, proline substitutions of the cleavable linkage with proline analogs have led to the development of specific inhibitors of PACE as described herein. Thus, the present invention contemplates APCE inhibitor peptidomimetics having up to 28 amino acids or more, and comprising proline derivatives and analogs for use as a substitute for proline Pi, which includes, but is not limited to, analogues and proline derivatives shown in Table I. Peptidomimetics preferably comprise 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, not including separating compounds to separate certain amino acids from the compound, or compounds that can bind or complex to these to extend the serum life of the peptide, such as PEG or other carriers or polymeric materials. Preferably, an APCE inhibitor having a positively charged residue at a distance corresponding to the length of four to seven residues in the 5 'direction of the cleavable link P1-P1' (3.5-24 A, i.e. 35 nm - 2.4 nm) in this manner can be used as a rapid, effective, tight binding inhibitor of PACE. In addition, these inhibitors may be useful for the inhibition of the closely related enzyme, fibroblast activation protein (FAP) and thus for the treatment of disorders that relate to FAP, for example as described in the United States patent. United number 6,949,514. The spacing of the positive charge at any of the positions P5, P6 or P7 of the cleavable union Pi-Pi 'is a relevant determinant and therefore any amino acid or other separator constructed of inert or neutral substances that fill this space to achieve Approximate length (ie, .35 nm - 2.4 nm) will be effective in the construction of an APCE / FAP inhibitor. In a particularly favorable mode of the FRET-peptide or APCE inhibitor, arginine (or another positively charged amino acid) is within 6-21 A, (.6 nm - 2.1 nm) of the proline (or residue substitute of Pi) in the cleavable union.
The inhibitor in a preferred embodiment comprises a sequence having 2-6 amino acids in the N-terminal direction of the ??? ?? 'junction, including a positively charged amino acid (eg, arginine, lysine, or histidine), and preferentially but not necessarily, it includes a pro-asn link in ?? - ?? ' and preferably a glycine in P2 and preferably a negatively charged or aromatic amino acid (eg, asp, glu, trp, tyr, and phe) at a position in the 3 'direction (C-terminal direction) from the junction ?? - ?? ' In a preferred embodiment of an inhibitor or FRET-peptide of the present invention, the compound may comprise a spacer group (linker or filler) between the cleavable linkage and the positively charged amino acid on the N-terminal side, wherein the amino acid is positively loaded, for example, arg, his, or lys, is in a position equivalent to P7, P6 or P5 (for example, see Figure 14). The spacer (i.e., linker or bulking agent) between the cleavable linkage group and the positively charged amino acid at the P5, P6 or P7 position may for example comprise a plurality of neutral, uncharged amino acids (eg, glycine, alanine) , leucine, isoleucine, valine, proline, methionine, tryptophan, tyrosine, threonine, serine, and more preferably serine, glycine, alanine, β-alanine, α-aminobutyric acid, epsilon-amino-caproic acid, or derivatives of amino-PEG such as 8-, 11-, or 14-amino-3, 6, 9, 12-tetraoxatetradecanoic acid and PEG oligomers (eg, n = 2-6)). These spacers can be homogeneous (e.g., all, glycine, alanine, etc., or other individual amino acid) or heterogeneous (e.g., more than one type of amino acid or a hybrid amino acid / PEG molecule), and is preferably 3.5 -21 Á (.35 nm - 2.1 nm) (or 1 to 7 amino acids) in length. The separator may be comprised of neutral monomers such as ethylene glycol, for example, or other similar monomer units (e.g., propylene glycol), having a length of 3.5-21 A (.35 nm - 2.1 nm) such that the separator places the arginine (or another positively charged amino acid) within about 5 - 25 A (.5 nm - 2.5 nm) of the proline or proline substitute or analog of the cleavable linkage. Therefore, the presence of a positively charged amino acid within the distance as defined herein is an aspect of the invention that specifically contributes to the specificity of binding to the APCE sequence. In addition, when the amino acids within this space are substituted with polyethylene glycol of lengths of 3.5-21 A (.35 nm - 2.1 nm), the cleavage rate of the P12-N13 binding remains maximal for the next peptide, X-R6 - (PEG3) -Gii-Pi2-N13-Qi -Ei5 (SEQ ID NO: 10), where (PEG3) represents polyethylene glycol containing three units of ethylene glycol and X is any amino acid. Given the specificity of the positive charge effect that must be within a certain distance from P12 (Pi), this pegylated peptide is the basis for the development of the inhibitor through selective modifications or substitutions of proline, P12, to inhibit the activity of proteinase of APCE. Figure 14 shows the inhibitory effect of PACE of several peptides (SEQ ID NO: 11-13) with the indicated sequences in 20 μ of each peptide when Prol2 is substituted with pipecolic acid, a proline analogue. In a preferred embodiment, the invention comprises a compound having the formula: Xaai-Spi-Gly-Cyc-Xaa2-Sp2-Xaa3 (Formula I) · In this embodiment, Xaai is a positively charged amino acid, including but not limited to, arginine, histidine, or lysine. Spi is a spacer molecule comprising from 1 to 6 amino acids, preferably neutral amino acids including serine, glycine, alanine, or β-alanine, or gamma-amino-butyric acid, epsilon-amino-caproic acid, 8-amino-3 acid , 6, 9, 12-tetraoxatetradecanoic, ll-amino-3, 6, 9, 12-tetraoxatetradecanoic acid, 14-amino-3, 6, 9, 12-tetraoxatetradecanoic acid, or PEGn or PPGn, where n = 1- 6 units of ethylene glycol or propylene glycol, and where Spiit has a length from 3 Á to 21 Á (.3 nm - 2.1 nm). Gly is glycine. Cyc is a carbocyclic or heterocyclic ring. The carbocyclic ring may comprise 4, 5, 6, or 7 carbon atoms, and the heterocyclic ring may comprise 4, 5, 6, or 7 atoms, for example wherein at least one atom is a heteroatom such as nitrogen. Xaa2 is preferably glutamine, asparagine, serine, histidine, tyrosine, alanine, or phenylalanine, or any natural amino acid that can serve as the Pi 'atom in the cleavable linkage - ??' of the compound of the present invention. Sp2 is a spacer molecule and may be absent, or may comprise from 1 to 6 amino acids, preferably neutral amino acids including serine, glycine, alanine, β-alanine, aspartic acid, glutamic acid, or gamma-amino-butyric acid, epsilon acid -amino-caproic 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 PEGn or PPGn, where n = 1-6 units of ethylene glycol or propylene glycol, and where Spi has a length of 3 Á to 2.1 Á (.3 nm - 2.1 nm). Xaa3 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 from 1 to 10 amino acids extending in an N-terminal direction from Xaai, and a C-terminal oligopeptide having 1-10 amino acids. which extend in the C-terminal direction from Xaa3, wherein the N-terminal oligopeptide and the C-terminal oligopeptide may comprise one or more of the 20 naturally occurring amino acids. The compound is preferably placed in a pharmaceutically acceptable carrier as described elsewhere herein. In one embodiment, the invention is a method for altering a plasma relationship of a2-antiplasmin in a subject having a pre-treatment level of et-a2-antiplasmin in plasma and a pre-treatment level of Asn-a2-antiplasmin in plasma. In particular, the method comprises treating the subject with an inhibitor of the antiplasmin cleavage enzyme wherein the inhibitor specifically inhibits cleavage of the cleavage site of Pro-Asn Met-a2-antiplasmin by the antiplasmin cleavage enzyme, wherein after the treatment the subject has a post-treatment level of Met-a2-antiplasmin in plasma that is at least 5% higher than the pretreatment level of Met-a2-antiplasmin in plasma, and has a post-treatment level. treatment of Asn-a2-antiplasmin in plasma that is at least 5% lower than the level of pretreatment of Asn-a2-antiplasmin, thereby altering the ratio of a2-antiplasmin in plasma in the subject. In the method, the alteration of the a2-antiplasmin ratio in the subject preferably improves fibrinolysis in the subject. In