Technical Field

This invention relates to the preparation of compounds containing sulfolithocoiic acid residues linked to polyamines, suitable for use as pharmaceutical agents to inhibit cellular proliferation and responses to VEGF and FGF. These compounds are useful as antiproliferative agents against smooth muscle and endothelial cells, as antipermeability agents against endothelial cells, and as inhibitors of blood flow chains against endothelial cells. Thus, these compounds are useful for the treatment of diseases or disorders that are characterized by excessive smooth muscle cell proliferation leading to numerous pathological states, including but not limited to vascular stenosis and post-angioplasty restenosis. In addition, these compounds exhibit antiproliferative and antipermeability activity against human vascular endothelial cells and thus are useful for the treatment of diseases or disorders characterized by excessive neovascularization and permeability including, but not limited to diabetes, neovascularizing ocular diseases, rheumatoid arthritis and cancer.

Background of the Invention

Cell-cell interactions within the blood vessel wall are important in the maintenance of normal vascular structure and function. The regulation of vascular smooth muscle cell (VSMC) and endothelial cell (EC) proliferation within the blood vessel wall is usually under very tight temporal and spatial control and results from a net balance of different growth stimulators and inhibitors that are produced by different cell types. A common feature underlying numerous vascular-related diseases is that of abnormal smooth muscle or endothelial cell proliferation. Vascular cell proliferation/migration in response to injury

results from a complex and intricate series of events involving the expression of growth factor(s)/growth factor receptor(s), modulation of VSMC or EC phenotype and remodeling of extracellular matrix. Uncontrolled VSMC proliferation results in the thickening and narrowing of blood vessels and is a key factor underlying vascular diseases, such as atherosclerosis, post-angioplasty restenosis, vascular graft failure, post-transplant accelerated atherosclerosis, and hemodialysis shunt failure. Angiogenesis, the sprouting of new blood vessels from pre-existing vessels, is a complex, multicellular phenomenon involving capillary endothelial cell (EC) proliferation, migration, and tissue infiltration. Abnormal angiogenesis plays a major role in the pathogenesis of tumor growth, rheumatoid arthritis, atherosclerosis and various neovascularizing ocular diseases.

Fibroblast Growth Factor

The fibroblast growth factor (FGF) family of heparin-binding polypeptide growth factors contains eight different members of which FGF-1 (acidic FGF) and FGF-2 (basic FGF) are the best-characterized. The understanding of FGF pathobiology is even more complicated by the observation that at least five different receptor isoforms exist. In the case of basic FGF, it is well-documented that these mitogens can be localized intracellularly, can be stored within the extracellular matrix of the blood vessel wall and can exert direct mitogenic actions on VSMCs and ECs. A large body of existing evidence suggests that increased basic FGF and FGF receptor (fig) expression play a predominant role in stimulating VSMC proliferation following mechanical injury of the blood vessel wall, i.e. restenosis following balloon angioplasty (Lindner V., Lappi D.A., Baird A., Majack R.A., Reidy M.A. Role of basic FGF in vascular lesion formation. Circ. Res. 1991 ;68:106-113. Cassells W., Lappi D.A., Olwin B.B., Wai C, Siegman M., Speir E.H., Sasse J., Baird A. Elimination of smooth muscle cells in experimental restenosis:

Targeting of fibroblast growth factor receptors. Proc. Natl. Acad. Sci. USA 1992;89:7159-7163. Reidy M.A. Neointimal proliferation: The role of bFGF on VSMC proliferation. Thrombosis and Haemostasis 1993;70:172-176. Lindner V., Reidy M.A. Expression of basic fibroblast growth factor and its receptor by smooth muscle cells and endothelium in injured rat arteries: an en face study. Circ. Res. 1993;73:589-595.), and in cancer (Yan G., Fukabori Y., McBride G., Nikolaropolous S., McKeehan W.L. Exon switching and activation of stromal and embryonic fibroblast growth factor (FGF)-FGF receptor genes in prostrate epithelial cells accompany stromal independence and malignancy. Molec. Cell Biol. 1993;13:4513-4522. Kurobe M., Takei Y., Ezawa H., Hayashi K. Increased level of basic fibroblast growth factor (bFGF) in sera of patients with malignant tumors. Horm. Metab. Res. 1993;25:395-396. Morrison R.S., Yamagucki F., Bruner J.M., Tang M., McKeehan W.L., Berger M.S. Fibroblast growth factor receptor gene expression and immunoreactivity are elevated in human glioblastoma multiforme, Cancer Res. 1994;54:2794-2799.). Given the central role of FGF-2 as a mitogen, antagonists of FGF action would be advantageous as interventional therapeutic agents in different vascular and non-vascular pathologies characterized by excessive cell proliferation.

Vascular Endothelial Growth Factor

In 1983, Dvorak and colleagues (Senger D.L., Galli S.J., Dvorak A.M., Peruzzi V.C.A., Harvey V.S., Dvorak H.F. Tumor cells secrete a vascular permeability factor that promotes accumulation of ascites fluid,

Science 1983;219:983-985. Senger D.R., Perruzzi C.A., Feder J.,

Dvorak H.F. A highly conserved vascular permeability factor secreted by a variety of human and rodent tumor cell lines. Cancer Res. 1986;46:5629-5632) first described an unidentified tumor-derived vascular permeability factor which was a potent stimulus for increasing

blood vessel permeability and promoting fluid accumulation and named this factor vascular permeability factor (VPF). Subsequently, Connolly et al. (Connolly D.T., Olander J.V., Heuvelman D., et al. Human vascular permeability factor: Isolation from U937 cells. J. Biol. Chem. 1989;264:20017-20024.) found VPF to be a potent, selective mitogen for ECs, thus raising the possibility that VPF contributes to tumor cell-stimulated angiogenesis. At the same time, proteins referred to as vascular endothelial growth factor (VEGF; Ferrara N., Henzel W.J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem. Biophy. Res. Commun. 1989;161 :851-858.), vasculotropin (Plouet, Schilling J., Gospodarowicz, D. Isolation and characterization of a newly identified endothelial cell mitogen by AtT-20 cells. EMBO. J. 1989;8:3801-3806.) and glioma-derived vascular EC growth factor (GD-ECGF; Conn G., Sodermann D.D., Schaeffer M.T., Wile M., Hatcher V.B., Thomas K.A. Purification of a glycoprotein vascular endothelial cell mitogen from a rat glioma-derived cell line. Proc. Natl. Acad. Sci. USA 1990;87:1323- 1327.) were isolated and exhibited functional and structural properties similar to VPF. Molecular cloning of the human, bovine and rat cDNAs for VPF (Connolly D.T., Olander J.V., Heuvelman D., et al. Human vascular permeability factor: Isolation from U937 cells. J. Biol. Chem. 1989;264:20017-20024.), VEGF (Leung D.W., Cachianes G., Kuang W.J., Goeddel D.V., Ferrara N. Vascular endothelial growth factor is a secreted angiogenic mitogen. Science 1989;246:1306-1309.) and GD- ECGF (Conn G., Bayne M.L., Soderman D.D., et al. Amino acid and cDNA sequences of a vascular endothelial cell mitogen that is homologous to platlet-derived growth factor. Proc. Natl. Acad. Sci. USA 1990;87:2628-2632.) has demonstrated that these proteins are encoded by the same gene. Of the angiogenic factors described to date, VEGF is believed to be the most EC specific mitogen. Vascular endothelial cell growth factor is a member of the heparin-binding family

