text:
|Publication number
|Publication type
|Application number
|Publication date
|May 7, 1996
|Filing date
|May 2, 1995
|Priority date
|Aug 18, 1989
|Fee status
|Also published as
|Publication number
|John M. Maraganore, Paul R. Bourdon, Jo-Ann M. Jablonski
|Original Assignee
|Biogen, Inc.
|Export Citation
|BiBTeX, EndNote, RefMan
|Patent Citations (3), Referenced by (9), Classifications (27), Legal Events (5)
|External Links: USPTO, USPTO Assignment, Espacenet
This application is a divisional of application Ser. No. 924,549, filed Jul. 31, 1992, now U.S. Pat. No. 5,425,936, is said Jun. 20, 1995 divisional of application Ser. No. 652,929, filed Feb. 8, 1991, now U.S. Pat. 5,240,913, issued Aug. 31, 1993, which is a continuation-in-part of application Ser. No. 549,388, filed Jul. 6, 1990, now U.S. Pat. No. 5,196,404, issued Mar. 23, 1993, which is a continuation-in-part of application Ser. No. 395,482, filed Aug. 18, 1989, now abandoned.
This invention relates to coating invasive devies with novel biologically active molecules which bind to and inhibit thrombin. These molecules comprise a catalytic site directed moiety (CSDM) of the formula: ##STR3## wherein X is hydrogen or is characterized by a backbone chain consisting of from 1 to 100 atoms; R1 is selected from the group consisting of unsubstituted, mono-substituted, di-substituted and tri-substituted saturated ring structures; R2 is a bond or is characterized by a backbone chain consisting of from 1 to 5 atoms; R3 is a bond or is characterized by a backbone chain consisting of from 1 to 3 atoms; R4 is any amino acid; R5 is any L-amino acid which comprises a guanidinium- or amino-containing side chain group; R6 is a non-amide bond; and Y is a bond or is characterized by a backbone chain consisting of from 1 to 9 atoms; or the formula: ##STR4## wherein R1 ' is selected from the group consisting of unsubstituted, mono-substituted, di-substituted and tri-substituted ring structures; R4 ' is any amino acid comprising a side chain group characterized by the capacity to accept a hydrogen bond at a pH of between about 5.5 and 9.5; and X, R2, R3, R5, R6 and Y are defined as above. Preferred thrombin inhibitors are further characterized by a anion binding exosite associating domain (ABEAM) and a linker portion of between 18Å and 42Å in length which connects Y to the ABEAM.
Acute vascular diseases, such as myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, and other blood system thromboses constitute major health risks. Such diseases are caused by either partial or total occlusion of a blood vessel by a blood clot, which contains fibrin and platelets.
Thrombin is the naturally occurring protein which catalyzes the conversion of fibrinogen to fibrin, the final step in blood clot formation. In addition to catalyzing the formation of a fibrin clot, thrombin also activates platelet aggregation and release reactions. This means that thrombin plays a central role in both acute platelet-dependent (arterial) thrombosis S. R. Hanson and L. A. Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginylchloromethylketone", Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)! and fibrin-dependent (venous) thrombosis.
Thrombin has several other bioregulatory roles J. W. Fenton, II, "Thrombin Bioregulatory Functions", Adv. Clin. Enzymol., 6, pp. 186-93 (19988)!. For example, thrombin can directly activate an inflammatory response by stimulating the synthesis of platelet activating factor (PAF) in endothelial cells S. Prescott et al., "Human Endothelial Cells in Culture Produce Platelet-Activating Factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) When Stimulated With Thrombin", Proc. Natl. Acad. Sci. USA, 81, pp. 3534-38 (1984)!. PAF is exposed on the surface of endothelial cells and serves as a ligand for neutrophil adhesion and subsequent degranulation G. M. Vercolletti et al., "Platelet-Activating Factor Primes Neutrophil Responses to Agonists: Role in Promoting Neutrophil-Mediated Endothelial Damage", Blood, 71, pp. 1100-07 (1988)!. Alternatively, thrombin may promote inflammation by increasing vascular permeability which can lead to edema P. J. Del Vecchio et al., "Endothelial Monolayer Permeability To Macromolecules", Fed. Proc., 46, pp. 2511-15 (1987)!. Reagents which block the active site of thrombin, such as hirudin, interrupt the activation of platelets and endothelial cells C. L. Knupp, "Effect of Thrombin Inhibitors on Thrombin-Induced Release and Aggregation", Thrombosis Res., 49, pp. 23-36 (1988)!.
Thrombin has also been implicated in promoting cancer, based on the ability of its native digestion product, fibrin, to serve as a substrate for tumor growth A. Falanga et al., "Isolation and Characterization of Cancer Procoagulant: A Cysteine Proteinase from Malignant Tissue", Biochemistry, 24, pp. 5558-67 (1995); S. G. Gordon et al., "Cysteine Proteinase Procoagulant From Amnion-Chorion", Blood, 66, pp. 1261-65(1995); and A. Falanga et al., "A New Procoagulant In Acute Leukemia", Blood, 71, pp. 870-75 (1988)!. And thrombin has been implicated in neurodegenerative diseases based on its ability to cause neurite retraction D. Gurwitz et al., "Thrombin Modulates and Reverses Neuroblastoma Neurite Outgrowth", Proc. Natl. Acad. Sci. USA, 85, pp. 3440-44 (1988)!. Therefore, the ability to regulate the in vivo activity of thrombin has many important clinical implications.
One route to the successful treatment or prevention of acute vascular disease is the inhibition of thrombin. Many types of thrombin inhibitors are already known in the art. Heparin, an indirect inhibitor of thrombin, is widely used to treat venous thrombosis. Although effective against fibrindependent clot formation, heparin has little efficacy in inhibiting thrombin-induced activation of platelets. Therefore, this drug is not utilized in the treatment of arterial thromboses. Moreover, heparin produces many undesirable side effects, including hemorrhaging and thrombocytopenia.
Hirudin is a naturally occurring polypeptide which is produced by the blood sucking leech Hirudo medicinalis. This compound, which is synthesized in the salivary gland of the leech, is the most potent natural inhibitor of coagulation known. Hirudin prevents blood from coagulating by binding tightly to thrombin (Kd =2�10-11) in a 1:1 stoichiometric complex S. R. Stone and J. Hofsteenge, "Kinetics of the Inhibition of Thrombin by Hirudin", Biochemistry, 25, pp. 4622-28 (1986)!. This, in turn, inhibits thrombin from catalyzing the conversion of fibrinogen to fibrin (clot), as well as inhibiting all other thrombin-mediated processes J. W. Fenton, II, "Regulation of Thrombin Generation and Functions", Semin. Thromb. Hemost., 14, pp. 234-40 (1988)!.
Hirudin inhibits thrombin by binding to the latter at two separate sites. Initially, the C-terminus of hirudin interacts with an "anion-binding exosite" (ABE) in thrombin J. W. Fenton, II et al., "Thrombin Anion Binding Exosite Interactions with Heparin and Various Polyanions", Ann. New York Acad. Sci., 556, pp. 158-65 (1989)!. Following this low affinity binding, the hirudin-thrombin complex undergoes a conformational change and amino terminal portion of hirudin is able to bind to the catalytic site of thrombin S. Kono et al., "Analysis of Secondary Structure of Hirudin and the Conformational Change Upon Interaction with Thrombin", Arch. Biochem. Biophys., 267, pp. 158-66 (1988)!.
The isolation, purification and amino acid sequence of hirudin are known in the art P. Walsmann and F. Markwardt, "Biochemical and Pharmacological Aspects of the Thrombin Inhibitor Hirudin", Pharmazie, 36, pp. 653-60 (1981); J. Dodt et al., "The Complete Covalent Structure of Hirudin: Localization of the Disulfide Bonds", Biol. Chem. Hoppe-Seyler, 366, pp. 379-85 (1995); S. J. T. Mao et al., "Rapid Purification and Revised Amino Terminal Sequence of Hirudin: A Specific Thrombin Inhibitor of the BloodSucking Leech", Anal. Biochem, 161, pp. 514-18 (1987); and R. P. Harvey et al., "Cloning and Expression of a cDNA Coding for the Anti-Coagulant Hirudin from the Bloodsucking Leech, Hirudo medicinalis", Proc. Natl. Acad. Sci. USA, 83, pp. 1084-88 (1986)!.
In animal studies, hirudin, purified from leeches, has demonstrated efficacy in preventing venous thrombosis, vascular shunt occlusion and thrombininduced disseminated intravascular coagulation. In addition, hirudin exhibits low toxicity and a very short clearance time from circulation F. Markwardt et al., "Pharmacological Studies on the Antithrombotic Action of Hirudin in Experimental Animals", Thromb. Haemost., 47, pp. 226-29 (1982)!.
Hirudin has more recently been cloned and expressed in E.coli European patent applications 58,564, 168,342 and 171,024!and yeast European patent application 200,655!. Despite these advances, hirudin is still moderately expensive to produce and it is not widely available commercially.
Recently, efforts have been made to identify peptide fragments of native hirudin or derivatives thereof which are also effective in prolonging clotting times. Such compounds are described in European patent application Nos. 276,014, 291,982, 333,356, 341,607 and 372,670. The molecules described in these patent applications demonstrated varying efficacy in inhibiting clot formation, but were all 2 to 4 orders of magnitude less potent than hirudin. Such peptide fragments, therefore, may not be fully satisfactory to dissolve blood clots in on-going therapy regimens.
More recently, compounds which mimic the action of hirudin by binding to both the anion binding exosite and the catalytic site of thrombin have been described U.S. Pat. No. 5,196,404!. These compounds demonstrate thrombin inhibitory activity equal to or greater than native hirudin. They are also smaller than hirudin and therefore less antigenic. These inhibitors are also produced synthetically, allowing for the production of commercially feasible quantities at reasonable costs.
Despite the developments to date, there is an ongoing need for even more potent thrombin inhibitors which can be produced inexpensively and in commercially feasible quantities. Such inhibitors would not only be effective in treating and preventing vascular disease, but may also be therapeutically useful in treating cancer, neurodegenerative disease and inflammation.
The present invention provides methods of coating invasive devices with molecules which are potent inhibitors of thrombin. These molecules have been designed based on the three-dimensional X-ray crystallographic structure of a thrombin-inhibitor complex. Because of this, the inhibitors of the present invention are spatially configured to provide the best fit into the three-dimensional spaces in and around the catalytic site of thrombin. This results in molecules which have optimal thrombin inhibitory activity.