in particular, the method is a treatment to inhibit or treat atherosclerosis, arterial thrombosis, venous thrombosis, stroke, or pulmonary embolism. In one embodiment of the method, the post-treatment level of et-a2-antiplasmin in plasma is at least 10% higher than the pretreatment level of Met-a.2-antiplasmin in plasma, and the post-treatment level of Asn- a2-antiplasmin in plasma is at least 10% lower than the level of pretreatment of Asn-a2-antiplasmin in plasma. In another embodiment, the post-treatment level of Met-a2-antiplasmin in plasma is at least 15% higher than the level of pretreatment of Met-a2-antiplasmin in plasma and the post-treatment level of Asn-a2-antiplasmin in plasma is at least 15% lower than the level of pretreatment of Asn-ct2-antiplasmin in plasma. In another embodiment, the level of post-treatment of Met-a, 2-antiplasmin in plasma is at least 20% higher than the level of pretreatment of Met-a, 2-antiplasmin in plasma and the post-treatment level of Asn-a2 -antiplasmin in plasma is at least 20% lower than the level of pretreatment of Asn-a, 2-antiplasmin in plasma. In another embodiment of the method, the post-treatment level of Met-a2-antiplasmin in plasma is at least 25% higher (and may be, for example, any greater percentage) than the level of pretreatment of Met-a2-antiplasmin in plasma and the post-treatment level of Asn-a2-antiplasmin in plasma is at least 25% lower (and may be, for example, any lower percentage) than the level of As-pretreatment ot2-antiplasmin in plasma. The invention is preferably, in one embodiment, a method of treating or inhibiting atherosclerosis, arterial thrombosis, venous thrombosis, stroke, or pulmonary embolism in a subject having a pretreatment level of Met-a2-antiplasmin in plasma and a pretreatment level. of Asn-a2-antiplasmin by altering the level of a2-antiplasmin in the subject. The method comprises treating the subject with an inhibitor of the antiplasmin cleavage enzyme wherein the inhibitor specifically inhibits cleavage of the Pro-Asn cleavage site of Met-a2-antiplasmin by the antiplasmin cleavage enzyme, wherein after treatment the subject has a post-treatment level of Met-a2-antiplasmin in plasma that is at least 5%, 10%, 15%, 20%, or 25% higher (or any higher percentage) than the level of pretreatment of Met-a2-antiplasmin in plasma, and has a post-treatment level of Asn-a2-antiplasmin in plasma that is at least 5%, 10%, 15%, 20%, or 25% lower (or any lower percentage) respectively, than the pretreatment level of Asn- a2-antiplasmin, thereby altering the a2-antiplasmin ratio in plasma in the subject. The alteration of the ratio of a2-antiplasmin in the subject is preferably presented by improvement of fibrinolysis in the subject. The present invention further contemplates therapeutic compounds for treating cancers and disorders comprising FAP such as those described in U.S. Patent No. 6,949,514, which is expressly incorporated herein by reference. Therapeutic compounds comprise the APCE inhibitors of the present invention as described herein that are conjugated to carrier compounds that are capable of passing through the cell membrane, including, but not limited to, protein transduction domains (PTD) ). PTDs are positively charged peptides or peptide-like molecules that permeate the lipid bi-layers of the cell membrane. Typically, PTDs contain various arginine residues and can be used to distribute other agents, such as peptides, proteins, oligonucleotides or small molecules through a cell membrane and into the cytosol. A PTD is a highly efficient molecular transporter formed by synthesizing an oligomer of arginines that are altered with eACA. Examples of PTD that can be used in the present invention are shown in U.S. Patent Nos. 7,166,692; 7,217,539; 7,053,200; 6,835,810; 6,645,501; and in published U.S. Patent Applications Nos. 2002/0009491; 2003/0032593; 2003/0162719; 2006/0159719; 2006/0293234; and 2007/0105775, each of which is expressly incorporated herein by reference in its entirety. Utility The utilities of the present invention include, but are not limited to: (1) prevention or reduction of atherosclerotic plaque development and progress, especially in high-risk patients; (2) prevention or reduction of the development of arterial or venous blood clot formation (atherothrombotic and venous thrombus disorders), especially in conditions recognized as high risk for these clots, that is, cardiac apoplexy or stroke; (3) improved maintenance of vessel opening by continuous administration of the APCE inhibitor, possibly in association with the simultaneous administration of low doses of plasminogen activator drugs; (4) prevention or reduction of fibrin formation where it can cause persistent chronic or acute symptoms in association with inflammatory conditions such as all forms of arthritis, organ fibrosis, scarring undesirable, and cancer and its metastasis; (5) reduction of the risk of bleeding is induced as a hyperfibrinolytic state, since a2APpr0 inhibits plasmin in the solution state as well as a2APact, when it is not reticulated in fibrin; (6) aid in the prevention and therapy of fibrin deposits that interfere with the function of the organs as can be seen in atherothrombotic disease, such as coronary artery thrombosis, stroke, pulmonary embolism, all other forms of arterial and venous thrombosis , inflammatory conditions, and cancer and their metastases; (7) determination if a subject has a Trp6 or Arg6 polymorphism in a2-antiplasmin; and (8) detection of inhibitors of the antiplasmin cleavage enzyme. Therefore, various embodiments of the present invention include, but are not limited to: (1) a therapeutic method for promoting fibrin digestion in vivo, which comprises administering to a subject an effective amount of an inhibitor of the cleavage enzyme of antiplasmin; (2) fluorescent resonance energy transfer peptides (FRET) to act as an APCE substrate; (3) an inhibitor of the antiplasmin cleavage enzyme, wherein the inhibitor can comprise, or is linked to, a polymeric binder, linker or carrier; (4) an inhibitor of the antiplasmin cleavage enzyme, which is effective in binding to or blocking the a2-antiplasmin binding site, or a2-antiplasmin pro-asn cleavage site of the antiplasmin cleavage enzyme; (5) a method for improving fibrin digestion in vivo, which comprises providing a subject in need of clot digestion or coagulum prevention, simultaneously or in sequence, an amount of plasminogen activator and an inhibitor of cleavage enzyme of antiplasmin, wherein the amount of plasminogen activator is less than the amount provided in the normal therapeutic protocol absent from the inhibitor of the antiplasmin cleavage enzyme; and (6) a DNA encoding the FRET-peptide or inhibitor peptide contemplated herein, and a plasmid or host vector containing the DNA. Other embodiments of the present invention provide pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the compounds described herein, formulated together with one or more carriers (additives) and / or pharmaceutically acceptable diluents. As described in detail below, the pharmaceutical compositions of the present invention can be formulated in a special manner for administration in solid or liquid form, including those adapted for the following: (1) oral administration, eg, aqueous solutions or suspensions or non-aqueous, tablets, for example, those directed for buccal, sublingual and systemic absorption, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular, intravenous or epidural injection such as for example a sterile solution or suspension, or sustained release formulation; (3) topical application, for example, as a cream, ointment, or controlled release patch or spray applied to the skin; (4) intravaginally or intra-rectally, for example, as a cream or foam; (5) sublingually; (6) ocularly; (7) transdermally; u (8) nasally. The phrase "therapeutically effective amount" as used herein means that amount of a compound, material, or composition comprising a compound of the present invention that is effective to produce the same desired therapeutic effect in at least one sub-population of cells in an animal at a reasonable benefit / risk ratio applicable to any medical treatment.