H

of growth factors, which also includes the fibroblast growth factor family (FGF) (Ferrara N., Henzel W.J. Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem. Biophys. Res. Commun. 1989;161:851-858. Yayon, A., Klagsbrun M., Esko, J.D., Leder P., Ornitz D.M. Cell surface, heparin- like molecules are required for binding of basic fibroblast growth factor to its high affinity receptor. Cell. 1991 ;64:841-848.). The biologically active, native protein exists as a disulfide-bonded homodimer of 34-42 kDa molecular weight suggesting that multiple isoforms can be processed (see below). This polypeptide growth factor is heat- and acid-stabile, exhibits a basic pH (=8.5) and is glycosylated at Asn-74.

Vascular endothelial growth factor is a product of several different tumor cell types, including glioblastomas, sarcomas, carcinomas and histiocytic lymphomas. Since these are tumor-derived cell lines, VEGF has been postulated to play roles in mediating tumor- associated angiogenesis and the abnormal increase in capillary permeability seen in tumor vessels. Subsequently, VEGF has been isolated or detected in several normal tissues, including lung, kidney, liver, brain, adrenal gland, and heart, as well as different cell types using immunohistochemical and in situ hybridization techniques. Non- tumor cultured cells that express VEGF include smooth muscle cells, keratinocytes, and retinal pigment epithelial cells.

In general, it has been known for some time that angiogenesis occurs in hypoxic tissues (Shweiki D., Itin A., Neufeld G., Gitay-Goren H., Keshet E. Patterns of expression of vascular endothelial growth factor (VEGF) and VEGF receptors in mice suggest a role in hormonally regulated angiogenesis. J. Clin. Invest. 1993;91 :2235-2243.). Thus, the most interesting and perhaps clinically relevant observation is that

VEGF mRNA levels can be regulated in numerous cell types (Pe'er J.,

Shweiki D., Itin A., Hemo I., Gnessin H., Keshet E. Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases. Lab. Invest. 1995;72:638-645. Murata T., Ishibashi T., Khalil T., Hata Y., Yoshikawa H., Inomata H. Vascular endothelial growth factor plays a role in hyperpermeability of diabetic retinal vessels. Opthalmic. Res. 1995;27:48-52. Ladoux A., Frelin C. Hypoxia is a strong inducer of vascular endothelial growth factor mRNA expression in the heart. Biochem. Biophy. Res. Commun. 1993;195:1005-1010.) and animals (Brogi E., Wu T., Namiki A., Isner J.M. Indirect angiogenic cytokines upregulate VEGF and bFGF gene expression in vascular smooth muscle cells, whereas hypoxia upregulates VEGF expression only. Circulation 1994;90(2):649-652. Kim K.J., Li B., Winer J., Armanini M., et al. Inhibition of vascular endothelial growth factor-induced angiogenesis suppresses tumour growth in vivo. Nature

1993;362:841-844. Thieme H., Aiello L.P., Takagi H., Ferrara N., King G.L. Comparative analysis of vascular endothelial growth factor receptors on retinal and aortic vascular endothelial cells. Diabetes 1995;44:98-103.) in response to hypoxia. Treatment with a monoclonal antibody directed against VEGF reduces the vascular density and inhibits the growth of several human tumors in nude mice without affecting the growth rate of tumor cells in vitro (Adamis A.P., Miller J.W., Bernal M.T., et al. Increased vascular endothelial growth factor levels in the vitreous of eyes with proliferative diabetic retinopathy. Am. J. Opthamol. 1994;118:445-450.). Interestingly, hypoxia has also been shown to be a strong stimulus to induce VEGF mRNA levels in retinal pigment epithelial cells (Pe'er J., Shweiki D., Itin A., Hemo I., Gnessin H., Keshet E. Hypoxia-induced expression of vascular endothelial growth factor by retinal cells is a common factor in neovascularizing ocular diseases. Lab. Invest. 1995;72:638-645.) and increase the number of VEGF receptors in retinal endothelial cells (Aiello L.P., Avery

R.L., Arrigg P.G., et al. Vascular endlothelial growth factor in ocular fluid of patients with diabetic retinopathy and other retinal disorders. N. Engl. J. Med. 1994;331 :1480-1487.). There are now numerous studies showing that increased ocular expression of VEGF mRNA and protein levels is closely correlated with neovascularization in patients with proliferative diabetic retinopathy (Miller J.W., Adamis A.P., Shima D.T., et al. Vascular endothelial growth factor/vascular permeability factor is temporally and spatially correlated with ocular angiogenesis in a primate model. Am. J. Pathol. 1994;145:574-584. Pierce E.A., Avery R.L., Foley E.D., Aiello L.P., Smith L.E.H. Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization. Proc. Natl. Acad. Sci. USA 1995;92:905- 909. Adamis A.P., Shima D.T., Tolentino M.J., et al. Inhibition of vascular endothelial growth factor prevents retinal ischemia-associated iris neovascularization in a nonhuman primate. Arch. Opthamol.

1996;114:66-71.) and in different animal models of ischemia-induced retinal neovascularization, including retinal vein occlusion and retinopathy of prematurity (Aiello L.P., Pierce E.A., Foley E.D., et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. USA 1995;92:10457-10461. Colville-Nash P.R., and Scott D.L. Angiogenesis and rheumatoid arthritis: pathological and therapeutic implications. Ann. Rheum. Disease 1992;51 :919-925.). Application of anti-VEGF antibodies following retinal vein occlusion prevented iris neovascularization in non-human primates (Fava R.A., Olsen N.J., Spencer-Green G., et al. Vascular permeability factor/endothelial growth factor (VPF/VEGF): Accumulation and expression in human synovial fluids and rheumatoid synovial tissue. J. Exp. Med. 1994;180:341-346.). Furthermore, Aiello et al. (Aiello L.P., Pierce E.A., Foley E.D., et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth

factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. USA 1995;92:10457-10461.) demonstrated that a soluble VEGF receptor chimeric protein was capable of suppressing neovascularization in a mouse model of retinopathy of prematurity. Cellular hyperplasia, hypoxia, angiogenesis, and fluid accumulation are also changes that are correlated closely with the development of the destructive synovial pannus in rheumatoid arthritis (Olander J.V., Connolly D.T., DeLarco J.E. Specific binding of vascular permeability factor to endothelial cells. Biochen. Biophys. Res. Commun. 1991 ;175:68-76.). Not surprisingly, increased levels of VEGF have been detected in the synovial fluid of patients with rheumatoid arthritis (Bikflvi A., Sauzeau C, Moukadiri H., et al. Interaction of vasculotropin/vascular endothelial cell growth factor with human umbilical vein endothelial cells: Binding, internalization, degradation and biological effects. J. Cell. Physiol. 1991 ;149:50-59. Satoh H., Yoshida M.C., Matsushime H., Shibuya M., Sasaki M. Regional localization on the human c-ros-1 on 6q22 and W on 13q12. Jpn. J. Cancer Res. 1987;78:772-775.). Thus, antagonists of VEGF action would be advantageous as interventional therapeutic agents in different pathologies characterized by excessive endothelial cell proliferation which is a common mechanism underlying diverse, yet inter-related pathologies such as tumor growth, retinal neovascularization and rheumatoid arthritis where tissue hypoxia is a central component.