The invention further provides coating invasive devices with thrombin inhibitors which additionally comprise a moiety that binds to the anion binding exosite of thrombin. These inhibitors qualitatively mimic the action of hirudin. Because these molecules are designed for optimal spatial configuration, they are more potent than hirudin. The high potency of the inhibitors of this invention allows them to be administered to patients in dosages which are comparatively lower than those required in hirudinbased therapy regimens.
Due to the fact that the molecules of the present invention may be prepared by chemical synthesis techniques, commercially feasible amounts may be produced inexpensively. Moreover, because the molecules of the present invention are significantly smaller than the thrombin inhibitors presently employed in medical treatment, they are less likely to stimulate an undesirable immune response in patients implanted with invasive devices coated with them. The molecules of the present invention may also be utilized in place of known thrombin inhibitors, especially heparin or hirudin.
As will be appreciated from the disclosure to follow, the inhibitors of this invention are useful in reducing the risk of thrombin-induced clot formation and/or platelet activation in patients receiving devices coated with them.
FIG. 1 depicts a space filling model of the Hirulog-8-thrombin complex.
FIG. 2 is a schematic depiction of the interaction between the 1-position D-Phe and the 2-postion proline of Hirulog-8 and the hydrophobic pocket adjacent the catalytic site of thrombin.
FIG. 3 depicts the comparative anticoagulant activity of hirugen, recombinant hirudin, Hirulog-8 and D-Cha-hirulog.
The following common abbreviations of the amino acids are used throughout the specification and in the claims:
______________________________________Orn - ornithine Gly - glycineAla - alanine Val - valineLeu - leucine Ile - isoleucinePro - proline Phe - phenylalanineTrp - tryptophan Met - methionineSer - serine Thr - threonineCys - cysteine Tyr - tyrosineAsn - asparagine Gln - glutamineAsp - aspartic acid Glu - glutamic acidLys - lysine Arg - arginineHis - histidine Nle - norleucineNpa - napthylalanine Cha - cyclohexylalanineHyp - hydroxyproline TPro - thioprolineAc - acetyl Suc - succinylBOC - tertButoxycarbonyl Tos - paraToluenesulfonylCbz - Carbobenzyloxy Inp - isonipecotic acid3,4,-dehydroPro - 3,4,- Sar - sarcosinedehydroproline (N-methylglycine)Tyr(OSO3 H) - tyrosine-O- Tyr(SO3 H) - 3-sulfo-sulfate tyrosine3-,5-diiodoTyr - 3-,5-diiodotyrosine______________________________________
The term "any amino acid" as used herein includes the L-isomers of the naturally occurring amino acids, as well as other "non-protein" α-amino acids commonly utilized by those in the peptide chemistry arts when preparing synthetic analogs of naturally occurring amino peptides. The naturally occurring amino acids are glycine, alanine, valine, leucine, isoleucine, serine, methionine, threonine, phenylalanine, tyrosine, tryptophan, cysteine, proline, histidine, aspartic acid, asparagine, glutamic acid, glutamine, γ-carboxyglutamic acid, arginine, ornithine and lysine. Examples of "non-protein" α-amino acids include norleucine, norvaline, alloisoleucine, homoarginine, thioproline, dehydroproline, hydroxyproline (Hyp), isonipecotic acid (Inp), homoserine, cyclohexylglycine (Chg), α-amino-n-butyric acid (Aba), cyclohexylalanine (Cha), aminophenylbutyric acid (Pba), phenylalanines substituted at the ortho, meta, or para position of the phenyl moiety with one or two of the following: a (C1 -C4) alkyl, a (C1 -C4) alkoxy, halogen or nitro groups or substituted with a methylenedioxy group; β2- and 3-thienylal-alanine, β2- and 3-furanylalanine, β2-, 3- and 4-pyridylalanine, β-(benzothienyl-2- and 3-yl)alanine, β-(1- and 2-naphthyl)alanine, O-alkylated derivatives of serine, threonine or tyrosine, S-alkylated cysteine, S-alkylated homocysteine, O-sulfate, O-phosphate and O-carboxylate esters of tyrosine, 3- and 5-sulfo tyrosine, 3- and 5-carboxy-tyrosine, 3- and 5-phosphotyrosine, 4-methane sulfonic acid ester of tyrosine, 4-methane phosphonic acid ester of tyrosine, 4-phenylacetic acid, 3,5-diiodotyrosine, 3- and 5-nitrotyrosine, ε-alkyl lysine, delta-alkyl ornithine, and the D-isomers of any of the above amino acids. Unless specifically indicated, all amino acids referred to in this application are in the L-form.
The compounds referred to herein as tyrosine-O-sulfate, Tyr(OSO3 H) and O-sulfate ester of tyrosine are identical and have the structural formula: ##STR5##
The compounds referred to herein as Tyr(SO3 H), 3-sulfo-tyrosine and 5-sulfo-tyrosine are identical and have the structural formula: ##STR6##
The term "patient" as used in this application refers to any mammal, especially humans.
The term "anionic amino acid" as used herein means a meta, para or ortho, mono- or di-substituted phenylalanine, cyclohexylalanine or tyrosine containing a negatively charged moiety, as well as S-alkylated cysteine, S-alkylated homocysteine, γ-carboxyglutamic acid, ε-alkyl lysine, delta-alkyl ornithine, glutamic acid, and aspartic acid. Examples of anionic amino acids are O-sulfate, O-phosphate and O-carboxylate esters of tyrosine, 3- and 5-sulfo-tyrosine, 3- and 5-carbo-tyrosine, 3- and 5-phospho- tyrosine, 4-methane sulfonic acid ester of tyrosine, 4-methane phosphonic acid ester of tyrosine, 4-phenylacetic acid, 3,5-diiodotyrosine and 3- and 5-nitrotyrosine.
The terms "catalytic site", "active site" and "active site pocket" as used herein, each refer to any or all of the following sites in thrombin: the substrate binding or "S1 " site; the hydrophobic binding or "oily" site; and the site where cleavage of a substrate is actually carried out ("charge relay site").
The term "backbone chain" as used herein, refers to the portion of a chemical structure that defines the smallest number of consecutive bonds that can be traced from one end of that chemical structure to the other. The atomic components that make up a backbone chain may comprise any atoms that are capable of forming bonds with at least two other atoms.
For example, each of the following chemical structures is characterized by a backbone chain of 7 atoms (the atoms which comprise the backbone chain are indicated in boldface): ##STR7##
The term "calculated length" as used in this application, refers to a predicted measurement derived by summing up the bond lengths between the atoms that comprise the backbone chain. Bond lengths between any two given atoms are well known in the art see, for example, CRC Handbook of Chemistry and Physics, 65th Edition, R. C. Weist, ed., CRC Press, Inc., Boca Raton, Fla. pp. F-166-70 (1984)!.
Applicants have analyzed the structure of a thrombin-Hirulog-8 complex by three-dimensional X-ray crystallography. Hirulog-8 is an inhibitor that binds to both the anionic binding exosite and the catalytic site of thrombin. It has the formula: (D-Phe)-Pro-Arg-Pro-(Gly)4 -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu- Tyr-Leu. The synthesis of this compound is described in Example 2 of this application. This crystallographic data revealed several structural features in and around the active site of thrombin that were crucial in designing the improved thrombin inhibitors of this invention.
One of these structural features is a hydrophobic pocket in thrombin adjacent to its catalytic center. In Hirulog-8 , the N-terminal D-Phe residue, specifically the phenyl ring of that amino acid, occupies this space. Substituting a saturated ring for the unsaturated ring increases the lipophilic interactions with thrombin, thus increasing inhibitory potency. Therefore, according to one embodiment of this invention, the thrombin inhibitor has the formula: ##STR8## wherein X is hydrogen or is characterized by a backbone chain consisting of from 1 to 100 atoms; R1 is selected from the group consisting of unsubstituted, mono-substituted, di-substituted and tri-substituted saturated homocyclic or heterocyclic ring structures; R2 is a bond or is characterized by a backbone chain consisting of from 1 to 5 atoms; R3 is a bond or is characterized by a backbone chain consisting of from 1 to 3 atoms; R4 is any amino acid; R5 is any L-amino acid which comprises a guanidinium- or amino-containing side chain group; R6 is a non-amide bond; and Y is characterized by a backbone chain consisting of from 1 to 9 atoms. Examples of L-amino acids which comprise a guanidinium- or amino-containing side chains are arginine, lysine and ornithine.
Preferably, the saturated homocyclic or heterocyclic ring structure is supplied by a D-cyclohexyl-alanine (D-Cha), a mono-substituted D-Cha, a di-substituted D-Cha or a tri-substituted D-Cha residue (i.e., X is H2 N; R1 is selected from the group consisting of unsubstituted, mono-substituted, di-substituted and tri-substituted cyclohexane; R2 is CH2 -CH; and R3 is C═O). Most preferably, X is H.sub. N; R1 is cyclohexane; R2 is CH2 --CH; R3 is C═O; R4 is proline; R5 is arginine and Y is proline.
The presence of a non-amide bond at R6 retards or prevents cleavage of the inhibitor by thrombin. The non-amide bond component may be formed by chemically modifying an amide bond. This may be achieved by methods well known in the art M. Szelke et al., "Potent New Inhibitors of Human Renin" Nature, 299, pp. 555-57 (1982); D. H. Coy et al., "Facile Solid Phase Preparation of Proteins containing the CH2 --NH Peptide Bond Isostere and Application to the Synthesis of Somatostatin (SRIF) Octapeptide Analogues" Peptides 1986, D. Theodropoulos, Ed., Walter Gruyter & Co., Berlin, pp. 143-46 (1987)!. When a non-amide bond is formed in this manner, it is preferable that the chemical modification be performed prior to the addition of the portion of the molecule containing this bond to the rest of the thrombin inhibitor. In this manner, the portion containing this non-amide bond can be added en bloc, in a single synthesis step, to the rest of the inhibitor.
In the most preferred embodiment R5 is Arg and Y is Pro. In this embodiment R6 is a naturally occurring imide bond, which is slowly cleaved by thrombin. This avoids the necessity of pre-forming the non-amide bond and allows Y and R5 to be added to the rest of the inhibitor sequentially rather than en bloc.
