阅读说明：本技术 用于抑制细胞粘附至rgd结合位点或引导诊断剂或治疗剂至 rgd结合位点的方法 (Methods for inhibiting cell adhesion to RGD binding sites or for directing diagnostic or therapeutic agents to RGD binding sites ) 是由 M·J·麦克尔 J·帕克 于 2010-11-10 设计创作，主要内容包括：本发明涉及用于抑制细胞粘附至RGD结合位点或引导诊断剂或治疗剂至RGD结合位点的组合物和方法。本发明公开了包括R-G-磺基丙氨酸(即R-G-NH-CH(CH<Sub>2</Sub>-SO<Sub>3</Sub>H)COOH或Arg-Gly-NH-CH(CH<Sub>2</Sub>-SO<Sub>3</Sub>H)COOH)和其衍生物(包括药学上可接受的盐、水合物、立体异构体、多聚体、环状形式、线性形式、药物缀合物、前药及其衍生物)的化合物。还公开了制备和使用这种化合物的方法,包括在人或动物受试者中抑制细胞粘附到RGD结合位点上或将其它诊断剂或治疗剂递送到RGD结合位点的方法。(The present invention relates to compositions and methods for inhibiting cell adhesion to RGD binding sites or directing diagnostic or therapeutic agents to RGD binding sites. The present invention discloses compositions comprising R-G-cysteic acid (i.e., R-G-NH-CH (CH) 2 ‑SO 3 H) COOH or Arg-Gly-NH-CH (CH) 2 ‑SO 3 H) COOH) and its derivatives (including pharmaceutically acceptable salts, hydrates, stereoisomers, and polyacids)Mer, cyclic form, linear form, drug conjugate, prodrug, and derivatives thereof). Also disclosed are methods of making and using such compounds, including methods of inhibiting cell adhesion to RGD binding sites or delivering other diagnostic or therapeutic agents to RGD binding sites in a human or animal subject.)

1. use of a pharmaceutical composition comprising i) a peptide comprising glycine-arginine-glycine-cysteic acid-threonine-proline-COOH, or ii) a peptide having the formula:

X1-R-G-cysteic acid-X

Wherein X and X1 are selected from: Phe-Val-Ala, -Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Ala, -Phe-Val; or selected from Arg, Gly, cysteic acid, Phe, Val, Ala, Leu, Pro, Thr and salts, combinations, D-isomers and L-isomers thereof.

2. The use according to claim 1, wherein the peptide has the following structural formula:

3. Use according to any of claims 1 or 2, wherein the medicament is administered by injection into the eye.

4. use according to any of claims 1 or 2, wherein the medicament is administered by topical application.

5. Use according to claim 4, wherein the medicament comprises a liquid or gel for topical administration to the eye.

6. Use according to any one of claims 1 to 5, wherein the medicament is for preventing the development of vitreous macular traction, proliferative diabetic retinopathy, macular hole, macular degeneration or flotage.

7. The use according to any one of claims 1-5, wherein the medicament is for inducing vitrectomy-induced vitrolysis or posterior vitrectomy detachment.

8. Use according to any one of claims 1 to 5, wherein the medicament is for the treatment of retinopathy, diabetic retinopathy, wet macular degeneration, dry macular degeneration, glaucoma, neovascular glaucoma.

9. Use according to any one of claims 1 to 5, wherein the medicament is for the treatment of an eye disease that causes abnormal adhesion to a cell adhesion motif selected from fibronectin, vitronectin, laminin, fibrinogen, thrombospondin and von Willebrand factor.

Technical Field

The present invention relates generally to the fields of chemistry and medicine, and more particularly to compositions and methods useful for inhibiting cell adhesion to the Arg-Gly-Asp ("RGD" tripeptide) binding site and related treatments for: such as inflammation, wound healing, prevention of scarring, thrombosis, cancer metastasis and tumors, proliferative or nonproliferative diabetic retinopathy, vitreous humor liquefaction, induction of Posterior Vitreoretinal Detachment (PVD), pathogenesis of vitreoretinal diseases, such as floaters, idiopathic macular holes, vitreous macular traction, age-related macular degeneration, wet macular degeneration, choroidal neovascularization, vitreoretinal surgery, vein occlusion, corneal neovascularization, ischemic optic nerve, rubeosis (rubeosis iridis), and prevention of scarring in glaucoma surgery.

Background

The RGD tripeptide sequence is present in many proteins, where it plays a role in cell adhesion. Examples of proteins in which the RGD tripeptide sequence is present include collagen, fibronectin, vitronectin, Von Willebrand Factor (VWF), certain disintegrins, and certain dictyostelins.

Integrins are heterodimeric cell surface receptors that mediate adhesion between cells and the extracellular matrix (ECM) by binding to ligands with exposed RGD sequences. Normal integrin-RGD binding is thought to play a role in gene expression involved in cell growth, migration and survival. This misregulation of cell growth, migration and survival can lead to a number of disease states, including thrombosis, inflammation and cancer. Therefore, RGD peptides have been studied as potential mimics of cell adhesion proteins and have been studied for their ability to bind to integrins, for therapeutic purposes (e.g. inhibition of apoptosis, angiogenesis, tumorigenesis), for their use in multimeric form as internal radiotherapeutic agents as well as cancer imaging agents and their ability to carry anticancer drugs.

