JP2003500341A - Long-lasting anti-angiogenic peptide - Google Patents

Abstract

  (57) [Summary] Disclosed are modified anti-angiogenic peptides. This modified peptide may form a peptidase stabilized anti-angiogenic peptide. Modified anti-angiogenic peptides, particularly modified kringle 5 peptides, can form conjugates with blood proteins. Conjugates are prepared from anti-angiogenic peptides, especially kringle 5 peptides, by combining a peptide with a reactive functional group with a blood protein. The conjugate can be formed in vivo or ex vivo. The conjugate is administered to a patient to provide an anti-angiogenic effect.

Description

Detailed Description of the Invention

FIELD OF THE INVENTION The present invention relates to modified anti-angiogenic peptides. In particular, the invention relates to modified kringle 5 peptides with long-term action for the treatment of diseases associated with angiogenesis.

BACKGROUND OF THE INVENTION Angiogenesis, the development of new blood vessels, is a highly regulated and essential process of endothelial cell proliferation. Although angiogenesis is a highly regulated process under normal conditions, many diseases (characterized as "angiogenic disorders") have persistently unregulated angiogenesis. Caused by. Unregulated angiogenesis can either directly cause certain diseases or exacerbate existing pathological conditions. For example, ocular neovascularization has been implicated as the most common cause of blindness and accounts for about 20 eye diseases. In certain current situations such as arthritis,
The newly formed capillaries penetrate the joints and destroy the cartilage. In diabetes, new capillaries formed in the retina invade the vitreous, bleed, and cause blindness. Solid tumor growth and metastasis are also angiogenesis-dependent (Folkman, J., Cancer Research, 46: 467-4.
73 (1986), Folkman, J. et al. , Journal of the N
national Cancer Institute, 82: 4-6 (1989).
)).

Much work has been done to identify anti-angiogenic molecules. One angiogenic molecule of particular interest is plasminogen. Of particular interest are the kringle 5 region of plasminogen and the various peptides within that kringle 5 region. Both plasminogen and the kringle 5 region of plasminogen have been shown to interfere with the angiogenic process and are therefore known as anti-angiogenic peptides.

[0004] Although useful, the Kringle 5 peptide, like other peptides, undergoes rapid renal excretion, liver metabolism, and degradation of endogenous peptidases, leading to a very short plasma half-life, thereby leading to anti-antibody activity. Their utility as angiogenic agents is diminished. As a result of their short half-life, peptides such as kringle 5 require continuous infusion to reach adequate plasma levels sufficient for effective treatment.

As a result, there is a need for long-lasting anti-angiogenic peptides such as kringle 5. Such long lasting peptides are useful in treating angiogenesis-related diseases in mammals.

SUMMARY OF THE INVENTION To achieve these needs, the present invention relates to modified anti-angiogenic peptides. In particular, the invention relates to modified kringle 5 peptides. The present invention relates to novel chemically reactive derivatives of anti-angiogenic peptides that can react with useful functional groups on mobile blood proteins to form covalent bonds. In particular, the present invention relates to novel chemically reactive derivatives of angiogenic peptides, such as kringle 5 peptide, that can react with useful functional groups on mobile blood proteins to form covalent bonds. Chemically reactive derivatives of anti-angiogenic peptides can form peptidase-stable anti-angiogenic peptides.

The present invention relates to derivatives of anti-angiogenic peptides such as kringle 5 peptide, wherein the derivatives react with amino, hydroxyl or thiol groups on blood proteins to form stable covalent bonds. And includes a reactive group. In a preferred form, the anti-angiogenic peptide comprises a succinimidyl-reactive group or a maleimide-reactive group.

The present invention relates to modified kringle 5 peptides, and their derivatives, and their use as anti-angiogenic agents. The kringle 5 peptide contains a reactive group capable of forming a covalent bond with mobile blood proteins.

[0009]   In particular, the invention relates to the following modified kringle 5 peptides:

[0010]

[Chemical 1] And other modified kringle 5 peptides.

These modified anti-angiogenic peptides find use in the treatment of angiogenesis in humans.

DETAILED DESCRIPTION OF THE INVENTION To ensure a complete understanding of the invention, the following definitions are provided: Reactive group: A reactive group is a chemical group capable of forming a covalent bond. . Such reactive groups are linked or attached to the anti-angiogenic agent, ie, more specifically, the kringle 5 polypeptide of interest. The reactive group is generally stable in aqueous environment and is usually a carboxy group, a phosphoryl group, or a conventional acyl group,
Present as either an ester or mixed anhydride, or as an imidate, which may form a covalent bond with a functional group, such as an amino group, a hydroxy or a thiol, at the target site of a mobile blood component. To a large extent, these esters include phenolic compounds or are thiol esters, alkyl esters, phosphate esters and the like. As the reactive group, succinimidyl (succ
imidyl) groups and maleimide groups.

Functional group: A functional group is a group on a blood component on which a reactive group reacts to form a covalent bond. Functional groups include hydroxyl groups for attachment to ester-reactive groups, thiol groups for attachment to imidate and thiol ester groups; attachment to carboxyl groups, phosphoryl groups or acyl groups on reactive groups It includes an amino group and a carboxyl group for coupling with the amino group.

Blood component: The blood component may be fixed or movable.
Fixed blood components are non-moving blood components and are tissues, membrane receptors, intervening proteins, fibrin proteins, collagen, platelets, endothelial cells, epithelial cells and the membrane and membrane receptors to which they bind, body cells. (Somat
ic body cells), skeletal and smooth muscle cells, neural components, osteocytes and osteoclasts and all body tissues, especially those associated with the circulatory and lymphatic systems. A mobile blood component is a blood component that does not have a fixed position for any extended period of time (typically no more than 5 minutes, more usually 1 minute). These blood components are not membrane bound and are present in blood for an extended period of time and are present at a minimum concentration of at least 0.1 μg / ml.
Mobile blood components include serum albumin, transferrin, ferritin, and immunoglobulins such as IgM and IgG. The mobile blood component has a half-life of at least about 12 hours.

Protecting Group: A protecting group is a chemical moiety utilized to protect a peptide derivative from reacting with itself. Various protecting groups are described in US Pat. No. 5,493,
No. 007, which is incorporated herein by reference. Such protecting groups include acetyl, fluorenylmethyloxycarbonyl (Fmo
c), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (C
bz) and the like. The specific protected amino acids are shown in Table 1.

[0016]

[Table 1] Sensitive Functional Groups: Sensitive functional groups are groups of atoms that provide potential reactive sites on anti-angiogenic peptides. When present, sensitive functional groups can be selected as attachment points for linker-reactive group modification. Sensitive functional groups include, but are not limited to, carboxyl groups, amino groups, thiol groups, and hydroxyl groups.

Modified peptide: A modified anti-angiogenic peptide is a peptide modified by the attachment of a reactive group and can form a peptidase-stabilizing peptide by binding to a blood component. The reactive group can be attached to the anti-angiogenic peptide either through a linking group or optionally without a linking group. It is also contemplated that one or more additional amino acids may be added to the anti-angiogenic peptide to facilitate attachment of reactive groups. The modified peptides may be administered in vivo such that conjugation with blood components occurs in vivo, or they are first conjugated with blood components in vitro and stabilized against the resulting peptidase. The peptide as defined (as defined below) can be administered in vivo. the term"
"Modified anti-angiogenic peptide" and "modified peptide" may be used interchangeably herein.

Anti-angiogenic peptide stabilized against peptidase: An anti-angiogenic peptide stabilized against peptidase refers to the reactivity of the modified peptide with or without a linking group. A modified peptide conjugated to a blood component via a covalent bond formed between the group and a functional group of the blood component. Peptidase stabilized peptides are more stable in vivo in the presence of peptidases than non-stabilized peptides. Anti-angiogenic peptides that are stabilized against peptidases generally have at least a 10-50% increased half-life compared to unstabilized peptides of the same sequence. Peptidase stability is
It is determined by comparing the half-life of the unmodified anti-angiogenic peptide in serum or blood with the half-life of the corresponding modified anti-angiogenic peptide in serum or blood. Half-life is determined by sampling serum or blood after administration of the modified and unmodified peptides and determining the activity of the peptide. In addition to determining activity, the length of anti-angiogenic peptides can also be measured by HPLC and mass spectrometry.

Linking Group: A linking group is a chemical moiety that attaches or links a reactive group to an anti-angiogenic peptide. The linking group is one or more alkyl groups (eg, methyl group, ethyl group, propyl group, butyl group, etc.), alkoxy group, alkenyl group, alkynyl group, amino group substituted with alkyl group, cycloalkyl group, polycyclic group. It may include formula groups, aryl groups, polyaryl groups, substituted aryl groups, heterocyclic groups, and substituted heterocyclic groups. The linking group may also include a polyethoxy amino acid, such as AEA ((2-amino) ethoxyacetic acid) or the preferred linking group AEEA ([2- (2-amino) ethoxy)] ethoxyacetic acid). A preferred linking group is AEEA ([2-
(2-amino)] ethoxyacetic acid).

DETAILED DESCRIPTION OF THE INVENTION In view of these definitions, the focus of the present invention is to focus on anti-angiogenesis by binding peptides to protein carriers without altering anti-angiogenic properties. Modifying the forming peptides, especially the Kringle 5 peptide, to improve bioavailability and prolong the half-life and distribution of the peptide in vivo. The carrier of choice in the present invention, but not limited to the present invention, is albumin linked via a free thiol with a kringle 5 peptide derivatized with a maleimide moiety.

1. Kringle 5 Peptide The term “kringle 5” as used herein contributes to a particular three-dimensional structure defined by the fifth kringle region of the mammalian plasminogen molecule. It refers to a region of mammalian plasminogen that has three disulfide bonds. 1
One such disulfide bond connects cysteine residues located at amino acid positions 462 and 541, a second disulfide bond connects cysteine residues located at amino acid positions 483 and 524, and a third disulfide bond. The bond is
The cysteine residues located at amino acid positions 512 and 536 are linked. The amino acid sequence of the complete mammalian plasminogen molecule (human plasminogen molecule) (including its kringle 5 region) is represented by (SEQ ID NO: 1).

The term "kringle 5 peptide fragment" has the following substantial sequence homology to the corresponding peptide fragment of mammalian plasminogen:
Between 10 and 104 amino acids (including 4 and 104) having anti-angiogenic activity: amino acid position 443 of intact mammalian plasminogen
Of the intact mammalian plasminogen at the amino acid position of about 513, and the amino acid position of about 523 of the amino acid position of SEQ ID NO: 1 of about 523 of SEQ ID NO: 1. α-C-terminal; the α-N-terminal of amino acid position about 525 of intact mammalian plasminogen, and the α-C-terminal of amino acid position of about 535 of SEQ ID NO: 1; amino acid position of about 529 of intact mammalian plasminogen. an α-N-terminal and an α-C-terminal at about amino acid position 535 of SEQ ID NO: 1; an α-N-terminal at about amino acid position 529 of intact mammalian plasminogen,
And the α-C-terminus of amino acid position 534 of SEQ ID NO: 1; and the α-N-terminus of amino acid position 150 of intact mammalian plasminogen and the α-C-terminus of amino acid position 156 of SEQ ID NO: 1.

In a preferred form, the kringle 5 peptides of the invention have one or more of the following sequences:

[0024]

[Chemical 2] Accordingly, the present invention is intended to encompass any derivative or modification of a kringle 5 peptide fragment having anti-angiogenic activity, and the entire class of kringle 5 peptide fragments described herein, It should be understood that as well as derivatives and modifications of these kringle 5 peptide fragments are included.

(2. Modified Kringle 5 Peptide) The present invention relates to a modified anti-angiogenic peptide, particularly a modified kringle 5 peptide. The modified kringle 5 peptides of the present invention can react with available reactive functional groups of blood components via covalent bonds. The invention also relates to such modifications, such combinations with blood components and methods of their use. These methods involve extending the effective therapeutic in vivo half-life of the modified kringle 5 peptide.

Chemically reactive groups (reactants) to form covalent bonds with functionalities on proteins
A wide variety of active carboxyl groups, especially esters, can be used as
The hydroxyl moiety is physiologically acceptable at the level required to modify the kringle 5 peptide. Many different hydroxyl groups can be used in these linking agents, but the most common are N-hydroxysuccinimide (
NHS), N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimido-benzoyl-succinimide (MBS), γ-maleimido-butyryloxysuccinimide ester (GMBS) and maleimidopropionic acid (M).
PA).

Primary amines are important targets for NHS esters as illustrated in the scheme below. The accessible α-amine group present at the N-terminus of the protein reacts with the NHS ester. However, the α-amino group of the protein is
It may not be desirable or available for coupling. Although 5 amino acids have a nitrogen in their side chain, only the ε-amine of lysine significantly
Reacts with HS ester. The amide bond is formed when the NHS ester reacts with a primary amine to release N-hydroxysuccinimide in a ligation reaction, as shown in the scheme below. Succinimides having these reactive groups are referred to herein as succinimidyl groups.

[0028]

[Chemical 3] In a preferred embodiment of the invention, the functional group of the protein is a thiol group and the chemically reactive group is a maleimide-containing group such as γ-maleimido-butylamide (GMBA) or MPA. Such maleimide-containing reactive groups are referred to herein as "maleimide groups". The maleimide group is the pH of the reaction mixture.
Is maintained between 6.5 and 7.4, it is most selective for the sulfhydryl group of the peptide. At pH 7.0, the reaction rate of maleimide groups with sulfhydryls is 1000 times faster than the reaction with amines. A stable thioether bond is formed between the maleimide group and the sulfhydryl, which cannot be cleaved under physiological conditions as shown in the scheme below.