Further analysis of the Hirulog-8-thrombin crystallographic structure revealed that the proline bound to the D-Phe of Hirulog-8 was within 3.46 Å of the hydroxyl group of Tyr76 of thrombin. Because this distance was close enough to form hydrogen bonds, the substitution of an amino acid comprising a side chain group characterized by the capacity to accept a hydrogen bond at a pH of between about 5.5 and 9.5 for Pro at this position should increase the binding affinity of the inhibitor. Accordingly, another embodiment of this invention is a thrombin inhibitor of the formula: ##STR9## wherein R1 ' is selected from the group consisting of unsubstituted, mono-substituted, di-substituted and tri-substituted ring structures; R2 is a bond or is characterized by a backbone chain consisting of from 1 to 5 atoms; R3 is a bond or is characterized by a backbone chain consisting of from 1 to 3 atoms; R4 ' is any amino acid comprising a side chain group characterized by the capacity to accept a hydrogen bond at a pH of between about 5.5 and 9.5 ; R5 is any L-amino acid which comprises a guanidinium- or amino-containing side chain group; R5 is a non-amide bond; and Y is characterized by a backbone chain consisting of from 1 to 9 atoms.
Amino acids comprising a side chain group characterized by the capacity to accept a hydrogen bond at a pH of between about 5.5 and 9.5 are well known in the art. For example, histidine (which contains an imidazolium nitrogen), thioproline (which contains a thiol group) and isonipecotic acid (which contains a carboxylate side group) are each known to be hydrogen bond acceptors at pH 5.5 to 9.5.
More preferred thrombin inhibitors according to this embodiment comprise an unsubstituted, mono-substituted, di-substituted and tri-substituted cyclohexane at the R1 ' position. Most preferably X is H2 N, R1 ' is cyclohexane, R2 is CH2 --CH═O and the resulting amino acid formed by X R1 ' R2 ' and R3 is in the D-configuration (i.e., D-Cha)
In another preferred embodiment the thrombin inhibitor of this invention additionally consists of an anion binding exosite associating moiety (ABEAM), and a linker bound to Y at one end and the ABEAM at the other. Similar thrombin inhibitors have been described in U.S. Pat. No. 5,196,404, which is herein incorporated by reference, but the present preferred inhibitors are surprisingly and unexpectedly more potent.
In this embodiment, the linker region of the inhibitor provides a bridge between the CSDM and the ABEAM. Accordingly, it is the length of the linker, rather than its structure, that is of prime importance. The calculated length of the backbone chain which characterizes the linker must be at least about 18 Å--the distance between the catalytic site and the anion binding exosite of thrombin - and less than about 42 Å.
The backbone chain of the linker may comprise any atoms which are capable of bonding to at least two other atoms. Preferably, the backbone chain consists of any chemically feasible combination of atoms selected from oxygen, carbon, nitrogen and sulfur. Those of skill in the art are aware of what combination of the above backbone chain atoms falls within the required length based on known distances between various bonds see, for example, R. T. Morrison and R. N. Boyd, Organic Chemistry, 3rd Edition, Allyn and Bacon, Inc., Boston, Mass. (1977)!. According to a preferred embodiment, the linker is a peptide which comprises the amino acid sequence Gly-Gly-Gly-Asn-Gly-Asp-Phe. Preferably, the amino acid bound to the ABEAM component is Phe.
The third domain of these preferred thrombin inhibitors is the ABEAM which binds to the anion binding exosite of thrombin. Preferably the ABEAM has the formula:
wherein W is a bond; B1 is an anionic amino acid; B2 is any amino acid; B3 is Ile, Val, Leu, Nle or Phe; B4 is Pro, Hyp, 3,4-dehydroPro, thiazolidine-4-carboxylate, Sar, any N-methyl amino acid or D-Ala; B5 is an anionic amino acid; B6 is an anionic amino acid; B7 is a lipophilic amino acid selected from the group consisting of Tyr, Trp, Phe, Leu, Nle, Ile, Val, Cha, Pro, or a dipeptide consisting of one of these lipophilic amino acids and any amino acid; B8 is a bond or a peptide containing from one to five residues of any amino acid; and Z is OH or is characterized by a backbone chain consisting of between 1 and 6 atoms.
Peptides which are homologous to the carboxy terminal portion of hirudin have been shown to bind to the anion binding exosite on thrombin J. M. Maraganore et al., "Anticoagulant Activity of Synthetic Hirudin Peptides", J. Biol. Chem., 264, pp. 8692-98 (1989); both of which is herein incorporated by reference!.
According to a preferred embodiment of this invention, ABEAM is homologous to amino acids 56-64 of hirudin, i.e., B1 is Glu; B2 is Glu; B3 is Ile; B4 is Pro; B5 is Glu; B6 is Glu; By is Tyr-Leu, Tyr(SO3 H)-Leu or Tyr (OSO3 H) -Leu, or (3-, 5-diiodoTyr) -Leu; B8 is a bond; and Z is OH. It should be noted that native hirudin contains Tyr(OSO3 H) at position 63. However, carboxy terminal hirudin peptides which contain Tyr(SO3 H) have identical anticoagulant activity as those which contain the native Tyr(OSO3 H) see U.S. patent application Ser. No. 314,756, now abandoned!.
Other ABEAM components within the scope of this invention may comprise those portions of any molecule known to bind to the anion binding site of thrombin. These include amino acids 1675-1686 of Factor V, amino acids 272-285 of platelet glycoprotein lb, amino acids 415-428 of thrombomodulin, amino acids 245-259 of prothrombin Fragment 2 and amino acids 30 to 44 of fibrinogen Aα chain. In addition, the ABEAM component may be selected from any of the hirudin peptide analogues described by J. L. Krstenansky et al., "Development of MDL-28,050, A Small Stable Antithrombin Agent Based On A Functional Domain of the Leech Protein, Hirudin", Thromb. Haemostas., 63, pp. 208-14 (1990), particularly those comprising the sequence Asp-Tyr-Glu-Pro-Ile-Pro-Glu-Glu-Ala-Cha-(D-Glu).
The thrombin inhibitors of the present invention may be synthesized by various techniques which are well known in the art. These include organic chemical synthesis techniques, solid-phase peptide synthesis, solution-phase peptide synthesis or a combination of these techniques. Portions of some of the inhibitors of this invention may also be produced by other methods, such as enzymatic cleavage of natural or recombinant hirudin or recombinant DNA techniques. These portions may then be bound to the synthetically produced portions of the inhibitor to produce the final product according to this invention. The choice of synthesis technique will, of course, depend upon the composition of the particular inhibitor.
In a preferred embodiment of this invention, the thrombin inhibitor is synthesized via a mixed heterologous/solid phase technique. This technique involves the solid-phase synthesis of all or most of the peptide portion of the molecule, followed by the addition of the non-amino acid components which are synthesized by solution phase techniques. The non-amino acid may be coupled to the peptidic portion via solid-phase or solution-phase methods. Similarly, any remaining peptidic portions may also be added via solid-phase or solution phase methods. This constitutes the most cost-efficient procedures for producing commercial quantities of these molecules.
When "non-protein" amino acids are contained in the thrombin inhibitor of this invention, they may be either added directly to the growing chain during peptide synthesis or prepared by chemical modification of the complete synthesized peptide, depending on the nature of the desired "non-protein" amino acid. Those of skill in the chemical synthesis art are well aware of which "non-protein" amino acids may be added directly and which must be synthesized by chemically modifying the complete peptide chain following peptide synthesis.
The molecules of the present invention display potent anticoagulant activity. This activity may be assayed in vitro using any conventional technique. Preferably, an assay for anticoagulant activity involves direct determination of the thrombin-inhibitory activity of the molecule. Such techniques measure the inhibition of thrombin-catalyzed cleavage of colorimetric substrates or, more preferably, the increase in thrombin times or increase in activated partial thromboplastin times of human plasma. The latter assay measures factors in the "intrinsic" pathway of coagulation. Alternatively, the assay employed may use purified thrombin and fibrinogen to measure the inhibition of release of fibrinopeptides A or B by radioimmunoassay or ELISA.
The antiplatelet activity of the molecules of this invention may also be measured by any of a number of conventional platelet assays. Preferably, the assay will measure a change in the degree of aggregation of platelets or a change in the release of a platelet secretory component in the presence of thrombin. The former may be measured in an aggregometer. The latter may be measured using RIA or ELISA techniques specific for the secreted component.
The molecules of the present invention are useful in compositions, combinations and methods for the treatment and prophylaxis of various diseases attributed to thrombin-mediated and thrombin-associated functions and processes. These include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, restenosis following arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, various cell regulatory processes (e.g. secretion, shape changes, proliferation), cancer and metastasis, and neurodegenerative diseases.
The thrombin inhibitors of the present invention may be formulated using conventional methods to prepare pharmaceutically useful compositions, such as the addition of a pharmaceutically acceptable carrier. These compositions and the methods employing them may be used for treating or preventing thrombotic diseases in a patient.
According to an alternate embodiment of the present invention, the thrombin inhibitors may be employed in combinations, compositions, and methods for treating thrombotic disease, and for decreasing the dosage of a thrombolytic agent required to establish reperfusion or prevent reocclusion in a patient. Additionally, the thrombin inhibitors of this invention may be used in combinations, compositions, and methods for decreasing reperfusion time or increasing reocclusion time in a patient treated with a thrombolytic agent. These combinations and compositions comprise a pharmaceutically effective amount of a thrombin inhibitor of the present invention and a pharmaceutically effective amount of a thrombolytic agent.
In these combinations and compositions, the thrombin inhibitor and the thrombolytic agent work in a complementary fashion to dissolve blood clots, resulting in decreased reperfusion times and increased reocclusion times in patients treated with them. Specifically, the thrombolytic agent dissolves the clot, while the thrombin inhibitor prevents newly exposed, clot-entrapped or clot-bound thrombin from regenerating the clot. The use of the thrombin inhibitor in the combinations and compositions of this invention advantageously allows the administration of a thrombolytic reagent in dosages previously considered too low to result in thrombolytic effects if given alone. This avoids some of the undesirable side effects associated with the use of thrombolytic agents, such as bleeding complications.
Thrombolytic agents which may be employed in the combinations and compositions of the present invention are those known in the art. Such agents include, but are not limited to, tissue plasminogen activator purified from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, prourokinase, anisolated streptokinase plasminogen activator complex (ASPAC), animal salivary gland plasminogen activators and known, biologically active derivatives of any of the above.
The term "combination" as used herein, includes a single dosage form containing at least one thrombin inhibitor of this invention and at least one thrombolytic agent; a multiple dosage form, wherein the thrombin inhibitor and the thrombolytic agent are administered separately, but concurrently; or a multiple dosage form wherein the two components are administered separately, but sequentially. In sequential administration, the thrombin inhibitor may be given to the patient during the time period ranging from about 5 hours prior to about 5 hours after administration of the thrombolytic agent. Preferably, the thrombin inhibitor is administered to the patient during the period ranging from 2 hours prior to 2 hours following administration of the thrombolytic agent.