The phrase "pharmaceutically acceptable" is used herein to refer to those compounds, materials, compositions and / or dosage forms that are, within the scope of reasonable medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problem or complication, in proportion to a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein means a pharmaceutically acceptable composition, material or carrier, such as a liquid or solid filler, diluent, excipient, or solvent encapsulating material, comprised in carrying or transporting the present composed from one organ, or a portion of the body, to another organ, or a portion of the body. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not harmful to the patient. Some examples of materials 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, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as coconut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) damping agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline solution; (18) Ringer's solution; (19) ethyl alcohol; (20) solutions buffered in pH; (21) polyesters, polycarbonates and / or polyanhydrides; and (22) other non-toxic compatible substances employed in pharmaceutical formulations. As set forth above, certain embodiments of the present compounds contain a basic functional group, such as amino or alkylamino, and thus are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable acids. The term "pharmaceutically acceptable salts" in this regard refers to relatively non-toxic inorganic and organic acid addition salts of the compounds of the present invention. These salts can be prepared in situ in the delivery vehicle or the process of making the dosage form, or by reacting separately a purified compound of the invention in its free base form with a suitable organic or inorganic acid, and by isolating salt formed in this manner during the subsequent purification. Representative salts include the salts of hydrobromide, hydrochloride, sulfate, bisulfate, phosphate, nitrate, acetate, valerate, oleate, palmitate, stearate, laurate, benzoate, lactate, phosphate, tosylate, citrate, maleate, fumarate, succinate, tartrate, naphthylate. , mesylate, glucoheptonate, lactobionate, and laurylsulfonate, and the like. The pharmaceutically acceptable salts of the present compounds include the conventional non-toxic salts or quaternary ammonium salts of the compounds, for example, of non-toxic organic or inorganic acids. For example, these conventional non-toxic salts include those derived from inorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic, phosphoric, nitric, and the like; and the prepared salts of organic acids such as acetic, propionic, succinic, glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, palmitic, maleic, hydroximic, phenylacetic, glutamic, benzoic, salicyclic, sulfanilic, 2-acetoxybenzoic, fumaric , toluenesulfonic, methanesulfonic, ethane-disulfonic, oxalic, isothionic, and the like. In other cases, the compounds of the present invention may contain one or more acid functional groups and thus are capable of forming pharmaceutically acceptable salts with pharmaceutically acceptable bases. The term "pharmaceutically acceptable salts" in these cases refers to the relatively non-toxic inorganic and organic base addition salts of the compounds of the present invention. These salts can also be prepared in situ in the delivery vehicle or in the process of preparing dosage forms, or by reacting separately the purified compound in its free acid form with a suitable base, such as hydroxide, carbonate or bicarbonate of a pharmaceutically acceptable metal cation, with ammonia, or with a pharmaceutically acceptable primary, secondary or tertiary organic amine. Representative alkaline or alkaline earth salts include the lithium, sodium, potassium, calcium, magnesium and aluminum salts and the like. Representative organic amines useful for the formation of the base addition salts include ethylamine, diethylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, and the like. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate and magnesium stearate, as well as coloring agents, release agents, coating agents, sweetening, flavoring and flavoring agents, preservatives and antioxidants may also be present in the compositions. Examples of pharmaceutically acceptable antioxidants include: (1) water soluble antioxidants, such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, and the like; and (3) metal chelating agents, such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like. As noted above, the formulations of the present invention include those suitable for oral, nasal, topical (including buccal and sublingual), rectal, vaginal and / or parenteral administration. The formulations can be conveniently presented in the unit dosage form and can be prepared by any method well known in the pharmacy art. The amount of active ingredient that can be combined with a carrier material to produce a dosage form will vary depending on the hosts being treated, the particular mode of administration. The amount of active ingredient that can be combined with a carrier material to produce a single dose form will generally be that amount of the compound that produces a therapeutic effect. In general, one hundred percent, this amount will vary from about 1 percent to about ninety-nine percent active ingredient, preferably about 5 percent. percent to about 70 percent, more preferably from about 10 percent to about 30 percent. In certain embodiments, a formulation of the present invention comprises an excipient selected from the group consisting of cyclodextrins, liposomes, micelle-forming agents, e.g., bile acids, and polymeric carriers, e.g., polyesters and polyanhydrides. Methods for preparing these formulations or compositions may include the step of bringing into association a compound of the present invention with the carrier and, optionally, one or more auxiliary ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association a compound of the present invention with liquid carriers, or finally divided, solid carriers, or both, and then, if necessary, forming the product. Formulations of the invention suitable for oral administration may be in the form of capsules, amylaceous capsules, pills, tablets, lozenges (using a flavored base, usually sucrose and acacia gum or tragacanth), powders, granules, or as a solution or a suspension in an aqueous or non-aqueous liquid, or as an oil in water or water in oil emulsion, or as a elixir or syrup, or as tablets (using an inert base, such as gelatin and glycerin, or sucrose and acacia) and / or as buccal washes and the like, each containing a predetermined amount of a compound of the present invention as a active ingredient. A compound of the present invention can also be administered as a bolus, or a paste. In the solid dosage forms of the invention for oral administration (capsules, tablets, pills, powders, granules, and the like), the active ingredient is mixed with one or more pharmaceutically acceptable carriers, such as sodium citrate or dicalcium phosphate, and / or any of the following: (1) fillers or extenders, such as starches, lactose, sucrose, glucose, mannitol, and / or silicic acid; (2) binders, such as, for example, carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose and / or acacia; (3) humectants, such as glycerol; (4) disintegrating agents, such as agar-agar, calcium carbonate, potato starch or tapioca, alginic acid, certain silicates, and sodium carbonate; (5) agents that retard the solution, such as paraffin; (6) absorption accelerators, such as quaternary ammonium compounds; (7) wetting agents, such as, for example, cetyl alcohol, glyceryl monostearate, and nonionic surfactants; (8) Absorbents, such as kaolin and clay bentonite; (9) lubricants, such as talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents. In the case of capsules, tablets and pills, the pharmaceutical compositions may also comprise buffering agents. Solid compositions of a similar type can also be used as filling agents in soft gelatine gelatin capsules using excipients such as lactose or milk sugars, as well as high molecular weight polyethylene glycols, and the like. A tablet may be made by compression or molding, optionally with one or more auxiliary ingredients. Compressed tablets can be prepared using binder (e.g., gelatin or hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (e.g., sodium starch glycolate or cross-linked sodium carboxymethyl cellulose), dispersing or active agent on the surface. Molded tablets can be made by molding in a suitable machine a mixture of the wetted powder compound with an inert liquid diluent. Tablets, and other solid dosage forms of the pharmaceutical compositions of the present invention, such as dragees, capsules, pills and granules, may optionally be scored or prepared with coatings and shells, such as synthetic coatings and other coatings either known in the pharmaceutical formulating art. They may also be formulated to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethyl cellulose in varying proportions to provide the desired release profile, other polymer matrices, liposomes and / or microspheres. They can be formulated for rapid release, eg, freeze drying. They can be sterilized for example by filtration through a bacteria retention filter, or by incorporating sterilizing agents in the form of sterile solid compositions which can be dissolved in sterile water, or some other sterile injectable medium immediately before use. These compositions may also optionally contain opacifying agents and may be of a composition that they release the active ingredients only, or preferentially, in a certain portion of the gastrointestinal tract, optionally, in a delayed manner. Examples of embedding compositions that may be used include light polymeric substances. The active ingredient may also be in microencapsulated form, if appropriate, with one or more of the excipients described above. 