Rationale for Biological Assays

Numerous studies have described specific binding sites for [ ¹²⁵ I]VEGF on cultured ECs in vitro (Jakeman L.B., Winer J., Bennett G.L., Altar C.A., Ferrara N. Binding sites for vascular endothelial growth factor are localized on endothelial cells in adult rat tissues. J. Clin.

Invest. 1992;89:244-253. Shibuya M., Yamaguchi S., Yamane A., et al.

Nucleotide sequence and expression of a novel human receptor-type tyrosine kinase gmr (fit) closely related to the fms family, Oncogene. 1990;8:519-524. DeVries C, Escobedo J.A., Ueno H., Houck K., Ferrara N., Williams, L.T. The ms-like tyrosine kinase, a receptor for vascular endothelial growth factor. Science 1992;255:989-991. )and to recombinant fusion proteins composed of the extracellular domain of VEGF receptors fused to the heavy chain of IgG (Aiello L.P., Pierce E.A., Foley E.D., et al. Suppression of retinal neovascularization in vivo by inhibition of vascular endothelial growth factor (VEGF) using soluble VEGF-receptor chimeric proteins. Proc. Natl. Acad. Sci. USA

1995;92:10457-10461.). Previous studies have shown that these binding sites exhibited high affinity (16-35 pM) and low capacity (2-7 fmol/mg protein) for [ ¹²⁵ I]VEGF. One important effector protein that is activated in response to certain growth factors is phospholipase C (PLC) (Lopez-Rivas A., Mendoza S.A., Nanberg E., Sinnett-Smith J., Rozengurt E. Ca2+ Mobilizing actions of platelet-derived growth factor differ from those of bombesin and vasopressin in Swiss 3T3 mouse cells. Proc. Natl. Acad. Sci. USA 1987;84:5768-5772). In general, a rise in [Ca ²⁺ ], is thought to result from the hydrolysis of phosphatidylinositol-4,5-bisphosphate by different isoforms of either PLC-β or PLC-γ to generate inositol-1,4,5-trisphosphate (IP3) and sn1 ,2-diacylglycerol (Blakely D.M., Corps A.N., Brown K.D. Bombesin and platelet-derived growth factor stimulated formation of inositol phosphates and Ca2+ mobilization in Swiss 3T3 cells by different mechanisms. Biochem. J. 1989;258:177-185.). This burst of IP3 release acts to mobilize intracellular calcium mobilization from the endoplasmic reticulum. Brock et al. (Hepler J.R., Jeffs R.A., Huckle W.R., et al. Evidence that the epidermal growth factor receptor and nontyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways. Biochem. J. 1990;270:337-344.) demonstrated that VEGF stimulation of human umbilical vein ECs

produces a three- to four-fold increase in intracellular free calcium concentration ([Ca ²⁺ ] _j ). Half-maximal increases in [Ca ² ^ were seen using highly-purified VEGF (-0.4 pM) isolated from guinea pig line 10 tumor cells or recombinant VEGF165 indicating that VEGF is a very potent EC agonist. Unlike the peak rise in [Ca ²⁺ ], seen following EC stimulation by thrombin or histamine, peak changes in [Ca ²⁺ ], following VEGF addition were slower (~ 60 sees) and were always preceded by a 10-15 second delay. Other growth factors, such as FGF, PDGF and epidermal growth factor (Hepler J.R., Jeffs R.A., Huckle W.R., et al. Evidence that the epidermal growth factor receptor and nontyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways. Biochem. J. 1990;270:337-344. Johnson R.M., Garrison J.C. Epidermal growth factor and angiotensin II stimulate formation of inositol 1 ,4,5- and inositol 1 ,3,4-trisphosphate in hepatocytes. J. Biol. Chem. 1987;262:17285-17293. Seetharam L., Gotoh N., Maru Y., Neufeld G., Yamaguchi S., Shibuya M. A unique signal transduction from FLT tyrosine kinase, a receptor for vascular endothelial growth factor VEGF. Oncogene. 1995;10:135-147.) also induce [Ca ²⁺ ], transients which are slow in onset and preceded by brief delays.

In addition to being a major growth factor in newly developing blood vessels, it is well-documented that VEGF can exert actions on quiescent ECs in culture or in intact arteries that may be independent of its mitogenic effects. As discussed previously, VEGF applied intradermally is a potent vascular permeability factor acting on post- capillary venules in vivo (Ziche M., Mobidelli L., Masini E., Granger H.J., Ledda F., Ziche M. Nitric oxide formation promotes DNA synthesis and cyclic GMP formation in endothelial cells from postcapillary venules. Biochem. Biophys. Res. Commu. 1993;192:1198-1203). However, recent studies have demonstrated saturable, displaceable [ ¹²⁵ I]VEGF

binding in situ on endothelium in several adult tissues and large vessels from normal animals (Stavri G.T., Zachary I.C., Baskerville P.A., Martin J.F., Erusalimsky J.D. Basic fibroblast growth factor upregulates the expression of vascular endothelial growth factor in vascular smooth muscle cells: Synergistic interaction with hypoxia. Circulation 1995; 92:11-14.). Over the past decade, it has been widely documented that EC stimulation can lead to secretion of various substances that affect blood vessel structure or function. In this regard, VEGF stimulates the release of von Willebrand Factor (Hepler J.R., Jeffs R.A., Huckle W.R., et al. Evidence that the epidermal growth factor receptor and nontyrosine kinase hormone receptors stimulate phosphoinositide hydrolysis by independent pathways. Biochem. J. 1990;270:337-344.), a large glycoprotein involved in platelet adhesion EC injury at sites of vessel damage.

Summary of the Invention

The present invention provides compounds of the formula:

[RLys(R)] ₂ X or R ₂ X

and X is any diamine.

The present invention also provides compositions containing a compound of formula 1 and a pharmaceutically acceptable carrier and methods of inhibiting cellular proliferation by administering a compound of formula 1 to a host in need of such treatment.

/ /

Description of the Drawings

FIG. 1 is a graph that shows the inhibition of [ ¹²⁵ I]VEGF binding for representative compounds of the present invention.