Alternatively, the thrombin inhibitor and the thrombolytic agent may be in the form of a single, conjugated molecule. Conjugation of the two components may be achieved by standard cross-linking techniques well known in the art. The single molecule may also take the form of a recombinant fusion protein, if both the thrombin inhibitor and the thrombolytic agent are peptidic.
Various dosage forms may be employed to administer the compositions and combinations of this invention. These include, but are not limited to, parenteral administration, oral administration and topical application. The compositions and combinations of this invention may be administered to the patient in any pharmaceutically acceptable dosage form, including those which may be administered to a patient intravenously as bolus or by continued infusion, intramuscularly--including paravertebrally and periarticularly--subcutaneously, intracutaneously, intra-articularly, intrasynovially, intrathecally, intra-lesionally, periostally or by oral, nasal, or topical routes. Such compositions and combinations are preferably adapted for topical, nasal, oral and parenteral administration, but, most preferably, are formulated for parenteral administration.
Parenteral compositions are most preferably administered intravenously either in a bolus form or as a constant infusion. If the thrombin inhibitor is being used as an antiplatelet compound, constant infusion is preferred. If the thrombin inhibitor is being used as an anticoagulant, a subcutaneous or intravenous bolus injection is preferred. For parenteral administration, fluid unit dose forms are prepared which contain a thrombin inhibitor of the present invention and a sterile vehicle. The thrombin inhibitor may be either suspended or dissolved, depending on the nature of the vehicle and the nature of the particular thrombin inhibitor. Parenteral compositions are normally prepared by dissolving the thrombin inhibitor in a vehicle, optionally together with other components, and filter sterilizing before filling into a suitable vial or ampule and sealing. Preferably, adjuvants such as a local anesthetic, preservatives and buffering agents are also dissolved in the vehicle. The composition may then be frozen and lyophilized to enhance stability.
Parenteral suspensions are prepared in substantially the same manner, except that the active component is suspended rather than dissolved in the vehicle. Sterilization of the compositions is preferably achieved by exposure to ethylene oxide before suspension in the sterile vehicle. Advantageously, a surfactant or wetting agent is included in the composition to facilitate uniform distribution of its components.
Tablets and capsules for oral administration may contain conventional excipients, such as binding agents, fillers, diluents, tableting agents, lubricants, disintegrants, and wetting agents. The tablet may be coated according to methods well known in the art. Suitable fillers which may be employed include cellulose, mannitol, lactose and other similar agents. Suitable disintegrants include, but are not limited to, starch, polyvinylpyrrolidone and starch derivatives, such as sodium starch glycolate. Suitable lubricants include, for example, magnesium stearate. Suitable wetting agents include sodium lauryl sulfate.
Oral liquid preparations may be in the form of aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for reconstitution with water or another suitable vehicle before use. Such liquid preparations may contain conventional additives. These include suspending agents; such as sorbitol, syrup, methyl cellulose, gelatin, hydroxyethylcellulose, carboxymethylcellulose, aluminum stearate gel or hydrogenated edible fats; emulsifying agents which include lecithin, sorbitan monooleate, polyethylene glycols, or acacia; non-aqueous vehicles, such as almond oil, fractionated coconut oil, and oily esters; and preservatives, such as methyl or propyl p-hydroxybenzoate or sorbic acid.
Compositions formulated for topical administration may, for example, be in aqueous jelly, oily suspension or emulsified ointment form.
The dosage and dose rate of the thrombin inhibitor will depend on a variety of factors, such as the size of the patient, the specific pharmaceutical composition used, the object of the treatment, i.e., therapy or prophylaxis, the nature of the thrombotic disease to be treated, and the judgment of the treating physician.
According to the present invention, a preferred pharmaceutically effective daily dose of the thrombin inhibitor of this invention is between about 0.5 nmoles/kg body weight of the patient to be treated ("body weight") and about 2.5 μmoles/kg body weight. In combinations containing a thrombolytic agent, a pharmaceutically effective daily dose of the thrombolytic is between about 10% and 80% of the conventional dosage range. The "conventional dosage range" of a thrombolytic agent is the daily dosage used when that agent is employed in a monotherapy. Physician's Desk Reference 1989, 43rd Edition, Edward R. Barnhart, publisher!. That conventional dosage range will, of course, vary depending on the thrombolytic agent employed. Examples of conventional dosage ranges are as follows: urokinase - 500,000 to 6,250,000 units/patient; streptokinase - 140,000 to 2,500,000 units/patient; tPA--0.5 to 5.0 mg/kg body weight; ASPAC - 0.1 to 10 units/kg body weight.
Most preferably, the therapeutic and prophylactic compositions of the present invention comprise a dosage of between about 5 nmoles/kg body weight and about 250 nmoles/kg body weight of the thrombin inhibitor. Most preferred combinations comprise the same amount of the thrombin inhibitor and between about 10% and about 70% of the conventional dosage range of a thrombolytic agent. It should also be understood that a daily pharmaceutically effective dose of either the thrombin inhibitors of this invention or the thrombolytic agent present in combinations of the invention, may be less than or greater than the specific ranges cited above.
Once improvement in the patient's condition has occurred, a maintenance dose of a combination or composition of this invention is administered, if necessary. Subsequently, the dosage or the frequency of administration, or both, may be reduced, as a function of the symptoms, to a level at which the improved condition is retained. When the symptoms have been alleviated to the desired level, treatment should cease. Patients may, however, require intermittent treatment upon any recurrence of disease symptoms.
According to an alternate embodiment of this invention, thrombin inhibitors may be used in compositions and methods for coating the surfaces of invasive devices, resulting in a lower risk of clot formation or platelet activation in patients receiving such devices. Surfaces that may be coated with the compositions of this invention include, for example, prostheses, artificial valves, vascular grafts, stents and catheters. Methods and compositions for coating these devices are known to those of skill in the art. These include chemical cross-linking or physical adsorption of the thrombin inhibitor-containing compositions to the surfaces of the devices.
According to a further embodiment of the present invention, thrombin inhibitors may be used for ex vivo thrombus imaging in a patient. The term "ex vivo thrombus imaging", as used herein, refers to viewing, outside the human body, a blood clot in a patient. As explained below, this is achieved through the use of a radioactively labelled thrombin inhibitor which is administered to the patient and an external device that detects radioactivity and converts it to an image. In this embodiment, the thrombin inhibitor is labelled with a radioisotope. The choice of radioisotope is based upon a number of well-known factors, for example, toxicity, biological half-life and detectability. Preferred radioisotopes include, but are not limited to, 125 I , 123 I and 111 In. Techniques for labelling the thrombin inhibitor are well known in the art. Most preferably, the radioisotope is 123 I and the labelling is achieved using 123 I-Bolton-Hunter Reagent. The labelled thrombin inhibitor is administered to a patient and allowed to bind to the thrombin contained in a clot. The clot is then observed by utilizing well-known detecting means, such as a camera capable of detecting radioactivity coupled to a computer imaging system. This technique also yields images of platelet-bound thrombin and meizothrombin.
This invention also relates to compositions containing the thrombin inhibitors of this invention and methods for using such compositions in the treatment of tumor metastases. The efficacy of the thrombin inhibitors of this invention for the treatment of tumor metastases is manifested by the inhibition of metastatic growth. This is based upon the presence of a procoagulant enzyme in certain cancer cells. This enzyme activates the conversion of Factor X to Factor Xa in the coagulation cascade, resulting in fibrin deposition which, in turn, serves as a substrate for tumor growth. By inhibiting fibrin deposition through the inhibition of thrombin, the molecules of the present invention serve as effective anti-metastatic tumor agents. Examples of metastatic tumors which may be treated by the thrombin inhibitors of this invention include, but are not limited to, carcinoma of the brain, carcinoma of the liver, carcinoma of the lung, osteocarcinoma and neoplastic plasma cell carcinoma.
The invention also relates to methods and compositions employing the above-described thrombin inhibitors to inhibit thrombin-induced endothelial cell activation. This inhibition includes the repression of platelet activation factor (PAF) synthesis by endothelial cells. These compositions and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema, which is thought to be mediated by PAF. Such diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage.
Early stages of septic shock include discrete, acute inflammatory and coagulopathic responses. It has previously been shown that injection of baboons with a lethal dose of live E. coli leads to marked declines in neutrophil count, blood pressure and hematocrit. Changes in blood pressure and hematocrit are due in part to the generation of a disseminated intravascular coagulopathy (DIC) and have been shown to parallel consumption of fibrinogen F. B. Taylor et al., "Protein C Prevents the Coagulopathic and Lethal Effects of Escherichia coli Infusion in the Baboon", J. Clin. Invest., 79, pp. 918-25 (1987)!. Neutropenia is due to the severe inflammatory response caused by septic shock which results in marked increases in tumor necrosis factor levels. The thrombin inhibitors of this invention may be utilized in compositions and methods for treating or preventing DIC in septicemia and other diseases.
This invention also relates to the use of the above-described thrombin inhibitors, or compositions comprising them, as anticoagulants for extracorporeal blood. As used herein, the term "extracorporeal blood" includes blood removed in line from a patient, subjected to extracorporeal treatment, and then returned to the patient in such processes as dialysis procedures, blood filtration, or blood bypass during surgery. The term also includes blood products which are stored extracorporeally for eventual administration to a patient and blood collected from a patient to be used for various assays. Such products include whole blood, plasma, or any blood fraction in which inhibition of coagulation is desired.
The amount or concentration of thrombin inhibitor in these types of compositions is based on the volume of blood to be treated or, more preferably, its thrombin content. Preferably, an effective amount of a thrombin inhibitor of this invention for preventing coagulation in extracorporeal blood is from about 0.5 nmoles/60 ml of extracorporeal blood to about 2.5 μmoles/60 ml of extracorporeal blood.
The thrombin inhibitors of this invention may also be used to inhibit clot-bound thrombin, which is believed to contribute to clot accretion. This is particularly important because commonly used anti-thrombin agents, such as heparin and low molecular weight heparin, are ineffective against clot-bound thrombin.
Finally, the thrombin inhibitors of this invention may be employed in compositions and methods for treating neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process suggestive of the rounding in shape changes of brain cells and implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.
In order that the invention described herein may be more fully understood, the following examples are set forth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
Carboxy terminal hirudin peptides effectively block thrombin-catalyzed fibrinogen hydrolysis, but not chromogenic substrate hydrolysis J. M. Maraganore et al., J. Biol. Chem., 264, pp. 8692-98 (1989)!. In addition, hirudin peptides do not neutralize thrombin-catalyzed activation of Factors V and VIII J. W. Fenton, II, et al., "Hirudin Inhibition by Thrombin", Anglo. Archiv. Biol., (1989)!.