[0029]

[Chemical 4] The kringle 5 peptides and peptide derivatives of the invention can be modified for specific and non-specific labeling of blood components.

A. Specific Labeling Preferably, the modified angiogenic peptides of the present invention are designed to specifically react with thiol groups of mobile blood proteins. Such reactions are preferably anti-neoplastic modified using maleimide linkages (eg, prepared from GMBS, MPA or other maleimides) to thiol groups of mobile blood proteins such as serum albumin or IgG. Established by covalent attachment of angiogenic peptides.

Under specific conditions, the specific labeling with maleimide (maleimide group) is
It offers several advantages over non-specific labeling of mobile proteins with groups such as NHS and sulfo-NHS. Thiol groups are less abundant in vivo than amino groups. Therefore, the maleimide derivative of the present invention covalently binds fewer proteins. For example, in albumin, the most abundant blood protein, there is only one thiol group. Thus, peptide maleimide-albumin conjugates tend to contain peptide to albumin in a molar ratio of about 1: 1. In addition to albumin, IgG molecules (class II) also have free thiols. Since IgG molecules and serum albumin make up the majority of proteins soluble in blood, they also make up the majority of the free thiol groups in blood available for covalent attachment to maleimide modified peptides.

Furthermore, even among free thiol-containing blood proteins, the specific labeling with maleimide leads to the preferential formation of peptide-maleimide-albumin conjugates due to the unique characterization of albumin itself. Lead. The single free thiol group of albumin is highly conserved among species and is located at amino acid residue 34 (Cys ³⁴ ). Recently, it has been shown that Cys ^{34 of} albumin is highly reactive compared to the free thiols of other free thiol-containing proteins. This is C of albumin
In part due to the very low pK value for ys ³⁴ (5.5). This is generally much lower than the typical pK value for cysteine residues (typically about 8). Due to this low pK, under normal physiological conditions Cys ³⁴ of albumin is the predominantly ionized form, which dramatically increases its reactivity. In addition to the low pK value of Cys ^34, another factor which enhances the reactivity of Cys ³⁴ is its location, its position is a gap close to the surface of one loop of region V of albumin. This position makes Cys ³⁴ just available for ligands of all types and is an important factor in Cys ³⁴ 's biological role as a free radical trap and free thiol scavenger. These properties make Cys ³⁴ highly reactive to maleimides, and the acceleration of the reaction rate can be 1000-fold compared to the reaction rate of the reaction of other free thiol-containing proteins with maleimide peptides.

Another advantage of the peptide-maleimide-albumin conjugate is the reproducibility associated with a 1: 1 ratio of peptide to albumin (specifically Cys ³⁴ ). Other techniques, such as glutaraldehyde, DCC, EDC and other chemical activations for free amines, lack this selectivity. For example, albumin is 52
Lysine residues, 25-30 of which are located on the surface of albumin and are accessible for binding. Activation of these lysine residues, or modification of peptides that bind through these lysine residues, results in a heterogeneous population of conjugates. Even when a 1: 1 molar ratio of peptide to albumin is used, the product consists of multiple conjugate products, some of which contain 0, 1, 2 or more peptides per albumin, Each has a peptide randomly attached to any one of the 25-30 available lysine sites. When many combinations are possible, the characterization of the extraction composition and the properties of each batch becomes difficult and reproducibility between batches is almost impossible, making such conjugates an anti-angiogenic peptide. Make it less desirable. Furthermore, although conjugation of albumin via lysine residues appears to have at least the advantage of delivering more of the anti-angiogenic agent per albumin molecule, studies have shown that A 1: 1 ratio of albumin has been shown to be preferred. Stehle et al.
"The Loading Rate Determines Tumor T
targeting properties of Methotrexate-
Albumin Conjugates in Rats ", Anti-Can
In a paper by Cer Drugs, 8, 677-685 (1997), which is incorporated herein in its entirety, the authors report that the anti-cancer agent methotrexate versus albumin 1: We reported that a ratio of 1 gave the most promising results. While these conjugates were taken up by tumor cells, those with 5: 1 to 20: 1 methotrexate molecules had altered HPLC profiles and were rapidly taken up by the liver in vivo.
It is inferred that at these higher ratios, conformational changes to albumin reduce its effectiveness as a therapeutic carrier.

Through the controlled administration of maleimide-peptides in vivo, the specific labeling of albumin and IgG in vivo can be controlled. In a typical administration, 80-90% of the administered maleimide-peptide labeled albumin and 5
Less than% labels IgG. Microlabelling of free thiols such as glutathione also occurs. Such specific labeling is preferred for in vivo applications. This is because it allows an accurate calculation of the estimated half-life of the administered drug.

In addition to providing controlled and specific in vivo labeling, maleimide-peptides can provide ex vivo specific labeling of serum albumin and IgG. Such ex vivo labeling may be performed with serum albumin and / or IgG.
And addition of the maleimide-peptide to blood, serum or saline containing. Once modified ex vivo with a maleimide-peptide, blood, serum or saline can be readministered into the blood for in vivo treatment.

In contrast to NHS-peptides, maleimido-peptides are generally very stable in the presence of aqueous solutions and in the presence of free amines. Maleimide-
Protecting groups are generally not required to prevent the maleimido-peptide from reacting with itself, as the peptide reacts only with free thiols. Furthermore, the increased stability of the peptides allows the use of additional purification steps such as HPLC to prepare highly purified products suitable for in vivo applications. Finally,
The increased chemical stability provides a product with a longer shelf life. B. Non-Specific Labeling The kringle 5 peptides of the present invention can also be modified for non-specific labeling of blood components. Coupling with amino groups can be commonly used, especially with the formation of amide bonds for non-specific labeling. To form such a bond, a wide variety of active carboxyl groups, especially esters, can be used as chemically reactive groups linked to the kringle 5 peptide, where the hydroxyl moiety is at the level required. Physiologically acceptable. Although a number of different hydroxyl groups can be used in these binders, N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide (sulfo-NHS) are the most convenient and this use forms a succinimidyl group. .

Other binders that can be utilized are described in US Pat. No. 5,612,034, which is incorporated herein by reference.

The various sites to which the chemically reactive groups of the kringle 5 peptide derivatives of the present invention may react in vivo include cells, particularly red blood cells (mature red blood cells) and platelets, and proteins such as immunoglobulins (IgG and IgM. )), Serum albumin, ferritin, steroid binding protein, transferrin, thyroxine binding protein, α-2-macroglobulin, etc.). Those receptors to which the derivatized kringle 5 peptide reacts, which are not long-lived
Are generally cleared from human hosts within approximately 3 days. The proteins shown above (including cellular proteins), based on blood levels, particularly with respect to half-life, may remain for at least 3 days and may remain for 5 days or more (generally 60
No more than 30 days, more commonly no more than 30 days).

For the most part, the reaction involves mobile components in the blood, in particular blood proteins and cells, more particularly blood proteins and mature red blood cells. By "mobility" is meant that the component is not in place for any extended period of time (typically no more than 5 minutes, more typically 1 minute), but some Blood components can be relatively quiescent for long periods of time. Initially, there is a relatively heterogeneous population of functionalized proteins and cells. However, for the most part, within days after administration, the population depends on the half-life of the functionalized protein in the bloodstream,
It will be substantially different from the first group. Thus, typically within about 3 days or more, IgG becomes the predominant functionalized protein in the bloodstream.

Typically, by 5 days post-administration, IgG, serum albumin and mature erythrocytes will be at least about 60 mol% of the binding components in the blood, usually at least about 75 mol%, and IgG, IgM (to a substantially lesser extent) ) And serum albumin are at least about 50 mol% of the acellular binding component, usually at least about 75 mo.
It will be 1%, more usually about 80 mol%.

[0041]   Preferably, the kringle 5 peptide derivative is bound to albumin.

Desired conjugates of non-specific kringle 5 peptide to blood components can be prepared in vivo by administering to the patient the kringle 5 peptide derivative. The patient can be a human or other mammal. The administration can be in bolus form or can be introduced slowly and over an extended period of time, such as by metering the flow and injecting.

If desired, the conjugates of the invention can also be prepared ex vivo by combining blood with a derivatized kringle 5 peptide of the invention, which is derivatized with reactive functional groups on blood components. Allowing covalent attachment to the 5 peptide and then returning or administering the conjugated blood to the host. In addition, the above is to first purify individual blood components or a limited number of components (eg red blood cells, immunoglobulins, serum albumin, etc.) and then chemically purify that component or components in ex vivo. Can be achieved by combining with a specifically reactive kringle 5 peptide derivative. The functionalized blood or blood component is then
The therapeutically effective conjugate can be returned to the host to provide the subject in vivo.
The blood can also be treated to prevent coagulation during ex vivo manipulation.

3. Synthesis of Modified Kringle 5 Peptides A. Synthesis of Kringle 5 Peptides Kringle 5 peptide fragments can be synthesized by standard methods of solid phase peptide chemistry known to those skilled in the art. For example, the Kringle 5 peptide fragment can be applied to the Applied Biosystems Synthesizer (Applied Biosy).
Steward and You, using the system synthesizer)
ng (Steward, JM and Young, JD, Solid.
Phase Peptide Synthesis, Second Edition. , Pierce
Chemical Company, Rockford, III. , (1984
)) Can be synthesized by solid phase chemistry techniques following the procedure described by)). Multiple fragments are then similarly joined together and synthesized to form larger fragments. These synthetic peptide fragments can also be made with amino acid substitutions at specific positions.

A number of techniques overviews for solid phase peptide synthesis are provided in J. M. Stewart and J.M. D. Young, Solid Phase Peptide Synt
hesis, W.C. H. Freeman Co. (San Francisco)
1963 and J. et al. Meienhofer, Hormonal Protei
ns and Peptides, Vol. 2, p. 46, Academic Pre
ss (New York), 1973. For traditional solution synthesis, see G. Schroder and K.S. Lupke, The Pepti
See des, Volume 1, Acoustic Press (New York). Generally, these methods involve the sequential addition of one or more amino acids or appropriately protected amino acids to grow a peptide chain. Usually, either the amino or carboxyl group of the first amino acid is protected by a suitable protecting group. The protected amino acid or derivatized amino acid is then
Appropriately protected complimen, attached to an inert solid support or under suitable conditions to form an amide bond.
tary) is utilized in solution by adding the next amino acid in the sequence with the (amino or carboxyl) group. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is added and so on.

After all the desired amino acids have been joined in the appropriate sequence, all remaining protecting groups (and any solid support) are removed sequentially or simultaneously to give the final poly Nucleotides are formed. A simple modification of this general procedure allows for the addition of more than one amino acid at a time to grow a chain (eg, a protected tripeptide into a properly protected dipeptide (a chiral center). (Under non-racemization conditions), and after deprotection to form the pentapeptide).

A particularly preferred method of preparing compounds of the present invention involves solid phase peptide synthesis, where the amino acid α-N-terminus is protected by an acid or base sensitive group. Such protecting groups should have the property of being stable to the conditions of peptide bond formation, while facilitating without breaking the growing peptide chain or racemization of any chiral centers contained therein. Can be removed. A suitable protecting group is 9
-Fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc), benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl, t-amyloxycarbonyl, isobornyloxycarbonyl, α, α-dimethyl- 3,5-dimethoxybenzyloxycarbonyl, o-
Examples include nitrophenylsulfenyl and 2-cyano-t-butyloxycarbonyl. The 9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularly preferred for the synthesis of kringle 5 peptide fragments. Other preferred side chain protecting groups are 2,2 for side chain amino groups such as lysine and arginine.
5,7,8-Pentamethylchroman-6-sulfonyl (pmc), nitro group, p
-Toluenesulfonyl group, 4-methoxybenzene-sulfonyl group, Cbz group, B
oc group and adamantyloxycarbonyl group; for tyrosine,
A benzyl group, an o-bromobenzyloxycarbonyl group, a 2,6-dichlorobenzyl group, an isopropyl group, a t-butyl (t-Bu) group, a cyclohexyl group, a cyclopenyl group and an acetyl (Ac) group; t-butyl group,
Benzyl and tetrahydropyranyl groups; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyl and 2,4-dinitrophenyl groups; for tryptophan, formyl; aspartic acid. And for glutamic acid, benzyl and t-butyl groups; and for cysteine, triphenylmethyl (trityl) group.

In the solid phase peptide synthesis method, the α-C-terminal amino acid is attached to a suitable solid support or resin. Suitable solid supports useful for the above synthesis are materials that are inert to the reagents and reaction conditions of the stepwise condensation-deprotection reaction, and are insoluble in the medium used. A preferred solid support for the synthesis of α-C-terminal carboxy peptides is 4-hydroxymethylphenoxymethyl-copoly (styrene-1% divinylbenzene). A preferred solid support for the α-C-terminal amide peptide is 4- (2 ′, 4′-dimethoxyphenyl-Fmo.
c-aminomethyl) phenoxyacetamidoethyl resin (Applied)
Biosystems (available from Foster City, Calif.). This α-C-terminal amino acid is 4-dimethylaminopyridine (DMAP)
, 1-hydroxybenzotriazole (HOBT), benzotriazole-1-
N, N′-dicyclohexylcarbodiimide (DCC) with or without yloxy-tris (dimethylamino) phosphonium-hexafluorophosphate (BOP) or bis (2-oxo-3-oxazolidinyl) phosphine chloride (BOPCl) , N, N′-diisopropylcarbodiimide (DIC
) Or O-benzotriazol-1-yl-N, N, N ', N',-tetramethyluronium-hexafluorofluorophosphate (HBTU) is attached to the resin, the attachment being such as dichloromethane or DMF. It is mediated in a solvent at a temperature between 10 ° C and 50 ° C for about 1 to 24 hours.