In the eye, integrins affect many processes including eye development, cell migration, healing, and several pathological processes. Integrins also mediate inflammation and thrombosis in ocular tissues. It has also been reported that intravitreal injection of RGD peptides in animal models causes posterior vitreoretinal detachment and, therefore, may be useful in the treatment of certain retinal conditions and/or to facilitate vitrectomy procedures. [ see Olivera, L.B. et al, RGD Peptide-Assisted vitality to facility indication of a stereor vitamins detail: a New Principle in pharmaceutical vitriolysis (RGD peptide-assisted vitrectomy, which favors the induction of posterior vitreal detachment: a New Principle of pharmacological vitriolysis); current Eye Research (8): 333-40 (12 months and 25 days 2002).

Disclosure of Invention

The present invention provides compositions comprising R-G-cysteic acid (i.e., R-G-NH-CH (CH)₂-SO₃H) COOH or Arg-Gly-NH-CH (CH)₂-SO₃H) COOH) and derivatives thereof (including pharmaceutically acceptable salts, hydrates, stereoisomers, multimers, cyclic forms, linear forms, drug conjugates, prodrugs and derivatives thereof).

The invention also relates toCompositions are provided for inhibiting cell adhesion to an RGD binding site or delivering other diagnostic or therapeutic agents to an RGD binding site in a human or animal subject, and methods for inhibiting cell adhesion to an RGD binding site or delivering other diagnostic or therapeutic agents to an RGD binding site in a human or animal subject by administering to the subject an effective amount of a composition comprising an R-G-cysteic acid peptide or derivative thereof (including pharmaceutically acceptable salts, hydrates, stereoisomers, multimers, cyclic forms, linear forms, drug conjugates, prodrugs, and derivatives thereof). Specific examples of R-G-cysteic acid peptides of the present invention include Arg-Gly-NH-CH (CH)₂-SO₃H) The linear form of COOH (referred to herein as an example of Compound 1) and Arg-Gly-NH-CH (CH)₂-SO₃H) COOH) (referred to herein as an example of compound 2).

The general formula of the R-G-cysteic acid derivatives of the present invention includes, but is not limited to, compounds having the following general formulas I-VII:

General formula I:

Wherein X is selected from: H. c₁-C₆Alkyl, Ph or SO₃H, Y ═ OH or NH₂。

general formula II:

Wherein X is selected from: H. c₁-C₆Alkyl, Ph or SO₃H。

general formula III:

Wherein X is selected from: H. c₁-C₆Alkyl, Ph or SO₃H, wherein Z is selected from: h or SO₃H。

General formula IV:

Wherein X is selected from: H. c₁-C₆alkyl, Ph or SO₃h; y is selected from OH or NH₂。

General formula V:

Wherein X is selected from: H. c₁-C₆Alkyl, Ph or SO₃H。

General formula VI:

Wherein X is selected from: H. c₁-C₆Alkyl, Ph or SO₃H。

General formula VII:

X₁-R-G-cysteic acid-X

wherein X and X₁Selected from: cyclic or linear-Phe-Val-Ala, -Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Ala, -Phe-Val, or any salt of the D-isomer or any combination of the L-isomers of the following amino acids: arg, Gly, cysteic acid, Phe, Val, Ala, Leu, Pro, Thr.

Examples of cyclic forms of formula VII include, but are not necessarily limited to:

Wherein X' is selected from: H. c₁-C₆Alkyl, Ph or SO₃H; z is selected from H or Me; y is selected from OH and NH₂。

Sulfonic acids are acids that are stronger than the corresponding carboxylic acids. This higher polarity of the sulfonic acid group results in stronger intermolecular binding. For example, R-G-cysteic acid, which has more polarized O-H bonds, may form stronger hydrogen bonds with amide groups of proteins in the integrin binding site than R-G-aspartic acid (RGD peptide), which has relatively less polarized O-H bonds, and/or have stronger interactions with metal ions complexed in the integrin binding site.

As described in more detail elsewhere herein, a specific example glycyl (glycyl) -arginyl-glycyl-cysteic acid-threonyl-proline-COOH (GRG cysteic acid TP; hereinafter Compound 1) of general formula VII was synthesized and tested in animals and found to be effective in inducing Posterior Vitreal Detachment (PVD) on the retinal surface by inhibiting integrin-extracellular matrix (ECM) interactions. Compound 1 was also tested in a wound healing model using Human Umbilical Vein Endothelial Cells (HUVEC), as described in more detail elsewhere herein, and was shown to inhibit cell adhesion by 74% within 12 hours compared to cyclic-RGD peptide inhibition (40%). These studies indicate and confirm the rationale for glycyl-arginyl-glycyl-cysteic acid-threonyl-proline-COOH to bind to integrins even more strongly than the RGD peptide itself.

The R-G-cysteic acid peptide sequence, which may be in L-form or D-form, is a competitive inhibitor of integrin-ECM interactions. The R-G-cysteic acid peptide sequence may be a protease resistant derivative or a cyclic derivative or a prodrug derivative or associated with a drug delivery system or a monoclonal antibody.

The compositions of the invention are useful for inhibiting angiogenesis, which is useful for treating inflammation, wound healing, thrombosis, cancer metastasis and tumors. Other beneficial applications may exist in ophthalmology, including proliferative or non-proliferative diabetic retinopathy, vitreous liquefaction, induction of Posterior Vitreoretinal Detachment (PVD), pathogenesis of vitreoretinal diseases, such as idiopathic macular hole, vitreous macular traction, age-related macular degeneration, wet macular degeneration, choroidal neovascularization, vitreoretinal surgery, vein occlusion, and prevention of scarring in glaucoma surgery. Furthermore, the multimeric and/or radiolabeled compositions of the invention are useful as diagnostic/imaging agents for the detection of tumors and as radiotherapeutic agents for the treatment of tumors and as carriers for anticancer drugs due to their tumor-targeting properties.