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, liquid dosage forms may contain inert diluents commonly used in the art, such as, for example, water or other solvents, solubilizing agents and emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate. , benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils (in particular, cotton, peanut, corn, germ, olive, castor bean and sesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycols and sorbitan fatty acid esters, and mixtures thereof. In addition to the inert diluents, the oral compositions may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, coloring, flavoring and preservative agents. The suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, and mixtures thereof. same. The formulations of the pharmaceutical compositions of the invention for rectal or vaginal administration can be present as a suppository, which can be prepared by mixing one or more compounds of the invention with one or more suitable non-irritating excipients or carriers comprising, for example, cocoa butter, polyethylene glycol, a suppository wax or a salicylate, and They are solid at room temperature, but liquid at body temperature, and will therefore melt in the rectum or vaginal cavity and release the active compound. Formulations of the present invention that are suitable for vaginal administration also include buffers, creams, gels, pastes, foams or spray formulations containing carriers as are known in the art to be appropriate. Dosage forms for topical or transdermal administration of a compound 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 may contain, in addition to an active compound of this invention, excipients, such as animal and vegetable fats, oils, waxes, paraffins, starch, tragacanth, cellulose derivatives, polyethylene glycols, silicones, bentonites, acid silicic, talc and zinc oxide, or mixtures thereof. The powders and sprays may contain, in addition to a compound of this invention, excipients such as lactose, talc, silicic acid, aluminum hydroxide, calcium silicates and polyamide powder, or mixtures of these substances. Sprays may additionally contain customary propellants, such as chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, such as butane and propane. Transdermal patches have the additional advantage of providing controlled distribution of a compound of the present invention to the body. These dosage forms can be made by dissolving or dispersing the compound in the appropriate medium. Absorption enhancers may also be used to increase the flow of the compound through the skin. The velocity of this flow can be controlled either by providing a speed control membrane or by dispersing the compound in a gel or polymer matrix. Also contemplated, which are within the scope of this invention, ophthalmic formulations, eye ointments, powders, solutions, and the like. The pharmaceutical compositions of this invention suitable for parenteral administration comprise one or more compounds of the invention in combination with one or more sterile, pharmaceutically isotonic solutions, dispersions, suspensions or emulsions, aqueous or non-aqueous. acceptable, or sterile powders, which can be reconstituted in injectable solutions or dispersions, sterile just before use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes that render the formulation isotonic with the blood of the proposed recipient or agents of suspension or thickeners. Examples of suitable aqueous and non-aqueous carriers that can be employed in the pharmaceutical compositions of the invention include water, ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such such as olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. These compositions may also contain adjuvants such as preservatives, wetting agents, emulsifying agents and dispersing agents. The prevention of the action of microorganisms in the present compounds can be ensured by the inclusion of various antibacterial and anti-fungal agents, for example, paraben, chlorobutanol, phenol-sorbic acid, and the like. It may also be desirable to include isotonic agents, such as sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be caused by the inclusion of agents that delay absorption such as aluminum monostearate and gelatin. In some cases, in order to prolong the effect of a drug, it is desirable to delay the absorption of the subcutaneous or intramuscular injection drug. This can be achieved by the use of a liquid suspension of crystalline or amorphous material having poor water solubility. The rate of absorption of the drug then depends on its rate of dissolution which, in turn, may depend on the size of the crystal and crystalline form. Alternatively, delayed absorption of a parenterally administered drug form is achieved by dissolving or suspending the drug in an oil vehicle. Injectable depot forms are made by forming microencapsulation matrices of. the present compounds in biodegradable polymers such as polylactide-polyglycolide. Depending on the ratio of the drug to the polymer, and the nature of the particular polymer employed, the rate of drug release can be controlled. Examples of other biodegradable polymers include poly (orthoesters) and poly (anhydrides). Depot injectable formulations are also prepared by trapping the drug in liposomes or microemulsions that are compatible with body tissue. When the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given per se or as a pharmaceutical composition containing, for example, from 0.1 to 99.5% (more preferably from 0.5 to 90%) of the active ingredient in combination with a pharmaceutically acceptable carrier. The preparations of the present invention can be given orally, parenterally, topically or rectally. Of course, they are given in appropriate forms for each administration route. For example, they are administered in tablets or capsule form, by injection, inhalation, eye lotion, ointment, suppository, etc., administration by injection, infusion or inhalation; topical by lotion or ointment; and rectal by suppositories. Oral administrations are preferred. The phrases "parenteral administration" and "parenterally administered" as used herein means different modes of administration of enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intra-arterial, intrathecal, intracapsular injection. , intraorbital, intracardiac, intradermal, intraperitoneal, transtratecal, subcutaneous, subeuticular, intra-articular, subcapsular, subarachnoid, intraspinal and intrasternal, and infusion. The phrases "systemic administration", "systemically administered", "peripheral administration" and "peripherally administered" as used herein mean the administration of a compound, drug or other material other than directly in the central nervous system. , such that it enters the patient's system and in this way is subjected to metabolism and other similar processes, for example, subcutaneous administration. These compounds can be administered to humans and other animals for therapy by any suitable route of administration, including orally, nasally, as by, for example, a spraying, rectally, intravaginally, parenterally, intracisternally and topically , as for powders, ointments or drops, including buccal and sublingual. Despite the selected route of administration, the compounds of the present invention, which can be used in a suitable hydrated form, and / or the pharmaceutical compositions of the present invention, are formulated in pharmaceutically acceptable dosage forms by conventional methods known per se. those skilled in the art. The actual dose levels of the active ingredients in the pharmaceutical compositions of this invention can be varied to obtain an amount of the ingredient active that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected level of dose will depend on a variety of factors including the activity of the particular compound of the present invention, employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound used, duration of treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, age, sex, weight, condition, general health and previous medical history of the patient in question , and similar factors well known in medical techniques. A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of the required pharmaceutical composition. For example, the physician or veterinarian may start doses of the compounds of the invention used in the pharmaceutical composition at levels lower than those required in order to achieve the desired therapeutic effect and gradually increase the dose until the desired effect is achieved. In general, an adequate daily dose of a compound of the invention will be that amount of the compound that is the lowest effective dose to produce an effect therapeutic. This effective dose will generally depend on the factors described above. In general, the intravenous, intracerebroventricular and subcutaneous doses of the compounds of this invention for a patient, when used for the indicated analgesic effects, will vary from about 0.0001 to about 100 mg per kilogram of body weight per day. If desired, the effective daily dose of the active compound can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms . While it is possible for a compound of the present invention to be administered alone, it is preferred to administer the compound as a pharmaceutical formulation (composition). In another aspect, the present invention provides pharmaceutically acceptable compositions comprising a therapeutically effective amount of one or more of the present compounds, as described above, formulated with one or more pharmaceutically acceptable carriers (additives) and / or diluents. As described in detail below, the pharmaceutical compositions of the present invention can be formulated in a special manner for administration in solid or liquid form, including those adapted for the following: (1) oral administration, example, aqueous or non-aqueous solutions or suspensions, tablets, boluses, powders, granules, pastes for application to the tongue; (2) parenteral administration, for example, by subcutaneous, intramuscular or intravenous injection such as for example a sterile solution or suspension; (3) topical application, for example, as a cream, ointment or spray applied to the skin, lungs, or oral cavity; or (4) intravaginally or intravectally, for example, as a pessary, cream or foam; (5) sublingually; (6) ocular shape; (7) transdermally; u (8) nasally. The compounds according to the invention can be formulated for administration in any convenient way for use in human or veterinary medicine, by analogy with other pharmaceutical products. The term "treatment" is proposed to also encompass prophylaxis, therapy and cure. The patient receiving this treatment is any animal in need, including primates, in particular humans and other mammals such as equines, cattle, pigs and sheep; and poultry and pets in general. The compound of the invention can be administered as such or in mixtures with pharmaceutically acceptable carriers and can also be administered in conjunction with antimicrobial agents such as penicillins, cephalosporins, aminoglycosides