When the solid support is 4- (2 ′, 4′-dimethoxyphenyl-Fmoc-aminomethyl) phenoxy-acetamidoethyl resin, the Fmoc group is coupled to the α-C-terminal amino acid described above. Prior to cleavage with a secondary amine, preferably piperidine. Deprotected 4- (2 ', 4'-dimethoxyphenyl-F
The preferred method for coupling to the moc-aminomethyl) phenoxy-acetamidoethyl resin is O-benzotriazol-1-yl-N, N in DMF.
, N ', N',-Tetramethyluronium-hexafluorophosphate (HB
TU, 1 eq) and 1-hydroxybenzotriazole (HOBT, 1 eq)
Is. Sequential coupling of protected amino acids can be performed in automated polypeptide synthesizers well known in the art. In a preferred embodiment, the α-N-terminal amino acid of the growing peptide chain is Fmoc protected. Removal of the Fmoc protecting group from the α-N-terminal side of the growing peptide is achieved by treatment with a secondary amine, preferably piperidine. Each protected amino acid is then introduced in approximately 3-fold molar excess and preferably the coupling is carried out in DMF. The coupling agent is usually O-
Benzotriazol-1-yl-N, N, N ′, N ′,-tetramethyluronium-hexafluorofluorophosphate (HBTU, 1 eq) and 1-hydroxybenzotriazole (HOBT, 1 eq).

At the end of solid phase synthesis, the polypeptide is removed from the resin and deprotected, either continuously or in a single operation. Removal and deprotection of the polypeptide is accomplished by a cleavage reagent containing thioanisole, water, ethanedithiol and trifluoroacetic acid.
can be achieved in a single operation by treating the resin-bound polypeptide with the agent. When the α-C-terminus of the polypeptide is an alkylamide,
The resin is cleaved by aminolysis with an alkylamine. Alternatively, the peptide can be removed by, for example, a transesterification reaction with methanol followed by aminolysis or direct transamidation. The protected peptide can be purified at this point or used directly in the next step. Removal of side chain protecting groups is accomplished using the cleavage cocktail described above. Fully deprotected peptides can be of any or all of the following types: ion exchange on weak base resins (acetate form); hydrophobic adsorption chromatography on underivatized polystyrene-divinylbenzene (eg Amberlite XAD); Silica gel adsorption chromatography; ion exchange chromatography on carboxymethyl cellulose; partition chromatography (eg Sephadex G-25, LH-2
0) or countercurrent partitioning; purified by a series of chromatographic steps utilizing high performance liquid chromatography (HPLC) (particularly reverse phase HPLC on octyl- or octadecylsilyl-silica bonded phase packings).

The molecular weights of these kringle 5 peptides are determined using fast atom bombardment (FAB) mass spectrometry.

The kringle 5 peptides of the present invention can be synthesized with N-terminal and C-terminal protecting groups.

(1. N-terminal protecting group) The term “N-protecting group” refers to protecting the α-N-terminal of an amino acid or peptide, or otherwise the amino group of an amino acid or peptide may be desirable during synthetic procedures. Refers to those groups intended to protect against unreacted reactants. Typically,
The N protecting group used is Greene, "Protective Groups.
In Organic Synthesis "(John Wiley & So
ns, New York (1981)), which is incorporated herein by reference. In addition, protecting groups can be used, for example, as prodrugs that are readily cleaved in vivo by enzymatic hydrolysis to release the biologically active parent. The α-N protecting group is a lower alkanoyl group (
For example, formyl group, acetyl group (“Ac”), propionyl group, pivaloyl group, t-butylacetyl group and the like; 2-chloroacetyl group, 2-bromoacetyl group,
Other acyl groups including trifluoroacetyl group, trichloroacetyl group, phthalyl group, o-nitrophenoxyacetyl group, chlorobutyryl group, benzoyl group, 4-chlorobenzoyl group, 4-bromobenzoyl group, 4-nitrobenzoyl group and the like;
For example, a sulfonyl group such as benzenesulfonyl group, p-toluenesulfonyl group and the like; for example, benzyloxycarbonyl group, p-chlorobenzyloxycarbonyl group, p-methoxybenzyloxycarbonyl group, p-nitrobenzyloxycarbonyl group, 2 -Nitrobenzyloxycarbonyl group, p-bromobenzyloxycarbonyl group, 3,4-dimethoxybenzyloxycarbonyl group, 3,5
-Dimethoxybenzyloxycarbonyl group, 2,4-dimethoxybenzyloxycarbonyl group, 4-ethoxybenzyloxycarbonyl group, 2-nitro-4,5
-Dimethoxybenzyloxycarbonyl group, 3,4,5-trimethoxybenzyloxycarbonyl group, 1- (p-biphenylyl) -1-methylethoxycarbonyl group, α, α-dimethyl-3,5-dimethoxybenzyloxycarbonyl group Base,
Benzhydryloxycarbonyl group, t-butyloxycarbonyl group, diisopropylmethoxycarbonyl group, isopropyloxycarbonyl group, ethoxycarbonyl group, methoxycarbonyl group, allyloxycarbonyl group, 2,2,2-trichloroethoxycarbonyl group, phenoxycarbonyl group Group, 4-nitrophenoxycarbonyl group, fluorenyl-9-methoxycarbonyl group, cyclopentyloxycarbonyl group, adamantyloxycarbonyl group, cyclohexyloxycarbonyl group, phenylthiocarbonyl group and other carbamate-forming groups; for example, benzyl group, triphenylmethyl Groups, arylalkyl groups such as benzyloxymethyl group, 9-fluorenylmethyloxycarbonyl group (Fmoc) and the like, for example trimethyl Including silyl groups such as silyl.

Preferred N protecting groups are formyl group, acetyl group, benzoyl group, pivaloyl group, t-butylacetyl group, phenylsulfonyl group, benzyl group, t-butyloxycarbonyl (Boc) group and benzyloxycarbonyl (Cbz). It is a base. For example, lysine can be protected at the α-N-terminus with an acid labile group (eg, Boc) and at the N-terminus with a base labile group (eg, Fmoc), then during synthesis. It can be selectively deprotected.

(2. Carboxyl Protecting Group) The term “carboxyl protecting group” refers to a carboxylic acid used to block or protect a carboxylic acid functional group while performing a reaction involving another functional group site of the compound. Refers to a protected ester group or amide group. The carboxy protecting group is
Greene, "Protective Groups in Organic
Synthesis, pages 152-186 (1981), which is incorporated herein by reference. In addition, the carboxy protecting group can be used as a prodrug, which allows the carboxy protecting group to be readily cleaved in vivo, eg, by enzymatic hydrolysis, releasing the biologically active parent. Such carboxy protecting groups are well known to those skilled in the art and are described in US Pat. Nos. 3,840,556 and 3,719,667,
Widely used in the protection of carboxyl groups in the field of penicillins and cephalosporins. These disclosures are incorporated herein by reference. Representative carboxy protecting groups are C ₁ -C ₈ lower alkyl groups such as methyl, ethyl or t-butyl groups; arylalkyl groups such as, for example, phenethyl or benzyl, as well as alkoxybenzyl. Groups or their substituted derivatives such as nitrobenzyl groups; eg arylalkenyl such as phenylethenyl group; aryl and its substituted derivatives such as eg 5-indanyl group; eg dimethylaminoethyl group etc. Dialkylaminoalkyl groups such as, for example, acetoxymethyl group, butyryloxymethyl group, valeryloxymethyl group, isobutyryloxymethyl group, isovaleryloxymethyl group, 1- (propionyloxy) -1-ethyl group , 1- (pivaloyloxy)
Alkanoyloxyalkyl groups such as -1-ethyl group, 1-methyl-1- (propionyloxy) -1-ethyl group, pivaloyloxymethyl group, propionyloxymethyl group; for example, cyclopropylcarbonyloxymethyl group , Cyclobutylcarbonyloxymethyl group, cyclopentylcarbonyloxymethyl group, cyclohexylcarbonyloxymethyl group and the like cycloalkanoyloxyalkyl group; for example, benzoyloxymethyl group, benzoyloxyethyl group and the like aroyloxyalkyl group; For example, arylalkylcarbonyloxyalkyl groups such as benzylcarbonyloxymethyl group, 2-benzylcarbonyloxyethyl group and the like; for example, methoxycarbonylmethyl group, cyclohexyloxycarboxyl group Alkoxycarbonylalkyl group or cycloalkyloxycarbonylalkyl group such as nylmethyl group, 1-methoxycarbonyl-1-ethyl group and the like; for example, methoxycarbonyloxymethyl group, t-butyloxycarbonyloxymethyl group, 1-ethoxycarbonyloxy -1-ethyl group,
An alkoxycarbonyloxyalkyl group such as a 1-cyclohexyloxycarbonyloxy-1-ethyl group or a cycloalkyloxycarbonyloxyalkyl group; for example, a 2- (phenoxycarbonyloxy) ethyl group, 2- (5
An aryloxycarbonyloxyalkyl group such as an indanyloxycarbonyloxy) ethyl group; and an alkoxyalkylcarbonyloxyalkyl group such as a 2- (1-methoxy-2-methylpropane-2-oiloxy) ethyl group Group; for example, an arylalkyloxycarbonyloxyalkyl group such as a 2- (benzyloxycarbonyloxy) ethyl group and the like; for example, 2- (
An arylalkenyloxycarbonyloxyalkyl group such as a 3-phenylpropen-2-yloxycarbonyloxy) ethyl group; an alkoxycarbonylaminoalkyl group such as a t-butyloxycarbonylaminomethyl group; Alkylaminocarbonylaminoalkyl group such as carbonylaminomethyl group; alkanoylaminoalkyl group such as acetylaminomethyl group; heterocyclic carbonyl such as 4-methylpiperazinylcarbonyloxymethyl group An oxyalkyl group;
Dialkylaminocarbonylalkyl groups such as dimethylaminocarbonylmethyl group, diethylaminocarbonylmethyl group and the like; for example, (5-t-butyl-2
A (5- (lower alkyl) -2-oxo-1,3-dioxolen-4-yl) alkyl group such as -oxo-1,3-dioxolen-4-yl) methyl group; and, for example, (5 A (5-phenyl-2-oxo-1,3-dioxolen-4-yl) alkyl group such as -phenyl-2-oxo-1,3-dioxolen-4-yl) methyl group.

Representative amidocarboxy protecting groups are aminocarbonyl groups and lower alkylaminocarbonyl groups.

Preferred carboxy protected compounds of the present invention include multiple compounds in which the protected carboxy group is a lower alkyl ester, cycloalkyl ester or arylalkyl ester (eg, methyl ester, ethyl ester, propyl ester, isopropyl ester. Ester, butyl ester, sec-butyl ester, isobutyl ester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester, phenylethyl ester, etc.) or alkanoyloxyalkyl ester, cycloalkanoyloxyalkyl ester, aroyloxyalkyl ester or arylalkyl. It is a carbonyloxyalkyl ester. A preferred amidocarboxy protecting group is a lower alkylaminocarbonyl group. For example, aspartic acid can be protected at the α-C terminus by an acid labile group (eg, t-butyl group) and at the β-C terminus by a hydrogenation labile group (eg, benzyl group). It can then be selectively deprotected during synthesis.

B. Modification of the Kringle 5 Peptide The manner in which the modified kringle 5 peptide of the present invention is produced varies widely depending on the nature of the various elements, including the molecule. The synthetic procedures are chosen to be simple, to provide high yields, and to allow highly purified products. Typically, the chemically reactive group is made at the final stage, eg, with a carboxyl group, and esterification to form the active ester is the final step in the synthesis. Specific methods for the production of derivatized kringle 5 peptides of the invention are described in the examples below.

Each kringle 5 peptide selected to undergo derivatization with a linker and a reactive agent is modified according to the following criteria: Carboxylic group not important for retention of pharmacological activity. Is available in the original molecule and no other reactive functional groups are present in this molecule,
The carboxylic acid is chosen as the attachment site for linker reactive group modification. If no carboxylic acid is available, any other functional group that is not important for retention of pharmacological activity is selected as the attachment site for linker reactive group modification. If some functional groups are available in the Kringle 5 peptide, after addition of linker / reactive groups and deprotection of all protected functional groups, such that retention of pharmacological activity is still obtained, A combination of protecting groups is used. If reactive functional groups are not available in the therapeutic agent, synthetic attempts will be made to modify the original parent drug in such a manner as to retain biological activity and retain receptor or target specificity. To enable.

The chemically reactive group is present at one site so that when the peptide binds to blood components, the peptide retains a substantial proportion of the parent compound's inhibitor activity.

Even more particularly, each kringle 5 peptide selected to undergo derivatization with a linker and reactive group is modified according to the following criteria: The terminal carboxylic acid group is utilized in the kringle 5 peptide. If possible and the terminal carboxyl group is not important for retention of pharmacological activity, and no other sensitive functional groups are present in the kringle 5 peptide, the carboxylic acid is linked to the linker reactive group modification. Select as a part. When the terminal carboxylic acid group is involved in the pharmacological activity, or when the carboxylic acid is not available, any other sensitive functional group that is not important for retaining the pharmacological activity is modified with a linker reactive group. As the binding site for If some sensitive functional groups are available in the kringle 5 peptide, such a manner that retention of pharmacological activity is still obtained after addition of linker / reactive groups and deprotection of all protected sensitive functional groups Where a combination of protecting groups is used. Synthetic Attempts at Retaining Biological Activity and Receptor or Target Specificity No Sensitive Functional Group is Available in the Therapeutic Peptide (or if a Simpler Altered Pathway is Desired) It allows modification of the native kringle 5 peptide in such a manner. In this case, the modification occurs at the opposite end of the peptide.