FIG. 2 is a graph that shows VEGF-stimulated vWF release from

HUVECs for representative compounds of the present invention.

FIG. 3 is a graph that shows the effects of various concentrations of the compound of Example 1 upon VEGF-stimulated intracellular calcium release.

FIG. 4 and FIG. 5 are two graphs that show the effects of the compound of Example 1 upon VEGF-stimulated HUVEC growth.

Detailed Description of the Preferred Embodiments

In the compounds of the present invention X can be any diamine of the formula HN-Y-NH where Y is any suitable spacer such as an alkylene group, or an aromatic group. Representative alkylene groups include pentylene and dodecylene. Representative aromatic groups include p-xylylene. Preferred examples of diamines include lysine (acid or ester), and H ₂ N(CH ₂ ) ₅ NH ₂ .

A preferred method of preparing representative compounds of the present invention is illustrated below:

l-λ

Lithocholic acid, HOBT NMM, EDC, DMF

Lys-OMe-2HCI RLys(R)-OMe

H ₂ N(CH ₂ ) ₅ NH ₂ , HOBT

RLys(R)-OR' NMM, EDC, DMF

[RLys(R)]HN NH[(R)LysR]

96%

R' = Me LiOH«H ₂ 0 THF/H ₂ 0, 0 °C R =H-*-

dine

BocLys(Boc)-OH, DIPEA, HBTU, DMF

Lys-OMe»2HCl [RLys(R)]Lys[RLys(R)]-OMe

99%

,

[RLys(R)]Lys[RLys(R)]-OMe

X = H 20%

S0 ₃ «Pyr, Pyridine +

X = S0 H -

[RLys(R)]Lys[RLys(R)]-OH

12%

In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention and a physiologically tolerable diluent.

The present invention includes one or more compounds of the present invention as described above formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, or the tike.

The compositions can be administered to humans and animals either orally, rectally, parenterally (intravenous, by intramuscularly or subcutaneously), intracisternally, intravaginally, intraperitoneally, locally (powders, ointments or drops), or as a buccal or nasal spray.

The compositions can also be delivered through a catheter for local delivery at the site of vascular damage, via an intracoronary stent (a tubular device composed of a fine wire mesh), or via a biodegradable polymer. The compositions may also be complexed to ligands, such as antibodies, for targeted delivery of the compositions to the site of smooth muscle cell proliferation.

The compositions are, preferably, administered via parenteral delivery at the local site of smooth muscle cell proliferation. The parenteral delivery is, preferably, via catheter.

Compositions suitable for parenteral injection may comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for

reconstitution into sterile injectable solutions or dispersions. Examples of suitable aqueous and non-aqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants.

These compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention of the action of microorganisms can be ensured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example sugars, sodium chloride and the like. Prolonged absorption of the injectable pharmaceutical form can be brought about by the use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Besides such inert diluents, the composition can also include adjuvants, such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.

Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or mixtures of these substances, and the like.

Dosage forms for topical administration of a conjugate of this invention include ointments, powders, sprays and inhalants. The active

component is admixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers or propellants as may be required. Ophthalmic formulations, eye ointments, powders and solutions are also contemplated as being within the scope of this invention.

The agents can also be administered in the form of liposomes. As is known in the art, liposomes are generally derived from phospholipids or other lipid substances. Liposomes are formed by mono- or multilamellar hydrated liquid crystals that are dispersed in an aqueous medium. Any non-toxic, physiologically acceptable and metabolizable lipid capable of forming liposomes can be used. The present compositions in liposome form can contain stabilizers, preservatives, excipients, and the like in addition to the agent. The preferred lipids are phospholipids and phosphatidyl cholines (lecithins), both natural and synthetic.

Methods of forming liposomes are known in the art. See, for example, Prescott, Ed., Methods in Cell Biology _x Volume XIV, Academic Press, New York, N.Y. (1976), p. 33 et seq.

The present invention is further directed to a method of treating diseases or disorders that are characterized by excessive smooth muscle proliferation or diseases or disorders characterized by excessive neovascularization and permeability comprising administering an effective amount of a compound of the present invention to a patient in need of such treatment. An effective amount of a compound of the present invention will vary according to the desired therapeutic effect, the route of administration, the duration of treatment and other factors. The total daily dose of a compound of the present invention that is administered to a patient may be in single or divided

dose(s). The specific dosage will vary depending upon a variety of factors including body weight, general health, sex, age, diet, time and route of administration, rates of absorption and excretion and the severity of the disease being treated.

The present invention may be further illustrated by the following representative examples:

Example 1 N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysine methyl ester: To a suspension of 658 mg of lysine methyl ester dihydrochloride (2.82 mmol) in 9.4 ml DMF at room temperature under a dry nitrogen atmosphere, 2.13 g of lithocholic acid (5.64 mmol), 0.84 g of 1- hydroxybenzotriazole (6.2 mmol), 1.95 mL N-methylmorpholine (17.8 mmol) and 1.19 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (6.2 mmol) were added sequentially in that order. The resulting mixture was stirred for 10 min, and 3 drops of deionized water were added followed by enough methanol (approximately 3 mL) to give a clear solution. The resulting solution was stirred overnight at room temperature and diluted with 200 mL dichloromethane. This mixture was washed with a saturated aqueous NaHC0 ₃ solution, water and brine. The organic phase was dried over MgS0 ₄ and filtered. The filtrate was concentrated in vacuo Xo give 3.16 g of viscous oil. This material was purified by silica gel chromatography, eluting with 80:18:2 chloroform:acetonitrile:methanol to yield 1.92 g of N-α-N-ε-di(3α- hydroxycholan-24-oyl)lysine methyl ester as a foam (78%). ¹ H NMR (400 MHz, CDCl ₃ ): d 0.63 (s, 6H), 0.90-0.92 (s overlapping d, 12 H), 0.95-1.90 (m, 56H), 1.93 (br d, J = 12 Hz, 2 H), 2.0-2.35 (m, 4H), 3.23 (m, 2H), 3.62 (m, 2 H), 3.73 (s, 3H), 4.57 (m, 1H), 5.66 (br. t, J = 6.6 Hz, 1 H), 6.16 (d, J = 8.1 Hz, 1H).