and glycopeptides. Conjunctive therapy, it includes in this manner sequential, simultaneous and separate administration of the active compound in a way that the therapeutic cts of the first administered do not completely disappear. when the subsequent one is administered. As used herein, the term "subject" or "patient" refers preferentially to a warm-blooded animal such as a mammal that is afflicted with a particular inflammatory disease state. It is understood that guinea pigs, dogs, cats, rats, mice, horses, cattle, sheep and humans are examples of animals within the scope of the meaning of the term. A therapeutically ctive amount of the compound used in the treatment described herein can be readily determined by the attending physician, as one skilled in the art, by the use of conventional techniques and by observing the results obtained under analogous circumstances. In determining the therapeutically ctive dose, several factors are considered by the attending physician, including, but not limited to: the species of the mammal; its size, age, and general health; the specific disease or condition included; the degree of involvement or severity of the disease or condition; the response of the individual subject; the particular compound administered; the mode of administration; the characteristic bioavailability of the preparation administered; the selected dose regimen; he use of concomitant medication; and other relevant circumstances. A therapeutically ctive amount of the compositions of the present invention will generally contain sufficient active ingredient (i.e., PACE or inhibitor thereof) to distribute from about 0.1 g / kg to about 100 mg / kg (active ingredient weight / weight) patient's body). Preferably, the composition will distribute at least 0.5 μg / kg to 50 mg / kg, and more preferably at least 1 μg / kg to 10 mg / kg. The practice of the method of the present invention comprises administering to a subject a therapeutically ctive amount of the active ingredient, in any suitable local or systemic formulation, in an amount ctive to distribute the doses listed above. The dose may be administered on a one-time basis, or (for example) one to five times per day or once or twice a week, or continuously, by venous drip, or other means, depending on the therapeutic ct wanted. As noted, preferred amounts and preferred modes of administration are capable of being determined by one skilled in the art. One skilled in the art to prepare formulations can easily select the appropriate form and the mode of administration depending on the particular characteristics of the selected compound, the disease state being treated, the condition of the disease, and other pertinent circumstances using formulation technology known in the art, described, or for example, in Remington's Pharmaceutical Sciences, latest edition, Mack Publishing Co. The pharmaceutical compositions can be made using known techniques. Typically, the therapeutically ctive amount of the compound will be mixed with a pharmaceutically acceptable carrier. The half-lives of the molecules described herein can be extended by being conjugated to other molecules such as polymers using methods known in the art to form drug-polymer conjugates. For example, the molecules can be attached to inert polymer molecules known in the art, such as a polyethylene glycol (PEG) molecule in a method known as "pegylation". Therefore, the pegylation can extend the life in vivo and in this way the therapeutic ctiveness of the molecule. PEG molecules can be modified by functional groups, for example as shown in Harris et al., "Pegylation, A Novel Process for Modifying Phararmacokinetics ", Clin Pharmacokinet, 2001: 40 (7); 539-551, and the amino-terminal end of the molecule, or residue of cysteine if present, or another binding amino acid therein can be linked to this, wherein the PEG molecule may have one or a plurality of one or more types of molecules. By "polyethylene glycol" or "PEG" is meant a polyalkylene glycol compound or a derivative thereof, with or without coupling or derivatizing agents with coupling or activating portions (for example, with thiol, triflate, tresylate, azirdine, oxirane, or preferably with a portion of maleimide). Compounds such as maleimido-monomethoxy-PEG are exemplified or activated PEG compounds of the invention. Other polyalkylene glycol compounds, such as polypropylene glycol, can be used in the present invention. Other suitable polymer conjugates include, but are not limited to, non-polypeptide polymers, charged or neutral polymers of the following types: dextran, colominic acids or other carbohydrate-based polymers, biotin derivatives and dendrimers, by way of example. The term PEG is also proposed to include other polymers of the class of polyalkylene oxides. The PEG can be linked to any N-terminal amino acid of the molecule described herein and / or can be linked to an amino acid residue in the 3 'direction of the N-terminal amino acid, such as lysine, histidine, tryptophan, aspartic acid , glutamic acid, and cysteine, by way of example, or other amino acids known to those skilled in the art. The PEG carrier molecule attached to the peptide can vary in molecular weight from about 200 to 20,000 PM. Preferably, the PEG portion will be from about 1,000 to 8,000 MP, more preferably from about 3,250 to 5,000 MP, more preferably from about 5,000 PM. The actual number of PEG molecules covalently linked by. The molecule of the invention can vary widely depending on the desired stability (ie, serum half-life). The molecules contemplated herein can be linked to PEG molecules using known techniques, for example (but not limited to), in U.S. Patent Nos. 4,179,337; 5,382,657; 5,972,885; 6,177,087; 6,165,509; 5,766,897; and 6,217,869; the specifications and figures of which are expressly incorporated herein by reference herein. Alternatively, it is possible to trap the molecules in microcapsules prepared, for example, by co-accumulation techniques or by interfacial polymerization (for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmetacylate) microcapsules, respectively), in distribution of colloidal drugs (eg, liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in macroemulsions. These The techniques are described in the latest edition of Remington's Pharmaceutical Sciences. U.S. Patent No. 4,789,734 describes methods for encapsulating biochemicals in liposomes and is thus expressly incorporated by reference herein. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants, if required, and the material dialyzed or treated with ultrasound, as necessary. A review of the known methods is given by G. Gregoriadis, Chapter 14, "Liposomes", Drug Carriers in Biology and Medicine, PP. 287-341 (Academic Press, 1979). Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be adapted for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the agents can be incorporated and the microspheres, or products composed of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, United States Patent Nos. 4,906,474; 4,925,673; and 3,625,214 which are incorporated herein by reference. When the composition is to be used as an injectable material, it can be formulated in a conventional injectable carrier. Suitable carriers include solutions phosphate-buffered saline, biocompatible and pharmaceutically acceptable, which are preferably isotonic. For reconstitution of a lyophilized product according to this invention, a sterile diluent may be employed, which may contain materials generally recognized to approximate physiological conditions and / or as required by governmental regulation. In this regard, the sterile diluent may contain a buffering agent to obtain a physiologically acceptable pH, such as sodium chloride, saline, phosphate buffered saline, and / or other substances that are physiologically acceptable and / or safe for use . In general, the material for intravenous injection in humans must comply with the regulations established by the Food and Drug Administration of the United States, which are available to those in the field. The pharmaceutical composition may also be in the form of an aqueous solution containing many of the same substances as described above for the reconstitution of a lyophilized product. The compounds may also be administered as a pharmaceutically acceptable acid or base addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and acid fumaric, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-alkyl and aryl-amines and substituted ethanolamines. As mentioned above, the compounds of the invention can be incorporated into pharmaceutical preparations that can be used for therapeutic purposes. However, the term "pharmaceutical preparation" is proposed in a broader sense herein to include preparations containing a glycosulpeptide composition according to this invention, used not only for therapeutic purposes but also for reagent or diagnostic purposes as it is known in the art, or for tissue culture. The proposed pharmaceutical preparation for therapeutic use should contain a "pharmaceutically acceptable" or "therapeutically effective amount" of a molecule as defined herein. All of the test methods listed herein are well within the ability of one skilled in the art, given the teachings provided herein. All references, patents and applications for patent cited herein are hereby incorporated in their entireties as a reference. In particular, U.S. patent applications serial numbers 10 / 774,242, 60 / 445,774, 60 / 811,568, and 60 / 836,365 are hereby expressly incorporated by reference in their entireties. Although the present invention and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations may be made herein without departing from the spirit and scope of the invention as defined by the appended claims. However, the scope of the present application is not intended to be limited to the particular modalities of the. process, elaboration, compositions of matter, means, methods and steps described in the specification. As will be readily appreciated by a person skilled in the art from the description of the present invention, the processes, processing, compositions of matter, media, or steps, which currently exist or are developed below, form substantially the same function or achieve substantially the same results as the corresponding embodiments described herein may be used according to the present invention. Accordingly, it is proposed that the appended claims include within their scope, these processes, elaboration, compositions of matter, means, methods or steps.