NHS derivatives can be synthesized from carboxylic acids in the kringle 5 peptide in the absence of other sensitive functional groups. In particular, such kringle 5 peptides react with N-hydroxysuccinimide in anhydrous CH ₂ Cl ₂ and EDC, and the product is purified by chromatography or a suitable solvent to give the NHS derivative. Recrystallized from the system.

Alternatively, NHS derivatives can be synthesized from kringle 5 peptides containing amino and / or thiol groups, and carboxylic acids. If free amino or thiol groups are present in the molecule, it is preferable to protect these sensitive functional groups before carrying out the addition of the NHS derivative. For example, if the molecule contains a free amino group, conversion of the amine to an Fmoc or preferably tBoc protected amine is required before carrying out the above chemistry. The amine functionality is not deprotected after the NHS derivative preparation. Therefore, this method applies only to peptides for which the amine groups are not required to be free to induce the desired pharmacological effect.

In addition, NHS derivatives can be synthesized from kringle 5 peptides containing amino or thiol groups and no carboxylic acids. If the molecule of choice does not contain a carboxylic acid, a series of bifunctional linkers can be used to convert the molecule into a reactive NHS derivative. For example, ethylene glycol bis (succinimidyl succinate) (EGS) and triethylamine (in a 10: 1 ratio, with EGS being preferred) dissolved in DMF and added to free amino-containing molecules. Produces a mono-NHS derivative. N- [γ-maleimidobutyryloxy] succinimide ester (GMBS) and triethylamine in DMF can be used to generate NHS derivatives from thiol derivatized molecules. The maleimide group reacts with the free thiol and the NHS derivative is purified from the reaction mixture by chromatography on silica or by HPLC.

NHS derivatives can also be synthesized from kringle 5 peptides containing multiple sensitive functional groups. In each case, the analysis and elucidation must be done in different ways. However, due to the large amount of commercially available protecting groups and bifunctional linkers as described above, the present invention preferably derivatizes the peptide, preferably in only one chemical step, or in advance of sensitive groups. It can be applied to any peptide in 2 or 3 steps (protection, activation, and deprotection) with the protection of. Only under exceptional circumstances is it necessary to use multiple (more than three) synthetic steps to convert the kringle 5 peptide to the active NHS or maleimide derivative.

Maleimide derivatives can also be synthesized from kringle 5 peptides containing free amino groups and free carboxylic acids. N- [γ-maleimidobutyryloxy] succinimide ester (GMBS) and triethylamine in DMF can be used to generate maleimide derivatives from amino derivatized molecules. The succinimide ester group reacts with the free amino and the maleimide derivative is purified from the reaction mixture by crystallization or by chromatography on silica or by HPLC.

Finally, maleimide derivatives can be synthesized from kringle 5 peptides containing multiple other sensitive functional groups and no free carboxylic acids. If the molecule of choice does not contain a carboxylic acid, a series of bifunctional cross-linking reagents can be used to convert the molecule to a reactive NHS derivative. For example, HBTU / HOBt / DI in DMF
EA activation can be used to couple maleimidopropionic acid (MPA) to a free amine and generate a maleimide derivative through reaction of the free amine with the carboxylic acid group of MPA.

A large number of bifunctional compounds are available for cross-linking to the entity. Exemplary entities include: azidobenzoylhydrazide, N- [4- (p-azidosalicylamino) butyl] -3 '-[2'-pyridyldithio) propionamide), bis-sulfosuccin. Imidil suberate (su
berate), dimethyl adipimidate, disuccinimidyl tartrate, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N
-Succinimidyl [4-azidophenyl] -1,3'-dithiopropionate, N-succinimidyl [4-iodoacetyl] aminobenzoate, glutaraldehyde, and succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate .

4. Use of Modified Kringle 5 Peptides As described above, angiogenesis is associated with tissue neovascularization (“sprouti”).
ng) ”), and various processes including angiogenesis or vasodilation. With the exception of traumatic wound healing, corpus leuteum formation and embryogenesis, most angiogenic processes are associated with disease processes, thus the use of the therapeutic methods of the invention is selective for the disease, And it may not have harmful side effects.

There are various diseases in which angiogenesis is thought to be important, and these diseases are
It may be treatable with the modified peptides of the invention. These diseases include, but are not limited to, for example, immune and non-immune inflammation, rheumatoid arthritis and inflammatory disorders such as psoriasis, eg, diabetic retinopathy, neovascular glaucoma, Restenosis, atherosclerotic plaque
disorders associated with vascular instability or inadequate infiltration such as capillary proliferation and osteoporosis in erotic plaques) Cancer-related disorders such as Kaposi's sarcoma and similar cancers that require neovascularization and support tumor growth.

The modified kringle 5 peptides of the invention are used in methods that inhibit angiogenesis in diseased tissues, ameliorate the symptoms of the disease, and, depending on the disease, may contribute to the cure of the disease. Find out. The modified peptides of the invention are more stable in vivo, and as such, smaller amounts of the modified peptides can be administered for effective treatment. In one embodiment, the invention by itself contemplates inhibition of angiogenesis in tissues. The degree of angiogenesis in the tissue, and thus the degree of inhibition achieved by the method of the invention, is determined by immunohistochemistry by α ₅
B ₃ - it can be evaluated by a variety of methods for detecting the vasculature of immature vasculature and nascent immunopositive.

As described herein, any of a variety of tissues, or organs composed of organized tissues, include skin, muscle, digestive tract, connective tissue, joints, bones and blood vessels.
It may support angiogenesis in disease states involving similar tissues that may be infiltrated by angiogenic stimuli.

In one related embodiment, the tissue treated with the modified kringle 5 peptide of the invention is inflammatory tissue and the angiogenesis to be inhibited is the presence of neovascularization of the inflammatory tissue. Angiogenesis of inflammatory tissue. In this class, the method contemplates inhibition of angiogenesis of arthritic tissue, eg, in patients with rheumatoid arthritis, immune or non-immune inflammatory tissue, psoriatic tissue, and the like.

While the principles of the present invention are understood to indicate that the present invention is effective with respect to all mammals, in many embodiments of the present invention the patient treated with the present invention is preferably Is a human patient and is intended to be included in the term “patient”. In this context, mammals are understood to include any mammalian species in which it is desired to treat diseases associated with angiogenesis, in particular mammalian and domestic mammalian species. .

In another related embodiment, the tissue treated with the modified kringle 5 peptide of the present invention is retinal tissue of a patient with diabetic retinopathy, macular degeneration or neovascular glaucoma, which is angiogenesis inhibited. Formation is angiogenesis of retinal tissue in which there is neovascularization of retinal tissue.

In a further related embodiment, the tissue treated with the modified kringle 5 peptide of the present invention is used in patients with solid tumors, metastases, skin cancers, breast cancers, hemangiopathies or angiofibromas and other cancers. Angiogenesis that is tumor tissue and that is inhibited is neovascularization of tumor tissue where there is neovascularization of the tumor tissue. Representative solid tumor tissues treatable by the methods of the present invention include lung, pancreas, breast, colon,
These include the pharynx, ovaries, and other tissues.

Due to the important role neovascularization plays in tumor growth, inhibition of tumor tissue angiogenesis is a particularly preferred embodiment. In the absence of neovascularization of the tumor tissue, the tumor tissue fails to obtain the required nutrients, slows growth, arrests further growth, recedes, and eventually necroses, and the tumor dies.

The invention thus provides a method of inhibiting tumor neovascularization by using the modified kringle 5 peptide of the invention and inhibiting tumor angiogenesis according to the methods of the invention. Similarly, the invention provides a method of inhibiting tumor growth by performing an angiogenesis inhibition method. This method is also particularly effective for the formation of metastases. Because the formation of metastases requires angiogenesis of the primary tumor so that (1) metastatic cancer cells can leave the primary tumor, and (2) at a second site to support the growth of the metastases. This is because neovascularization is required for the establishment of the metastasis.

In a related embodiment, the present invention provides for this method in combination with other therapies, such as, for example, conventional chemotherapy for targeting solid tumors and controlling the establishment of metastases. Is intended to be implemented. Administration of the modified kringle 5 peptides of the present invention is typically performed during or after chemotherapy, but after several chemotherapy regimens.
Tumor tissue responds to toxin attack by the induction of angiogenesis, which inhibits angiogenesis, where it is restored by providing blood supply and nutrients to the tumor tissue. Furthermore, as a prophylaxis against metastases, it is preferred that the modified kringle 5 peptide be administered after surgery in which the solid tumor has been removed. As long as the method of the invention applies to the inhibition of tumor neovascularization, using the modified kringle 5 peptide of the invention,
This method may also be applied to the inhibition of tumor tissue growth, the inhibition of tumor metastasis formation, and regression of established tumors.

Restenosis is a process of smooth muscle cell (SMC) migration and proliferation at the site of percutaneous transluminal coronary angioplasty that impedes successful angioplasty. Migration and proliferation during restenosis of SMCs can be considered as a process of angiogenesis that is inhibited by the modified kringle 5 peptides of the invention. Accordingly, the present invention also contemplates inhibiting restenosis by inhibiting angiogenesis in a patient according to an angioplasty procedure. For inhibition of restenosis, the modified kringle 5 peptide is typically administered according to this procedure for about 2 to 28 days after the angioplasty procedure, and more typically about the first about 1 day.
It was administered over 4 days.

The method of the present invention for inhibiting angiogenesis in a tissue comprises contacting a tissue at which or at risk for angiogenesis with a composition comprising a therapeutically effective amount of a modified kringle 5 peptide. Including steps. As further described herein, the dose range for administration of the modified Kringle 5 peptide depends on the form of the peptide, and its potency, and is angiogenesis and diseases mediated by angiogenesis. The amount is large enough to produce the desired effect, which improves symptoms. The dose should not be so high as to cause adverse side effects (eg hyperviscosity syndrome, pulmonary edema, congestive heart failure, etc.). Generally, the dose will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of ordinary skill in the art. The dose can also be adjusted by the individual physician in the event of any complication.

As inhibitors of neovascularization, such modified kringle 5 peptides are useful in the treatment of both early and metastatic solid tumors and the following cancers: breast; colon; rectum; lung; oropharyngeal. Hypopharynx; esophagus; stomach; pancreas; liver; gallbladder; bile duct; small intestine; urinary tract including kidney, bladder and urothelium; female reproductive with cervical, uterine, ovarian, choriocarcinoma and gestational trophoblastic disease Ducts; male reproductive tracts including prostate, seminal vesicles, testes and germ cell tumors; endocrine glands including thyroid, adrenal gland, and pituitary gland; including hemangiomas, melanomas, sarcomas of bone or parenchyma, and Kaposi's sarcoma Skin; brain, nerve, eye, and astrocytoma, glioma, glioblastoma, retinoblastoma,
Meningeal tumors, including neuromas, neuroblastomas, schwannomas and meningiomas; solid tumors resulting from hematopoietic malignancies such as leukemia and of chloroma, plasmacytoma, mycosis fungoides and cutaneous Including T-cell lymphoma / leukemia plaques and tumors; Lymphomas including both Hodgkin and non-Hodgkin lymphomas; Prevention of autoimmune diseases, including rheumatoid arthritis, immune arthritis and osteoarthritis; diabetic retinopathy , Immature retinopathy, corneal transplant rejection, post lens fibroplasia, neovascular glaucoma, rubeosis, ocular diseases including neovascularization and hypoxia due to macular degeneration; abnormal neovascularization of the eye; skin including psoriasis Diseases; hemangiomas in atherosclerotic plaques and vascular diseases including capillary growth; Osler-Weber syndrome; myocardial Angiogenesis; plaque neovascularization; telangiectasia; hemophilic joints; vascular fibroma; granulation of wounds; adhesions of the intestine, Crohn's disease, atherosclerosis,
Diseases characterized by excessive or aberrant stimulation of endothelial cells, including scleroderma and hyperplastic scars (ie keloids), and new veins as pathological consequences including Nekoscratch disease and ulcers (Helicobacter pylori) Diseases with tube formation (Rochelle). Another use is as a birth control drug, which inhibits ovulation and placenta establishment.

The modified kringle 5 peptides of the present invention may also be used alone or in combination with radiotherapy and / or other chemotherapeutic treatments that are routinely administered to patients to treat angiogenic diseases. If used, it may be useful for the prevention of metastases from the tumors described above. For example, when used in the treatment of solid tumors, the modified kringle 5 peptides of the invention may be used as chemotherapeutic agents (eg, alpha interferon, COMP (cyclophosphamide, vincristine, methotrexate and prednisone), etoposide, mBACOD). (Methotrexate, bleomycin, doxorubicin, cyclophosphamide, vincristine and dexamethasone), PRO-MACE / MOPP (prednisone, methotrexate (w / leucovin rescue), doxorubicin, cyclophosphamide, taxol, etoposide / mecroletamine,
Vincristine, prednisone and procarbazine), vincristine, vinblastine, vascular inhibin, TNP-470,
Pentosan polysulfate, platelet factor 4, angiostatin (angio
statin), LM-609, SU-101, CM-101, techgalan (T
echgalan), thalidomide, SP-PG and the like. Other chemotherapeutic agents include alkylating agents such as mechlorethamine, melphalan, chlorambucil, cyclophosphamide and ifosfamide.
Nitrogen mustard containing ide); nitrosoureas containing carmustine, lomustine, semustine and streptozocin; alkyl sulfonates containing busulfan; triazines containing dacarbazine; ethyleneimine containing thiotepa and hexamethylmelamine; folic acid containing methotrexate Analogs; 5-fluorouracil, pyrimidine analogs including cytosine arabinoside; purine analogs including 6-mercaptopurine and 6-thioguanine;
Antitumor antibiotics including actinomycin D; doxorubicin, bleomycin,
Anthracyclines containing mitomycin C and metramycin; tamoxifen and corticosteroids
Included are miscellaneous agents including hormones including eroids and hormone antagonists and cisplatin and brequinars. For example, tumors may be routinely treated with surgery, radiation or chemotherapy and administration of kringle 5, followed by prolonging the cessation of micrometastases and stabilizing and inhibiting any residual primary tumors. Kringle 5 can be administered.