Hirudin peptides, such as Tyr63 -O-sulfate-N-acetyl-hirudins53-64 ("hirugen"), exhibit potent inhibitory effects toward thrombin-induced platelet activation in vitro J. A. Jakubowski and J. M. Maraganore, "Inhibition of Thrombin-Induced Platelet Activities By A Synthetic 12 Amino Acid Residue Sulfated Peptide (Hirugen)", Blood, p. 1213 (1989)!. Nevertheless, a thrombin inhibitor capable of blocking the active site may be required for inhibition of platelet thrombosis in vivo, if activation of Factors V and VIII are critical and rate-limiting steps. This conclusion is warranted from results obtained with the irreversible thrombin inhibitor (D-Phe)-Pro-Arg-CH2 Cl S. R. Hanson and L. A. Harker, "Interruption of Acute Platelet-Dependent Thrombosis by the Synthetic Antithrombin D-Phenylalanyl-L-Prolyl-L-Arginyl Chloromethyl Ketone", Proc. Natl. Acad. Sci. USA, 85, pp. 3184-88 (1988)!and other reversible thrombin inhibitors J. F. Eidt et al., "Thrombin is an Important Mediator of Platelet Aggregation in Stenosed Canine Coronary Arteries with Endothelial Injury", J. Clin. Invest., 84, pp. 18-27 (1989)!.
Using the above knowledge that the NH2 -terminus of hirudin peptides is proximal to Lys-149, we employed a three-dimensional model of thrombin B. Furie, et al., "Computer-Generated Models of Blood Coagulation Factor Xa, Factor IXa, and Thrombin Based Upon Structural Homology with Other Serine Proteases", J. Biol. Chem., 257, pp. 3875-82 (1982)! to design an agent which: 1) binds to the anion binding exosite of thrombin; and, 2) is capable of blocking the active site pocket of thrombin and inhibiting the function of catalytic residues contained therein.
We determined that the minimal distance from the ε-NH2 of Lys-149 to the β-hydroxylate of Ser-195 is 18-20 Å. Based on a 3 Å/amino acid residue length, we calculated that at least about 4-7 amino acids would be required to link a hirudin peptide, such as Tyr63 -O- sulfate-hirudin53-64, to a domain comprising an active-site inhibitor structure. The composition of the linker was designed to be glycine. Glycine was chosen in order to engineer the greatest flexibility of a linker for these preliminary investigations. It should be understood, however, that other, more rigid biopolymer linkers may also be employed.
We chose the sequence (D-Phe)-Pro-Arg-Pro as the active site inhibitor because thrombin exhibits specificity for Arg as the P1 amino acid in the cleavage of substrates. A Pro following the Arg (the P1 ' amino acid) yields a bond that is cleaved very slowly by thrombin. We designed alternate peptides by replacing that Pro with a sarcosyl- or N-methyl-alanine amino acid or by chemical reduction of an Arg-Gly scissile bond.
Hirulog-8 has the formula: H-(D-Phe)-Pro-Arg-Pro-(Gly)4 -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-OH. We synthesized Hirulog-8 by conventional solid-phase peptide synthesis employing an Applied Biosystems 430 A Peptide Synthesizer. This peptide was synthesized using BOC-L-Leucine-O-divinylbenzene resin. Additional t-BOC-amino acids (Peninsula Laboratories, Belmont, Calif.) used included BOC-O-2,6-dichlorobenzyl tyrosine, BOC-L-glutamic acid (γ-benzyl ester), BOC-L-proline, BOC-L-isoleucine, BOC-L-phenylalanine, BOC-L-aspartic acid (β-benzyl ester), BOC-glycine, BOC-L-asparagine, BOC-D-phenylalanine, and BOC-L-arginine. In order to achieve higher yields in synthesis, the (Gly)4 linker segment was attached in two cycles of manual addition of BOC-glycylglycine (Beckman Biosciences, Inc., Philadelphia, Pa.). After completion of synthesis, the peptide was fully deprotected and uncoupled from the divinylbenzene resin by treatment with anhydrous HF: R-cresol: ethylmethyl sulfate (10:1:1 , v/v/v). Following removal from the resin, the peptide was lyophilized to dryness.
Crude Hirulog-8 was purified by reverse-phase HPLC employing an Applied Biosystems 151A liquid chromatographic system and a Vydac C18 column (2.2�25 cm). The column was equilibrated in 0.1% TFA/water and developed with a linear gradient of increasing acetonitrile concentration from 0 to 80% over 45 minutes in the 0. 1% TFA at a flow-rate of 4.0 ml/min. The effluent stream was monitored for absorbance at 229 nm and fractions were collected manually. We purified 25-30 mg of crude Hirulog-8 by HPLC and recovered 15-20 mg of pure peptide.
We confirmed the structure of purified Hirulog-8 by amino acid and sequence analyses. Amino acid hydrolysates were prepared by treating the peptide with 6 N HCl, in vacuo, at 110� C. for 24 hrs. We then analyzed the hydrolysates by ion-exchange chromatography and subsequent ninhydrin derivatization/detection using a Beckman 6300 automated analyzer. We performed sequence analysis using automated Edman degradation on an Applied Biosystems 470A gas-phase sequencer equipped with a Model 900A data system. Phenylthiohydantoin (PTH) amino acids were analyzed on-line using an Applied Biosystems 120A PTH-analyzer and a PTH-C18 column (2.1�220 mm).
Substituted Hirulogs
We obtained the X-ray crystallographic structure of the Hirulog-8: thrombin complex by the following steps. First, we generated Hirulog-8:thrombin complex crystals of suitable quality to obtain a high resolution diffraction pattern. We next collected diffractometer data employing these crystals. Finally, we determined the 3-dimensional structure of the Hirulog-8:thrombin complex using molecular replacement rotation/translation methods employing the coordinates of the PPACK:thrombin W. Bode et al., "The Refined 1.9 Å Crystal Structure of Human α-Thrombin: Interaction With D-Phe-Pro-Arg-Chloromethylketone and Significance of the Tyr-Pro-Pro-Trp Insertion Segment", EMBO J., 8, pp. 3467-75 (1989)!and hirudin:thrombin T. J. Rydel et al., "The Structure of a Complex of Recombinant Hirudin and Human α-Thrombin", Science, 49, pp. 277-80, (1990)!complexes. As shown in FIG. 1, the structure of Hirulog-8 bound to thrombin was resolved allowing resolution of the D-Phe-Pro-Arg sequence of the CSDM and the Asp-Phe-Glu-Glu-Ile segment of the ABEAM.
In FIG. 1, thrombin is displayed in white, except for its active site, which is shown in dense dots. Hirulog-8 is depicted in sparse dots. The left-hand portion of Hirulog-8 nearest the thrombin active site is the CSDM. The right-hand portion is the ABEAM. Other amino acids of Hirulog-8 are not shown in Figure 1 because electron densities corresponding to them could not be assigned.
Examination of the ABEAM portion of the Hirulog-8:thrombin structure showed the positioning of the 1-position amino acid (D-Phe) in a hydrophobic pocket formed by His57, Tyr60A, Trp60D, Leu99, Ile174, and Trp215 of thrombin. The D-Phe residue formed close van der Waals contacts with Leu99, Ile174, and Trp215 (FIG. 2). In FIG. 2, thrombin is displayed in soid lines and Hirulog-8 is in dashed lines. The positioning of the D-Phe residue within the pocket suggested that substitutions at the 1 -position which enhance lipophilic contacts would lead to an increased binding affinity of the CSDM moiety in the thrombin inhibitors of this invention. Accordingly, we replaced the D-Phe residue of Hirulog-8 with either D-napthylalanine (D-NPA) or D-cyclohexylalanine (D-Cha) to form D-NPA-Hirulog-8 and D-Cha-Hirulog-8, respectively.
The binding of the CSDM of Hirulog-8 to the catalytic site of thrombin was also found to include apolar interactions between the first proline of the inhibitor (adjacent to D-Phe) and a pocket defined by His57, Tyr60A and Trp60D of thrombin (FIG. 2). Furthermore, that proline was found to be within 3.46 Å of the phenolic hydroxyl group of thrombin Tyr60A (FIG. 2). In FIG. 2, thrombin is depicted in solid lines and Hirulog-8 in dashed lines.
The proximity of this proline to Tyr60A of thrombin suggested the potential for hydrogen bond formation between the two. By substituting proline with an amino acid capable of forming hydrogen bonds, the stability of the CSDM binding to the thrombin active site can be increased. This, in turn, would increase the inhibitory activity of such a molecule. Therefore, we replaced the proline of Hirulog-8 with either L-histidine (His2 -Hirulog-8), L-thioproline (TPro2 -Hirulog-8) or isonipecotic acid (Inp2 -Hirulog-8). Each of these substitutions created a hydrogen bond acceptor at the 2-position (R4 ' component) of the thrombin inhibitors of this invention (i.e., an imidazolium nitrogen, a thiol and a carboxylate, respectively).
D-Npa-Hirulog-8 was synthesized in the same manner as Hirulog-8 (Example 2), except that Boc-D-napthylalanine (Bachem Inc., Torrance, Calif.) was employed in place of D-Phe at the last cycle of synthesis. D-Cha-Hirulog-8 was similarly prepared using Boc-D-cyclohexylalanine (Bachem Biosciences, Pa.) at the last cycle of synthesis.
Both 1-position substituted peptides were purified as described for Hirulog-8 in Example 2. The purified peptides were characterized by amino acid analysis and by FAB-MS.
Substitutions at the 2-position were designed having the formula: (D-Cha)-X-Arg-HPro- (Gly)4 -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu or (D-Phe)-X-Arg-HPro-(Gly)4 -Asn-Gly-Asp-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu, wherein X is either histidine, thioproline or isonipecotic acid. These peptides are synthesized essentially as described in Example 2 and Example 4 except for incorporation of Boc-L-hydroxyproline (Bachem, Inc.) instead of Boc-L-proline at cycle 16 of the synthesis and either Boc-N-im-CBZ-L-histidine, Boc-L-thioproline, or Boc-isonipecotic acid (all obtained from Bachem, Inc.) instead of Boc-L-proline at cycle 18. HPro is used at the 4-position to slow down the rate of cleavage of the inhibitor by thrombin. The peptides are purified and characterized as described in Example 2.