References Collen D. The plasminogen (fibrinolytic) system. Thromb.Haemost. 1999; 82: 259-270. Wiman B, Collen D. On the kinetics of the reaction between human antiplasmin and plasmin. Eur.J Biochem. 1978; 84: 573-578. Mullertz S, Clemmensen I. The primary inhibitor of plasma in human plasma. Biochem. J. 1976; 159: 545-553. Collen D. Identification and some properties of a new fast-reacting plasmin inhibitor in human plasma. Eur. J. Biochem. 1976; 69: 209-216. Bangert K, Johnsen AH, Christensen U, Thorsen S. Different N-terminal forms of alpha 2-plasmin inhibitor in human plasma. Biochem. J. 1993; 291 (Pt 2): 623-625. Koyama T, Koike Y, Toyota S et al. Different NH2-terminal form with 12 additional residues of alpha 2-plasmin inhibitor from human plasma and culture media of Hep G2 cells. Biochem. Biophys. Res. Commun. 1994; 200: 417-422. Lee KN, Jackson KW, Christiansen VJ, Chung KH, McKee PA. A novel plasma proteinase potentiates alpha2-antiplasmin inhibition of fibrin digestion. Blood 2004; 103: 3783-3788. Lee KN, Jackson KW, Christiansen VJ et al. Antiplasmin-cleaving enzyme is a soluble form of fibroblast activation protein. Blood 2006; 107: 1397-1404. Hirosawa S, Nakamura Y, Yura O, Sumi Y, Aoki N.
Organization of the human alpha 2-plasmin inhibitor gene. Proc.Natl.Acad.Sci. U.S. To 1988; 85: 6836-6840. Holmes WE, Nelles L, Lijnen HR, Collen D. Primary structure of human alpha 2-antiplasmin, to serine protease inhibitor (serpin). J. Biol. Chem.1987; 262: 1659-1664. Tone M, Kikuno R, Kume-Iwaki A, Hashimoto-Gotoh T. Structure of human alpha 2-plasmin inhibitor deduced from the cDNA sequence. J. Biochem. (Tokyo) 1987; 102: 1033-1041. Lind B, Thorsen S. A novel missense mutation in the human plasmin inhibitor (alpha2-antiplasmin) gene associated with a bleeding tendency. Br. J. Haematol. 1999; 107: 317-322. Wiman B. Affinity-chromatographic purification of human alpha 2-antiplasmin. Biochem. J. 1980; 191: 229-232. Beebe DP, Aronson DL. An automated fibrinolytic assay performed in microtiter plates. Thromb.Res. 1987; 47: 123-128. Jones AJ, Meunier AM. A precise and rapid microtiter píate clot lysis assay: methodology, kinetic modeling and measurement of catalytic constants for plasminogen activation during fibrinolysis. Thromb. Haemost. 1990; 64: 455-463. Lee KN, Lee SC, Jackson KW et al. Effect of phenylglyoxal-modified alpha2-antiplasmin on urokinase-induced fibrinolysis. Thromb. Haemost. 1998; 80: 637-644. Tam JP. Synthetic peptide vaccine design: synthesis and properties of a high-density multiple antigenic peptide system. Proc. Nati Acad. Sci. OR . S. A 1988; 85: 5409-5413. von Rokitansky C. Abnormal conditions of the arteries. A Manual of Pathological Anatomy. London: Sydenham Society; 1852: 261-275. Virchow R. Phlogose and Thrombose im Gefasssystem. In: Virchow R, ed. Gesammelte Abhandlungen zur issenschatflichen Medizin. Frankfurt: Meidinger Sohn & Co.; 1856: 458-636. Smith EB. Fibrinogen, fibrin and fibrin degradation products in relation to atherosclerosis. Clin. Haematol 1986; 15: 355-370. Jorgensen L. The role of platelets in the initial stages of atherosclerosis. J. Thromb. Haemost. 2006; 4: 1443-1449. Schwartz CJ, Valente AJ, Kelley JL, Sprague EA, Edwards EH. Thrombosis and the development of atherosclerosis: Rokitansky revisited. Semin. Thromb. Hemost. 1988; 14: 189-195. Fuster V, Badimon L, Badimon JJ, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes (1). N. Engl. J. Med. 1992; 326: 242-250. Bini A, Kudryk BJ. Fibrinogen in human atherosclerosis. Ann. . Y . Acad. Sci. 1995; 748: 461-471. f Bini A, Fenoglio JJ, Jr., Mesa-Tejada R, Kudryk B, Kaplan KL. Identification and distribution of fibrinogen, fibrin, and tibrin (ogen) degradation products in atherosclerosis. Use of monoclonal antibodies. Arteriosclerosis 1989; 9: 109-121.