/ 7

N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24-oyl)lysine methyl ester: To a solution of 100 mg of N-α-N-ε-di(3α-hydroxycholan-24- oyl)lysine methyl ester (0.11 mmol) in 1.1 mL DMF at room temperature under a dry nitrogen atmosphere, 40 mg of sulfur trioxide dimethylformamide complex (0.26 mmol) was added at once. The resulting mixture was stirred at room temperature overnight and 0.06 mL pyridine (0.78 mmol) was added followed by an aqueous quench. The mixture was concentrated in vacuo and the residue was taken up in 50 mL methanol and 1.5 mL of a 0.1 M aqueous Na ₂ C0 ₃ solution was added. The resulting mixture was again concentrated in vacuo and the residue was taken up in methanol to give a suspension. The suspension was centrifuged and the centrifugate was concentrated in vacuo to give 150 mg of a white solid. This material was purified by reverse phase HPLC (semi-preparative column; 2 mL/min flow rate; 10- 60% acetonitrile in 0.01 M aqueous ammonium formate in 30 min; 214 nm) to yield 52 mg of N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24- oyl)lysine methyl ester as a white solid (46%). ¹ H NMR (400 MHz, CD ₃ OD): d 0.68 (s, 3H), 0.68 (s, 3H), 0.94 (s, 6H), 0.94-0.99 (d overlapping d, 6H), 1.0-1.96 (m, 56H), 2.01 (br. d, J = 10.2 Hz, 2 H), 2.05-2.35 (m, 4H), 3.15 (t, J = 6.6 Hz, 2 H), 3.69 (s, 3H), 4.27 (m, 2 H), 4.34 (dd, 9.2, 5.1 Hz, 1H).

N-α-N-ε-Di(3α-hydroxycholan-24-oyl)lysine: To a solution of 943 mg of N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysine methyl ester (1.07 mmol) in 5.4 mL THF at 0°C, 2.0 mL of a 0.75M aqueous solution of lithium hydroxide monohydrate (1.5 mmol) was added dropwise. The resulting solution was stirred at 0°C for 45 min and then 1 mL of 2N HCI followed immediately by 25 ml EtOAc. Methanol was added dropwise until all solid had dissolved and the mixture was poured into a separatory funnel containing 200 ml of EtOAc and 100 mL of 2N HCI. The organic phase was washed with deionized water and brine, then

dried over MgS0 ₄ and filtered. The filtrate was concentrated in vacuo to yield 920 mg of N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysine as a white powder (99%). This material was used without purification. ¹ H NMR (400 MHz, 9:1 CDCI ₃ :CD ₃ OD): d 0.59 (s, 6H), 0.84-0.90 (s overlapping pair of d, 12 H), 0.90-1.85 (m, 56H), 1.91 (br. d, J = 12.1 Hz, 2H), 1.97-2.3 (m, 4H), 3.15 (t, J = 6.8 Hz, 2H), 3.55 (m, 2H), 4.43 (dd, J = 8.0, 4.8 Hz, 1 H).

N,N'-Bis[N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysine]-1 ,5- diaminopentane: To a solution of 1.16 g of N-α-N-ε-di(3α- hydroxycholan-24-oyl)lysine (1.34 mmol) in 13 mL DMF at room temperature under a dry nitrogen atmosphere, 181 mg of 1- hydroxybenzotriazole (1.34 mmol), 0.29 mL N-methylmorpholine (2.68 mmol), 1.20 mL of a 0.56M solution of 1 ,5-diaminopentane in DMF (0.67 mmol) and 321 mg of 1-(3-dimethylaminopropyl)-3- ethyicarbodiimide hydrochloride (1.68 mmol) were added sequentially in that order. The reaction was stirred for 1 hour and analysis by TLC indicated incomplete conversion so an additional 0.08 mL N- methylmorpholine (0.67 mmol) and 80 mg of 1-(3- dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.42 mmol) were added. The mixture was stirred for an additional 30 min. and then diluted with 200 mL dichloromethane. This mixture was washed with a saturated aqueous NaHC0 ₃ solution, water and brine. The organic phase was dried over MgS0 ₄ and filtered. The filtrate was concentrated in vacuo to give 1.9 g of viscous oil. This material was purified by silica gel chromatography, eluting with 92:8 chloroform:methanol to yield 1.15 g of N,N'-bis[N-α-N-ε-di(3α- hydroxycholan-24-oyl)lysine]-1 ,5-diaminopentane as a foam (96%). ¹ H NMR (400 MHz, 9:1 CDCI ₃ :CD ₃ OD): d 0.55 (s, 12H), 0.82-0.84 (s overlapping d, 24 H), 0.84-1.82 (m, 118H), 1.87 (br. d, 12.0 Hz, 4H),

1.90-2.09 (m, 4H), 2.10-2.25 (m, 4H), 3.20-3.00 (m, 8H), 3.55 (m, 4H), 4.16 (t, J = 6.8 Hz, 2H).

N,N'-Bis[N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24-oyl) lysine]- 1 ,5-diaminopentane: To a solution of 888 mg of N,N'-bis[N-α-N-ε- di(3α-hydroxycholan-24-oyl)lysine]-1 ,5-diaminopentane (0.50 mmol) in 5 mL pyridine at room temperature under a dry nitrogen atmosphere, 477 mg of sulfur trioxide pyridine complex (0.88 mmol) was added at once. The resulting mixture was heated to 80°C overnight, cooled to room temperature and quenched with 22 mL of a 0.1 M aqueous

Na ₂ C0 ₃ solution. The resulting mixture was diluted with 100 mL water and concentrated in vacuo. The residue was taken up in methanol to give a suspension, which was filtered through Celite. The filtrate was concentrated in vacuo to give 900 mg of a light yellow solid. This material was purified by reverse phase silica gel chromatography, eluting with 1 :1 methanol:water increasing to 4:1 methano water to yield 690 mg of N,N'-bis[N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24- oyl)lysine]-1 ,5-diaminopentane as a white solid(63%). 'H NMR (400 MHz, DMSO): d 0.61 (s, 12H), 0.88 (br. s, 24H), 0.94-1.87 (m, 118H), 1.87-2.20 (m, 12 H), 2.99 (m, 8H), 3.95 (m, 4H), 4.14 (m, 2H), 7.66 (t, J = 5.5 Hz, 2H), 7.73 (dd, J = 10.3, 7.0 Hz, 4 H).

Example 2

N-α-N-ε-Bis[N-α-N-ε-di(tert-butoxycarbonyl)lysine]lys ine methyl ester: To a separatory funnel containing 2.65 g of N-α-N-ε-di(tetf- butoxycarbonyl)lysine dicyclohexylammonium salt (5.0 mmol) suspended in 20 mL ethyl acetate, 6.0 mL of an ice cold 0.50M aqueous sulfuric acid solution was added. The mixture was shaken until all the solid had dissolved. The aqueous phase was diluted with 10 mL ice cold water and then extracted twice with 20 mL ethyl acetate. The organic phases were combined, washed twice with 20 mL water,