We compared the inhibition of thrombincatalyzed hydrolysis of Spectrozyme TH (tosyl-Gly-Pro-Arg-p-nitroanilide; American Diagnostica, New York, N.Y.) of Hirulog-8, D-Cha-Hirulog-8 and D-Npa-Hirulog-8 in an assay. Specifically, we measured the initial rate velocities in the presence or absence of each inhibitor over a range of substrate concentrations from 2.2 to 22 μM. The thrombin-catalyzed rate was monitored in a Cary 19 spectrophotometer at 405 nm and recorded continuously as a function of time. Kinetics were performed at room temperature (25�1� C. ) in a 0.05 M sodium borate buffer, pH 8.4, containing 0.1M NaCl.
For a typical enzyme reaction, 1.0 ml of buffer was added to both the sample and reference cuvettes. Thrombin (3.2�10-9 M, final concentration) and the hirulog (0-4�10-8 M) were added to the sample cuvette prior to addition of Spectrozyme TH (2.2-22μM). Immediately following addition of substrate, the contents of the sample cuvette were mixed by use of a plastic pipette. The reaction was monitored spectrophotometrically for 5-15 minutes.
Initial rate velocities at each substrate concentration were expressed as moles Spectrozyme TH hydrolyzed/sec/mole thrombin. This was determined during the initial linear phase of the reaction (≦15% total hydrolysis of substrate) by measuring the slope of the hydrolytic reaction. Lineweaver-Burke plots were constructed accordingly, by plotting the inverse of the initial velocity against the inverse of the substrate concentration. Shown below are the inhibitory constants obtained for Hirulog-8 and derivatives of the present invention.
______________________________________Derivative Ki, nM______________________________________Hirulog-8 1.4D-Cha-Hirulog-8 0.12D-Npa-Hirulog-8 4.3______________________________________
As can be seen from these results, substitution of D-Phe in Hirulog-8 with D-Cha results in a surprising and unexpected decrease in Ki by one order of magnitude. This finding indicates that substitution of D-Phe by D-Cha increases the binding affinity of the CSDM in the inhibitors of this invention. The failure of D-Npa-Hirulog-8 to decrease Ki indicates that the presence of a saturated ring structure at this position causes the increased binding affinity. D-Cha contains such a saturated ring, while D-Npa contains an unsaturated ring.
Molecules containing the 2-position substitutions described in Example 5 will display similar surprising and unexpected decreases in Ki.
Anticoagulant Activity Of A1 -Substituted Hirulogs
We compared the anticoagulant activity of Tyr63 -O-sulfate-N-acetyl-hirudin53-64 ("hirugen"), recombinant hirudin (American Diagnostica), Hirulog-8 , and the 1 -position substituted hirulogs of the present invention using pooled, normal human plasma (George King Biomedical, Overland Park, Kans.) and a Coag-A-Mate XC instrument (General Diagnostics, Organon Technica, Oklahoma City, Okla.). Activity was monitored using the activated partial thromboplastin time (APTT) assay with CaCl2 and phospholipid solutions obtained from the manufacturer. Recombinant hirudin (American Diagnostica), Hirulog-8, D-Cha-Hirulog-8 or hirugen was then added to the APTT determination wells at a final concentrations of 10 to 32,300 ng/ml in a total volume of 25 μl prior to addition of 100 μl of plasma.
As shown in FIG. 3, D-Cha-Hirulog-8 prolonged APTT to 470% of control values at a concentration of 1 μg/μ1 . This increase was significantly greater than the increases in APTT caused by hirugen, recombinant hirudin or Hirulog-8 at the same concentration. Thus, in addition to showing enhanced antithrombin activities in vitro over Hirulog-8, D-Cha-Hirulog-8 also showed a significantly increased anticoagulant effect in plasma assays over Hirulog-8.
Molecules containing the 2-position substitutions described in Example 5 will display increases in APTT that are greater than Hirulog-8.
While we have hereinbefore presented a number of embodiments of this invention, it is apparent that our basic construction can be altered to provide other embodiments which utilize the molecules, compositions, combinations and methods of this invention. Therefore, it will be appreciated that the scope of this invention is to be defined by the claims appended hereto rather than the specific embodiments which have been presented hereinbefore by way of example.
|Cited Patent
|Filing date
|Publication date
|Apr 3, 1990
|Nov 20, 1990
|Merrell Dow Pharmaceuticals
|Anticoagulant peptide alcohols
|Feb 8, 1991
|Aug 31, 1993
|Biogen, Inc.
|Inhibitors of thrombin
|Jul 31, 1992
|Jun 20, 1995
|Biogen, Inc.
|Inhibitors of thrombin
|Citing Patent
|Filing date
|Publication date
|Jun 10, 1997
|Jul 21, 1998
|Brother Kogyo Kabushiki Kaisha
|Positively chargeable single-component developer and image-forming apparatus for using the same
|Jul 1, 1998
|Aug 24, 1999
|Conjuchem, Inc.
|Methods for producing novel conjugates of thrombin inhibitors
|Jun 11, 1999
|Jul 11, 2000
|Conjuchem, Inc.
|Methods of treating or preventing thromboses
|Jun 22, 2000
|Aug 21, 2001
|Conjuchem, Inc.
|Methods and compositions for producing novel conjugates of thrombin inhibitors and endogenous carriers resulting in anti-thrombins with extended lifetimes
|Sep 3, 2009
|Aug 28, 2012
|ScnioPharm Taiwan, Ltd.
|Process for making bivalirudin
|Sep 3, 2009
|Mar 4, 2010
|Scinopharm Taiwan Ltd.
|Process for Making Bivalirudin
|Dec 29, 1995
|Jul 10, 1997
|Thomas W Dr Stief
|Use of guanidine or its derivatives to inhibit activation of haemostasis
|Dec 29, 1995
|Jul 13, 2000
|Thomas W Stief
|H�mostaseaktivierungs-Inhibitor und Verfahren zum Hemmen der H�mostaseaktivierung in Blut oder anderen biologischen Fl�ssigkeiten
|Sep 3, 2009
|Mar 11, 2010
|Scinopharm Taiwan Ltd.
|Process for making bivalirudin
|U.S. Classification
|427/2.24, 530/330, 424/1.69, 427/2.3, 514/14.7, 514/21.9
|International Classification
|Cooperative Classification
|European Classification
|Nov 8, 1999
|Fee payment
Year of fee payment: 4
|Nov 6, 2003
|Fee payment
Year of fee payment: 8
|Apr 15, 2004
Owner name: BIOGEN IDEC MA, INC., MASSACHUSETTS
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|Nov 7, 2007
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|Nov 12, 2007
|Maintenance fee reminder mailed


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- Brand Name: Nikon
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Nikon COOLPIX S6200 16 MP Digital Camera with 10x Optical Zoom NIKKOR ED Glass Lens and HD 720p Video (Silver)
- 10x optical Zoom-NIKKOR ED glass lens in a compact body design
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If you haven't been saving memories with movies, get started now with the S6200. With a single touch of a button, you'll capture every sight and sound of a baby's first steps, a bride's first kiss or the laughter over a friend's most embarrassing moment in HD (720p) with stereo sound. The S6200's powerful 10x zoom lets you bring the action closer for dramatic close-ups. Then plug the mini HDMI connector into an HDTV and easily share those moments. It's your life—start putting memories in motion.
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It's easy to add pizzazz to your photos and movies. Touch a button and get creative by shooting in black and white, a soft sepia for a nostalgic mood, high key or even low key. The S6200 does all the work, so you don't have to spend any time using special processing software later on. And you'll love incorporating filter effects such as fisheye, soft focus or even miniaturization. Unleash your creative side and experiment with all the creative tools the S6200 offers. Start spicing up your world.
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Convenient features for optimal shooting
Your life is complicated; your camera shouldn't be. With the S6200, just point and shoot to capture the smiles—near and far. With the help of 18 scene modes, including Auto Scene Selector and Nikon's amazing Smart Portrait System, you'll never have to guess how to configure the camera to an environment. Just aim on a grin and the S6200 does all the work for you. It automatically sets the optimum Scene Mode, finds and focuses on the face, fixes red eye and even warns you if your subject blinks! The S6200 keeps photography fun, easy and enjoyable.
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The S6200's Vibration Reduction (VR) Image Stabilization features automatically reduce the streaky images associated with shaky cameras.
Strength in numbers
You'll always get a great shot with Nikon's Best Shot Selector. Press the shutter once, and the S6200 takes up to ten shots and then selects and saves only the sharpest image.

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Today's Events
Movies in the Park: Cinderella at North Domingo Baca Multigenerational Center
Ella is at the mercy of her stepfamily, who reduce her to their personal maid. Despite the situation, she refuses to despair in hopes that true love will save her. Bring your lawn chairs and blankets.
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Anxiety seems to grip our society these days. September 11 brought fears of flying and of the mail. Airlines heighten security and try to attract passengers with low fares and bonus frequent flyer miles. Homeland security alerts heighten tensions. Should we worry about this bridge, that building or tonight's sporting event? A necessary war with …
Tag Archive: terror
Sep 25 2013
High anxiety: Had your dose of peace today?
September 25, 2013
Tags: 9-11, 9/11, air, airplanes, anxiety, chemical, Christ, Christian, chrstianity, death, dying, economic, economy, emotion, emotional paralysis, exercize, fear, fearful, fearful world, God, grave, greastest fear, guilt, harmonal, homeland, Homeland Security, illness, imbalance, Jesus, lack of exercise, lack of friendship, lack of meaning, life, lor, lord, lord jesus, mail, pizza, planes, poor diet, poor diet or exercise, post 9-11 world, prince of peace, problems, relationships, Rusty Wright, scare, sep 11, September 11, shakey, sick, Taliban, terrified, terror, terrorist, troubled mind, uncertain, uncertainty, war, what if, what ifs what if's, what is life about, world's troubles, worried, worry
Permanent link to this article: http://www.easttexasreview.com/high-anxiety-had-your-does-of-peace-today/
Jun 21 2013
Metadata mining is mega awful
It's good to know that our friendly, über-secret National Security Agency is out there every day, protecting our freedom. By violating it. A whistleblower has blown the lid off the NSA's super-snoop program of rummaging electronically through about a billion phone calls made every day by us average Americans. This revelation prompted Al Gore to …
Tags: Al Gore, America, authority, average americans, Barak Obama, big brother, big brother government, blanket, blanket surveillance, call, complaint, Congress, data, democrat, Democratic, federal judge, Fourth Amendment, freedom, Georgia Republican, government, Hightower Lowdown, illegal, Jim Hightower, Lindsey Graham, mega aweful, meta data, metasurveillance, metea data mining, national, national security, national security agency, nsa, Obama, outrageous, phone, phone call, phone calls, phone line, President Obama, private, private call, private lives, problem, protect, protection, Republican, Saxby Chambliss, secret, security, senator, Senator Graham, snoop, snooping, South Carolina Republican, spy, spy court, spying, surveillance, terror, terrorist, united states, usa, watching, whistle blower, whistleblower, White House, "It doesn't bother me one bit that NSA has my number
Permanent link to this article: http://www.easttexasreview.com/metadata-mining-is-mega-awful/
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Credit Markets Weak with Outflows Increasing 0 comments
This morning credit is open weaker across the board.