Loscalzo J. The relation between atherosclerosis and thrombosis. Circulation 1992; 86: 11195-11199. Smith EB, Keen GA, Grant A, Stirk C. Fate of fibrinogen in human arterial intima. Arteriosclerosis 1990; 10: 263-275. Smith EB, Thompson WD. Fibrin as a factor in atherogenesis. Thromb.Res. 1994; 73: 1-19. White JG. Platelets and atherosclerosis. Eur. J.Clin. Invest 1994; 24 Suppl 1: 25-29. Xiao Q, Danton MJ, Witte DP et al. Fibrinogen deficiency is compatible with the development of atherosclerosis in mice. J. Clin. Invest 1998; 101: 1184-1194. Marutsuka K, Hatakeyama K, Yamashita A, Asada Y. Role of thrombogenic factors in the development of atherosclerosis. J. Atheroscler. Thromb. 2005; 12: 1-8. Kathiresan S, Yang Q, Larson MG et al. Common genetic variation in five thrombosis genes and relations to plasma hemostatic protein level and cardiovascular disease risk. Arterioscler. Thromb. Vasc. Biol. 2006; 26: 1405-1 12. Spurlock BO, Chandler AB. Adherent platelets and surface microthrombi of the human aorta and left coronary artery: a scanning electron microscopy feasibility study. Scanning Microsc. 1987; 1: 1359-1365. Meade TW, Ruddock and, Stirling Y, Chakrabarti R, Miller GJ. Fibrinolytic activity, clotting factors, and long- term incidence of ischaemic heart disease in the Northwick Park Heart Study. Lancet 1993; 342: 1076-1079. Xiao Q, Danton MJ, Witte DP et al. Plasminogen deficiency accelerates vessel wall disease in mice predisposed to atherosclerosis. Proc. to you . Acad. Sci. OR . S. A 1997; 94: 10335-10340. Kwaan HC. Physiologic and pharmacologic implications of fibrinolysis. Artery 1979; 5: 285-290. Smith EB. Haemostatic factors and atherogenesis. Atherosclerosis 1996; 124: 137-143. Juhan-Vague I, Collen D. On the role of coagulation and fibrinolysis in atherosclerosis. Ann. Epidemiol. 1992; 2: 427-438. Tanaka K, Sueishi K. The coagulation and fibrinolysis systems and atherosclerosis. Lab Invest 1993; 69: 5-18. Favier R, Aoki N, MP. Congenital alpha (2) -plasmin inhibitor deficiencies: a review. Br. J. Haematol. 2001; 114: 4-10. Eda K, Ohtsuka S, Seo Y et al. Conservative treatment of hemolytic complication following coil embolization in two adult cases of patent ductus arteriosus. Jpn.Circ.J. 2001; 65: 834-836. Levi M, Roem D, Kamp A et al. Assessment of the relative contribution of different protease inhibitors to the inhibition of plasmin in vivo. Thromb. Haemost. 1993; 69: 141-146. Harpel PC, osesson W. Degradation of human fibrinogen by plasmids alpha2-macroglobulin-enzyme complexes. J.Clin. Invest 1973; 52: 2175-2184. Aoki N, Harpel PC. Inhibitors of the fibrinolytic enzyme system. Semin. Thromb. Hemost. 1984; 10: 24-41. Table I. Cyclic Amines and Analogs of Proline 4-Hydroxypyrrolidine-2-carboxylic acid (cis and trans) 3-Phenylpyrrolidine-2-carboxylic acid (cis and trans) 3-hydroxypyrrolidine-2-carboxylic acid (cis and trans) 4-Hydroxypyrrolidine-2-carboxylic acid (cis and trans) 2-Ethylthiazolidine-4-carboxylic acid (cis and trans) 2-Methylthiazolidine-4-carboxylic acid (cis and trans) 2-phenylthiazolidine-4-carboxylic acid (cis and trans) trans) 5,5-dimethylthiazolidine-4-carboxylic acid thiazolidine-2-carboxylic acid (cis and trans) thiazolidine-4-carboxylic acid (cis and trans) azetidine-2-carboxylic acid (cis and trans) thiazolidine-2-acid carboxylic (cis and trans) thiazolidine-4-carboxylic acid (cis and trans) amino-L-proline methyl ester cyano-L-proline methyl ester 4-cyano-L-proline 3,4-dehydro-L-proline 4-fluoro-L-proline Lisyl-piperidide N- (4-chlorobenzyl) 4-0x0-4- (1-piperidinyl) -1, 3- ( s) -butane diamine bromocyclopentylcarboxylic acid chlorocyclopentylcarboxylic acid fluorocyclopentylcarboxylic acid cis-3-methylproline cis-3-ethylproline cis-3-isopropylproline cis-3-isopentanilproline homoproline benzyl-proline (2-fluoro-benzyl) -proline (3-fluoro-benzyl) -proline (-fluoro-benzyl) -proline (2-chloro-benzyl) -proline (3-chloro-benzyl) -proline (4-chloro-benzyl) -proline (2-bromo-benzyl) ) -proline (3-bromo-benzyl) -proline (4-bromo-benzyl) -proline phenethyl-proline (2-methyl-benzyl) -proline (3-methyl-benzyl) -proline (4-methyl-benzyl) -proline (2-nitro-benzyl) -proline (3-nitro-benzyl) -proline-nitro -benzyl) -proline (1-Naphthalenylmethyl) -proline (2-Naphthalenylmethyl) -proline (2,4-dichloro-benzyl) -proline-dichloro-benzyl) -proline (3,4-difluoro-benzyl) -proline -trifluoromethyl-benzyl-proline (3-trifluoromethyl-benzyl) -proline -trifluoromethyl-benzyl) -proline -cyano-benzyl) -proline-cyano-benzyl) -proline-cyano-benzyl) -proline (-iodo-benzyl) -proline (3-phenyl-allyl) -proline-phenyl-allyl) -prolone-phenyl-propyl) -proline (4-tert-Butyl-benzyl) -proline Benzhidril-proline (4-Biphenylmethyl) -proline (4-thiazolylmethyl) -proline (3-Benzo [b] thiophenylmethyl) -proline (2-thiophenylmethyl) -proline (5-Bromo-2-thiophenylmethyl) -proline (3-thiophenylmethyl) -proline (2-Furanylmethyl) -proline (2-pyridinylmethyl) -proline (3-pyridinylmethyl) -proline (4-pyridinylmethyl) -proline Allyl-proline Propinyl-proline 4-phenyl-pyrrolidine-3-carboxylic acid 4- (2- fluoro-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-Fluoro-phenyl) -pyrrolidine-3-carboxylic acid trans-4- (4-fluoro-phenyl) -pyrrolidine-3-carboxylic acid trans-4- ( 2-chloro-phenyl) -pyrrolidine-3-carboxylic acid trans-4- (3-chloro-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-chloro-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-Bromo-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-Bromo-phenyl) -pyrrolidine-3-carboxylic acid Trans-4- (4-bromo-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-Methyl-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-Methyl-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-methyl-phenyl) -pyrrolidin-3-carboxylic acid 4- (2-nitro-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-nitro-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-nitro-phenyl) -pyrrolidine-3-carboxylic acid 4- (1-naphthyl) -pyrrolidine-3-carboxylic acid 4- (2-naphthyl) -pyrrolidine-3-carboxylic acid 4- (2, 5-dichloro-phenyl) -pyrrolidine-3-carboxylic acid 4- (2,3-dichloro-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-trifluoromethyl-phenyl) -pyrrolidine-3 acid -carboxylic acid 4- (3-trifluoromethyl-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-trifluoromethyl-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-cyano-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-Cyano-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-Cyano-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-methoxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-methoxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-methoxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-Hydroxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (3-Hydroxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (4-hydroxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (2,3-dimethoxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (3,4-Dimethoxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (3,5-Dimethoxy-phenyl) -pyrrolidine-3-carboxylic acid 4- (2-pyridinyl) -pyrrolidine-3-carboxylic acid 4- (3-pyridinyl) -pyrrolidine-3-carboxylic acid 4- (6-methoxy-3-pyridinyl) -pyrrolidine-3-carboxylic acid 4- ( 4-pyridinyl) -pyrrolidine-3-carboxylic acid 4- (2-thienyl) -pyrrolidine-3-carboxylic acid 4- (3-thienyl) -pyrrolidine-3-carboxylic acid 4- (2-furanyl) -pyrrolidine-3 -carboxylic acid 4-isopropyl-pyrrolidine-3-carboxylic acid Piperidine Pyrrolidine Azetidine Pipecolic acid Piperidide 3-carboxy-l, 2,3, 4-tetrahydro-isoquinoline 2-carboxy-2, 3-dehydroindole acetyl-gli-valboropro cyclopentyl N-substituted cyclopentyl derivatives Cyclohexyl N-substituted cyclohexyl derivatives val-boropro glu-boropro oxazolidine and proline analogs and derivatives as defined in U.S. Patent Nos. 4,428,939; 6,890,904; 4,762,821 and Published Requests No. 2003/0158114; 20050272703; and 2006/0287245.