(5. Administration of Modified Kringle 5 Peptide) The modified kringle 5 peptide is a physiologically acceptable medium (eg, deionized water, phosphate buffered saline (PBS), saline, aqueous ethanol). Or other alcohol, plasma, proteinaceous solution, mannitol, aqueous glucose, alcohol, vegetable oil, etc.). Other additives that may be included include buffers (the medium is generally buffered at a pH in the range of about 5-10, the buffers generally about 50
˜250 mM concentration range, salt (concentration of salt is generally about 5-500 mM)
, And physiologically acceptable stabilizers and the like. The composition may be lyophilized for convenient storage and shipping.

The modified kringle 5 peptides of the invention are, for the most part, orally, parenterally (eg, intravascularly (IV), intraarterial (IA), intramuscularly (IM), subcutaneously). (SC) etc.). Administration will be by infusion, where appropriate. In some cases, where the reaction of the functional groups is relatively slow, the administration may be oral, nasal, rectal, transdermal or aerosol, where the nature of the conjugate is , Allows transfer to the vascular system. Usually a single injection will be used, but more than one injection can be used if desired. The modified kringle 5 peptide can be administered by any convenient means, including syringes, trocars, catheters and the like. The particular mode of administration will vary depending on the amount to be administered, whether it is a single bolus, continuous administration, or the like. Preferably, administration is intravascular (the site of this introduction is not critical to the invention), preferably where there is rapid blood flow (eg, intravenous, peripheral vein or central vein). ). Other routes may find use in which administration is associated with a delayed release technique or a protective matrix. The intent is that the kringle 5 peptide, analog or derivative is effectively distributed in the blood, so that it may react with blood components. Conjugate concentrations generally vary from about 1 pg / ml to 50 mg / ml and vary widely. The total amount administered intravascularly will generally range from about 0.1 mg / ml to about 10 mg / ml, more usually from about 1 mg / ml to about 5 mg / ml.

The binding of persistent components of blood such as immunoglobulins, serum albumin, red blood cells and platelets results in a number of advantages. The activity of the modified Kringle 5 peptide compound is extended for days to weeks. Only one dose needs to be given during this period. Greater specificity may be achieved because the active compound is predominantly bound to large molecules, where it is less likely to be vascularly uptake to interfere with other physiological processes.

The formation of covalent bonds between blood components can occur in vivo or ex vivo. For ex vivo covalent bond formation, the modified kringle 5 peptide is added to blood, serum or saline solution containing human serum albumin or IgG to allow covalent bond formation between the modified kringle 5 peptide and blood components. In a preferred manner, the kringle 5 peptide is modified with maleimide and reacted with human serum albumin in saline solution. Once the modified kringle 5 peptide reacts with blood components to form a kringle 5 peptide-protein conjugate, the conjugate can be administered to a patient.

Alternatively, the modified kringle 5 peptide can be administered directly to the patient, resulting in
A covalent bond forms in vivo between the modified kringle 5 peptide and the binding moiety.

(6. Monitoring the Presence of Modified Kringle 5 Peptide) The blood of a mammalian host was tested for the presence of the modified Kringle 5 peptide compound by 1
It can be monitored more than once. By taking a portion or sample of the host's blood, one of skill in the art can determine whether the Kringle 5 peptide is bound to a permanent blood component in an amount sufficient to be therapeutically active, and The level of the Kringle 5 peptide compound in the blood can then be determined. If desired, one of skill in the art can also determine to which of the blood components the kringle 5 peptide derivative molecule is attached. This is especially important when using non-specific kringle 5 peptides. Calculating the half-life of serum albumin and IgG for a particular maleimido-kringle 5 peptide is much easier.

Modified kringle 5 peptides can be monitored using HPLC-MS or antibodies against kringle 5 peptides.

A. HPLC-MS Mass spectrometry (MS) coupled HPLC can be utilized in assays for the presence of peptides and modified peptides, as is well known to those of skill in the art. Two mobile station phases are typically utilized: 0.1% TFA / water and 0.1% TFA / acetonitrile. Column temperature and gradient conditions can be varied. Specific details are outlined in the Examples section below.

B. Antibodies Another aspect of the invention is the use of antibodies specific for kringle 5 peptides or peptide analogs or their derivatives and conjugates to biological samples (eg blood). As a method for determining the concentration of kringle 5 peptides and / or analogs, or their derivatives and conjugates, and as a treatment for toxicity potentially associated with such kringle 5 peptides and / or their derivatives or conjugates. It relates to the use of such antibodies. This is advantageous in patients, where increased stability and longevity of the kringle 5 peptide may lead to new problems during treatment, including increased potential for toxicity, in vivo. The use of antibodies with specificity for their analogs or derivatives, either monoclonal or polyclonal, may aid in mediating any such issues. Antibodies may be generated from a host immunized with a particular modified kringle 5 peptide, or with an immunogenic fragment of the factor, or a synthetic immunogen corresponding to an antigenic determinant of the factor, Or it may be derived from them. Preferred antibodies have high specificity and affinity for the native, derivatized, and conjugated forms of modified kringle 5 peptides.
Such antibodies can also be labeled with enzymes, fluorescent dyes, or radioactive labels.

Antibodies specific for the modified kringle 5 peptide can be produced by using the purified kringle 5 peptide for the induction of derivatized kringle 5 peptide-specific antibodies. The induction of antibodies contemplates not only stimulation of the immune response by injection into animals, but also similar steps in the production of synthetic antibodies or other specific binding molecules (eg, screening of recombinant immunoglobulin libraries). . Both monoclonal and polyclonal antibodies can be produced by procedures well known in the art.

These antibodies can be used to monitor the presence of kringle 5 peptide in the bloodstream. Blood and / or serum samples can be analyzed by SDS-PAGE and Western blotting. Such techniques allow blood or serum analysis to determine the binding of modified kringle 5 peptides to blood components.

Anti-therapeutic agent antibodies may also be used to treat toxicity induced by administration of kringle 5 peptides, and may be used ex vivo or in vivo. The ex vivo method involves immunodialysis treatment for toxicity using anti-therapeutic antibody immobilized on a solid support. In vivo methods include the administration of an anti-therapeutic agent antibody in an amount effective to induce clearance of the antibody-drug complex.

The antibody can be used to remove the modified kringle 5 peptide and its conjugates from a patient's blood by contacting the blood with the antibody ex vivo under sterile conditions. For example, the antibody can be immobilized or otherwise immobilized on a column matrix, and patient blood can be removed from the patient and passed through the matrix. The modified kringle 5 peptide binds to the antibody, and blood containing low concentrations of kringle 5 peptide can then be returned to the patient's circulatory system. The amount of modified kringle 5 peptide removed can be controlled by adjusting the pressure and flow rate. Preferentially removing the modified kringle 5 peptide from the plasma component of the patient's blood may be accomplished, for example, by the use of a semipermeable membrane, or otherwise prior to passing the plasma component over a matrix containing anti-therapeutic antibodies, It can be carried out by first separating the plasma component from the cellular components by methods known in the art. Alternatively, preferential removal of kringle 5 peptide bound blood cells (including red blood cells) collects and concentrates blood cells in the patient's blood and immobilizes until the serum component of the patient's blood is removed. It can be carried out by contacting these cells with an anti-therapeutic antibody.

Anti-therapeutic antibodies can be administered parenterally in vivo to patients receiving the modified kringle 5 peptide or conjugate for treatment. The antibody binds to the Kringle 5 peptide compound and the conjugate. Once combined, the Kringle 5
Peptide activity is hindered, if not completely blocked, thereby reducing the biologically effective concentration of kringle 5 peptide compound in the patient's bloodstream and minimizing adverse side effects. Furthermore, the bound antibody-kringle 5 peptide complex facilitates clearance of the kringle 5 peptide compound and conjugate from the patient's bloodstream.

The fully described invention will now be illustrated by the following non-limiting examples.

EXAMPLES General Description Solid phase peptide synthesis of kringle 5 peptide on a 100 μmol scale was performed using manual solid phase synthesis and Symphony Peptide Syn.
performed using the thesizer: N, N-dimethylformamide (DMF
) Fmoc-protected Rink Amide MBHA resin, Fmoc-protected amino acid, O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and N- in solution. Activation with methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1). If required, selective deprotection of the Lys (Aloc) group was performed manually and the resin was loaded with 5 mL of CHCl ₃ : NMM: HOAc (1
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in 8: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL)
, 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). In some examples, the synthesis is then 1
Add one AEEA (aminoethoxyethoxyacetic acid) group, add acetic acid, or 3
-Re-automated to the addition of maleimidopropionic acid (MPA) (step 3)
. Resin cleavage and product isolation was performed using precipitation with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product is Varian (Rainin) preparative binary H
Purified by preparative reverse phase HPLC using PLC system: 1 at 9.5 mL / min.
Gradient elution 80 30-55% over minutes B (0.045% in H ₂ O TFA (A) and CH ₃ CN 0.045% in TFA (B)) (Phenomen
ex Luna 10 μ Phenyl-hexyl, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax U
VD II) is used). Purity, Hewlett Packard LCMS with diode array detector and using electrospray ionization
95% by RP-HPLC mass spectrometry using a -1100 series analyzer
I decided.

[0100]   (Example 1)   (NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys-NH ₂ ． Preparation of 3TFA)   The following protected amino acids were linked to Rink Amide using automated peptide synthesis:   Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH, Fmoc
-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pb
f) -OH, Fmoc-Pro-OH. N-terminal Fmoc group of resin-bound amino acid
Deblocking was performed with 20% piperidine in DMF for about 15-20 minutes.
did. Acetic acid coupling was performed under conditions similar to amino acid coupling
. The final cleavage from the resin was performed using the cleavage mixture as above. So
Of the desired product was isolated by precipitation and purified by preparative HPLC to give the desired product.
The product was obtained as a white solid upon lyophilization.

[0101]   (Example 2)   (NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys-NH ₂ ． Preparation of 3TFA)   The following protected amino acids were linked to Rink Amide using automated peptide synthesis:
  Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH. N-terminal F of resin-bound amino acid
Deblocking the moc group was performed with 20% piperidine in DMF to about 15-2.
It was carried out for 0 minutes. Acetic acid coupling under similar conditions to amino acid coupling
Carried out. Final cut from resin is performed using the cut mixture as above
did. The product was isolated by precipitation and purified by preparative HPLC.
The desired product was obtained as a white solid on lyophilization.

Example 3 (Nac-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Le
u-Try-Asp-Tyr- Lys-NH 2. Preparation of 3TFA) The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fm
oc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc
-Thr (tBu) OH, Fmoc-Tyr (tBu) OH. Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Example 4 (NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gl)
y-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pr
o-Arg-Lys-Leu- Tyr-Asp-Tyr-Lys-NH 2. 4T
Preparation of FA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH Fmoc-Lys (Boc)
-OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fmo
c-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc-
Thr (tBu) OH, Fmoc-Tyr (tBu) OH, Fmoc-Ala-
OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-
OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)-
OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc
-Arg (Pbf) -OH. Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product,
Obtained as a white solid on lyophilization.

Example 5 (NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gl)
y-Gly-Pro-Trp- Lys-NH 2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fm
oc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu)
-OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (
Pbf) -OH. Deblocking of the N-terminal Fmoc group of resin-bound amino acids
Performed for about 15-20 minutes with 20% piperidine in MF. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Example 6 (NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Ly
_{s- (Nε-MPA) -NH 2} . Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH. Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and the resin
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in L of CHCl ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimidopropionic acid (step 3). Resin cleavage and product isolation was performed using 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product was subjected to Varian (Rainin) preparative binary HPLC.
It was purified by preparative reverse-phase HPLC using the system: 9.5 mL / min from 30 to 55% over 180 minutes in B (H ₂ O 0.045% in TFA (A) and CH ₃ 0 in CN. 045% TFA (B)) gradient elution (Phenomenex L)
una 10 μ Phenyl-hexyl, 21 mm × 25 cm column and λ214
And UV detector at 254 nm (Varian Dynamax UVD I
I)).

Example 7 ((MPA-AEEA) -Pro-Arg-Lys-Leu-Tyr-Asp
-Tyr-NH _2. Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed with: Fmoc-protected R in N, N-dimethylformamide (DMF) solution
Activity with ink Amide MBHA resin, Fmoc protected amino acids, O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM). And piperidine deprotection of the Fmoc group (step 1).

The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH. Deprotection of the terminal Fmoc group was performed by 20% piperidine (step 2) followed by Fmoc-AEE.
Achieved using the coupling of A. Deprotection of the resulting Fmoc-AEEA-peptide with 20% piperidine in DMF allows subsequent addition of 3-MPA (step 3). 86% TFA / 5% TI for resin cleavage and product isolation
Performed using precipitation with S / 5% H ₂ O / 2% thioanisole and 2% phenol, followed by dry ice cold Et ₂ O (step 4). The product is
Purified by preparative reverse phase HPLC using an ian (Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 m
m × 25 cm column and UV detector at λ 214 and 254 nm (Vari
Dynamax C ₁₈ , 60Å with an Dynamax UVD II)
, 8 μm, 21 mm × 25 cm column). This product is a Hewlett equipped with a diode array detector and using electrospray ionization.
RP-HP using t Packard LCMS-1100 series analyzer
It had a> 95% purity as determined by LC mass spectrometry.