Biomaterials incorporating R-G-cysteic acid peptides can also provide a synthetic viscous microenvironment for long-term cell survival and growth and for living tissue engineering for applications in tissue engineering and regenerative medicine. By its integrin adhesion receptor binding properties, the R-G-cysteic acid peptide, when attached to a biomaterial or scaffold, can provide a pro-adhesion signal. R-G-cysteic acid based materials mediate cell adhesion, cell diffusion and migration. In addition, integrin-mediated cell adhesion promotes cell proliferation and survival, and plays a key role in the assembly and organization of multicellular structures and tissues.

Drugs used to treat macular degeneration like Lucentis and avastin are based on inhibiting VEGF, which otherwise introduces new blood vessel growth, angiogenesis, and consequent macular edema. It is known that the RGD small peptide can be bound by competition, and inhibit the cell from extracellular matrix¹attachment to induce apoptosis is shown in U.S. patent No. 6,500,924 to Brooks et al.

the RGD peptide binding motif or recognition motif may be present in proteins of the extracellular matrix and in integrins that link the intracellular cytoskeleton of cells to the ECM by recognizing the RGD adhesion epitope. See, e.g., Foos, R y., invest, opthalmol, vis.sci. (1972)11, 801-808. Cells that do not attach to the ECM typically undergo apoptosis.

In general, the interaction between fibroblasts and glycoprotein components of the extracellular matrix causes severe scarring mediated primarily by RGD-containing amino acid sequences interacting on cell surface integrins. The RGD sequence is also known to be involved in cell-ECM interactions during inflammatory and homeostatic responses (see Hershkoviz, s.m. et al, invest, ophthalmol. vis. sci., (1994), 35, 2585-2591), and integrins play an important role in cell migration during wound healing or pathology as well as in regulating inflammation and thrombosis. Thus, potent integrin antagonists, like the RGD peptide, can be very useful as pharmacological agents such as anti-inflammatory, anti-metastatic or anti-thrombotic agents (see Elner, s.g. and Elner, v.m., IOVS (1996) 37: 696-701. the literature also reports that the CD44 receptor of hyaluronic acid mediates cell-cell and cell-matrix interactions through its affinity for hyaluronic acid, and possibly also through its affinity for other ligands such as osteopontin, collagen and Matrix Metalloproteinases (MMPs)).

Adhesion to hyaluronan plays an important role in cell migration, tumor growth and progression, and is also involved in lymphocyte activation, recirculation and homing, and hematopoiesis. Altered expression or dysfunction causes a variety of pathogenic phenotypes (see, e.g., Jiang D., Annu.Rev.cell.Dev.biol. (2007) 23: 435-461; and Knudson, W., et al, matrixBio. (2002), 21: 15-23).

Recently, studies have shown that the interaction of CD44 with cell matrix components (e.g., HA) plays an important role in the development of various inflammatory diseases, and disruption of the hyaluronan-CD 44 interaction can lead to improvement in choroidal neovascularization (see Hiroshi Mochimaru et al, invest. ophthalmol. vis. sci. (2009) 50: 4410-4415).

these evidence suggests that adhesion molecules like RGD peptide or CD44 in cell-cell and cell-ECM interactions play an important role in the development of various pathogenic diseases, and inhibition of the interactions may be a new therapeutic target in the treatment and cure of diseases.

Synthetic peptides have also been shown to bind to integrins and growth factors. It has been found that the RGD sequence containing the cyclized pentapeptide inhibits vitronectin from alpha_vβ₃Integrin binding (see Michael A. Dechantsreiter et al, J.Med. chem. (1999) 42: 3033-3040) and both vitronectin and fibronectin binding to alpha_vβ₃And alpha_IIbβ₃Integrin binding (see Roland Haubner et al, J.Am.chem.Soc., (1996) 118: 7461-7472). This inhibition has been shown to be useful in the treatment of a variety of unrelated diseases. In hamster studies, cyclic pentapeptides delay tumor growth and metastasis compared to control animals (see M.A. Buerkle et al, British J.cancer (2002) 86: 788-795). Pentapeptides have also been shown to reduce the binding of sickle red blood cells to the vascular endothelium and to improve hemodynamic performance (see eireen m. finnegan et al,Am.j.physiol.heart circ.physiol., (2007) 293: H1038-H1045). Another cyclic peptide containing the RGD sequence was shown to bind strongly to α 4 β 1, α 4 β 1 being an integrin known to play a role in leukocyte binding in inflammatory responses and immune responses (see Pina M. Cardarelli et al, J.biol. Chem. (1994) 269: 18668-18673). Studies have shown that a synthetic sulfated tetrapeptide binds strongly to VEGF (see Maynard, J.A. Hubbell, AcaBiomaterialia (2005) 1: 451-459).

In addition, in one important and useful application, dimeric RGD peptide-drug conjugates have been shown to be useful for integrin-targeted drug delivery for tumor targeting (see Chen et al, J.Med.chem., (2005)48 (4): 1098-1106).

In another equally important and useful application, it has been shown that multimeric radiolabeled RGD peptides can be targeted to integrin α_vβ₃But as diagnostic/imaging agents for tumor detection and as radiotherapeutic agents for tumor-specific targeting and therapy (see Zi-Bo Li et al, j.nuclear. medicine, (2007) 48: 1162-1171).