dried over MgS0 ₄ and filtered. The filtrate was concentrated in vacuo to yield 1.87 g of the free acid as a white solid. To a mixture of 1.12 g of N-α-N-ε-di(tert-butoxycarbonyl)lysine (3.23 mmol) and 357 mg of lysine methyl ester dihydrochloride (1.53 mmol) in 15 mL DMF at room temperature under a dry nitrogen atmosphere, 1.6 mL diisopropylethylamine (9.2 mmol) and 1.23 g of 2-(1H-benzotriazole-1- yl)-1 ,1 ,3,3-tetramethyluronium hexafluorophosphate (3.23 mmol) were added sequentially. The resulting mixture was stirred at room temperature for 40 hours and was then diluted with 200 mL ethyl acetate. This mixture was washed with saturated aqueous NH ₄ CI, water (twice), 5% aqueous NaHC0 ₃ and brine. The organic phase was dried over MgS0 ₄ and filtered. The filtrate was concentrated in vacuo to yield 1.31 g of light yellow solid. This material was filtered through silical gel, eluting with 19:1 chloroform:methanol to yield 1.24 g of N-α- N-ε-bis[N-α-N-ε-di(tert-butoxycarbonyl)lysine]lysine methyl ester as a light yellow solid (99%). ¹ H NMR (400 MHz, CDCI ₃ ): d 1.40 (s, 9H), 1.41 (s, 9H), 1.42 (s, 18H), 1.20-1.85 (m, 18H), 2.94 (m, 1H), 3.09 (m, 4H), 3.53 (m, 1H), 4.04 (m, 1H), 4.26 (m, 1H), 4.38 (br. s, 1H), 4.74 (br. s, 1 H), 5.51 (d, J = 8.1 Hz, 1 H), 5.90 (d, J = 6.6 Hz), 6.85 (br. s, 1 H), 7.34 (br. s, 1H).

N-α-N-ε-Bis[N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysin e]lysine methyl ester: To a flask containing 131 mg of N-α-N-ε-bis[N-α-N-ε- di(fert-butoxycarbonyl)lysine]lysine methyl ester (0.16 mmol), 1.6 mL of a 1 :1 mixture of dichloromethane:trifluoroacetic acid was added. The resulting solution was stirred for 20 min. and was then concentrated in vacuo. The residue was washed four times with 2 mL ether and was then taken up in 1.6 mL DMF and placed under a dry nitrogen atmosphere at room temperature. To this solution, 0.17 mL diisopropylethylamine(0.96 mmol), 301 mg of lithocholic acid (0.80 mmol) and 303 mg of 2-(1 H-benzotriazole-1-yl)-1 ,1 ,3,3- 1

tetramethyluranium hexafluorophosphate (0.80 mmol) were added sequentially in that order. The resulting mixture was stirred at room temperature overnight and was then diluted with 200 mL ethyl acetate. This mixture was washed with saturated aqueous NH ₄ CI, water, saturated aqueous NaHC0 ₃ and brine. The organic phase was dried over MgS0 ₄ and filtered. The filtrate was concentrated in vacuo to yield 500 mg of white solid. This material was purified by silical gel chromatography, eluting with 9:1 chloroform:methanol to yield 271 mg of N-α-N-ε-bis[N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysine]l ysine methyl ester as a white solid (92%). ¹ H NMR (400 MHz, 9:1

CDCI ₃ :CD ₃ OD): d 0.54 (s, 12H), 0.83 (s overlapping m, 24H), 0.86-1.82 (m, 126 H), 1.87 (br. d, J = 12.1 Hz, 4H), 1.91-2.07 (m, 4H), 2.08-2.24 (m, 4H), 3.09 (m, 4H), 3.50 (m, 4H), 3.64 (s, 3H), 4.22 (m, 2H), 4.36 (q, J = 7.2 Hz, 1 H).

N-α-N-ε-Bis[N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24 - oyl)lysine]lysine and N-α-N-ε-bis[N-α-N-ε-di(3-0-sulfo-3α- hydroxycholan-24-oyl)lysine]lysine methyl ester: To a solution of 100 mg of N- -N-ε-bis[N-α-N-ε-di(3α-hydroxycholan-24-oyl)lysine]lysin e methyl ester (0.146 mmol) in 1.5 mL pyridine at room temperature under a dry nitrogen atmosphere, 140 mg of sulfur trioxide pyridine complex (0.88 mmol) was added at once. The resulting mixture was heated to 80°C overnight, cooled to room temperature and quenched with 6 mL of a 0.1 M aqueous Na ₂ C0 ₃ solution. The resulting mixture was concentrated in vacuo and the residue was taken up in methanol to give a suspension. The suspension was filtered through Celite and the filtrate was concentrated in vacuo to give 312 mg of a white solid. This material was purified by reverse phase HPLC (semi-preparative column; 2 mlJmin. flow rate; 20-60% acetonitrile in 0.01 M aqueous ammonium formate in 30 min.; 214 nm) to yield 40 mg of N-α-N-ε-bis[N- α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24-oyl)lysine]lysine as a white

solid (12%) and 65 mg of N-α-N-ε-bis[N-α-N-ε-di(3-0-sulfo-3α- hydroxycholan-24-oyl)lysine]lysine methyl ester as a white solid (20%).

N-α-N-ε-bis[N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24 - oyl)lysine]lysine: ¹ H NMR (400 MHz, CD ₃ OD) d 0.68 (s, 12 H), 0.96(m, 24H), 1.00-1.98 (m, 118H), 2.01 (d, J = 9.9 Hz, 4H), 2.05-2.35 (m, 8H), 3.15 (m, 6H), 4.20-4.33 (m, 7H).

N-α-N-ε-bis[N-α-N-ε-di(3-0-sulfo-3α-hydroxycholan-24 - oyl)lysine]lysine methyl ester: Η NMR (400 MHz, CD ₃ OD) d 0.68 (s, 12H), 0.96 (m, 24H), 1.00-1.97 (m, 118H), 2.01 (d, J = 11.3 Hz, 4H), 2.05-2.36 (m. 8H), 3.15 (m, 6H), 3.70 (s, 3H), 4.20-4.4 (m, 7H).

The present invention is also illustrated by the following in vitro and in vivo biological data that is generated pursuant to the hereinafter described protocols:

Biological Assays

Methods:

[ ¹²⁵ I]VEGF binding to f/MgG fusion proteins:

The seven loop ectodomain of the human fit receptor was cloned into a vector containing the heavy chains of the mouse lgG2a for use in a baculovirus expression system. Insect cells (s/-21) were grown on T- 175 flasks (Falcon) in SFM-900 II serum-free growth media for 2-3 days following infection with virus, lmmulon-4 plates were coated overnight at 4°C with 100-ng/well of goat-antimouse lgG2a specific antibodies diluted in PBS. Wells were washed 3 x PBS containing 0.05% Tween- 20 then 3 x PBS. Wells were blocked with 1% BSA in PBS for 1 hour at room temperature. Wells were again washed 3 x PBS Tween-20 then

3 x PBS. Conditioned media from infected cells was added to the wells

- 3

and allowed to incubate for 2-4 hours at 37°C (or overnight at 4°C). The wells were then washed 3 x PBS Tween-20 then 3 x PBS. Antagonists were routinely screened at 10 μM and were added simultaneously with 4 ng/ml [ ¹²⁵ I]VEGF (Biotechnology Technologies, Inc.) in a total volume of 0.15-ml/well. Nonspecific binding was determined as the amount of [ ¹²⁵ I]VEGF bound in the presence of 800 ng/ml VEGF. After 1 hour at room temperature, the wells were washed 1 x 400-μl PBS then 2 x 200-μl PBS followed by addition of 160-μl/well of PBS containing 1% Triton X-100. After 15 minutes at room temperature, the wells were separated and added to vials containing scintillation fluid. The radioactivity was determined by scintillation spectroscopy and the results were expressed as the percentage inhibition of total specific counts in control wells.