Example 8 ((MPA) -Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-
NH ₂ . 2TFA Preparation) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using Symphony Peptide Synthesizer
Performed above: Fmoc protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl-uro. Activation with N-hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1). The following protected amino acids were sequentially added to the Rink Amide MBHA resin using automated peptide synthesis: Fmoc-Lys (Boc) -OH, Fmoc-Tyr (tBu).
OH, Fmoc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH
, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-A
rg (Pbf) -OH, Fmoc-Pro-OH. Deprotection of the terminal Fmoc group,
Achieved using 20% piperidine (step 2), followed by coupling of 3-MPA (step 3). 86% TFA / 5% TIS / for resin cleavage and product isolation
5% H ₂ O / _2% thioanisole and 2% phenol, followed was performed using precipitation by dry-ice cold Et ₂ O and (Step 4). The product is Varia
Purified by preparative reverse phase HPLC using a n (Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm ×
UV detector (Varian at 25 cm column and λ 214 and 254 nm)
Dynamax C ₁₈ , 60Å, 8 with Dynamax UVD II)
μm, using a 21 mm × 25 cm column). This product is a Hewlett equipped with a diode array detector and uses electrospray ionization.
RP-HPLC using a Packard LCMS-1100 series analyzer
It had a> 95% purity, determined by mass spectrometry.

Example 9 (NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Le
u-Tyr-Asp-Tyr- Lys- (Nε-MPA) -NH 2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, F
moc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmo
c-Thr (tBu) OH, Fmoc-Tyr (tBu) OH. Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and preparative HPL
Purified by C to give the desired product as a white solid upon lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and the resin
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in L of CHCl ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimidopropionic acid (step 3). Resin cleavage and product isolation was performed using precipitation with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system: 30-55% B (0.045 in H ₂ O over 180 min at 9.5 mL / min). % TFA (A)
And CH ₃ gradient elution of 0.045% TFA in CN (B)) (Phenom
enex Luna 10μ Phenyl-hexyl, 21 mm x 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax).
UVD II) is used).

Example 10 ((MPA-AEEA) -Tyr-Thr-Thr-Asn-Pro-Arg
-Lys-Leu-Tyr-Asp- Tyr-NH 2. Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed with: Fmoc-protected R in N, N-dimethylformamide (DMF) solution
ink Amide MBHA resin, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium (uron
ium) Activation with hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).

[0113]   The following protected amino acids were linked to Rink Amide using automated peptide synthesis:
  Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fm
oc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc
-Thr (tBu) OH, Fmoc-Tyr (tBu) OH. Of the terminal Fmoc group
Deprotection was performed with 20% piperidine (step 2), followed by coupling with Fmoc-AEEA.
To achieve. Obtained Fmoc with 20% piperidine in DMF
Deprotection of the -AEEA-peptide allows subsequent addition of 3-MPA (engineering).
3). 86% TFA / 5% TIS / 5% H for resin cleavage and product isolation ₂ O / 2% thioanisole and 2% phenol, followed by dry ice cooled Et₂ Performed using precipitation with O (step 4). The product was converted into Varian (Rai
Purified by preparative reverse-phase HPLC using a (nin) preparative binary HPLC system (D
ynamax C₁₈, 60Å, 8μm guard module, 21mm × 25cm module
Lamb and UV detector at λ 214 and 254 nm (Varian Dyna)
Dynamax C with max UVD II)₁₈, 60Å, 8μm, 21
mm using a 25 cm column). This product is a diode array detector
Hewlett Packard equipped and equipped with electrospray ionization
RP-HPLC mass spectrometry using rd LCMS-1100 series analyzer
Had> 95% purity as determined by.

Example 11 ((MPA) -Tyr-Thr-Thr-Asn-Pro-Arg-Lys-
Leu-Tyr-Asp-Tyr- NH 2. Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed with: Fmoc-protected R in N, N-dimethylformamide (DMF) solution
Activation with ink Amide MBHA resin, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM). , And piperidine deprotection of the Fmoc group (step 1). The following protected amino acids were continuously added to the Rink Amide MBHA resin using automated peptide synthesis: Fmoc-Lys (Boc) -OH, Fmoc-Tyr (tBu) O.
H, Fmoc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH,
Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Ar
g (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt)-
OH, Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -OH
, Fmoc-Tyr (tBu) OH. Deprotection of the terminal Fmoc group is achieved using 20% piperidine (step 2) followed by coupling of 3-MPA (step 3). 86% TFA / 5% TIS / 5% H2O for resin cleavage and product isolation
/ 2% thioanisole and 2% phenol, followed by dry-ice cold Et ₂ O
Was carried out using the precipitation according to (Step 4). The product was converted into Varian (Rain
in) purified by preparative reverse phase HPLC using a preparative binary HPLC system (Dy
namax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynam.
Dynamax C ₁₈ , 60Å, 8 μm, 21 m with ax UVD II)
m × 25 cm column is used). This product is a Hewlett Packard equipped with a diode array detector and using electrospray ionization.
had a purity of> 95% as determined by RP-HPLC mass spectrometry using a LCMS-1100 series analyzer.

Example 12 (NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gl)
y-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pr
o-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys- (Nε-MP
A) -NH _2. Preparation of 3TFA) The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, F
moc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmo
c-Thr (tBu) -OH, Fmoc-Tyr (tBu) OH, Fmoc-A
la-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-G
ly-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-A
sp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtB
u) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, F
moc-Arg (Pbf) -OH. Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and the resin
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in L of CHCl ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimidopropionic acid (step 3). Resin cleavage and product isolation was performed using precipitation with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system: 30-55% B (0.045 in H ₂ O over 180 min at 9.5 mL / min). % TFA (A)
And CH ₃ gradient elution of 0.045% TFA in CN (B)) (Phenom
enex Luna 10μ Phenyl-hexyl, 21 mm x 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax).
UVD II) is used).

Example 13 ((MPA-AEEA) -Arg-Asn-Pro-Asp-Gly-Asp
-Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr
-Asn-Pro-Arg-Lys- Leu-Tyr-Asp-Tyr-NH 2
． Preparation of 3TFA) Solid phase peptide synthesis of modified Kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed above: Fmoc protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Activation with uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).

The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH, Fm
oc-Asn (Trt) -OH, Fmoc-Thr (tBu) -OH, Fmoc
-Thr (tBu) OH, Fmoc-Tyr (tBu) OH, Fmoc-Ala
-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly
-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp
(OtBu) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu)
-OH, Fmoc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmo
c-Arg (Pbf) -OH. Deprotection of the terminal Fmoc group was carried out using 20% piperidine (
This is achieved using step 2), followed by Fmoc-AEEA coupling. DM
Deprotection of the resulting Fmoc-AEEA-peptide with 20% piperidine in F allows subsequent addition of 3-MPA (step 3). Resin cleavage and product isolation 86% TFA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed was performed using precipitation by dry-ice cold Et ₂ O and (Step 4 ). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å).
, 8 μm guard module, 21 mm × 25 cm column and λ 214 and 2
Dynamax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column with UV detector (Varian Dynamax UVD II) at 54 nm). The product was determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series analyzer equipped with a diode array detector and using electrospray ionization,> 95.
It had a% purity.

Example 14 ((MPA) -Arg-Asn-Pro-Asp-Gly-Asp-Val-
Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-
Pro-Arg-Lys-Leu- Tyr-Asp-Tyr-NH 2. 3TFA
Preparation of Synthetic Peptide Synthesizer using the following solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale:
Performed with: Fmoc-protected R in N, N-dimethylformamide (DMF) solution
Activity with ink Amide MBHA resin, Fmoc protected amino acids, O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM). And piperidine deprotection of the Fmoc group (step 1). The following protected amino acids were sequentially added to the Rink Amide MBHA resin using automated peptide synthesis: Fmoc-Lys (Boc) -OH, Fmoc-Tyr (tBu).
OH, Fmoc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH
, Fmoc-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-A
rg (Pbf) -OH, Fmoc-Pro-OH, Fmoc-Asn (Trt)
-OH, Fmoc-Thr (tBu) -OH, Fmoc-Thr (tBu) -O
H, Fmoc-Tyr (tBu) OH, Fmoc-Ala-OH, Fmoc-T
rp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-G
ly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH,
Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fmoc-P
ro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf)
-OH.

Deprotection of the terminal Fmoc group was accomplished by 20% piperidine (step 2) followed by 3-MPA.
This is accomplished using the coupling of (step 3). 8 for resin cleavage and product isolation
6% TFA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed was performed using precipitation by dry-ice cold Et ₂ O and (Step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å, 8 μm).
Guard module, 21 mm x 25 cm column and λ214 and 254 nm
Dy with UV detector (Varian Dynamax UVD II) at
namax C ₁₈ , 60Å, 8 μm, using a 21 mm × 25 cm column).
The product had> 95% purity as determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series analyzer equipped with a diode array detector and using electrospray ionization.

Example 15 (NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gl)
y-Gly-Pro-Trp- Lys- (Nε-MPA) -NH 2. Preparation of TFA) Symphony Peptide Synthesizer using solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using
Performed above: Fmoc protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Activation with uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).

[0122]   The following protected amino acids were linked to Rink Amide using automated peptide synthesis:
  Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, F
moc-Gly-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu
) -OH, Fmoc-Gly-OH, Fmoc-Asp (OtBu) -OH, F
moc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg
(Pbf) -OH. Selective deprotection of the Lys (Aloc) group was performed manually and
The resin to 5 mL of CHCl₃: Dissolved in NMM: HOAc (18: 1: 0.5)
3 equivalents of Pd (PPh₃)_FourAchieved by treating with the above solution for 2 hours
(Step 2). This resin is then treated with CHCl₃(6 x 5 mL), 20% in DCM
  HOAc (6 x 5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL)
) Was washed with. The synthesis is then re-run on addition of 3-maleimidopropionic acid.
Automated (step 3). 85% TFA / 5% T for resin cleavage and product isolation
IS / 5% thioanisole and 5% phenol, followed by dry ice cooled Et ₂ Performed using precipitation with O (step 4). The product was converted into Varian (Ra
inin) Purified by preparative reverse phase HPLC using a preparative binary HPLC system:
30-55% B (H) over 180 min at 9.5 mL / min₂0.045 in O
% TFA (A) and CH₃Gradient dissolution of 0.045% TFA (B) in CN
Release (Phenomenex Luna 10μ Phenyl-hexyl, 21mm x
UV detector (Varian at 25 cm column and λ 214 and 254 nm)
  Dynamax UVD II) is used).

Example 16 ((MPA-AEEA) -Arg-Asn-Pro-Asp-Gly-Asp
-Val-Gly-Gly-Pro- Trp-NH 2. Preparation of TFA) Symphony Peptide Synthesizer using solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using
Performed with: Fmoc-protected R in N, N-dimethylformamide (DMF) solution
ink Amide MBHA resin, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ', N'-tetramethyl-uronium (uron
ium) Activation with hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).

The following protected amino acids were linked to the Rink Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fm
oc-Gly-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Gly
-OH, Fmoc-Val-OH, Fmoc-Asp (OtBu) -OH, Fm
oc-Pro-OH, Fmoc-Asn (Trt) -OH, Fmoc-Arg
(Pbf) -OH. Deprotection of the terminal Fmoc group was carried out with 20% piperidine (step 2),
This is subsequently achieved using the Fmoc-AEEA coupling. Deprotection of the resulting Fmoc-AEEA-peptide with 20% piperidine in DMF was performed with 3-M
Allows subsequent addition of PA (step 3). 86 for resin cleavage and product isolation
% TFA / 5% TIS / 5 % H 2 O / 2% thioanisole and 2% phenol, followed was performed using precipitation by dry-ice cold Et ₂ O and (Step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm ( Dyn with Varian Dynamax UVD II)
amax C ₁₈ , 60Å, 8 μm, using a 21 mm × 25 cm column). The product had> 95% purity as determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series analyzer equipped with a diode array detector and using electrospray ionization.

Example 17 ((MPA) -Arg-Asn-Pro-Asp-Gly-Asp-Val-
Gly-Gly-Pro-Trp- NH 2. Preparation of TFA) Symphony Peptide Synthesizer using solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using
Performed above: Fmoc protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Activation with uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1). The following protected amino acids were sequentially added to the Rink Amide MBHA resin using automated peptide synthesis: Fmoc-Lys (Boc) -OH, Fmoc-Trp-OH,
Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,
Fmoc-Asp (OtBu) -OH, Fmoc-Gly-OH, Fmoc-V
al-OH, Fmoc-Asp (OtBu) -OH, Fmoc-Pro-OH,
Fmoc-Asn (Trt) -OH, Fmoc-Arg (Pbf) -OH. Deprotection of the terminal Fmoc group is achieved using 20% piperidine (step 2) followed by coupling of 3-MPA (step 3). 86% for resin cleavage and product isolation
TFA / 5% TIS / 5% H 2 O / 2% thioanisole and 2% phenol, followed was performed using precipitation by dry-ice cold Et ₂ O and (Step 4)
. The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and U at λ 214 and 254 nm.
Dynam with V detector (Varian Dynamax UVD II)
ax C ₁₈ , 60Å, 8 μm, 21 mm × 25 cm column). The product had> 95% purity as determined by RP-HPLC mass spectrometry using a Hewlett Packard LCMS-1100 series analyzer equipped with a diode array detector and using electrospray ionization.