Scarring by fibroblasts is one of the major problems in ophthalmology in wound healing, particularly in glaucoma. This results from the interaction between the fibroblasts and the glycoprotein components of the ECM. Recognition of ECM glycoproteins occurs through cell surface integrins specific for adhesion epitopes such as the Arg-Gly-Asp (or RGD) sequence. The RGD sequence, present in several matrices of plasma proteins, including Fibronectin (FN) and Vitronectin (VN), is involved in cell-ECM interactions that occur during inflammatory and homeostatic responses. Inhibition of the interaction between fibroblasts and glycoproteins of the ECM ameliorates scarring (see Rami Hershkoviz et al, invest. ophthalmol. vis. sci. (1994) 35: 2585-2591).

Collagen fibrils of the posterior vitreocortical adhere to the vitreoretinal interface, specifically the inner limiting layer of the retinal surface (see Sebag j., Eye (1992), 6: 541-552). Adhesion to the entire posterior pole, on the vitreous base, optic disc, along the major retinal blood vessels and in a superficial manner, plays an important role in the pathogenesis of vitreoretinal diseases: such as idiopathic macular hole, vitreous macular traction, proliferative diabetic retinopathy, etc. (Akiba j. Quiroz m.a. et al, Ophthalmol. (1990)97, 1619-1655).

Angiogenesis inhibition shows early promise for diabetic retinopathy and macular degeneration, both of which are caused by ocular vascular proliferation. In these conditions, blood vessels interfere with normal structures in the eye, or they block light from reaching the back of the eye. The new blood vessels themselves are the main pathologies, and the termination of blood vessel growth prevents blindness. Thus, vascular inhibition (angiointerference) may not only lead to the treatment of these conditions; it can also lead to healing. In addition, it has been postulated that separation of the vitreous from the retina may reduce macular traction, reducing the risk of macular hole formation. Retinal neovascularization can thus be prevented in eyes with diabetic retinopathy and retinal vein occlusion by inhibiting posterior vitreal detachment by fibronectin and laminin binding to integrins at the vitreoretinal interface (see Akiba j. quiiroz MA, Ophthalmol. (1990)97, 1619-1655; Kelly n.e. et al, arch. Ophthalmol. (1991)109, 654-659; and Kado M et al, am.j. Ophthalmol. (1988) 105: 20-24).

In recent years, vitrectomy procedures have been greatly improved, reducing vitreoretinal traction and reducing retinal edema. Despite the ongoing improvements in surgical techniques and instrumentation, in some patients, particularly diabetic retinopathy and pediatric patients, non-invasive extraction of the vitreous cortex has been difficult to achieve due to complications such as retinal breaks, retinal detachments, and retinal nerve fiber damage (see Sebag J., Arch. Clin. exp. Ophthalmol. (1987) 225: 89-93; and HanD. P. et al, Arch. Ophthalmol. (1998) 106: 998-1000), among others. Thus, there is a great need for less invasive methods of selectively cutting the vitreous interface without damaging the retina. In recent years, reports have been made in the literature on various pharmacological agents for isolating the vitreoretinal interface (see Trese m.t., Eye (2002) 16: 365-368; Gandorfer a. et al, am.j. ophthalmol (2002) 133: 156-159; and Hesse l. et al, Eye Res. (2000) 70: 31-39). In the past, pharmacological vitrolysis using enzymes such as hyaluronidase (see U.S. Pat. No. 6,863,886 to Karageozian et al) and autologous plasmin (Sakuma T et al, Nippon Ganka Gakkai Zassi (2003) 107: 709-718) has been developed to facilitate digestion of extracellular matrix and induce posterior vitreal detachment. However, nonspecific destruction of adjacent tissues by the enzymes used has hindered the success of their therapeutic applications. In recent years, new approaches using non-enzymatic pharmacological agents like urea (see Nickerson, C. et al, J. Biomechanics, (2008) 41: 1840-1846 and Macromol. Symp. (2005): 183-189) and RGD peptides (see Leonardo B. Oliviera et al, curr. Eye Res. (2002) 25: 333-340) have been investigated by focusing on the separation of the vitreoretinal interface. Studies have shown that synthetic analogs of RGD peptides compete with the RGD motif of ECM proteins to disrupt integrin-ECM interactions and loosen attachment in vitro (Williams J.A., Pathol. Bio. (1992) 40: 813-821; Gehlsen K.R. et al, J.cell.biol. (1988) 106: 925-930; Pierschbacher, M.D. et al, J.biol. CHEm. (1987) 262: 17294-17298; and Zhon L.L. et al, IOVS. (1996) 37: 104-113) and in vivo. Thus, in the rabbit model, intravitreal injection of soluble RGD peptides results in release of RGD-epitopes from soluble ECM proteins on the retinal surface, thus favoring non-enzymatic PVD. Apparently, these results indicate that the vitreoretinal interface is involved in the attachment of integrins to the RGD motif of the ECM and the adhesion of vitreocortical collagen to the Inner Limiting Layer (ILL). RGD peptides and their derivatives promote the migration of epithelial cells in wounds (see P.M.Mertz et al, J.Burn Care Res. (1996) 17: 199-206) and retain their biological activity when incorporated into synthetic Biomaterials such as hydrogels (see MP Lutolf et al, Proc.Nat.Acad.Sci. (2003) 100: 5413-5418; and MP Lutolf et al, Nature Biotechnol. (2003) 21: 513-518), other polymer matrices (see Horng-Ban Lin et al, J.biomed.Material. Res. (2004) 28: 329-342) or as surface membranes on hard substrates (D.M.Ferris et al, Biomaterials (1999) 20: 2323-2331). RGD peptides also promote enhanced adhesion of epithelial or endothelial cells to Vascular prostheses coated with peptide sequences (see K.Wallusscheck et al, Eur.J. Vascaler and Enovascal surgery (1996) 12: 321-330) and other artificial organs (see Jeschke, Brigette, Biomaterials (2002) 23: 3455-3463) and have been shown to support nerve regrowth (see M.Rafiddin Ahmed et al, Brain Res. (2003) 993: 208-216). The bioactive surface of the prosthesis may comprise synthetic resin fibers or polymers.