[ ¹²⁵ I]VEGF binding to HUVECs:

HUVECs are plated at 12500 cells/cm ² in gelatin coated 48-well plates (Falcon) and maintained in M199/15% FBS/10 μM thymidine/2 μM glutamine/100 U/ml penJ100 ug/ml strep, containing 50ug/ml endothelial cell mitogen (BTI) and 100 ug/ml heparin for 3-5 days prior to binding. Cells are used at 80-90% confluence. Cells are washed 2 x 500-μl binding buffer (M199/25μM HEPES (pH 7.4) + 0.1 % BSA. Antagonists are mixed with [ ¹²⁵ I]VEGF for 30 minutes at 37°C prior to addition to the cells. After 60 minutes at room temperature, the wells were washed 3 x 500-μl with PBS. Then 160-μl of PBS containing 1% Triton X-100 was added to each well. After 15 minutes at room temperature, the cell lysates were collected into scintillation vials and each well was washed with an additional 160-μl of PBS/Triton X-100. The washes were combined with the original lysates and scintillation cocktail was added. The radioactivity was determined by scintillation spectroscopy. Antagonist concentration response curves were run

against 4 ng/ml [ ¹²⁵ I]VEGF and the data was expressed as a percentage of control wells.

von Willebrand Factor release: VEGF-stimulated release of von Willebrand Factor (vWF) was measured by capture ELISA (American Diagnostica, Inc.) from HUVECs. HUVECs were plated on gelatin coated 35-mm dishes (Falcon) at 8000 cells/cm ² and maintained as described above. Cells were used at 80-90% confluence. Cells were washed with 2 x 500-μl with PBS then 800-μl /dish of PBS containing antagonists were added. After 15 minutes at 37°C, the media was removed then replaced with PBS containing antagonists at the same concentration with or without 30 ng/ml rhVEGF165 (R&D Systems). After 10 minutes, the media was removed the kept at 4°C until analyzed by ELISA and the results expressed as percentage of control.

VEGF-stimulated increases of intracellular calcium:

HUVECs were grown and maintained as describe above on 100-mm gelatin coated plates (Falcon) to 80-90% confluence. Cells were removed with PBS (-Ca ² 7Mg ²⁺ ) containing 2 μM EDTA. After washing with PBS/0.1% BSA cells were loaded with 1μM Fura-2/AM in PBS(+Ca ² 7Mg ²⁺ ) for 30 minutes at 37°C. Cells were washed 1x with PBS/0.1% BSA then resuspended in PBS/0.1% BSA. Cells were routinely treated with various concentrations of antagonists for 2 minutes prior to the addition of 10 ng/ml rhVEGFI 65 (R&D Systems). Peak Ca ²⁺ levels were determined and the data was presented as a percentage from basal Ca ²⁺ levels.

Inhibition of VEGF-stimulated HUVEC growth: HUVECs are plated at 12500 cells/cm ² on gelatin coated 48-well plates (Falcon). The following day, the cells are placed in a defined

AS

media of M 199/15% FBS/10 μM thymidine and 10 ng/ml rhVEGFI 65 with or without various concentrations of antagonist. After four days in culture, the cells were removed from the well with 100-μl of 0.5% trypsin and counted with a Coulter cell counter. Data is presented as the number of cells/cm ² .

[ ¹²⁵ I]FGF2 binding: a). Binding to flg-\gG fusion proteins:

The two loop ectodomain of the human / -receptor was cloned into a vector containing the heavy chains of the mouse lgG2a for use in a baculovirus expression system. Insect cells (sf-21) were grown on T- 175 flasks (Falcon) in SFM-900 II serum-free growth media for 2-3 days following infection with virus, lmmulon-4 plates were coated overnight at 4°C with 100-ng/well of goat-antimouse lgG2a specific antibodies diluted in PBS. Wells were washed 3 x PBS containing 0.05% Tween- 20 then 3 x PBS. Wells were blocked with 1% BSA in PBS for 1 hour at room temperature. Wells were again washed 3 x PBS/Tween-20 then 3 x PBS. Conditioned media from infected cells was added to the wells and allowed to incubate for 2-4 hours at 37°C (or overnight at 4°C). The wells were then washed 3 x PBS/Tween-20 then 3 x PBS. Antagonists were routinely screened at 10 μM and were added sequentially after addition of 300 pg/ml [ ¹²⁵ I]FGF2 (Biomedical Technology, Inc.) in a total volume of 0.10-ml/well. Nonspecific binding was determined as the amount of [ ^1Z5 I]FGF2 bound in the presence of 300 ng/ml FGF2. After 90 minutes at room temperature, the wells were washed 3 x 200-μl PBS then the wells were separated and added to vials containing scintillation fluid. The radioactivity was determined by scintillation spectroscopy and the results were expressed as the percentage inhibition of total specific counts in control wells. Similar protocols were used for cross screening against EGF, TNF-a, HB-EGF, and FGF1. For TNF-a, the ectodomain was captured with an anti-

human TNF receptor antibody. Labeled ligands were all used at 300 pg/ml and nonspecific binding was defined as counts bound in the presence of a 1000-fold excess of unlabeled ligand.

b). [ ¹²⁵ I]FGF2 binding to A431/bA1 cells:

A431/βA1 cells are plated at 8000 cells/cm ² on 24-well plates (Falcon). Cells are used at 80-90% confluence. Cells are washed 2 x 500-μl binding buffer (M199/25μM HEPES (pH 7.4) + 0.1% BSA. Antagonists were added sequentially following the addition of 300 pg/ml [ ¹²⁵ I]FGF2 in a total volume of 0.3-ml/well. After 90 minutes at room temperature, the wells were washed 3 x 500-μl with PBS then with 250 ug/ml heparin in PBS. The heparin wash was collected into scintillation vials as a measure of low affinity HSPG binding. Cells were then washed with PBS followed by the addition of PBS containing 1% Triton X-100. After 15 minutes at room temperature, the cell lysates were collected into scintillation vials, each well was washed with an additional 160-μl of PBS/Triton X-100 and each wash was combined with the original lysate as a measure of high affinity receptor binding. Scintillation cocktail was added and the radioactivity wasdetermined by scintillation spectroscopy. The data is expressed as a percentage of control wells.

The results of these in vitro assays are set forth in Table 1 and

FIGS. 1-5.