It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (1)

  1. CLAIMS Having described the invention as above, the claim contained in the following claims is claimed as property: 1. Method for altering a plasma relationship of a2-antiplasmin in a subject having a pretreatment level of Met-a2-antiplasmin in plasma and a plasma pre-treatment level of Asn-a2-antiplasmin, characterized in that it comprises: treating the subject with an inhibitor of antiplasmin cleavage enzyme where the inhibitor specifically inhibits cleavage of the Pro-Asn cleavage site of Met-a2- antiplasmin by the antiplasmin cleavage enzyme, where after the treatment the subject has a post-treatment level of Met-a2-antiplasmin in plasma which is at least 5% higher than the level of pretreatment of Met-a2-antiplasmin in plasma, and has a level of post-treatment of Asn-a2-antiplasmin in plasma that is at least 5% lower than the level of pretreatment of Asn-a2-antiplasmin, thus altering the relac plasma ion of 2-antiplasmin in the subject. Method according to claim 1, characterized in that the alteration of the ratio of a2- antiplasmin in the subject improves fibrinolysis in the subject. Method according to claim 1, characterized in that the treatment of the subject with the inhibitor of the antiplasmin cleavage enzyme is a 5 treatment to inhibit or treat atherosclerosis, arterial thrombosis, venous thrombosis, stroke, or pulmonary embolism. 4. Method according to claim 1, characterized in that the post-treatment level of Met-a2-0 antiplasmin in plasma is at least 10% higher than the level of pretreatment of Met-a2-antiplasmin in plasma and the level of post-treatment of Asn-a2-antiplasmin in plasma is at least 10% lower than the pretreatment level of Asn-a2-antiplasmin in plasma. 5. Method according to claim 1, characterized in that the post-treatment level of Met-a2-antiplasmin in plasma is at least 15% higher than the level of pretreatment of Met-a2-antiplasmin in plasma and the level of Q post-treatment of Asn-a2-antiplasmin in plasma is at least 15% lower than the pretreatment level of Asn-a2-antiplasmin in plasma. 6. Method according to claim 1, characterized in that the post-treatment level of Met-a2-5 antiplasmin in plasma is at least 20% higher than the level of Pretreatment of Met-a2-antiplasmin in plasma and the post-treatment level of Asn-012-antiplasmin in plasma is at least 20% lower than the level of pretreatment of Asn-a2-antiplasmin in plasma. Method according to claim 1, characterized in that the level of post-treatment of Met-a2-antiplasmin in plasma is at least 25% higher than the level of pretreatment of Met-a2-antiplasmin in plasma and the level of post - Treatment of Asn-a2-antiplasmin in plasma is at least 25% lower than the pretreatment level of Asn-a2-antiplasmin in plasma. 8. Compound having the formula: Xaai-Spi-Gly-Cyc-Xaa2-Sp2-Xaa3 characterized in that: Xaai is arginine, histidine or Usin; Spi is a separator comprising at least one of 8-, 11-, or 14-amino-3, 6, 9, 12-tetraoxatetradecanoic acid, PEGn or PPGn, wherein n = 1-6, gamma-amino acid butyric, epsilon-amino-caproic acid, serine, glycine, alanine, or β-alanine, or a combination thereof, wherein Spi has a length of 0.3 nm - 2.1 nm; Gly is glycine; Cyc is a carbocyclic or heterocyclic compound; Xaa2 is glutamine, asparagine, serine, histidine, tyrosine, alanine, or phenylalanine; Sp2 is a separator comprising at least one of PEGn or PPGn, wherein n = 1-5, 8-, 11-, or 14-amino-3, 6, 9, 12-tetraoxatetradecanoic acid, gamma-amino-butyric acid , epsilon-amino-caproic acid, serine, glycine, alanine, β-alanine, aspartic acid, glutamic acid, or a combination thereof, or is absent; and Xaa3 is glutamic acid, aspartic acid, tryptophan, tyrosine, or phenylalanine. Compound according to claim 8, characterized in that Cyc is a carbocycle of 4, 5, 6 or 7 carbon members. Compound according to claim 8, characterized in that Cyc is a heterocycle of 4, 5, 6 or 7 carbon members. 11. Compound according to claim 10, characterized in that the carbon heterocycle comprises a nitrogen heteroatom. Compound according to claim 8, characterized in that Spi is PEGn and where n = 2-5. 13. Compound according to claim 8, characterized in that Spi has a length of .6 nm to 17.5 nm. 14. Compound according to claim 8, characterized in that it comprises an oligopeptide l-8mer that it extends from Xaai in the N-terminal direction. 15. Compound according to claim 8, characterized in that it comprises a 2-10mer oligopeptide extending from Xaa3 in the C-terminal direction. 16. Compound according to claim 8, characterized in that it is placed inside a pharmaceutically acceptable carrier. 17. Compound according to claim 8, characterized in that it is linked by an amino acid residue to the carrier molecule of polyethylene glycol. 18. Method for selecting inhibitors of the antiplasmin cleavage enzyme, characterized in that it comprises: providing a fluorescent resonance energy transfer peptide that is cleavable by the a.2-antiplasmin cleavage enzyme, and which comprises a binding? - ?? ', and comprises a fluorophore and an extinction group separated by the union ?? - ??', where ?? comprises a proline, Pi 'comprises an asn, phe, gln, ser, tyr, his or ala, and having a residue P2 comprising gly; providing an amount of a2-antiplasmin cleavage enzyme; exposing the a2-antiplasmin cleavage enzyme to an a2-antiplasmin cleavage enzyme inhibitor candidate to form a test mixture; combine the test mixture with the fluorescent resonance energy transfer peptide; and measuring the fluorescent emission from the test mixture to identify when the candidate inhibitor of a.2-antiplasmin cleavage enzyme inhibits the activity of the o2-antiplasmin cleavage enzyme. 19. Method according to claim 18, characterized in that the fluorescent resonance energy transfer peptide comprises the extinction group in the 5 'direction of the binding of proline-asparagine and the fluorophore in the 3' direction of the binding? ? - ?? ' 20. Method according to claim 18, characterized in that the fluorescent resonance energy transfer peptide comprises the extinction group in the 3 'direction and the fluorophore in the 5' direction of the coupling ?? - ?? ' 21. Method according to claim 18, characterized in that the fluorescent resonance energy transfer peptide comprises SEQ ID NO: 9. 22. A2-antiplasmin cleavage enzyme inhibitor, characterized in that it is identified by the method of detection of claim 18. 23. Oligopeptide substrate of the a2-antiplasmin cleavage enzyme, characterized in that it comprises: Xi - X2 - X3 - X4 - 5 - Gly - Pro - X8 (SEQ ID NO: 9) where: Xi is selected from arg, his, lys, thr, ser, trp, gly, ala, gln, asn, ile , leu, met, phe, val, pro, and tyr; X2 is selected from arg, his, lys, thr, ser, trp, gly, ala, gln, asn, ile, leu, met, phe, val, pro, and tyr; X3 is selected from arg, his, lys, thr, ser, trp, gly, ala, gln, asn, ile, leu, met, phe, val, pro, and tyr; X4 is selected from thr, ser, trp, gly, ala, gln, ile, leu, met, phe, val, pro, tyr, asn, and his; X5 is selected from thr, ser, trp, gly, ala, gln, ile, leu, met, phe, val, pro, tyr, asn, and his; XQ is selected from asn, ser, his, tyr, ala, phe, gln; and wherein at least one of Xi, X2, and X3 is selected from arg, his, and lys; and wherein one or two of Xi, X2, and X3 may be absent and wherein the oligopeptide consists of up to 28 amino acids. 24. Oligopeptide according to claim 23, characterized in that it also comprises an oligopeptide l-8mer extending from ?? in the N-terminal direction. 25. Oligopeptide according to claim 23, characterized in that it also comprises a 2-10mer oligopeptide extending from X8 in the direction C-terminal. 26. Oligopeptide according to claim 23, characterized in that it also comprises an extinction group and a fluorophore on opposite sides of the Gly-Pro junction.
MX2008015649A 2006-06-07 2007-06-06 Substrates and inhibitors of antiplasmin cleaving enzyme and methods of use. MX2008015649A (en)

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