drawings

FIG. 1 shows the effect of 3 peptides, cyclic-RGD-peptide, R-G-cysteic acid peptide (Compound 1), and RGE peptide, on the kinetics of wound healing;

FIG. 2 shows an HPLC chromatogram of R-G-cysteic acid peptide (Compound 1);

FIG. 3 shows an electrospray mass chromatogram of R-G-cysteic acid peptide (Compound 1);

FIG. 4 shows an HPLC chromatogram of cyclic-R-G-cysteic acid peptide (Compound 2); and

FIG. 5 shows an electrospray mass chromatogram of cyclic-R-G-cysteic acid peptide (Compound 2).

Detailed Description

The present invention provides novel compounds, including compounds of the general formulas I-VII described above. Specific examples include Arg-Gly-NH-CH (CH)₂-SO₃H) The linear form of COOH (referred to herein as an example of Compound 1) and Arg-Gly-NH-CH (CH)₂-SO₃H) COOH) and derivatives thereof, including pharmaceutically acceptable salts, hydrates, stereoisomers, multimers (mutimers), cyclic forms, linear forms, multimeric forms, drug conjugates, prodrugs, and derivatives thereof.

Synthesis of Compounds 1 and 2

Conventional solid phase peptide synthesis known to those of ordinary skill in the art can be performed (SPPS; see R.B.Merrifield, J.Am.chem.Soc. (1963)85 (14): 2149-2154). SPPS is the preferred method of synthesis due to its high yield. In general, the first stage of the solid phase peptide synthesis technique involves peptide chain assembly with protected amino acid derivatives on a polymeric support. The second stage of the technique is to cleave the peptide from the resin support, wherein all side chain protecting groups are cleaved simultaneously, yielding the crude free peptide. The general principle of SPPS is that of repeated cycles of coupling-deprotection. The free N-terminal amine of the solid phase-linked peptide is coupled to a single N-protected amino acid unit. The unit is then deprotected, which exposes a new N-terminal amine to which another amino acid can be attached. See Asymmetric Synthesis by Von g.m. copperol and h.f. schuster for Synthesis, protection and deprotection strategies; john Wiley & Sons, New York 1987, for protection and deprotection strategies see Peter G.M.Wuts and Theodora W.Greene, Greene's Protective Groups in organic Synthesis, (2 nd edition) J.Wiley & Sons, 1991.

Two main use forms of solid phase peptide synthesis-Fmoc(9-fluorenylmethyloxycarbonyl; base-labile alpha-amino protecting group) and t-Boc (t-butyloxycarbonyl; acid-labile protecting group), Fmoc can be preferably used in the peptide synthesis of the invention. Each method involves different resins and amino acid side chain protection, followed by cleavage/deprotection steps. After cleavage from the resin, the peptide is usually purified by reverse phase HPLC using columns such as C-18, C-8 and C-4.

One example of solid phase peptide synthesis

The following is an overview of the synthetic procedure for peptide synthesis using a base-labile 9-fluorenylmethyloxycarbonyl (Fmoc) protecting group on Wang resin as a solid support.

Fmoc deprotection

0.08mmol of Fmoc-Pro-Wang resin was loaded into a sintered column equipped with a plastic cap. The resin was washed with 2X 3-ml portions of DMF (dimethylformamide) for 1 minute each. Next, about 3ml of 20% piperidine/DMF was added and Fmoc deprotection was allowed to continue for 15 minutes. During this time, the column is gently swirled or agitated to ensure complete mixing. After completion of the reaction (about 15 min), the reaction column was drained and the resin was washed again with DMF (4X 3 ml).

amide bond coupling

The desired Fmoc-protected amino acid Fmoc-Thr-tBu (3 equivalents; relative to the resin loading indicated by the supplier) and DIEA (6 equivalents)/DCM (0.5M relative to the amino acid) were then added to the resin. The mixture was allowed to cool at-20 ℃ for 20 minutes. Next, the peptide coupling reagent benzotriazol-1-yl-oxytripyrrolidine hexafluorophosphate for solid phase peptide synthesis was used(PyBOP) (3 equiv.) was added to the reaction. After shaking for 8 h at-20 deg.C, the reaction mixture was drained and the resin washed with DCM (3X).

after Fmoc deprotection with 20% piperidine/DMF (15 min) and washing with DMF (3X), the next Fmoc-protected amino acid (3 equivalents; relative to resin loading) was PyBop coupled in the same manner as above.

Cracking

To obtain the peptide in free acid form, the ester bond is cleaved using strongly acidic conditions such as TFA (trifluoroacetic acid). The resin was treated with 2-3ml of a 95: 5 solution of trifluoroacetic acid and water. The resin was gently agitated over a 25 minute period. Next, the column was drained and the filtrate carefully collected in a glass collection vessel.

Synthesis of compound 1(GRG cysteic acid TP):

Compound 1

Step 1, resin loading: proline-preloaded o-chlorotrityl resin was used as starting material.