IG16 which had eked out a 1 bp tightening on Friday is right back out 1 bp and trading at 100.50. It is basically the same story across the CDX indices where they are retracing Friday's gains.
SOVX is actually outperforming. It is only out 8 bps today, to 229, which is only half of the 16 bps it tightened on Friday. This relative outperformance could be healthy if it signified an improvement in sovereign debt, but it seems that it is an indication that some fear is slipping into the real economy and there is pressure on corporate credit. With big sell offs over the past month in sovereigns and CMBS it is not surprising that corporate spreads are widening.
(NYSEARCA:HYG) and (NYSEARCA:JNK) both saw shares outstanding decline of Friday in spite of the bounce in price. The daily decline in shares for (HYG) was the most since the decline started in early June. If withdrawals are accelerating, it will have an outsized impact on this market that is already shaky.
Asides from the obvious declines in HYG and JNK and widening in the CDX indices, the HYG NAV is another sign of how shaky the market is. The NAV did not drop as much as HYG on Thursday, but also showed a small decline in value of Friday. This is a sign, to me, that liquidity is drying up in the HY market. Bonds where the average bid/offer typically ranges from 1/2 pt to 1 pt, it widened to 1 to 2 pts. This means that calculating the NAV is as much art as science when bid/offer is so wide. According to TRACE volumes on the HY market plunged to the second lowest full day volume on the year. Another worrisome sign and an indication that price discovery is low.
The single name CDS market rolls today. So far it looks like it is creating a bit more activity, and might encourage some new shorts as they don't have to worry about another roll for 3 months.
I remain short (NYSEARCA:SPY), though down to about 1/2 position as I want to see how the market trades near the 200 day moving average. I had cut my (HYG) short last week - long (NYSEARCA:SJB), but may open that again depending on how the market trades. I thought we could get a bounce more significant than we got on Friday, but the combination of withdrawals, volumes, NAV calculations, CDS roll, and sovereign debt contagion in the forefront after this week's failure to come up with a Greek plan, I am more likely to go short HYG again.Back To Peter Tchir's Instablog HomePage »
Instablogs are blogs which are instantly set up and networked within the Seeking Alpha community. Instablog posts are not selected, edited or screened by Seeking Alpha editors, in contrast to contributors' articles.Full index of posts »
$VZ this week the bond deal was more important than the equity and still looks cheap http://bit.ly/1evGIMF Sep 14, 2013
$HYG $JNK $LQD $TLT $EMB get your weekly fixed income (with an ETF focus) update at http://bit.ly/1evGIMF tapering to tightening Sep 14, 2013
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text: Sunday Comics: Eating Before BedtimeOwen Good7/15/12 10:00amFiled to: Funniessunday comicsComicsHumorWebcomicsTop1EditInvite manuallyPromoteDismissUndismissHideShare to KinjaToggle Conversation toolsGo to permalink Welcome to your Sunday read of the week's best in web comics. Make sure to click on the expand button in the bottom right to enlarge each comic. Brawl In The Family by Matthew Taranto published July 10.—Read more of Brawl In The Family Awkward Zombie by Katie Tiedrich published July 9.—Read more of Awkward Zombie Advertisement Nerf NOW!! by Josué Pereira published July 12.—Read more of Nerf NOW!! Penny Arcade by Jerry Holkins and Mike Krahulik published July 11.—Read more of Penny Arcade Sponsored Manly Guys Doing Manly Things by Kelly Turnbull published July 9.—Read more of Manly Guys Doing Manly Things Virtual Shackles by Jeremy Vinar and Mike Fahmie published July 9.—Read more of Virtual Shackles Another Videogame Webcomic by Phil Chan and Joe Dunn published July 13.—Read more of Another Videogame Webcomic ActionTrip by Borislav Grabovic and Ure Paul published July 9.—Read more of ActionTrip Advertisement Legacy Control by Javis Ray published July 11.—Read more of Legacy ControlReplyLeave a reply


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Allenspark CO Real Estate & Homes for Sale
17 Properties Found
The median home value in Allenspark, CO is $477,500.
This is
higher than
the county median home value of $345,300.
The national median home value is $177,414.
The average price of homes sold in Allenspark, CO is $477,500.
Approximately 23.52% of Allenspark homes are owned,
compared to 4.47% rented, while
72.01% are vacant.
Allenspark real estate listings include condos, townhomes, and single family homes for sale.
Commercial properties are also available.
If you see a property you're interested in, contact a Allenspark real estate agent to arrange a tour today!
Sort Results:
1 - 17 of 17 Properties Displayed
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Courtesy: Re/Max Alliance
View additional info
Inexhaustible beauty and sunshine will fill your life in this wonderfully remodeled home. Exquisite 180 degree views of James Peak, Mt Thorodin and the surrounding peaks and valleys. Expansive decks that you will never want to leave.2500 sq ft. 3 beds, 2 baths. Private, silent, yet convenient to Nederland and Boulder, hiking, biking and skiing (10 minutes to Eldora). Kitchen boasts Corian counters, beautiful hardwood cabinets, stainless steel appliances. 256 addl sq ft could be added to south br. porperty abuts national forest on north end
Courtesy: Re/Max Mountain Brokers Peggy Lynch
View additional info
Rare find 26 acres bordered 3 sides by National Park and Forest. Meticulously maintained fully furnished, full-log, open floor plan intimate, yet spacious. Stunning 2 story great room, kitchen, covered deck enjoy magnificent views over Wild Basin and the Divide. Concrete walk out basement ideal for bedroom/bath expansion with views. Easy 1 mile private road access from highway 7. Pond with 5 acre-foot water rights fed directly from RMNP. A protected, secluded haven 20 minutes from Estes Park.
Courtesy: Re/Max Mountain Brokers Peggy Lynch
View additional info
Quality custom home on 2 lots totaling 5.7 acres Borders Rocky Mountain national Park!Complete privacy, yet easy drive to Estes,Longmont orBoulder.Massive Moberg mossrock fireplace extends up the 27' vaulted ceiling in the great room/dining room/kitchen with nice tile and carpeted floors.Main level living with extra guest room.Upstairs has 26' recreation room, Guest Bedroom, bath and large office with great views. 725 sq. ft heated shop/garage ready for your hobby. Meeker and Twin Sister views.
12650 Hwy 7 Allenspark, CO 80510
Courtesy: Colorado Landmark Realtors
View additional info
Bordered by Park Service land at the Wild Basin entrance to the Rocky Mountain National Park, enjoying Mt. Meeker as backdrop, this retreat offers lux living coupled with the utmost privacy. The 36 acre gently sloping lot is dense with Pine & Aspen trees complimented by many moss rock outcroppings. The home has been custom built with attention paid to the most finite detail. Mortis & Tenon timbers from British Columbia, custom walnut cabs, 12â antique oak flrs from a farm in Ohio. Itâs Perfect! The location is ideal: 15 min to Estes Park, 20 mins to Lyons, 45 mins to Boulder
12650 Highway 7 Allenspark, CO 80510
Courtesy: Colorado Landmark-Boulder Stephen Remmert
View additional info
Bordered by Park Service land at the Wild Basin entrance to Rocky Mountain National Park, enjoying Mt. Meeker as backdrop, this retreat offers lux living coupled with the utmost privacy. The 36 acre gently sloping lot is dense with Pine & Aspen trees complimented by many moss rock outcroppings. The home has been custom built with attention paid to the most finite detail. Mortis & Tenon timbers from British Columbia, custom walnut cabs, 12" antique oak flrs from a farm in Ohio. It's Perfect!
Courtesy: Rocky Mountain Property Inc. Tammy Ackerman
View additional info
On a tranquil setting this cabin sits. Walking or skiing distance to St Vrain & Meadow Mountain trail head in Roosevelt National Forest. Offering two spacious bedrooms, one full bath, a large eat in kitchen with flagstone floor, a living room fireplace and more. Complete with an over sized deck for outdoor living, beautiful views of the Ironclads, meadow, Aspen and evergreens. Sellers use the cabin as a year around vacation home. Shared well and septic.
Courtesy: Range Realty Ann Racine
View additional info
Just a little cabin with a big Mt. Meeker view and a lot that is heavily treed with Aspen! Located at the base of the mountain and backing up to Rocky Mountain National Park, you can start your hiking adventures right out the back door. This cabin is summer only; the property has an outhouse and will need a vault. The water is from Mt. Meeker Lodge.
11983 Highway 7 Allenspark, CO 80510
Courtesy: First Colorado Realty Christian J. Collinet
View additional info
Spectacular 38.6-acre setting within alpine forest, aspen groves & wildflower meadows along peaceful creek! Enjoy this historic, 1924 log home with private hiking that leads directly into the Nat'l Forest. Rich in history, the home offers a grand Rocky Mountain greatroom with native stone fireplace, 3 bedrooms and classic bath with claw-foot tub. Waterfall views, year-round creek and pristine forest, plus water rights. A magical mountain property of natural beauty, privacy and serenity.
2633 Big Owl Rd Allenspark, CO 80510
Courtesy: Rocky Mountain Property Inc. Tammy Ackerman
View additional info
Beautiful Rusticity- This property located between Allenspark and Estes Park offers a lovely log cabin and guest house with Fawn Creek running through. The main house has hardwood floors, spacious remodeled kitchen with french doors opening to an outdoor deck for summer entertaining, custom lighting, and more. Frontage on Highway 7 brings the possibility of a home based business allowed by Boulder County in Forestry zoning with one 2'x2' sign.