Example 18 (NAc-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys- (N
ε-MPA) -NH _2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH. Deblocking of the N-terminal Fmoc group of resin-bound amino acids was carried out with 20% piperidine in DMF to give about 15-
It was carried out for 20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above. The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and the resin
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in L of CHCl ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimidopropionic acid (step 3). Resin cleavage and product isolation was performed using precipitation with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system: 30-55% B (0.045 in H ₂ O over 180 min at 9.5 mL / min). % TFA (A)
And CH ₃ gradient elution of 0.045% TFA in CN (B)) (Phenom
enex Luna 10μ Phenyl-hexyl, 21 mm x 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax).
UVD II) is used).

Example 19 ((MPA-AEEA) -Arg-Lys-Leu-Tyr-Asp-Tyr
-NH _2. Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed above: Fmoc-protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc-protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Uronium
activation with hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).

The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc
-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc
) -OH, Fmoc-Arg (Pbf) -OH. Deprotection of the terminal Fmoc group is 2
Achieved using 0% piperidine (step 2), followed by Fmoc-AEEA coupling. Obtained Fmoc-AEE with 20% piperidine in DMF
Deprotection of the A-peptide allows subsequent addition of 3-MPA (step 3).
86% TFA / 5% TIS / 5% H ₂ O / 2 for resin cleavage and product isolation
Performed using precipitation with% thioanisole and 2% phenol, followed by dry ice cold Et ₂ O (step 4). The product was transformed into Varian (Rainin
) Purified by preparative reverse phase HPLC using a preparative binary HPLC system (Dyna
max C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax
Dynamax C ₁₈ with UVD II), 60Å, 8 μm, 21 mm ×
Use a 25 cm column). The product is a Hewlett Packard equipped with a diode array detector and using electrospray ionization.
It had a> 95% purity as determined by RP-HPLC mass spectrometry using a LCMS-1100 series analyzer.

Example 20 ((MPA) -Arg-Lys-Leu-Tyr-Asp-Tyr-NH ₂ ..
Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed above: Fmoc-protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc-protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Uronium
activation with hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).
The following protected amino acids were linked to Rink Amide using automated peptide synthesis:
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fmo.
c-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmoc-
Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Boc)
-OH, Fmoc-Arg (Pbf) -OH. Deprotection of the terminal Fmoc group is
% Piperidine (step 2), followed by coupling of 3-MPA (step 3). 86% TFA / 5% TIS / 5% for resin cleavage and product isolation
Performed using precipitation with H ₂ O / 2% thioanisole and 2% phenol, followed by dry ice cold Et ₂ O (step 4). The product was
Purified by preparative reverse phase HPLC using a Rainin) preparative binary HPLC system (Dynamax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25.
cm column and UV detector at λ 214 and 254 nm (Varian D
Dynamax C ₁₈ , 60Å, 8 μm with ynamax UVD II)
, Using a 21 mm x 25 cm column). This product is equipped with a diode array detector and uses Hewlett Pa using electrospray ionization.
It had a> 95% purity as determined by RP-HPLC mass spectrometry using a ckard LCMS-1100 series analyzer.

Example 21 (NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Lys- (N
ε-MPA) -NH _2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH, Fmo
c-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (P
bf) -OH, Fmoc-Pro-OH. N-terminal Fmoc of resin-bound amino acid
Deblocking of the groups was performed with 20% piperidine in DMF for about 15-20 minutes. The coupling of acetic acid was performed under similar conditions to the amino acid coupling. The final cleavage from the resin was performed using the cleavage mixture as above.
The product was isolated by precipitation and purified by preparative HPLC to give the desired product as a white solid upon lyophilization.

Selective deprotection of the Lys (Aloc) group was performed manually and the resin
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in L of CHCl ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of 3-maleimidopropionic acid (step 3). Resin cleavage and product isolation was performed using precipitation with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product was purified by preparative reverse phase HPLC using a Varian (Rainin) preparative binary HPLC system: 30-55% B (0.045 in H ₂ O over 180 min at 9.5 mL / min). % TFA (A)
And CH ₃ gradient elution of 0.045% TFA in CN (B)) (Phenom
enex Luna 10μ Phenyl-hexyl, 21 mm x 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynamax).
UVD II) is used).

Example 22 ((MPA-AEEA) -Pro-Arg-Lys-Leu-Tyr-Asp
-NH _2. Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed above: Fmoc protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Activation with uronium hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).

The following protected amino acids were linked to the Link Amide using automated peptide synthesis.
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fm.
oc-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH, Fmoc
-Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pb
f) -OH, Fmoc-Pro-OH (step 1). Deprotection of the terminal Fmoc group is achieved using 20% piperidine (step 2), followed by Fmoc-AEEA coupling. Fmoc-A obtained with 20% piperidine in DMF
Deprotection of EEA-peptide allows subsequent addition of 3-MPA (step 3
). 86% TFA / 5% TIS / 5% H ₂ O for resin cleavage and product isolation
/ 2% thioanisole and 2% phenol, followed by dry-ice cold Et ₂ O
Was carried out using the precipitation according to (Step 4). The product was converted into Varian (Rain
in) purified by preparative reverse phase HPLC using a preparative binary HPLC system (Dy
namax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynam.
Dynamax C ₁₈ , 60Å, 8 μm, 21 m with ax UVD II)
m × 25 cm column is used). This product is a Hewlett Packard equipped with a diode array detector and using electrospray ionization.
had a purity of> 95% as determined by RP-HPLC mass spectrometry using a LCMS-1100 series analyzer.

Example 23 ((MPA) -Pro-Arg-Lys-Leu-Tyr-Asp-NH ₂ ..
Preparation of 2TFA) Solid phase peptide synthesis of modified kringle 5 peptide on a 100 μmol scale using the following Symphony Peptide Synthesizer.
Performed above: Fmoc-protected Rink Amide MBHA resin in N, N-dimethylformamide (DMF) solution, Fmoc-protected amino acid, O-benzotriazol-1-yl-N, N, N ′, N′-tetramethyl- Uronium
activation with hexafluorophosphate (HBTU) and N-methylmorpholine (NMM), and piperidine deprotection of the Fmoc group (step 1).
The following protected amino acids were linked to Rink Amide using automated peptide synthesis:
Sequentially added to MBHA resin: Fmoc-Lys (Boc) -OH, Fmo.
c-Asp (OtBu) -OH, Fmoc-Tyr (tBu) OH, Fmoc-
Leu-OH, Fmoc-Lys (Boc) -OH, Fmoc-Arg (Pbf
) -OH, Fmoc-Pro-OH. Deprotection of the terminal Fmoc group is achieved using 20% piperidine (step 2) followed by coupling of 3-MPA (step 3). 86% TFA / 5% TIS / 5% H ₂ O for resin cleavage and product isolation
/ 2% thioanisole and 2% phenol, followed by dry-ice cold Et ₂ O
Was carried out using the precipitation according to (Step 4). The product was converted into Varian (Rain
in) purified by preparative reverse phase HPLC using a preparative binary HPLC system (Dy
namax C ₁₈ , 60Å, 8 μm guard module, 21 mm × 25 cm column and UV detector at λ 214 and 254 nm (Varian Dynam.
Dynamax C ₁₈ , 60Å, 8 μm, 21 m with ax UVD II)
m × 25 cm column is used). This product is a Hewlett Packard equipped with a diode array detector and using electrospray ionization.
had a purity of> 95% as determined by RP-HPLC mass spectrometry using a LCMS-1100 series analyzer.

Example 24 (NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Ly
s- (Nε-AEEA-MPA) -NH 2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH (step 1). Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. Acetic acid coupling,
It was performed under conditions similar to amino acid coupling. Selective deprotection of the Lys (Aloc) group was performed manually and the resin was added to 5 mL CHCl ₃ : NMM: HO
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in Ac (18: 1: 0.5) for 2 hours (step 2). This resin was then treated with CHCl ₃ (6
X5 mL), 20% HOAc in DCM (6 x 5 mL), DCM (6 x 5 mL
), And DMF (6 × 5 mL). The synthesis was then re-automated for the addition of AEEA (aminoethoxyethoxy acetic acid) groups and 3-maleimidopropionic acid (MPA) (step 3). 85% TF for resin cleavage and product isolation
Performed using precipitation with A / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product is
Purified by preparative reverse phase HPLC using an ian (Rainin) preparative binary HPLC system: 30-55% B (0.045% TFA (A in H ₂ O over 180 min at 9.5 mL / min. ) and CH ₃ CN 0.045% in TFA (B
)) Gradient elution (Phenomenex Luna 10μ phenyl-hexyl, 21 mm x 25 cm column and UV detector at λ 214 and 254 nm (
Varian Dynamax UVD II) is used).

Example 25 (NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-Ly
_{ε- (N-AEEA n -MPA)} -NH 2. Preparation of 2TFA) Using automated peptide synthesis, the following protected amino acids were added to the Link Amide
Sequentially added to MBHA resin: Fmoc-Lys (Aloc) -OH, F
moc-Tyr (tBu) OH, Fmoc-Asp (OtBu) -OH, Fmo
c-Tyr (tBu) OH, Fmoc-Leu-OH, Fmoc-Lys (Bo
c) -OH, Fmoc-Arg (Pbf) -OH, Fmoc-Pro-OH (step 1). Deblocking of the N-terminal Fmoc group of resin bound amino acids was performed with 20% piperidine in DMF for about 15-20 minutes. Acetic acid coupling,
It was performed under conditions similar to amino acid coupling.

Selective deprotection of the Lys (Aloc) group was performed manually and the resin
This was achieved by treatment with a solution of ₃ equivalents of Pd (PPh ₃ ) ₄ dissolved in L of CHCl ₃ : NMM: HOAc (18: 1: 0.5) for 2 hours (step 2). The resin was then loaded with CHCl ₃ (6 × 5 mL), 20% HOAc in DCM (6 × 5 mL).
5 mL), DCM (6 x 5 mL), and DMF (6 x 5 mL). The synthesis was then re-automated for the addition of n AEEA (aminoethoxyethoxyacetic acid) groups and 3-maleimidopropionic acid (MPA) (step 3). Resin cleavage and product isolation was performed using precipitation with 85% TFA / 5% TIS / 5% thioanisole and 5% phenol, followed by dry ice cold Et ₂ O (step 4). The product was subjected to Varian (Rainin) preparative binary HPLC.
It was purified by preparative reverse-phase HPLC using the system: 9.5 mL / min from 30 to 55% over 180 minutes in B (H ₂ O 0.045% in TFA (A) and CH ₃ 0 in CN. 045% TFA (B)) gradient elution (Phenomenex L)
una 10 μ Phenyl-hexyl, 21 mm × 25 cm column and λ214
And UV detector at 254 nm (Varian Dynamax UVD I
I)).

Example 26 Peptide Stability Assay A peptide stability assay was performed. (MPA) -Pro-Arg-Lys-
Leu-Tyr-Asp-Lys- NH 2. 2TFA was synthesized as above and identified as MPA-K5. Unmodified corresponding peptide Pro-Arg-
The Lys-Leu-Tyr-Asp- Lys-NH 2 The, 3-MPA was synthesized as described above without adding, and then K5 and identification.

K5 (MW = 1260.18, 918.12 free base) was prepared as a 100 mM stock solution in water. MPA-K5 (MW = 1411.17, 106
9.11. Free base) was prepared as a 100 mM stock solution in water. Human serum albumin (HSA) is a 25% solution (about 250 mg / ml, as Albutein®) available from Alpha Therapeutic.
3.75 mM). Human plasma from Golden West Biolo
Obtained from Gicals.

A. Stability of K5 in Human Plasma K5 was prepared as a 1 μM solution and dissolved in 25% human serum albumin. This mixture was then incubated at 37 ° C. in the presence of human serum to give 16
The final concentration of K5 was 0 mM. Aliquots of 100 μl were removed from plasma at 0, 4 and 24 hours. This 100 μl aliquot was added to 100 μl of blocking solution (5 volumes of 5% Z to precipitate all proteins).
NSO _4/3 was mixed with methanol) acetonitrile / 2 volumes of. This sample was centrifuged at 10,000 g for 5 minutes to collect the peptide-containing supernatant,
Then, it was filtered through a 0.22 μm filter. Free intact K5
The presence of peptides was assayed by HPLC / MS. The HPLC parameters for the detection of K5 peptide in serum were as follows.

The HPLC method was as follows: Vydac C18 250X4.6.
mm, 5μ particle size column was used. The column temperature is 30 ° C and the flow rate is 0.
． It was 5 ml / min. Mobile phase A was 0.1% TFA / water. Mobile phase B was 0.1% TFA / acetonitrile. The injection volume was 10 μl.

[0143]   The gradient was as follows:           Time (minutes)% A% B             0 95 5           20 70 30           25 10 90           30 10 90           35 95 5           45 95 5.

These proteins were detected at 214, 254 and 334 nm. For mass spectral analysis, the ionization mode was API-electrospray (positive mode) in the M / Z range 300-2000. Gain is 3.0,
The fragmentor was 120 v, the threshold was 20, and the step size was 0.1. The gas temperature was 350 ° C. and the dry gas volume was 10.0 l / min.
The tip (Neb) pressure was 24 psi and the Vcap was 3500V. The HPLC method was as follows: Vydac C18 250X4.
． A 6 mm, 5μ particle size column was used. The column temperature was 30 ° C. and the flow rate was 0.5 ml / min. Mobile phase A was 0.1% TFA / water. Mobile phase B was 0.1% TFA / acetonitrile. The injection volume was 10 μl.

[0145]   The gradient was as follows:           Time (minutes)% A% B               0 95 5             20 70 30             25 10 90             30 10 90             35 95 5             45 95 5.