Step 2. peptide assembly: fmoc synthesis was used to assemble the peptides. The protected amino acid was activated with PyBOP and the terminal Fmoc group was removed with 20% piperidine/DMF. The following protected amino acids were used in the order of their occurrence:

Fmoc-Thr-tBu (Fmoc threonine-tert-butyl ester)

Fmoc-cysteic alanine-Pfp (Fmoc cysteic acid-pentafluorophenyl ester)

Fmoc-Gly (Fmoc glycine)

Fmoc-Arg-Pbf (N α -Fmoc-N ω - (2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl) -L-arginine)

Fmoc-Gly (Fmoc glycine)

Step 3, cleavage of the peptide from the resin: the resulting peptide was cleaved from the solid support and the protecting groups were removed with a solution of 85.5% TFA, 5% phenol, 2% water, 5% phenylthiomethane, and 2.5% ethanedithiol.

And 4, purifying: the resulting R-G-cysteic acid peptide was purified using High Performance Liquid Chromatography (HPLC).

The amount of compound 1 prepared as described above was analyzed by high performance liquid chromatography to be > 98% area/area purity [ HPLC conditions: and (3) buffer solution A: 0.1% trifluoroacetic acid (TFA)/water, buffer B: 0.1% TFA/acetonitrile, Mobile Phase (MP) a: 97% buffer a and 3% buffer B, mobile phase B: 79% buffer a and 21% buffer B, mobile phase C: 50% buffer a and 50% buffer B; see table 1 below for gradients; flow rate: 1.0 mL/min; column: watersC18, 5 mu, 4.6X 250 mm; column temperature: 30 ℃; a detector: UV @220 nm; sample injection volume: 20.0 mu L; sample preparation: 20 μ L of sample diluted with 1.0mL of mobile phase A (about 0.5mg/mL)]. The corresponding HPLC chromatogram is shown in FIG. 2. In addition, the molecular weight of purified Compound 1 was determined by electrospray mass spectrometry to be 638.3amu (theoretical mass: 637.7amu) based on the stepwise addition of the corresponding amino acids used to synthesize the peptide sequence, confirming the identity of Compound 1. The electrospray mass spectrum of compound 1 is shown in figure 3.

table 1: pump gradient procedure for detection of Compound 1 by HPLC

compound 2 (Cyclic-R-G-cysteic acid fN (CH)₃) V) Synthesis:

Compound 2

Step 1, resin loading: an o-chlorotrityl resin was used as a starting material. Fmoc-N-methyl-L-Val was attached to the resin.

Step 2. peptide assembly: fmoc synthesis for peptide assembly. The protected amino acid was activated with PyBOP and the terminal Fmoc group was removed with 20% piperidine/DMF. The following protected amino acids were used in the order of their occurrence:

Fmoc-Phe (Fmoc-phenylalanine)

Fmoc-cysteic acid-PfP (Fmoc-cysteic acid pentafluorophenyl ester)

Fmoc-Gly (Fmoc-glycine)

d.Fmoc-Arg-Pbf(N_α-Fmoc-N ω - (2, 2, 4, 6, 7-pentamethyldihydrobenzofuran-5-sulfonyl) -L-arginine)

Step 3, cleavage of the peptide from the resin: cleavage of peptides from solid supports Using acetic acid/TFA/DCM (1: 3)

step 4. cyclization and deprotection to the desired cyclic peptide: cyclization was performed by in situ activation using diphenylphosphoryl azide and sodium bicarbonate at high dilution. The side chain was deprotected using 85.5% TFA, 5% phenol, 2% water, 5% phenylthiomethane, 2.5% ethanedithiol.

and 5, purifying: HPLC was used for purification.

The amount of Compound 2 prepared as above was analyzed by high performance liquid chromatography to a purity of > 99% area/area (HPLC conditions: mobile phase A: 0.1% trifluoroacetic acid/water, B: 0.1% TFA/(80% acetonitrile plus 20% water), gradient over 20 minutes 26% -36% B, flow rate: 1.0 mL/min; column: Phenomenex C18(2) 4.6X 150mm, 5. mu.100A; detector: UV @220 nm; sample injection volume: 100.0. mu.L). The corresponding HPLC chromatogram is shown in FIG. 4. In addition, the molecular weight of purified Compound 2 was determined by electrospray mass spectrometry to be 625.3amu (theoretical mass: 625.77amu) based on the stepwise addition of the corresponding amino acid for peptide sequence synthesis, confirming the identity of Compound 2. The electrospray mass spectrum of compound 2 is shown in figure 5.

Method of inhibiting cell adhesion

The invention also provides for the treatment of a disease or disorder by administering to a subject an effective amount of R-G-cysteic acid (i.e., R-G-NH-CH (CH)₂-SO₃H) Linear form of COOH or R-G-NH-CH (CH)₂-SO₃H) COOH cyclic form) or derivatives thereof (including pharmaceutically acceptable salts, hydrates, stereoisomers, multimers, cyclic forms, linear forms, drug conjugatessubstance, prodrug and derivatives thereof) to inhibit cell adhesion to the RGD binding site in a human or animal subject.

Applicants have discovered that the synthetic R-G-cysteic acid peptides of the present invention induce apoptosis by competitively inhibiting cell attachment to ECM components. Thus, the synthetic R-G-cysteic acid peptides and derivatives thereof of the present invention are useful as potent integrin antagonists, such as therapeutic agents for angiogenesis, inflammation, cancer metastasis, thrombosis, prevention and treatment of scarring, and pharmacological vitrolytic agents. Furthermore, in an important aspect of the present invention, an improved targeting method of α, β integrins using multimerized and radiolabeled R-G-cysteic acid peptides is contemplated for use as an internal radiotherapeutic agent for the detection (diagnostic or imaging) and treatment of tumors. In another important aspect, the improved R-G-cysteic acid peptide conjugates or multimeric R-G-cysteic acid peptide conjugates function as drug carriers, for example, as anti-cancer drug carriers that effectively target tumors.