S County Road 59 Allenspark, CO 80510
Courtesy: Hayden Outdoors - Windsor coires
View additional info
Old Elk Park Ranch consists of 548 acres. Here you will find limitless views of the surrounding mountains, open pastures full of wildlife, awe inspiring surroundings, and abundant water. Multiple well-built improvements are off-the-grid. 10 miles from Estes Park and RMNP. Two airstrips makes for unmatched access from anywhere in the country. Great hunting, horseback riding and off-road riding. For more info, pics and video visit http://www.haydenoutdoors.com/land-for-sale/old-elk-park-ranch
5150 County Road 59 Allenspark, CO 80510
Courtesy: Hayden Outdoors - Windsor coires
View additional info
Wild Basin Ranch is 154 acres offering an expansive valley with sub irrigated grass, numerous springs and an abundance of wildlife. Heated garage with a 2 bedroom apartment above. Building envelopes to transfer. Water rights could be used to develop future ponds/lakes. Year round access, 10 miles from Estes Park and RMNP. National Forest on 2 sides. Great hunting, horseback riding and trail riding! For more info, pics and video visit http://www.haydenoutdoors.com/land-for-sale/wild-basin-ranch
5240 County Road 59 Allenspark, CO 80510
Courtesy: Hayden Outdoors - Windsor coires
View additional info
80 acre mountain getaway only 10 miles from Estes Park and 1 hour from Front Range. Spectacular views, outdoor activities, year round accessibility, end of the road seclusion and 2 comfortable living cabins, with garage to store toys. Cabins are off grid and offer upscale finishes. Property has good grass and is fenced for horses. Historic barn. Bordered on 2 sides by national forest. great hunting! For more info, pics and video visit http://www.haydenoutdoors.com/land-for-sale/elk-park-retreat
1448 Le Cook Allenspark, CO 80510
Courtesy: Hayden Outdoors - Windsor coires
View additional info
ATTENTION AVIATORS! Unique opportunity for a pilot to own their very own high mountain ranch. 80 acres of exceptional high mountain acreage, a certified airstrip, and a large airplane hangar with lots of room for additional toys. Above hangar is a upscale 3 bed apartment. 10 miles south of Estes Park and Rocky Mountain National Park. Great hunting! Property is bordered on 3 sides by National Forest. For more info visit http://www.haydenoutdoors.com/land-for-sale/elk-park-airstrip
Courtesy: Greg Falconer
View additional info
REDUCED $35,000 & now below $585K appraisal. Highest quality custom home with many imported hardwoods for floors, custom handmade cabinets & doors. Beautiful open floor plan with a high vaulted ceiling. The 3 ceiling fans are antique replicas connected with a belt driven system. The kitchen is well appointed with granite countertops and the best of appliances and is ready for classy entertaining.Borders National Forest. View of Mt Meeker. A 4th garage space is in the walk out house basement.
303 Old State Hwy 7 Allenspark, CO 80510
Courtesy: Fuller Sotheby's Intl Realty Marie Jacobs
View additional info
Experience Colorado authentically. Serenity abounds on this 32-acre mtn property abutting National Forest. Located 15 min from Estes Park, this log home provides ample indoor/outdoor living and widespread mountain views. The fenced property includes a pond, pool, hot tub, barn, tack and corral. Main floor master, handicapped access, finished lower level walk-out, large loft and a separate studio w/bath complete this exceptional home. New kitchen to be included in price.
12 Clarabell Rd Allenspark, CO 80510
Courtesy: Greg Falconer
View additional info
A great 1 1/2 ac. with a stream building site near Allenspark, National Forest land and Rocky Mtn National Park. The existing cabin, well and septic system all contribute to getting a building permit for construction of a new residential dwelling. The existing 484 square foot cabin might be used for a rough weekend cabin. Consult Boulder County or improvement requirements.
County Road 82E Allenspark, CO 80510
Courtesy: Stroh & Company Dan Stroh
View additional info
The Trevarton Ranch has been family owned and operated since 1929. This is a 2100 acre ranch located between Estes and Allens Park. The ranch provides lush mountain grass,hay,lumber,and firewood,sawmill services,horse pasture/boarding and hunting.Beautiful high mountain meadows provides high quality mountain grass for horses and cattle.Property features historic 100 year old barn. Barn is beautiful and very functional. Live water throughout the ranch Little Thompson River.Breathtaking Property!
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Allenspark CO Real Estate Listings Provided By:
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Allenspark ZIPs80510 Homes for Sale


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Arab League should postpone action to establish a court of human rights
The ICJ and other rights groups today sent an open letter to the Ministers of Foreign Affairs of the member States of the League of Arab States (LAS) urging them to defer action on the adoption of the draft Statute of the proposed Arab Court of Human Rights (the Arab Court).
The organizations called on the LAS instead to allow for the draft Statute to be amended through a deliberative and transparent process.
The ICJ notes that the draft Statute was prepared by an LAS-appointed expert Committee, whose membership has remained a secret.
The entire drafting process was conducted behind closed doors. Despite repeated requests, civil society organizations and other stakeholders were excluded from the process.
The draft, if adopted, would reserve access to the Court primarily to States, and not to individual victims.
"While the ICJ welcomes, in principle, the idea of establishing the Arab Court, we are deeply concerned that the draft Statute falls woefully short of what would be required for a Court that is able to provide justice for victims of human rights violations," said Said Benarbia, Director of the ICJ Middle East and North Africa Programme. "The inability for individual victims of human rights violations to access the Arab Court would defeat its entire object and purpose."
A decision on the adoption of the draft Statute is due to take place at the upcoming session of the Ministerial Council, which is scheduled for 3 to 8 September 2014.
The ICJ and the other signatories to the letter said that the draft Statute needs to be amended significantly to allow for the establishment of an independent, impartial and effective judicial mechanism to protect rights and freedoms in the LAS member States.
The organizations called for a number of amendments aimed at ensuring:
- the independence and impartiality of the Arab Court and its judges, through nomination, election, suspension and removal procedures and conditions of tenure that are in line with international standards;
- that the jurisdiction of the Arab Court is restricted to the rights contained in the Arab Charter of Human Rights, interpreted consistently with States' other obligations under international law;
- that provisions on the requirement to exhaust local remedies are not overly restrictive and that the Arab Court has flexible discretion to decide on the admissibility of cases;
- that all individuals within the territory of a State party, or otherwise subject to its jurisdiction, have access to the Arab Court when they claim to be a victim of a violation of a right, and that obstacles that limit NGO access are removed, such as the requirement that the State against which a claim is made accept such access;
- that individuals and NGOs can also join proceedings as interested parties or as third party interveners;
- that the Arab Court is mandated to issue interim measures where the applicant faces an imminent risk of serious, irreversible or irreparable harm and to order protection measures necessary for witnesses;
- that mechanisms are in place to ensure that the Arab Court's judgments are effectively executed, including by providing for an independent and effective monitoring mechanism; and
- that the host State of the Court is determined based on that State's commitment to and compliance with universal human rights laws and standards, and that the host State provides the necessary guarantees for the Court, its judges and staff, and for the protection of victims and their representatives, witnesses and NGOs.
Said Benarbia, ICJ Director of the Middle East and North Africa Programme, tel: 41 22 979 38 17, e-mail: said.benarbia(a)icj.org
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Click here to list ALL Makes
20 ft box van with extended tail lift.
Ideal for sports/saloon, or easily converted to take 2 single seaters/caterhams.
Workshop area with workbench,vice, storage cupboards, tyre racks, etc
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Read-Along with Disney!
Mowgli is a little boy who has grown up in the jungle. Then one day the tiger Shere Khan returns, and Mowgli is no longer safe.
Follow along with the word for word narration in this storybook retelling of Mowgli's adventures from the classic Disney film!
Other Books in the Disney Series
The Haunted MansionBy: Jason Surrell
Liv and Maddie: Double TroubleBy: Lexi Ryals
The Hidden Mickeys of DisneylandBy: Bill Scollon
Live and Maddie: Sisters ForeverBy: Lexi Ryals
The Lone RangerBy: Elizabeth Rudnick
Poster Art of the Disney ParksBy: Daniel Handke, Vanessa Hunt
Be Our GuestBy: The Disney Institute, Theodore Kinni
Walt Disney ImagineeringBy: The imagineers
Other Books Ages 3-5
How This Book Was MadeBy: Mac BarnettIllustrated By: Adam Rex
Let Me Finish!By: Minh LêIllustrated By: Isabel Roxas
Rules of the HouseBy: Mac BarnettIllustrated By: Matt Myers
Explorers of the WildBy: Cale Atkinson
Good Night OwlBy: Greg Pizzoli
Walt Disney's Alice in Wonderland (Re-issue)By: Jon Scieszka
Leaps and BounceBy: Susan Hood
Bug ZooBy: Andy HarknessIllustrated By: Andy Harkness
That's Not Bunny!By: Chris BartonIllustrated By: Colin Jack


text:
Backport #7936
backport r39449
#1 [ruby-core:52783] Updated by Yusuke Endoh over 3 years ago
- Status changed from Open to Assigned
- Assignee changed from Yusuke Endoh to Tomoyuki Chikanaga
This is not a typo fix or format improvement, but includes (very small) semantic change.
Let's consider after the patch level release after p0.
Yusuke Endoh email@example.com
#2 [ruby-core:53267] Updated by Tomoyuki Chikanaga over 3 years ago
I think passing String to these methods is meaningful because it is a way to avoid unnecessary Symbol allocation. So I'll merge r39449.
mame san, do you have a concern?
#3 [ruby-core:53712] Updated by Nobuyoshi Nakada over 3 years ago
Yes, it's meaningful, but the documents are inaccurate on that point.
Any *_defined? methods don't convert the argument to a Symbol always.
I don't think the distinguishment between symbol and string here is important enough.
And the name symbol/string seems less meaningful.
I'd suggest to change the argument names and mention that it can be a symbol or a string.
#4 [ruby-core:53720] Updated by Zachary Scott over 3 years ago
nobu (Nobuyoshi Nakada) wrote:
Yes, it's meaningful, but the documents are inaccurate on that point.
Thank you for your feedback, always appreciated nobu-san.
I'd suggest to change the argument names and mention that it can be a symbol or a string.
Do you mean for *_defined? methods or for all methods mentioned in this patch?
#5 [ruby-core:54257] Updated by Tomoyuki Chikanaga over 3 years ago
- Assignee changed from Tomoyuki Chikanaga to Nobuyoshi Nakada
- Status changed from Assigned to Feedback
nobu, could you clarify about zzak's question?
#6 [ruby-core:54611] Updated by Nobuyoshi Nakada about 3 years ago
I meant for all methods.
Sorry for late reply.
#7 [ruby-core:54613] Updated by Zachary Scott about 3 years ago
@nobu np, you mean something like:
attr_accessor(string or symbol)
attr(string or symbol)
#8 Updated by Yui NARUSE about 2 months ago
- Status changed from Feedback to Rejected