These proteins were detected at 214, 254 and 334 nm. For mass spectral analysis, the ionization mode was API-electrospray (positive mode) in the M / Z range 300-2000. Gain is 3.0,
The fragmentor was 120 v, the threshold was 20, and the step size was 0.1. The gas temperature was 350 ° C. and the dry gas volume was 10.0 l / min.
Tip pressure was 24 psi and Vcap was 3500V.

[0147]             Time% K5 peptide in plasma           0 hours 100%           4 hours 9%         0% for 24 hours.

After only 4 hours of incubation in plasma, only 9% of the original K5 peptide remained. These results indicate that the unmodified K5 peptide is unstable in serum, probably as a result of protease activity.

B. MPA-K5-HSA Conjugate Stability in Plasma MPA-K5 (modified K5 peptide) was incubated with 25% HSA for 2 hours at room temperature. The MPA-K5-HSA conjugate was then incubated at 37 ° C. in the presence of human serum at a final concentration of 160 μm. After the specified incubation period (0, 4 and 24 hours), 100 μl aliquots were removed and filtered through a 0.22 μm filter. The presence of intact conjugate was assayed by HPLC-MS.

The column was an Aquapore RP-300, 250x4.6 mm, 7μ particle size. The column temperature was 50 ° C. Mobile phase A is 0.1% T
FA / water. Mobile phase B was 0.1% TFA / acetonitrile. The injection volume was 1 μl. The gradient was as follows: time (min)% A% B flow rate (ml / min) 0 66 34 0.700 1 66 34 0.700 25 58.8 41.2 0.700 30 50 50 0. 70 35 5 95 1.00 41 5 95 1.00 45 45 66 34 1.00 46 66 34 0.70.

Peptides were detected at 214 nm for quantification. For mass spectrometric analysis of this peptide, the ionization mode was AP in the range 1280-1500 m / z.
I-electrospray, gain 1.0, fragmentor 125V,
The threshold was 100 and the step size was 0.40. The gas temperature was 350 ° C. and the dry gas was 13.0 l / min. Pressure is 60 psi and V
The cap was 6000V. The results are shown below.

About 33% of albumin circulating in the bloodstream is mercaptoalbumin (SH-albumin), which is not blocked by endogenous sulfhydryl compounds (eg cysteine or glutathione), and thus is Can be used for reactions. The remaining 66% of circulating albumin is capped or blocked by the sulfhydryl compound. The HPLC MS assay allows the identification of capped HSA, SH-albumin and K5-MPA-albumin. MPA covalently binds to a free thiol on albumin. The stability of the three forms of albumin in plasma is shown below.

[0153]

[Table 2] The proportions of the three forms of human serum albumin remained relatively constant throughout the 24 hour assay period. In particular, the proportion of K5-MPA-HSA remained relatively constant throughout the 24 hour plasma assay. These results are dramatic compared to those obtained with unmodified K5, which reduces to 9% of the original amount of K5 in plasma in just 4 hours. These results indicate that K5-MPA-HSA is quite stable in plasma from peptidase activity, in contrast to K5, which is fairly unstable in plasma.

Example 27 Endothelial Cell Migration Assay Modified anti-neopoiesis peptide activity can be determined using an endothelial cell migration assay. This endothelial cell migration assay is described in Polverini, P .; J. Et al, Metho
can be carried out as described by ds Enzymol, 198: 440-450 (1991), which is incorporated herein by reference. Briefly, bovine capillary (pararenal) endothelial cells (BCE, which is the Judah Folkma
n, Harvard University Medical School) can be starved overnight in DMEM with 0.1% bovine serum albumin (BSA). The cells are then harvested with trypsin and 0.
Resuspend at a concentration of 1.5 × 10 ⁶ cells / mL in DMEM containing 1% BSA. The cells were placed in a 48-well modified Boyden chamber (eg, Nucleopo
re Corporation, Cabin John, Md. From the bottom). The chamber was collected and inverted, and cells were allowed to attach to a polycarbonate chemotaxis membrane (5 μm pore size) (soaked overnight in 0.1% gelatin and dried) for 2 hours at 37 ° C. . The chamber is then inverted again and the test substance is added to the wells of the upper chamber (50 μl).
Of the total volume); then the device is incubated at 37 ° C. for 4 hours. Membranes were harvested, fixed and stained (DiffQuick, Fisher Scient).
ific, Pittsburgh, Pa. ), And per 10 high power fields,
Count the number of cells transferred to the upper chamber. Background migration to DMEM + 0.1% BSA can be subtracted, and the data show 10 high power fields (40
It can be reported as the number of migrated cells per 0 ×), or as the% inhibition of migration compared to the positive control when the results from multiple experiments are combined.

Example 28 Preparation of HSA-Kringle 5 Conjugate The modified Kringle 5 peptide is dissolved in distilled water to a final concentration of 100 mM. For the conjugation reaction, 1 volume of 100 mM modified kringle 5 peptide was added to 99 volumes of 25% HSA (Albutein®, 25% solution,
Alpha Therapeutic inc. ) To 1 mM modified K5
: 3.75 mM HSA conjugate is obtained. This mixture is incubated at room temperature for 2 hours. The presence of conjugate and the absence of unreacted modified K5 peptide herein are determined by HPLC coupled with mass spectrometry.

Example 29 Effect of Modified Kringle 5 Peptide on Endothelial Cell Proliferation In Vitro The biological activity of free and HSA-conjugated kringle 5 peptide was determined in vitro using an endothelial cell proliferation assay. Can be determined. Bovine aortic endothelial cells,
Dulbecco's modified Eagle medium (DM containing 10% heat-inactivated calf serum)
EM, Gibco) at a density of 2500 cells per well in 96 well plates. The cells were placed in a 5% CO ₂ incubator,
Attach at 37 ° C. for 24 hours. The medium was then replaced with fresh DMEM (containing free K5 peptide and HSA-Kringle 5 peptide) at various concentrations.
(Without serum). After 30 minutes at 37 ° C., bFGF (basic fibroblast growth factor) can then be added to a final concentration of 1 ng / mL to stimulate proliferation. After 72 hours, the number of cells was determined by measuring the colorimetric substrate WST-1 (Boehrin
ger Mannheim) can be used to determine the effect of the modified K5 peptide on endothelial cell proliferation in vitro.

Although the present invention has been described in connection with its particular embodiments, further modifications are possible and this application is generally in accordance with the principles of the invention and within the art to which this invention belongs. Such developments from the disclosure of the invention which are within the known or conventional practice and which may be applied to the essential characteristics described herein and which are subject to the scope of the appended claims. Including any variations, uses of the invention,
Or, it is understood that it is intended to encompass adaptation.

[Sequence list]

─────────────────────────────────────────────────── ─── Continued front page    (81) Designated countries EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, I T, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, K E, LS, MW, SD, SL, SZ, TZ, UG, ZW ), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, C R, CU, CZ, DE, DK, DM, EE, ES, FI , GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, K Z, LC, LK, LR, LS, LT, LU, LV, MA , MD, MG, MK, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, S K, SL, TJ, TM, TR, TT, TZ, UA, UG , US, UZ, VN, YU, ZA, ZW (72) Inventor Rasamoeri Solo, Mikel             Canada H2P2XS               Quebec, Montreal, Aper             Tomento 405, Mistral Avenue             ー 1111 (72) Inventor Cibaudue, Karen             Canada H3 W2 L5               Quebec, Montreal, Bonavi             Star Street Number 407 4700 (72) Inventor Huan, Shikai             Canada H9J3X2               Quebec, Kirkland, Denaul             To 153 (72) Inventor Billiview, Richard             Canada H3E1L8 Kebe             Kook, Badan, Wilson Aveni             View 266 F-term (reference) 4H045 AA10 AA11 AA30 BA14 BA15                       BA16 BA17 BA18 BA50 EA20                       FA34

Claims (26)

[Claims] 1. A modified anti-angiogenic peptide having a reactive group that reacts with an amino group, a hydroxyl group, or a thiol group of a blood component to form a stable covalent bond. A modified anti-angiogenic peptide. 2. The modified peptide according to claim 1, wherein the peptide is kringle 5 peptide. 3. The kringle 5 peptide according to claim 2, wherein the derivative is reactive with blood proteins. 4. The kringle 5 peptide of claim 3, wherein the derivative is reactive with thiol groups of blood proteins. 5. The peptide according to claim 2, wherein the peptide is selected from the group consisting of:
Kringle 5 peptide described in: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 9. 6. The peptide according to claim 2, wherein the peptide is selected from the group consisting of:
Kringle 5 peptide described in: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12,
SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16. 7. A composition comprising a derivative or analog thereof in the Kringle 5 peptide, said derivative being for use in a method of treating angiogenesis in humans, an amino group, a hydroxyl group of a blood component, Or reacting with a thiol group,
A composition comprising a reactive group that forms a stable covalent bond. 8. The composition of claim 7, wherein the derivative is reactive with blood proteins. 9. The composition according to claim 7, wherein the derivative is reactive with thiol groups of blood proteins. 10. A derivative of kringle 5 peptide, said derivative comprising a maleimide group that reacts with a thiol group of human serum albumin to form a covalent bond. 11. A derivative according to claim 10, wherein said peptide is selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8. , And SEQ ID NO: 9. 12. The derivative according to claim 10, wherein the peptide is selected from: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO :. 16. 13. A composition comprising a derivative of an anti-angiogenic peptide, which derivative reacts with a thiol group of human serum albumin for use in a method of treating angiogenesis in humans. Including a maleimide group forming a covalent bond,
Composition. 14. The peptide according to claim 1, which is a kringle 5 peptide.
The composition according to 3. 15. The composition of claim 14, wherein the peptide is selected from: SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9. 16. The composition according to claim 14, wherein said peptide is selected from: SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and sequence. Number 16. 17. Use of a composition for a manufacturer of a medicament for prolonging the in vivo half-life of a kringle 5 peptide in a patient to provide an anti-angiogenic effect, said composition comprising: kringle. Uses, including derivatives of 5 peptides or analogs thereof, which contain reactive groups that react with amino, hydroxyl, or thiol groups of blood components to form stable covalent bonds. 18. The use of the composition of claim 14 for treating angiogenesis in humans, wherein the anti-angiogenic kringle 5 peptide derivative reacts with blood proteins. To use. 19. A modified kringle 5 peptide selected from the group consisting of: (MPA-AEEA) -Pro-Arg-Lys-Leu-Tyr-Asp-.
Tyr-NH ₂ and (MPA) -Pro-Arg-Lys -Leu-Tyr-
Asp-Tyr-NH _2. 20. A modified kringle 5 peptide selected from the group consisting of:
: NAc-Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu
-Tyr-Asp-Tyr-Lys- (N [epsilon] -MPA) -NH₂; (MPA-A
EEA) -Tyr-Thr-Thr-Asn-Pro-Arg-Lys-Leu.
-Tyr-Asp-Tyr-NH₂MPA) -Tyr-Thr-Thr-As
n-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂; NA
c-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-Gl
y-Pro-Trp-Ala-Tyr-Thr-Thr-Asn-Pro-Ar
g-Lys-Leu-Tyr-Asp-Tyr-Lys- (Nε-MPA) -N
H₂(MPA-AEEA) -Arg-Asn-Pro-Asp-Gly-As
p-Val-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Th
r-Asn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH ₂ And (MPA) -Arg-Asn-Pro-Asp-Gly-Asp-V
al-Gly-Gly-Pro-Trp-Ala-Tyr-Thr-Thr-A
sn-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-NH₂. 21. A modified kringle 5 peptide selected from the group consisting of: NAc-Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly.
-Gly-Pro-Trp-Lys- ( Nε-MPA) -NH 2; MPA-AE
EA) -Arg-Asn-Pro-Asp-Gly-Asp-Val-Gly-
_{Gly-Pro-Trp-NH 2} ; (MPA) -Arg-Asn-Pro-As
p-Gly-Asp-Val- Gly-Gly-Pro-Trp-NH 2; NA
c-Arg-Lys-Leu-Tyr-Asp-Tyr-Lys- (Nε-MP
_{A) -NH 2; (MPA-} AEEA) -Arg-Lys-Leu-Tyr-As
_{p-Tyr-NH 2; (} MPA) -Arg-Lys-Leu-Tyr-Asp-
Tyr-NH ₂ ; NAc-Pro-Arg-Lys-Leu-Tyr-Asp-
Lys- (Nε-MPA) -NH ₂ ; (MPA-AEEA) -Pro-Arg-
Lys-Leu-Tyr-Asp- NH 2; (MPA) -Pro-Arg-Ly
s-Leu-Tyr-Asp- NH 2; NAc-Pro-Arg-Lys-Le
u-Tyr-Asp-Tyr-Lys- (Nε-AEEA-MPA) -NH ₂ ;
And NAc-Pro-Arg-Lys-Leu-Tyr-Asp-Tyr-L
_{ys- (Nε-AEEA n -MPA)} -NH 2. 22. The modified anti-angiogenic peptide of claim 1, wherein the reactive group comprises a succinimidyl group or a maleimide group. 23. The composition according to claim 7, wherein the reactive group comprises a succinimidyl group or a maleimide group. 24. The use according to claim 17, wherein the reactive group comprises a succinimidyl group or a maleimide group. 25. Any one of claims 1 to 6, 10 to 12 and 19 to 21.
A conjugate comprising a modified peptide according to claim 6, wherein the peptide is covalently bound to a blood component and the conjugate is stable in vivo. 26. A method for extending the in vivo half-life of an anti-angiogenic peptide, which method comprises: attaching a reactive group to the peptide and attaching the reactive group to a blood component. Reacting with a functional group to form a covalent bond, thereby
A method comprising forming a stable in vivo conjugate having an in vivo half-life longer than the in vivo half-life of the anti-angiogenic peptide alone.