As described elsewhere herein, the sulfonic acid in the R-G-cysteic acid-peptide is a stronger acid than the corresponding carboxylic acid in the RDG-peptide. This higher polarity of the sulfonic acid group results in stronger intermolecular binding. For example, R-G-cysteic acid, which has more polarized O-H bonds, can form stronger hydrogen bonds with amide groups and/or side chains of amino acids of proteins in integrin binding sites than R-G-aspartic acid, which has relatively less polarized O-H bonds, and/or stronger interactions with metal ions complexed in integrin binding sites. Accordingly, the novel R-G-cysteic acid peptides and derivatives thereof of the present invention provide improved compounds and compositions that are superior to the corresponding RGD peptides in integrin receptor recognition and binding.

Furthermore, in diabetic patients with chronic hyperglycemia accompanied by elevated IOP, open-angle glaucoma has been particularly reported to be associated with the accumulation of fibronectin in the trabecular meshwork tissue, and fibronectin is considered to inhibit the outflow of aqueous humor in excess (see Oshitari, T et al, am.j. ophthalmol. (2007) 143: 363-365). Fibronectin, which is involved in cell-ECM interactions in the trabecular meshwork, suggests primary open angle glaucoma (see Mark s. filla et al, invest. ophthalmol. vis. sci. (2002) 43: 151-161; and Cheryl r. hann et al, ophthalmoc res. (2001) 33: 314-324). It has also been reported that endothelial cells of Schlemm's canal interact with the extracellular matrix in affecting outflow capacity (see Cindy K.Bahler et al, invest. Ophthalmol. Vis. Sci. (2004) 45: 2246-2254). The use of R-G-cysteic acid peptides and derivatives thereof to treat elevated IOP in diabetic patients may be highly beneficial in diabetic patients, depending on the inhibition of aqueous outflow by the presence of excess extracellular matrix components, such as fibronectin.

Preferred R-G-cysteic acid peptides may be fusion polypeptides, cyclic or linear polypeptides, derivatized polypeptides (including R-G-cysteic acid peptides derivatized with or associated with or coupled to drug delivery systems or other drugs (e.g., anti-cancer drugs)), multimerized R-G-cysteic acid peptides, monoclonal antibodies containing R-G-cysteic acid sequences immunoreactive with binding sites of integrins or functional fragments thereof.

The R-G-cysteic acid-containing polypeptide may have a sequence corresponding to the amino acid residue sequence of a native integrin adhesion binding domain, such as that found in ligands such as fibrinogen, fibronectin, vitronectin, von willebrand factor, laminin, thrombospondin, and the like.

The peptide sequences of the present invention consist of 3 amino acids with terminal guanidino, sulfonic and carboxyl groups and their derivatives coupled and/or associated with drug delivery systems including peptide fragments, glycoproteins and polymeric groups (e.g., PEG, Pluronic and other polymeric groups), as well as liposomes and nanoparticles. Pharmaceutical compositions comprising the peptide sequences for the treatment of various pathological conditions include injectables, gels, suspensions, ointments, solid dosage forms and liquid dosage forms.

Integrin receptors associated with cell adhesion motifs such as fibronectin, vitronectin, laminin, fibrinogen, thrombospondin, and von willebrand factor are target epitopes for R-G-cysteic acid peptides and derivatives thereof. The tripeptide R-G-cysteic acid has been found to be the smallest amino acid sequence that can be recognized by the cell binding domain. The sequences may also interfere with immune functions unrelated to integrins. Thus, the synthetic R-G-cysteic acid sequence was found to mimic the RGD cell binding domain, and substitution of the aspartate α -carbon provided a stronger binding affinity to the target integrin. The sulfonic acid in the R-G-cysteic acid-peptide is a stronger acid than the corresponding carboxylic acid in the RDG-peptide. This higher polarity of the sulfonic acid groups results in stronger intermolecular binding. For example, R-G-cysteic acid (which has more polarized O-H bonds) can form stronger hydrogen bonds with the amide group and/or side chain of an amino acid of the integrin binding site than R-G-aspartic acid (which has relatively less polarized O-H bonds) and/or have stronger interactions with metal ions complexed in the integrin binding site.

Most of the general formulae for the R-G-cysteic acid sequences of the present invention are as follows:

formula A:

Wherein X is-CH (R)₁)-S(＝O)₂-Y；

-CH(R₁)-SH；

-CH(R₁)-OZ；

-CH(R₁)S(＝O)Y；

-CH(R₁)-O-S(＝O)₂-OX₁(ii) a And

-CH(R₁)-O-P(＝O)₂-OX₁(ii) a And

Wherein Y is OX₁、NH₂；X₁＝-H、C₁-C₆straight chain alkyl, phenyl;

R₁＝H、C₁-C₆Straight chain alkyl, phenyl or SO₃H

Z＝H、SO₃H

Formula B:

Wherein X is-CH(R₁)-S(＝O)₂-Y；

-CH(R₁)-SH；

-CH(R₁)-OZ；

-CH(R₁)S(＝O)Y；

-CH(R₁)-O-S(＝O)₂-OX₁(ii) a And

-CH(R₁)-O-P(＝O)₂-OX₁(ii) a And

Wherein Y is OX₁、NH₂；X₁＝-H、C₁-C₆Straight chain alkyl, phenyl;

R₁＝H、C₁-C₆Straight chain alkyl, phenyl or SO₃H

Z＝H、SO₃H; and

A₂selected from: -Phe-Val-Ala, -Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Ala, -Phe-Val or salts or N-alkylated derivatives thereof. Any combination of the D-or L-forms of Arg, Gly, cysteic acid, Phe, Val, Ala, Leu, Pro, Thr, as well as cyclic forms of the above sequences, may be used.