In Vivo Data

Granulation tissue chamber model for diabetic vascular dysfunction:

Relevance of Model. Vascular hemodynamic (i.e., blood flow changes) and permeability changes are clinical hallmarks of diabetic vascular

disease (Williamson J.R., Chang K., Frangos M., Hasan K.S., Ido Y., Kawamura T., Nyengaard J.R., Van den Enden M., Kilo C, Tilton R.G. Hyperglycemic pseudohypoxia and diabetic complications. Diabetes 1993;42:801-813. Ruderman N.B., Williamson J.R., Brownlee M. Glucose and diabetic vascular disease. FASEB Journal 1992;6:2905- 2914.). Vasodilatation and increased blood flow are also characteristic vascular responses to tissue hypoxia. The diabetic milieu is characterized by numerous systemic hormonal and metabolic imbalances. However, studies in both humans and experimental animals have provided strong support for the hypothesis that diabetes- induced vascular changes are the direct result of hyperglycemia. Although considerable work has been done using cultured cells to examine the effects of glucose and various agents on endothelial function in vitro, extrapolation of these findings to intact tissue is often difficult. The granulation tissue chamber provides a simple model to investigate the direct effects of elevated glucose and growth factors on early disturbances in vascular cell function in vivo (Williamson J.R., Kilo C. Granulation tissue: a new model for studies of vascular complications of diabetes. Hormone and Metabolic Res. 1985; 15:27- 31. Wolf B.A., Williamson J.R., Easom R.A., Chang K., Sherman W.R., Turk J. Diacylglycerol accumulation and microvascular abnormalities induced by elevated glucose levels. J. Clin. Invest. 1990;87:31-38.). Previous experimental results obtained using this model system to examine glucose-induced vascular dysfunction have been demonstrated to mimic findings obtained in whole animals with diabetes and to reflect biologically relevant clinical findings observed in diabetic humans.

Male, Sprague-Dawley rats (250 g) are anesthetized with sodium pentobarbital (4.6 mg/100 g body weight; diabetic rats receive one half dose), hair is shaved from the back upper torso and 2-cm skin

circles are removed on either side of the mid-line. The flanged base of a plastic, granulation tissue chamber is sutured to the skin at the wound margins with the skin overlapping the flange to prevent re- epithelialization of the granulation tissue inside the chamber. Granulation tissue containing new blood vessels forms on the surface of the exposed fascia inside the chamber. Stainless steel screwcaps can be removed to add pharmacologic agents to the newly formed blood vessels inside the chamber.

Seven to 14 days after chamber placement, 1 ml of Hepes buffer

(25 μM Hepes, pH 7.4, 100 μg/ml penicillin G, 10 μg/ml gentamicin) containing one of the following reagents is added to each chamber: a) 5 μM D-glucose, b) 30 μM D-glucose, and c) 30 μM glucose + the product of Example 1. These solutions are added twice daily (8:00 a.m. and 4:00 p.m.) for seven days. Within an hour of the last treatment, albumin clearance and blood flow are assessed as detailed below.

Measurement of albumin permeation and blood flow in granulation tissue: Rats are anesthetized with Inactin (100 mg/kg body weight, i.p.), and core body temperature is maintained at 37°C. The left femoral vein (for tracer injection), left iliac artery (for reference sample withdrawal at a constant rate of 0.05 ml/min.). right subclavian artery (for blood pressure monitoring), and right carotid artery are cannulated with polyethylene tubing (0.58 mm i.d.) filled with heparinized saline (400 U heparin/ml). The tip of the right carotid artery cannula is placed in the left ventricle of the heart and is used for injecting microspheres. Vascular albumin permeation (μg plasma/g tissue wet weight/min.) is quantitated using a double isotope-dilution technique. ¹²⁵ I-BSA is used to quantify vascular albumin permeation after 10 min. of tracer circulation, while ¹³¹ I-BSA serves as a plasma volume marker to correct

¹²⁵ I-BSA tissue activity for tracer contained within vessels. Blood flow (ml/minJ g wet weight) is assessed by injecting 46Sc-labeled microspheres (15 μm diameter). Following injection of labeled albumin and microspheres, radioactive tracer activities in the granulation tissue and arterial plasma samples are counted in a g counter, respectively. A quantitative index of ¹²⁵ l-albumin clearance in granulation tissue is calculated as previously described [Diabetes 38:1258-1270, 1989; JCI 8.5:1167-1172, 1990]. Blood flow is calculated using conventional techniques (Pugliese G., Tilton R.G., Speedy A., Santarelli E., Eades D.M., Province M.A., Kilo C, Sherman W.R., Williamson J.R.

Modulation of hemodynamic and vascular filtration changes in diabetic rats by dietary myo-inositol. Diabetes 1990;39:312-322.).

3Z5

The results of these tests are set forth in Tables 2 and 3 below.

Table 2

Effect of Product of Example 1 to Inhibit Glucose-induced Permeability Changes

¹²⁵ l-albumin Permeation (μg/min/g wet tissue weight)

5 mM 30 mM 5 mM 30 mM 30 mM

Glucose Glucose glucose glucose glucose

+ 100 pM +VEGF +

VEGF Ab TBC1635

Mean _{1 g2 43β 4?0 234 1 Q3}

^{S D} 24 34 56 32 20 ⁿ 17 17 7 7 3

Table 3

Effect of Product of Example 1 to Inhibit Glucose-induced Blood Flow Changes

Blood Flow

(μl/min/g wet weight tissue)

5 mM 30 mM 5 mM 30 mM 30 mM

Glucose Glucose glucose glucose glucose

+ 100 pM +VEGF +

VEGF Ab TBC1635

Mean 160 450 481 233 153

S D 18 24 60 71 22 n 17 16 7 7 3

3^

Solubility of Product of Example I

Various bile salts and sterol-containing compounds have previously been found to be soluble in 45% aqueous 2-hydroxypropyl- γ-cyclodextrin (HPCD) ¹ . The product of Example 1 was found to be soluble in HPCD at a concentration of up to 30 μM (approximately 66 mg/ml). However, such a solution of product, when diluted in aqueous buffers, was inactive in various assays for bFGF and VEGF activity, including binding, and Ca ²⁺ and PLCγ signaling. Interestingly, when a 30 μM solution of the product of Example 1 in 45% aqueous HPCD was diluted in serum, it regained about one half its original activity in the bFGF binding assay (Table 4A). Furthermore, when diluted in culture medium containing 10% fetal bovine serum, it inhibited serum- stimulated proliferation of human aortic smooth muscle cells (Table 4B).

Table 4A: Inhibition of bFGF-binding to FGFR1-lgG fusion protein

Compound Solvent/Diluent % Inhibition

10μM Example 1 DMSO/DMSO 83 DMSO/Water 75 HPCD/Water 7 HPCD/Serum 40

Table 4B: Inhibition of Serum-stimulated HASMC Proliferation

Compound Solvent/Diluent % Inhibition

30μM Example 1 DMSO/DMEM/10%FBS 72 HPCD/DMEM/10%FBS 63

¹ De Caprio, J., Yun, J., and Javitt, N. B. 1992. Bile acid and sterol solubilization in 2-hydroxypropyl-γ-cyclodextrin. J. Lipid Research,

33:441 -443.