Examples of cyclic forms include:

Formula C:

Wherein X' is selected from: H. c₁-C₆Alkyl, Ph, SO₃H；Y＝OH、NH₂；Z＝H、CH₃。

Cyclic forms also include pentapeptides and heptapeptides, such as the specific compound of formula C, Compound 2 (Cyclic-R-G-cysteic acid fN (CH)₃) V), is represented as follows:

compound 2

Formula A includes formulae I-VI described elsewhere herein. Formula B includes formula VII described elsewhere herein.

Formula D:

A₁-Arg-Gly-NH-CH(X)-CO-A₂

Wherein X is-CH (R)₁)-S(＝O)₂-Y；

-CH(R₁)-SH；

-CH(R₁)-OZ；

-CH(R₁)S(＝O)Y；

-CH(R₁)-O-S(＝O)₂-OX₁(ii) a And

-CH(R₁)-O-P(＝O)₂-OX₁(ii) a And

Wherein Y is OX₁、NH₂；X₁＝-H、C₁-C₆straight chain alkyl, phenyl;

R₁＝H、C₁-C₆Straight chain alkyl, phenyl or SO₃H

Z＝H、SO₃H; and

A₁And A₂Selected from: -Phe-Val-Ala, -Phe-Leu-Ala, -Phe-Val-Gly, -Phe-Leu-Gly, -Phe-Pro-Ala, -Phe-Val or salts or N-alkylated derivatives thereof. Any combination of the D-or L-forms of Arg, Gly, cysteic acid, Phe, Val, Ala, Leu, Pro, Thr, as well as cyclic forms of the above sequences, may be used.

Substituted R-G-cysteic acid sequences include cyclic R-G-cysteic acid analogs.

Administration of R-G-cysteic acid and its derivatives can be performed subcutaneously, dermally, ocularly, and systemically by using any pharmaceutically acceptable dosage form of a drug delivery system or an injection formulation or a solid formulation or an ointment formulation.

The compounds of the present invention may be administered by any route suitable to produce the desired therapeutic effect, including but not limited to: oral, rectal, intravenous, intraarterial, intradermal, subcutaneous, intramuscular, intrathecal, sublingual, buccal, intranasal, transmucosal, transdermal, topical, intraocular, intravitreal, other enteral, other parenteral, and/or other feasible routes of administration.

The compounds of the present invention may be administered at any dose that provides the desired therapeutic effect while avoiding undue or toxic effects. Typical dosages of the compounds of the present invention that can be administered to a human subject range from about 1ng/kg to about 1.0 g/kg.

The compounds of the invention may optionally be prepared in the form of liposomes or nanoparticles (e.g., nanocapsules), as practicable and appropriate. The formation and use of liposomes is generally known to those skilled in the art. Liposomes are formed from phospholipids that are dispersed in aqueous media such that they spontaneously form multilamellar concentric bilayer vesicles, sometimes referred to as multilamellar vesicles (MLVs). MLVs typically have a diameter of 25nm to 4 μm. Upon sonication, MLVs form small multilamellar vesicles (SUVs) of about 200-500 angstroms in diameter, with a core containing an aqueous solution. In general, phospholipids can form various structures other than liposomes when dispersed in aqueous media, depending on the molar ratio of lipid to water. At low molar ratios of lipid to water, liposomes can be formed. The physical properties of liposomes depend on pH, tonicity and the presence or absence of divalent cations. Liposomes can interact with cells by different mechanisms including 1) endocytosis (e.g., phagocytosis of the liposome by cells such as macrophages and neutrophils), adsorption to the cell surface, 2) interaction with cell-surface components, 3) fusion with plasma cell membranes by insertion of the lipid bilayer of the liposome into the plasma membrane, or 4) transfer of the liposome lipids to the cell membrane or subcellular membrane, and vice versa. Altering liposome formulations can alter the mechanisms by which liposomes rely on interacting with cells in the paranasal sinuses, nasal mucosa, and the like.

Nanocapsules are any nanoparticles consisting of a shell and a space in which a desired substance can be placed. Techniques for forming nanocapsules are known in the art. The polymer nanocapsules can be prepared in a particular size and shape. They can be produced as monodisperse particles that have precisely defined physicochemical properties and, therefore, can be tailored to facilitate the release of therapeutic or diagnostic substances in response to specific bimolecular triggering mechanisms, such as the pH, mucus flow, or other conditions found in the paranasal sinuses or other regions of the ear, nose, or throat in which the device is implanted. Nanocapsules are useful in the present invention as "smart drugs" having specific chemical receptors or binding sites that can bind to specific target cells (e.g., cancer cells or cells associated with inflammatory conditions).

the following are non-limiting formulation examples of pharmaceutical preparations containing the compounds of the present invention. Also included are examples of safety and/or efficacy demonstrated using the exemplary R-G-cysteic acid peptides or derivatives in inhibiting cell adhesion. As used herein, the terms "R-G-cysteic acid peptide," "RGCys-peptide," and "compound of the present invention" shall mean, synonymously, compositions containing the sequence R-G-cysteic acid and derivatives thereof, including, but not limited to, the compositions defined by formulas I-VII and compounds 1 and 3-5 described herein.